Current Topics in Biotechnology & Microbiology

Dhingra, H. et al.
2011
Current Topics in Biotechnology & Microbiology
Narayan Hari
1
LAP LAMBERT ACADEMIC PUBLISHING AG & CO. KG, DUDWELLER LANDSTR, GERMANY
Dhingra, H. et al. 2011
CURRENT TOPICS IN BIOTECHNOLOGY AND MICROBIOLOGY

Edited by
Dr. Harish Kumar Dhingra

Ph.D. (Biotechnology & Molecular Biology) Assistant Professor in Science, Mody Institute of Technology & Science Laxmangarh Distt. Sikar Rajasthan, India
Dr. Prabhat Nath Jha

Assistant Professor Dept. of Biosciences Birla Institute of Technology and Science Pilani, -333 031 Rajasthan, India
Ms. Pratima Bajpai

M. Tech, M.Sc. (Biotechnology) Assistant Professor in Science, Mody Institute of Technology & Science Laxmangarh Distt. Sikar Rajasthan, India
2
Acknowledgement First of all, I would like to express my sincere gratitude to the almighty who has made me capable for preparing this manuscript. The completion of this book has come through the overwhelming help that came from many people whom mentioning each one may not be exhaustive. I wish to express my sincere gratitude to all the people who offered their kind help and guidance all the time till completion of this manuscript. I deeply appreciate the efforts of co-editors of this manuscript, Dr. Prabhat Nath Jha, Assistant Professor, Dept. of Biosciences, Birla Institute of Technology and Science, Pilani and Ms. Pratima Bajpai, Assistant Professor in Science, Mody Institute of Technology & Science, Laxmangarh, Distt. Sikar, Rajasthan whose help, guidance and experience, has assisted me in production of this book. I would also like to thank all the authors of different chapters included in this book for providing me the data of this manuscript. My sincere acknowledgements are due to Prof. Dr. S. Baijal, Dean FASC, MITS University, for her kindness and inspiration through out the course. My special gratitude also goes to R. K. Gaur (HOD, Department of Science, FASC, MITS University) for his immense support and help. I would like to pay special thanks to my wife for giving me inspiration for the writing of the manuscript. My heart felt gratitude to all my friends who helped me in completing this project. Last but not least, I thank all my family members for all the support during completion of this manuscript.
Dr. Harish Kumar Dhingra
3
Preface
Welcome to Current Topics in Biotechnology and Microbiology. Our book includes various chapters related to recent branches of Biotechnology and Microbiology like Nanobiotechnolgy, genetic engineering and agricultural as well as medical microbiology etc. We have tried to include the applied part of these brances, how knowledge of these branches can be helpful to the society, to agriculture and to medical fields etc. Science is experimentation to determine the evidences behind every truth and explore these evidences and the results, therein, for the welfare of the society. Exploitation of the biological agents or their components for the welfare of the human being can be evidenced with the advent of Biotechnology and Microbiology. The rapid pace of discovery in the field of Biotechnolgy and in its associated branches makes it most exciting of all the natural sciences. This field is having application in all fields of life viz. medical, agriculture, industry, environmental quality management and waste management. One of the main goals of this book is to fascinate with the living world and their capabilities, to introduce the dynamic nature of science by conveying the passion for biological research and to excite the students about the opportunities in this field. It was not possible to include all the applications and all the recent topics of Biotechnology & Microbiology; however we will try to include more on that in our future publications.
Dr. Harish Kumar Dhingra Dr. Prabhat Nath Jha Ms. Pratima Bajpai
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Contents
Chapter No. I.
Title & Authors Name
Page No.
BIOPESTICIDAL NEMATODES AND MOLECULAR TECHNIQUES INVOLVED FOR THEIR CHARACTERIZATION
7-36
Jola Dubey, B. N. Tiwary and Sudershan Ganguly II. III.

PROBIOTICS: POTENTIAL HEALTH TOOL Pratima Bajpai, Anamika Pandey and Harish Dhingra TISSUE ENGINEERING AND ITS THERAPEUTIC APPLICATION Atul K. Singh, Chandrabhan Seniya, Sharad S Lodhi, Sudhanshu Singh, G.J. Khan and, Jaykar Jha RNA INTERFERENCE: MOLECULAR MECHANISM AND THERAPEUTIC APPLICATIONS G.J.Khan, Chandrabhan Seniya, Prabhat N. Jha, Atul K. Singh, Jaykar Jha BIOTECHNOLOGICAL INTERVENTIONS TO COMBAT ABIOTIC STRESS IN PLANTS
37-45 46-69
IV.
70-92
V.
93-116
Rajesh Mehrotra, Uthra Balasubramaniyan, Purva Bhalothia, Bhavya Ravi and Sandhya Mehrotra VI.
SYNTHESIS AND APPLICATION OF MAGNETIC NANOPARTICLES: A BIOLOGICAL PERSPECTIVE Arpit Bhargava, Navin Jain and Jitendra Panwar ADVANCES IN NANOBIOTECHNOLOGY FOR AGRICULTURE Vinod Saharan IN SILICO ANALYSIS OF SEQUENCE VARIATIONS IN CHOLERA TOXIN B SUBUNIT AND THEIR BINDING WITH CARBOHYDRATE LIGANDS MHU Turabe Fazil, Sunil Kumar and Durg V singh STRUCTURE-BASED DRUG DESIGN APPROACHES IN DRUG DISCOVERY Pramod K. Yadav, Satendra Singh, Chandrabhan Seniya, Atul K. Singh, G. J. Khan NITROGEN ACQUISITION IN CYANOBACTERIA Pradeep kumar L and Vani B
117-155
VII.
156-167
VIII.
168-177
IX.
178-195
X.
196-216
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XI.
PLASTIDS IN Plasmodium SIGNIFICANCE IN MALARIA TREATMENT Vishal Saxena and Shilpi Garg THE ENIGMA OF Helicobacter pylori INFECTION AND GASTROINTESTINAL DESEASES Sushil Kumar, Shailesh K. Shahi, Ashok Kumar and V.K. Dixit TOXICITY OF SILVER NANOPARTICLES: THE FLIP SIDE OF THE COIN Madhu Yashpal, Brigesh Shahare and Gajendra Singh SUSPENSION ARRAY TECHNOLOGY: A MULTIPLEXING APPROACH IN IMMUNODIAGNOSTICS
217-236
XII.
237-267
XIII
268-287
XIV
288-310
Manoj Kumar and Subhash V. Kapre XV

CYANOTOXINS: PHARMACOLOGICAL AND ECOLOGICAL EFFECTS R. Ashwin Kumar, S.K. Verma
311-329
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Chapter I
BIOPESTICIDAL NEMATODES AND MOLECULAR TECHNIQUES INVOLVED FOR THEIR CHARACTERIZATION Jola Dubey1, B. N. Tiwary1 and Sudershan Ganguly2
1
Department of Biotechnology, Guru Ghasidas Vishwavidyalaya, Bilaspur, Chhattisgarh

2
EPN Genomics Laboratory, Division of Nematology, I.A.R.I., New Delhi
Corresponding Author: Jola Dubey, Email: pandeyjola@rediffmail.com
ABSTRACT: Entomopathogenic nematodes belonging to the families Steinernematidae and

Heterorhabditidae have tremendous biocontrol potential against several insect pests. These nematodes carry mutualistic bacteria of the genus Xenorhabdus for Steinernematidae and Photorhabdus for Heterorhabditidae. The infective juveniles (IJs) or dauer juveniles containing bacterial symbiont infect the host through natural openings or inter- segmental areas and release the bacterium in hemocoel where it propagates, causes septicemia and kills the host within 24-48 h. It also protects the cadaver from colonization by other micro-organisms. These nematodes being compatible with recommended doses of several pesticides and non target microorganisms can suitably be incorporated in the integrated pest management (IPM) schedules. Unlike other soil nematodes, the taxonomic study of these nematodes is very complex, involving morphological, biological, biochemical and molecular parameters. Gel diffusion and immunoelectrophoresis techniques is used to differentiate Steinernema isolates. Isozymic patterns may prove to be excellent tool for preliminary identification of newly isolated strains during the routine surveys. The polymerase chain reaction (PCR) in combination with restriction fragment length polymorphism (RFLP) of internal transcribed spacer (ITS) region of ribosomal DNA, is being used in the descriptions of new species of Steinernema and deducing their phylogenetic relationships with other species. Recently, the DNA sequences of ITS region of ribosomal DNA, have been found to be more useful for detailed information about variation within and among nematode species than PCR-RFLP approaches. Mitochondrial DNA (mtDNA) is also now widely used in studying speciation, phylogenetic relationships and molecular evolution.
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INTRODUCTION
In recent years, a large number of synthetic pesticides including insecticides and fungicides have been banned in the western world because of their undesirable attributes such as high toxicity, long degradation periods, accumulation in the food chain and extension of their influence to destroy both useful and harmful pests [1]. In the developing countries such as in India, these are still being used despite their detrimental effects [2,3]. It has been estimated that hardly 0.1% of the agrochemicals used in crop protection reach the target pest, leaving the remaining 99.9% to enter the environment to cause hazards to non-target organisms including humans [4]. On the other hand, many plant pathogenic micro-organisms have developed resistance against known chemical pesticides [5,6,7]. In addition, investigation in the area of chemical control has been slowed down due to the rising cost of production and sales of the limited fungicides available in the market, which the poor subsistent farmers are unable to afford. Basic research for over more than 40 years in biology and biochemistry has made it possible to envisage not only how new pesticides may be synthesized but also a completely new approach for the protection of plants using secondary plant products or microorganisms which may be toxic to a specific pest yet harmless to man. Indias vast potential of exporting agri-products will be successful only when we produce agri-products through minimum use of chemical pesticides or no pesticides. Environmental pollution and toxic hazards due to haphazard use of chemical pesticides have forced the scientists to look for safe and environmentalfriendly alternative strategies. Thus there is an all-round compulsion to go in for bio-rational alternative arsenals which can be ecofriendly and benign to environment. Bio-control agents are the best alternative available today. Microbial control envisages the use of bacteria, viruses, fungi and nematodes or their biproducts in disease and pest control. Research on the use of microorganisms i.e. Bacteria, Fungi, viruses and nematodes in disease and pest management during the past couple decades has led to the development of several microbial pesticides. It is now feasible to replace many of the highly toxic insecticides and fungicides by bio-pesticides which are safe and non hazardous to the environment, such as the use of microorganisms including entomopathogenic nematodes (EPN) [8]. The term entomopathogenic is being widely used exclusively for steinernematid and heterorhabditid nematodes which kill the insect host quickly i.e. within a day or 2 after the 8
invasion [9,10,11]. EPNs have emerged as suitable biocontrol agents for eco-friendly pest management [12,13,14,,15]. These are soil-inhabiting, lethal insect parasitoids that belong to the Phylum Nematoda, commonly called roundworms. Although many other parasitic nematodes cause diseases in plants, livestock, and humans, entomopathogenic nematodes (EPNs), as their name implies, only infect insects and produce harmful effects on insect- pests. EPNs live inside the body of their host, and so they are designated as endoparasitic. The nematodes of the families Steinernematidae and Heterorhabditidae, though highly pathogenic, are facultative parasites due to their capability to complete their life-cycle on the symbiotic bacteria without the insect host. However, the pathogenicity, host searching behaviour, and survivability of different nematode species are varied making them suitable in biological control programs [16]. They infect many different types of soil insects, including the larval forms of butterflies, moths, beetles, and flies, as well as adult crickets and grasshoppers [17]. EPNs have been found in all inhabited continents and a range of ecologically diverse habitats, from cultivated fields to deserts. EPNs have tremendous biological control potential for managing a wide range of insect pests of crops as well as some household pests. These nematodes along with their symbiotic bacteria kill the host within a day or two and ultimately emerge from the insect cadaver in millions to infect the other insect host. They have very high potentialities as bioagents against the insect pests, due to their several attributes like parasitoids / predators, they have chemoreceptors and are motile, like pathogens, they are highly virulent, killing their host within a day or two, active host seeking, can be cultured easily in vitro, have a high reproductive potential, have broad host range, safe to the plant and animal health, and the environment, can be easily applied using standard spray equipments, have potential to recycle in environment; compatible with many chemical pesticides,easy mass production, long term efficacy. It has also been established that EPNs are safe from the point of view of human health and environment and hence exempted from registration in USA and some other countries [18,19]. In some of the developed nations, EPNs are already being used commercially for managing insect pests of turf grass, fruit trees, mushrooms and other field crops [17]. However, India is yet to exploit this area of high biological control potential. There are several species of EPNs used around the world against a variety of pests in niche markets (e.g. fungus gnats in nurseries, hydroponics, and mushroom; weevils on 9
ornamentals, strawberries, cranberries, citrus and bananas; scarabs of turf, ornamentals and blue berries; cutworms, webworms, billbugs and mole crickets; termites in wooden articles and trees; peach borer moth in apples and carpenter worm in shade trees in China, and fig trees in USA) [20]. Recently, out of 13% bio-insecticide sale in industrialized countries, EPN sale was only second to Bacillus thuringiensis at 80% [21]. Considerable progress has also been made during the last 20 years on the subject dealing with taxonomy, biology, genetics, ecology, host range, production, application technologies, laboratory and field trials and commercialization of EPNs and their symbiotic bacteria resulting in over 2000 publications. This aspect was briefly reviewed by Friedman [17], Kaya and Gaugler [13], Akhurst [22], and more comprehensively by Gaugler [15] and Grewal et al. [23,24]. The entomopathogenic activity of steinernematid and heterorhabditid species has been documented against a broad range of insect pests in a variety of habitats [13]. They have been used inundatively in a number of high value cropping systems [25]. Various tests against mammals (mice, rabbits and monkeys) [26], earthworms and other non-target organisms including plants have shown that the EPNs are harmless and of course nonpolluting [27]. These nematodes have been produced in large scale in various countries for over 10 years and large numbers of production workers have been exposed without any adverse effects being recorded. The Environment Protection Agency (EPA) in the India, USA, Australia and many European countries has exempted EPNs from registration [18,19]. When environmental benefits including safety for humans and other non-target organisms, reduction of pesticide residues in food, increased activity of other natural enemies, and increased biodiversity in managed ecosystems are taken into account, the advantages are numerous. Till date, there are two genera listed under Steinernematidae Steinernema and Neosteinernema, with 67 species in the former and only one species in the latter [28] have been characterized. Under Heterorhabditidae, there is only one genus Heterorhabditis with only 18 known species. Some of the important EPN species belonging to Steinernema and Heterorhabditis with their original localities and sources of isolation are listed in the Table 1.
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Table 1: EPN species of Steinernema and Heterorhabditis with their original localities and sources of isolation
NEMATODE SPECIES Heterorhabditis Poinar 1976 1. H. argentinensis Stock 1993 2. H. bacteriophora Poinar 1976 3. H. brevicaudis Liu 1994 4. H. hawaiiensis Gardener et al. 1994 5. H. indica Poinar et al. 1992 6. H. marelata Liu & Berry 1996 7. H. megidis Poinar et al. 1987 8. H. zealandica Poinar 1990 Steinernema Travassos 1927 1. S. abbasi Elawad et al. 1997 2. S. affine (Bovien 1937) Wouts et al.1982 3. S. arenarium Wouts et al. 1982 4. S. bicornutum Tallosi et al. 1995 5. S. carpocapsae (Weiser 1955)Wouts et al. 1982 6. S. caudatum Xu et al. 1991 7. S. ceratophorum Jian et al. 1997 8. S. cubanum Mracek et al. 1994 9. S. feltiae (Filipjev 1934) Wouts et al.1982 10. S. glaseri (Steiner 1929) Wouts et al. 1982 11. S. intermedium (Poinar 1985) Mamiya 1988 12. S. karii Waturu et al. 1997 13. S. krausei (Steiner 1923) Travassos 1927 14. S. kushidai Mamiya 1988 15. S. longicaudatum Shen & Wang 1991 16. S. monticolum Stock et al. 1997 17. S. neocurtillae Nguyen & Smart 1992 18. 19. 20. 21. 22. 23. 24. S. orgonense Liu & Berry 1996 S. puertoricense Roman & Figueroa1994 S. rarum (de Doucet 1986) Mamiya 1988 S. riobrave Cabanillas et al. 1994 S. ritteri de Doucet & Doucet 1990 S. scapterisci Nguyen & Smart 1990 S. thermophilum Ganguly & Singh, 2000
ORIGINAL LOCALITY Rafaela, Argentina Brecon, South Australia Fujian Province, China Hawaii, USA Coimbatore, India Seaside, Oregon, USA Jeromesville, USA Auckland, NewZealand Sultanate of Oman Denmark Central Russia Strazilovo, Yugoslavia Czechoslovakia China Jining Province, China Western Cuba Denmark New Jersey, USA South Carolina, USA Central Province, Kenya Germany Hamikita, Guangdong, China Republic of Korea LaCrosse, Florida, USA Oregon, USA Puerto Rico Cordoba, Argentina Waslaco, Texas, USA Cordoba, Argentina Rivera, Uruguay IARI Farm, Delhi, India
ORIGINAL SOURCE Graphognathus sp. Heliothis punctigera Soil Soil Soil Soil Popillia japonica Heteronychus arator Soil Bibio sp. Soil Soil Cydia pomonella Soil Soil Soil Agrotis feltiae Popillia japonica Soil Soil Cephaleia abietis Anomala cuprea Soil Soil Neocurtilla hexadactylla Soil Soil Soil Soil Soil Scapteriscus vicinus Soil
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SYSTEMATIC POSITION OF EPN:

Phylum Class Order Superfamily Family : Nematoda : Secernentea : Rhabditida : Rhabditoidea : Steinernematidae, Heterorhabditidae
HABITAT
Steinernematid and heterorhabditid nematodes are microscopic soil dwelling organisms. They are ubiquitous, having been isolated from every inhabited continent from a wide range of ecologically diverse soil habitats including cultivated fields, forests, grasslands, deserts, and even ocean beaches. In New Jersey, entomopathogenic nematodes were recovered from 22% of sites sampled [29].
LIFE-CYCLE
The life-cycles of the genera grouped under Heterorhabditidae and Steinernematidae are particularly well known. Although not closely related, phylogenetically, both share similar life histories (Poinar, 1993). The cycle begins with an infective juvenile, whose only function is to seek out and infect new hosts. After entering an insect, infective juveniles release an associated mutualistic bacterium. The bacteria of the two genera Xenorhabdus and Photorhabdus, along with steinerernematides and heterorhabditids, respectivelycause host mortality within 48 hours. The nematodes provide shelter to the bacteria, which, in return, kill the insect host and provide nutrients to the nematode. Together, the nematodes and bacteria feed on the liquefying host, and reproduce for several generations inside the cadaver. Steinernematid infective juveniles may become males or females, whereas heterorhabditids develop into self-fertilizing hermaphrodites with later generations producing two sexes. When food resources in the host become scarce, the adults produce new infective juveniles adapted to withstand the outside environment. After about a week, hundreds of thousands of infective juveniles emerge and leave in search of new hosts, carrying with them an inoculum of mutualistic bacteria, received from the internal host environment [30,31,11] (Figs.1, 2).
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APPEARANCE
Nematodes are roundworms, colourless, unsegmented, lacking appendages, may be freeliving, predaceous or parasitic. Infective juveniles are the only free-living and therefore environmentally tolerant stage. Infective juveniles of entomopathogenic nematodes range from 0.5 to 1.5 mm in length and can be observed with a hand lens or microscope after separation from formulation materials. Disturbed nematodes move actively, however sedentary ambusher species (e.g. Steinernema carpocapsae, S. scapterisci) in water soon revert to a characteristic "J"shaped resting position. Low temperature or oxygen levels inhibit movement of even active cruiser species (e.g., S. glaseri, Heterorhabditis bacteriophora). In short, lack of movement is not always a sign of mortality; nematodes may have to be stimulated (by e.g., probes, acetic acid, gentle heat) to move before assessing viability. Good quality nematodes tend to possess high lipid levels that provide a dense appearance, whereas nearly transparent nematodes are often active but possess low powers of infection. Insects killed by most steinernematid nematodes become brownish-yellow, whereas insects killed by heterorhabditids become red and the tissue assumes a gummy consistency (Fig. 3). A dim luminescence given off by insects freshly killed by heterorhabditids is a foolproof diagnostic for this genus (the symbiotic bacteria provide the luminescence). Black cadavers with associated putrefaction indicate that the host was not killed by entomopathogenic species. Nematodes found within such cadavers tend to be free-living soil saprophages.
Figure1: Life cycle of Steinernema and Heterorhabditis [30] 13

Figure 2: Enmasse production of nematode progeny from the insect cadaver
HOST RANGE
The entomopathogenic activity of steinernematid and heterorhabditid species has been documented against a broad range of insect pests in a variety of habitats [13]. They have been used inundatively in a number of high value cropping systems [25]. The nematode-bacterium complex kills insects so rapidly that the nematodes do not form the intimate, highly adapted, host-parasite relationship characteristic of other insect nematode associations. This rapid mortality permits the nematodes to exploit a range of hosts that spans nearly all insect orders, a spectrum of activity well beyond that of any other microbial control agent. In laboratory tests, S. carpocapsae alone infected more than 250 species of insects from over 75 families in 11 orders [33]. The nematodes attack a far wider spectrum of insects in the laboratory where host contact is assured, environmental conditions are optimal, and no ecological or behavioural barriers to infection exist [34]. Some nematode species may search for hosts at or near the soil surface (e.g., S. carpocapsae and S. scapterisci), whereas others are adapted to search deeper in the soil profile (e.g., H. bacteriophora and S. glaseri). The former group has been referred to as ambusher, which remains nearly sedentary while waiting for the mobile surface dwelling hosts [35]. The latter group has been referred to as cruiser which is highly mobile, responds strongly to longrange host chemical cues, and is therefore best adapted to find sedentary hosts [36]
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Figure 3: (A) Insect larvae killed by Steinernema, (B) Insect larvae killed by Heterorhabditis Because the symbiotic bacterium kills insects so quickly, there is no intimate hostparasite relationship as is characteristic for other insect-parasitic nematodes. Consequently, EPNs are lethal to an extraordinarily broad range of insect pests in the laboratory. Field host range is considerably more restricted, with some species being quite narrow in host specificity. When considered as a group of nearly 30 species, however, EPNs are useful against a large number of insect pests, many of which are listed in the Table 2. As field research progresses and improved insect-nematode matches are made, this list is certain to expand. Regrettably, nematodes have yet to be found which are effective against several of the most important soil insects, including wireworms, grape phylloxera, fire ants, or corn rootworms.
IN VIVO PRODUCTION AND FORMULATION TECHNOLOGIES

It is far too expensive to rear EPNs by in vitro media as they require separate media source for nematodes growth and their associated bacterium with suitable fermentor. The in vivo process, however, lacks any economy of scale; the labour, equipment, and material (insect diet) costs increase as a linear function of production capacity. However, in India the first successful and commercial scale of mass production of EPN species; H. indica, S. carpocapsae, S. thermophilum and S. glaseri on larvae of greater wax moth, Galleria mellonella L. (Lepidoptera: Pyralidae) and formulation of these nematodes have been developed after three years of rigorous research by Multiplex Biotech Pvt. Ltd., Tumkur, Karnataka, India. This is a pioneered organization commercializing these entomopathogenic nematodes reared in vivo, to increase 15
their virulent and survival. Galleria mellonella are most commonly used host insect to mass produce these beneficial nematodes because of its rich nutrient source available in body and easy to multiply in economical semi-synthetic diet source containing wheat and corn flour based media. About 2.5 billion infective juveniles (IJs) of EPN are recommended to treat one hectare where the insect pests are highly infested on any crop [37]. The latest formulation developed by Multiplex Biotech Pvt. Ltd., is comprised of hydro gel based semi solid cream with IJs suspension containing 1 million nematodes in a polythene pouch that can be readily mixed in a water spray tank without blocking the nozzles. Fifty such pouches are recommended to spray in one acre against the highly infested crop. The nematode shelf-life of 2 to 3 months was achieved at room temperature (27-28C) with maximum of 80 % survival by increasing the nematodes metabolic rate by mixing silica powder (0.1 %). The company do have products of EPNs that are currently marketed satisfactorily in the name of Soldier recommended as soil application for soil dwelling insect pests and Bouncer as foliar application for leaf, flower, fruit, pods, stem and borer insect pests against agriculture, horticulture and forest insect pests. EPNs are often applied to sites and ecosystems that routinely receive other inputs including chemical pesticides, surfactants (e.g., wetting agents), fertilizers, and soil amendments that may interact with nematodes. Infective juveniles are tolerant of short exposures (2-8 h) to most agrochemicals including herbicides, fungicides, acaricides, and insecticides [38, 39, 40]. Some pesticides act synergistically with EPNs and therefore improve nematode efficacy in inundative applications [41,42]. Nematodes are also compatible with most inorganic fertilizers when they applied inundatively [43].
APPLICATIONS
Although the biological control industry has acknowledged EPNs since the 1980s, relatively little is understood about their biology in natural and managed ecosystems [44]. Nematode-host interactions are poorly understood, and more than half of the natural hosts for recognized Steinernema and Heterorhabditis species remain unknown [45]. Information is lacking because isolates of naturally infected hosts are rare, so native nematodes are often baited using Galleria mellonella, a lepidopteran that is highly susceptible to parasitic infection. Laboratory studies showing wide host ranges for EPNs were often overestimates, because in a laboratory, contact with a host is assured and environmental conditions are ideal; there are no 16
ecological barriers to infection [13,46]. Therefore, the broad host range initially predicted by assay results has not always translated into insecticidal success. Table 2: Current use of nematodes as biological insecticides
COMMODITY Artichokes Berries Citrus Cranberries
INSECT PEST Artichoke plume moth Root weevils Root weevils Root weevils Cranberry girdler
NEMATODE SPECIES S. carpocapsae H. bacteriophora S. riobrave H. bacteriophora, S. carpocapsae S. carpocapsae S. feltiae H. bacteriophora, H. megidis S. carpocapsae, H. bacteriophora S. feltiae H. bacteriophora S. riobrave, S. scapterisci H. bacteriophora, S. carpocapsae S. carpocapsae
Mushrooms Ornamentals
Sciarids Root weevils Wood borers Fungus gnats
Turf
Scarabs Mole crickets Billbugs Armyworm, Cutworm, Webworm
The lack of knowledge about nematode ecology has resulted in unanticipated failures to control pests in the field. For example, parasitic nematodes were found to be completely ineffective against blackflies and mosquitoes due to their inability to swim [47]. Efforts to control foliage-feeding pests with EPNs were equally unsuccessful, because nematodes are highly sensitive to UV light and desiccation [47]. Comparing the life-histories of nematodes and target pests can often explain such failures [46]. Each nematode species has a unique array of characteristics, including different environmental tolerances, dispersal tendencies, and foraging behaviors [47]. Increased knowledge about the factors that influence EPN populations and the
17
impacts they have in their communities will likely increase their efficacy as biological control agents. Entomopathogenic nematodes should be applied at the first sign that a pest population is initializing to cause damage. Reapplying nematodes depends on the success of the first nematodes released. Their survivorship and success are based on environmental condition, moisture and soil type and percentage of living nematodes actually released during the first application. Nematodes should be reapplied on 7 days intervals if damage continues. In order to ensure maximum effectiveness, it is crucial to apply them at the optimum environmental conditions needed for their better survival. Therefore, it is best to irrigate the target site, both before and after application because they need moist conditions to prevent desiccation and aid with movement to find hosts. Also, the best results are obtained when the relative humidity is high, ambient temperature is neither extremely hot or cold, soil temperature is between 10 to 35C, soil is moist and direct sunlight is minimal. All of these factors help to prevent the nematodes from drying out and increase their survival and virulence. Entomopathogenic nematodes are remarkably versatile in being useful against many soil and cryptic insect pests in diverse cropping systems, yet are clearly underutilized. Like other biological control agents, nematodes are constrained by being living organisms that require specific conditions to be effective. Thus, desiccation or ultraviolet light rapidly inactivates insecticidal nematodes; chemical insecticides are less constrained. Similarly, nematodes are effective within a narrower temperature range than chemicals, and are more impacted by suboptimal soil type, thatch depth, and irrigation frequency [48]. Nematode-based insecticides may be inactivated if stored in hot vehicles, cannot be left in spray tanks for long periods, and are incompatible with several agricultural chemicals. Certain species cannot be applied with highpressure application equipment; unused nematodes cannot be applied the following year; different species require different screen sizes. Chemicals also have problems (e.g., mammalian toxicity, resistance, groundwater pollution, etc.) but a large knowledge base has been developed to support their use. Accelerated implementation of nematodes into IPM (Integrated pest management) systems will require users to be more knowledgeable about how to use them effectively.
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NEMATODE-BACTERIUM SYMBIOSIS
These steinernematids and heterorhabditids have symbiotic relationships with the bacteria belonging to the genera Xenorhabdus and Photorhabdus, respectively, which are species specific, as for e.g. X. indica with S. thermophilum; X. bovieni with S. feltiae, X. nematophilus with S. carpocapsae, X. poinarii with S. glaseri and Photorhabdus luminescens with Heterorhabditis bacteriophora. These bacterial symbionts are gram negative, anaerobic rods, classified under the family Enterobacteraceae. Excellent mutualism occurs between the nematode and the bacterium [49, 31, 13]. The nematode is dependent on the bacterium for Killing its host; Creating a suitable environment for nematode development and preventing the infection by secondary microorganisms; Serving as a food source and; Breaking down the host tissues to serve as nutrient source for nematode. On the other hand, The bacterium needs the nematode for protection from the external environment; penetration into hosts haemocoel and; Inhibition of the hosts antibacterial proteins. The nematode-killed insects are flaccid and do not have a putrid odor because the mutualistic bacteria produce antibiotics which prevent the growth of secondary microorganisms [31,13]. These bacteria are known to exhibit peculiar behavior of phase transition [50, 51]. Although, both phases are equally pathogenic to the insects, the phase one produces better conditions for nematode reproduction, an important consideration in commercial use of these microbes as biopesticides [12,13,14,15].
NEMATODE-BACTERIUM COMPLEX AND ITS MODE OF ACTION

After entering through insect natural openings the EPNs deliver the symbiotic bacteria from their intestine to the haemocoel of the insect hosts [13]. The bacterium replicates logarithmically in the insect rich haemolymph, then causes a suppression to the host immune capacity against both the symbiotic nematodes and the bacteria themselves by inhibiting phospholipase A of the insect host [52,53]. At this immuno-compromised state, the infected insect can be susceptible to other saprophytic pathogens. To maintain monoxenic condition, bacterium synthesizes and secretes broad spectral antibiotics as well as narrow spectral bacteriocins [54,55] consequently inhibiting growth of other pathogens. The monoxenic state leads to lethal septicemia of the target insect, which is required for development of the symbiotic 19
nematodes in the cadaver [56]. Various kinds of antibiotics against bacteria and fungi are synthesized and secreted during the bacterial cultures growth of two related entomopathogenic bacteria, Xenorhabdus and Photorhabdus [57]. Among them, indoles [58], dithiolopyrrolones and xenocoumacins are the antibacterial compounds synthesized by Xenorhabdus spp [59]). The mechanism of antibacterial activity has not been elucidated, but primarily speculated as inhibition of RNA and protein synthesis by an accumulation of the regulatory nucleotide, guanosine-3 pyrophosphate (ppGpp) especially in indole compounds [60]. This antibiotic aspect of the entomopathogenic bacteria reflects the potential medical and agricultural importance of their metabolic products [61].
RESEARCH SCENARIO
The biological control potential of entomopathogenic nematodes of the families Steinernematidae and Heterorhabditidae is being exploited for managing the insect pests of crops as well as household pests in several parts of the world [62]. Presently, there are more than hundred companies in the world, mostly in USA, Canada and Europe, which are mass producing, formulating and marketing EPNs for managing insect pests of turf grass, mushrooms, ornamentals and some fruit crops [12,15]. Remarkable progress has been made on the techniques related to mass culturing, formulations and application technologies of these nematodes [63,64]. The bioefficacy of these nematodes as foliar spray as well as soil application against different insect pests have been demonstrated in at least in 44 trials in 12 countries, including USA, Japan, Argentina, Canada, New Zealand, China, England, France, Italy, Soviet Union, etc. [15]. Till now, about 67 species of Steinernema and 18 species of Heterorhabditis have been described from different parts of the world [65,66,29]. Some protocols for biosystematics of these nematodes have also been developed through multi-national COST (European cooperation in science and technology) Project involving working groups from 17 countries, wherein use of morphological, biological (life-cycle and interbreeding tests), biochemical and molecular tools (Base composition of rDNA) have been emphasized for the description and identification of species (67- 69]. The nematode-bacterium relationship is species-specific, and remarkable progress has also been made on the taxonomy and metabolites of the symbiotic bacteria associated with Steinernema and Heterorhabditis. There are now about 20 species of Xenorhabdus associated
20
with different species of Steinernema and 4 species of Photorhabdus with Heterorhabditis (10,70,57, 72]. Some efforts have also been made to identify the biochemical and molecular basis of nematode-bacterium-insect relationships, biotoxins and the genes involved, and also exploiting these for developing insect resistant transgenic plants and biopesticides [45,73,74]. The molecular approaches are very useful for differentiating the species, subspecies or strains; understanding their biology and evolution; identification of species and strains for registration, quarantine and proprietory protection purposes [75]. Molecular approaches have been applied in the taxonomy of not only entomopathogenic nematodes [76] but also their symbiotic bacteria [58]. The polymerase chain reaction (PCR) in combination with restriction fragment length polymorphism (RFLP) of ITS region of ribosomal DNA, have been used in the descriptions of new species of Steinernema and deducing their phylogenetic relationships with other species [7779]. Recently, the DNA sequences of internal transcribed spacer (ITS) region of ribosomal DNA, have been found to be more useful for detailed information about variation within and among nematode species than PCR-RFLP approaches (80-83].
MOLECULAR TECHNIQUES INVOLVED FOR CHARACTERIZATION

Various biochemical techniques which have been used in the molecular taxonomy of Entomopathogenic nematodes are:
PROTEIN ELECTROPHORESIS
Species-specific isozymic patterns revealed through polyacrylamide slab gel electrophoresis have been found to be useful for differentiating the species of Meloidogyne [84,85]. This biochemical technique was first applied by Herman and Jackson [86] wherein they examined the differences in alkaline phosphatase, esterase and lactate dehydrogenase in different cultures and life stages of S. glaseri and S. carpocapsae; later several workers studied isozymic profiles of different enzymes for resolving the taxonomic anomalies in steinernematids, especially in the identification of S. carpocapsae, S. glaseri and S. feltiae, and other native strains [87-90]. Sha [87] observed qualitative differences in esterase patterns of S. feltiae, S. glaseri and a group of 3 isolates of S. carpocapsae. The common esterase patterns of 3 isolates of S. carpocapsae 21
clarified precisely the species status of S. feltiae isolated by Stanuszek [91]. Some differences were also noted between juveniles and adult stages within S. feltiae and 2 isolates of S. carpocapsae. Kozodoi et al. [92] differentiated the Steinernema species on the basis of total protein patterns, non-specific esterase and alkaline phosphotase. Some ontogenetic variations in alkaline phosphatase were observed only in S. anomali as the isozyme pattern varied in its different life-stages (IJs, females and males). Similar results were obtained on the basis of morphological data as well as interbreeding tests. Later on, the taxonomic significance of total protein patterns, non-specific esterase, alkaline phosphatase, acid phophatase, malate dehydrogenase have been also worked out at least to differentiate the morphologically similar species of Steinernema. Akhurst [93] studied the isozyme patterns of 23 isolates of different species of Heterorhabditis. The dissimilarity matrix and cluster analysis of these isolates based on 10 enzymes could categorize them into 3 8 groups. Ganguly and Pandey [89] and Ganguly et al. [94] have demonstrated the utility of esterase, catalase and SOD isozymes of infective juveniles for the differentiation of some of the species of Steinernema, as evident from the species-specific enzyme phenotypes obtained for S. thermophilum, S. carpocapsae and S. glaseri, using both the enzymes (Fig 4,5 &6).
MITOCHONDRIAL DNA SEQUENCE ANALYSIS

Mitochondrial DNA (mtDNA) is now widely used in studying speciation, phylogenetic relationships and molecular evolution [95]. Divergence in mtDNA sequences may accumulate during a relatively short evolutionary time, a period in which traditional genetic and morphological markers might remain unchanged [96]. Numerous genetic and systematic studies have focused on mtDNA. Its High rate of evolution permits comparisons of specific or subspecific taxa [97]. However, there are currently not much published data on mtDNA sequences in Heterorhabditis species. Among the few mitochondrial genes investigated in other nematodes, the ND4 gene appeared to be a very useful marker for population genetics applications [98]. Some more studies were done to obtain partial DNA sequences of ND4 gene from some EPN isolates collected from different regions of the world. These isolates were analyzed for sequence variation. Phylogenetic relationships among these isolates were inferred from the ND4 gene sequences and compared with the relationships inferred from the ITS1 region of the ribosomal DNA gene [99]. Scientists also determined the complete sequence of the mitochondrial DNA of the entomopathogenic nematode Steinernema carpocapsae and analyzed 22
its structure and composition as well as the secondary structures predicted for its tRNAs and rRNAs. Almost the complete genome has been amplified in one fragment with long PCR and sequenced using a shotgun strategy (Montiel et al., 2006)
Figure 4: Esterase profiles of Infective Juveniles of Steinernema species. a: Steinernema thermophilum; b: S. carpocapsae; c:Steinernema simkayai; d: S. glaseri; e: IARI-EPN-mg1; f: IARI-EPN-gj1
Figure 5: Catalase isozymic profiles of infective juveniles of six Indian species of Steinernema. a: S. thermophilum; b: S. carpocapsae; c: S. siamkayai; d: S. glaseri; e: Steinernema sp. (strain IARI-EPN-mg1); f: S. riobrave (IARI-EPN-gj1). 23
Figure 6: Superoxide dismutase isozymic profiles of infective juveniles of six Indian species of Steinernema. a: S. thermophilum; b: S. carpocapsae; c: S. siamkayai; d: S. glaseri; e: Steinernema sp. (strain IARI-EPN-mg1); f: S. riobrave (IARI-EPN-gj1).
IMMUNOLOGICAL TECHNIQUES Gel diffusion and immuno-electrophoresis techniques, using antisera raised against nematode homogenates injected in to rabbits, were capable of discriminating three isolates of Steinernema [100]. DNA SEQUENCE ANALYSIS DNA sequence divergence between species of entomopathogenic nematodes was first studied by visualization of repetitive DNA restriction fragment length differences (RFLDs) in total genomic DNA of S. glaseri, S. feltiae, Steinernema spp, and Heterorhabditis isolates [101]. DNA sequence analysis was applied to Heterorhabditis isolates from North America by Curran and Webster [102]) and three genotypic groups based on RFLDs in repetitive DNA and rRNA gene, were identified. In the absence of supporting morphological or cross breeding data, it was not possible to determine whether these groupings represented species or intraspecific forms. 24
Hominick et al., [103] developed protocols and techniques under a multi-country project on entomopathogenic nematodes for the use of molecular data for identifying the species. As per the protocol, RFLPs should be obtained from total genomic DNA [77]) for knowing any intra population variations present in type sample. PCR reactions should be performed using ITS primers of Vrain et al [104] and digested with the 17 restriction enzymes (AluI, BSt O I, Dde I, EcoR I, Hae III, Hha I, Hind III, Hinf-I, Hpa II, Kpn I, Pst I, Pvu II, Rsa I, Sal I, Sau3A I, Sau96 I and Xba I). For screening the new isolates, they recommended a combination of four restriction enzymes (Alu I, Dde I, Hinf I and Rsa I) and if the results do not tally with the database of the protocol, then all the 17 enzyme profiles should be obtained. Dubey et al. [83] used Alu I, Hha I, Hinf I and Rsa I to differentiate some of the species of Steinernema and found AluI yielded polymorphism that could distinguish 8 out of 9 species (Figs. 7, 8 & 9). Recently, the DNA sequences of internal transcribed spacer (ITS) region of ribosomal DNA, have been found to be more useful for detailed information about variation within and among nematode species than PCR-RFLP approaches [80,81,105,82,83]
Figure 7: RFLPs yielded by digestion of - a: S. glaseri b: IARI-EPN- chh1, c: IARI-EPNmg1 with 4 different restriction enzymes: AluI, RsaI, HinfI, HhaI.
25
Figure 8: RFLPs yielded by digestion of a: S. thermophilum; b: IARI-EPN-dl1; c: IARI-EPNdl2 with 4 different restriction enzymes: AluI, RsaI, HinfI, HhaI
Figure 9: RFLPs yielded by digestion of a: S. carpocapsae b: IARI-EPN -gj1, c: IARIEPN-gj2 with 4 different restriction enzymes: AluI, RsaI, HinfI, HhaI.
26
CONCLUSION
The potential of entomopathogenic nematodes (steinernematids and heterorhabditids) for insect control has been now fully realized and well documented, and the number of researchers studying them has also increased considerably during the last one decade. Till to date, there are 67 valid species of Steinernema, one species of Neosteinernema and 18 species of Heterorhabditis. Besides, there are several strains isolated from different parts of the world, which are yet to be identified. The accurate identification of the strains must be done before taking up any further research on them. Therefore, there is an urgent need to have strong biosystematics base in every country for the identification of entomopathogenic nematodes. Morphological features of infective juveniles and males offer the best differentiating characters but the identification of these nematodes by these standard morphological criteria is rarely straightforward and for this reason scientists turned to gel diffusion, immuno-electrophoresis, enzyme analysis and DNA (genomic and mitochondrial) based molecular biological techniques. The isozyme analysis technique is simpler, less time-consuming and less costly and therefore can be widely applied for identifying the samples obtained from large surveys. Combination of esterase, catalase and superoxide dismutase isozymic profiles from infective juveniles, supplemented with morphological details, proved useful for preliminary screening and differentiation of this strain from other species of the genus Steinernema. Polymerase chain reaction made it possible to identify nematodes using small sample size and now Internal transcribed spacer region (ITS) of ribosomal DNA repeat unit is preferably used for molecular taxonomy as well as identification and characterization. The ribosomal genes flanking this region are highly conserved allowing the construction of primers that enable the PCR amplification of the highly variable ITS region between them. Sequence variation in this region yields many RFLPs which can be used for identification. Presentlly, sequencing of ITS region of rDNA and its alignment with the closely related species, has been found to be useful in describing the species. Numerous genetic and systematic studies have also focused on mtDNA. Conclusively, integration of morphological data together with DNA analysis is the best approach for the biosystematics of entomopathogenic nematodes and for accurate identification of the species, and this will have far reaching impact on all round developments in research for exploiting these bioagents in integrated pest management.
27
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73. Jagdale, G. B., Saeb, A.T., Somasekhar, N. & Grewal, P. S. (2006). Genetic variation and relationships between isolates and species of the entomopathogenic nematode genus Heterorhabditis deciphered through isozyme profiles. Journal of Parasitology, 92, 509- 516. 74. Schuh, R. T. & Brower, A. V. Z. (2009). Biological Systematics: Principles and Applications, 2nd edn. 380. 75. Curran, J. (1990). Molecular techniques in taxonomy. In: Gaugler, R. & Kaya, H.K. (Eds.), Entomopathogenic nematodes in biological control. Boca Raton, Florida, USA, CRC Press, 63-74. 76. Reid, A.P. & Hominick, W. M. (1992). Restriction fragment length polymorphism within the ribosomal DNA repeat unit of British entomopathogenic nematodes (Rhabditida: Steinernematidae)(I). Parasitology 105:317-323. 77. Hazir, S., Stock, S. P. & Keskin, N. (2003). A new entomopathogenic nematode, Steinernema anatoliense n. sp. (Rhabditida: Steinernematidae), from Turkey. Systematic Parasitology. 55, 211-220. 78. Alper, S. & Umut, T. (2006). Molecular identification of three entomopathogenic nematodes from turkey by PCR-RFLP of the ITS regions. Phytoparasitica, 34,17-20. 79. Nguyen, K.B., Maruniak, J. & Adams, B.J. (2001). Diagnostic and phylogenetic utility of the rDNA internal transcribed spacer sequences of Steinernema. Journal of Nematology, 33, 7382. 80. Nguyen, K.B. & Duncan, L.W. (2002). Steinernema diaprepesi n.sp. (Rhabditida: Steinernematidae), a parasite of the citrus root weevil Diaprepes abbreviatus (L) (Coleoptera: Curculionidae). Journal of Nematology, 32,159-170. 81. Phan, L.K., Subbotin, S.A., Waeyenberge, L. & Moens, M. (2005). A new entomopathogenic nematode, Steinernema robustispiculum n.sp. (Rhabditida: Steinernematidae), from Chumomray national park in Vietnam. Systematic Parasitology, 60, 23-32. 82. Dubey, J., Tiwary, B.N., Rathour, K.S. & Ganguly, S. (2009). Phylogeny of some Indian strains/species of Steinernema (Rhabditida; Steinernematidae) based on RFLPs of the ITS region of rDNA. International Journal of Nematology, 19, 182-188. 83. Esbenshade, P.R.; Triantaphyllou, A.C. (1985). Identification of major Meloidogyne species employing enzyme phenotypes as differentiatingcharacters. In: Sasser, J.N. & Carter, C.C. (Eds.), An Advanced Treatise on Meloidogyne Vol. 1. Biology and Control. Raleigh, U.S.A., North Carolina State University Graphics, 135-140. 34
84. Molinari, S., Lamberti, F., Crozzoli, R., Sharma, S.B. & Sanchez, P. (2005). Isozyme patterns of exotic Meloidogyne spp. populations. Nematologia Mediterranea, 33, 61-65. 85. Herman, I.W. & Jackson, G.J. (1963). Zymograms of the parasitic nematodes, Neoaplectana glaseri and N. carpocapsae, grown axenically. Journal Parasitology, 49, 392. 86. Sha, C.Y. (1985). A comaparative analysis of esterase of insect parasitic nematodes of the genus Neoplectana. Acta Zoologica Sinica, 10, 246-249. 87. Artyukhovsky, A.K., Kozodoi, E.M., Reid, A.P. & Spiridonov, S.E. (1997). Redescription of Steinernema arenarium (Artyukhovsky, 1967) topotypes from Central Russia and a proposal for S. anomalae (Kozodoi, 1984) as a junior synonym. Russian Journal of Nematology, 5, 31-37. 88. Ganguly, S. & Pandey, J. ( 2006). Esterase isozymes for differentiating some Indian species of Steinernema ( Nematoda: Steinernematidae ) International Journal of Nermatology, 16, 70-74. 89. Dubey, J., Tiwary, B.N. & Ganguly, Sudershan (2011). Differentiation of some native strains of Steinernema based on isozymic profiles International Journal of Nematology (accepted). 90. Stanuszesk (1974). Neoaplectana feltiae pieridarum, n. ecotype (Nematoda: Rhabditoidea, Steinemematidae) - aparasite of Pieris brassicae L. and Mamestra brassicae L. in Poland, Morphology and biology. Prob. lostepbw Nuuk Rolniczych, 154, 361-393. 91. Kozodoi, E.M., Voronov, D.A. & Spiridinov, S.E. (1986). The use of an electrophoretic method to determine the species association of Neoplectana specimens. Trudy Gel minthologicheskoi laboratorii. Voprosy biotsenologii gel mintov,34: 55. 92. Akhurst, R.J. (1987). Use of starch gel electrophoresis in the taxonomy of the genus Heterorhabditis (Nematoda: Heterorhabditidae). Nematologica, 33, 19.. 93. Ganguly, S., Pandey, J. & Rathour, K.S. (2006). Catalase and superoxide dismutase isozymes of Indian species of Steinernema (Nemattoda: Steinernematidae ). Nematologia Mediterranea, 36, 111-114. 94. Meyran, JC , Monnerot, M. & Taberlet, P. (1997). Taxonomic status and phylogenetic relationships of some species of the genus (Crustacea, Amphipoda) deduced from mitochondrial DNA sequences. Molecular Phylogenetics and Evolution, 8, 1-10. 95. Hyman, B. C., Beck J. L. & Weiss, K. C. (1988). Sequence amplification and gene rearrangement in parasitic nematode mitochondrial DNA. Genetics, 120, 707-712. 35
96. Powers, T. O. & Sandall, L. J. (1988). Estimation of genetic divergence in Meloidogyne mitochondrial DNA. Journal of Nematology, 20, 505-511. 97. Blouin, M. S., Yowell, C. A., Courtney, C.H. & DAME, J. B. (1995). Host movement and the genetic structure of populations of parasitic nematodes. Genetics, 141, 10071014 98. Adams, B.J., Burnell, A.M. & Powers, T.O. (1998). A phylogenetic analysis of Heterorhabditis (Nemata: Rhabditidae) based on internal transcribed spacer 1 DNA sequence data. Journal of Nematology, 30, 2239. 99. Jackson, G.J. (1965). Differentiation of three species of Neoplectana (Nematoda: Rhabditida), grown axenically. Parasitology, 55, 571-578. 100. Curran, J., Baillie, D.L. & Webster, J.M. (1985). Use of restriction fragment length differences in genomic DNA to identify nematode species. Parasitology, 90, 137-144. 101. Curran, J. & Webster, J.M. (1989). Genotypic analysis of Heterorhabditis isolates from North Carolina, USA. Journal of Nematology, 21, 140-147. 102. Hominick, W.M., Briscoe, B.R., Del Pino, F.G., Heng, J.A., Hunt, D.J., Kozodoy, E.,
Mracek, Z., Nguyen, K.B., Reid, A.P., Spiridonov, S.E., Stock, P. , Sturhan, D., Waturu, C. & Yoshida, M. (1997). Biosystematics of entomopathogenic nematodes: current status, protocols and definitions. Journal of Helminthology, 71, 271-298. 103. Vrain, T.C., Wakarchuk, D.A., Levesque, A.C. and Hamilton, R.I. (1992). Intraspecific americanum group.
rDNA restriction fragment length polymorphism in the Xiphinema Fundamental and Applied Nematology, 15, 563-574. 104.
Nguyen, K.B. & Adams, B.J. (2003). SEM and systematic studies of Steinernema abbasi
Elawad et al., 1997, and S. riobrave Cabanillas et al., 1994 (Rhabditida: Steinernematidae). Zootaxa, 179,1-10.
36
Chapter II
PROBIOTICS: POTENTIAL HEALTH TOOL

*1 1
Pratima Bajpai, 2Anamika Pandey and 1Harish Dhingra
Department of Science, FASC, MITS University, Lakshmangarh, Sikar-332311, Rajasthan, INDIA 2 ACTREC, Tata Memorial Centre, Kharghar, Navi Mumbai-410210
Coressponding author: PratimaBajpai, Assistant Professor of Biotechnology, Department of Science, FASC ,MITS University, Lakshmangarh, Sikar-332311, Rajasthan, INDIA E.mail:bajpai.pratima@rediffmail.com
ABSTRACT: Probiotics are refered to beneficial microbes, administration of which improves microfloaral balance of gastrointestinal tract. Probiotics consisted of Lactobacillus, Bifidobacter and certain other bacteria can be used as dietary supplement. Use of probiotics has been found to reduce risk of cancer and other serious ailments. In addition, prebiotic substance may be added which stimulates growth of beneficial bacteria. In essence, probiotics may reduce level of pathogenic microorganism, reduce level of toxic substances, strengthen immune system, assist in digestion of lactose and supply vitamins. Extensive research is required for exploration and optimization of microorganism which can be exploited as probiotics to improve health. Keywords; Probiotics, Lactobacillus and Bifidobacterium INTRODUCTION: The term probiotic refers to the bacteria which beneficial effects to humans and animals. Historically, the original observation of the positive effect exerted by some of the selected bacteria, was made by Eli Metchnikoff, the Russian born Nobel Prize winner, while working at the Pasteur Institute. He suggested that the dependence of the intestinal microbes on the food enable them to adopt measures to modify the flora in our bodies and to replace the harmful microbes by beneficial microbes [1]. Henry Tissier, a French paediatrician, noticed scanty number of bacteria characterized by a peculiar, Y shaped morphology in stools of children with diarrhea. These bifid bacteria were, on the contrary, abundant in healthy children [2]. Based on his observation and analysis, he suggested that these bacteria could be administered to 37
patients with diarrhea to help restore a healthy gut flora. The works of Metchnikoff and Tissier were the first to make scientific suggestions concerning the probiotic use of bacteria, even if the word "probiotic" was not coined until 1960, to name substances produced by microorganisms which promoted the growth of other microorganisms [3]. In order to point out the microbial nature of probiotics, redefined the word as "A live microbial feed supplement which beneficially affects the host animal by improving its intestinal balance" [4]. A similar definition was proposed by Havenaar and Huis in 't Veld [5], a viable mono or mixed culture of bacteria which, when applied to animal or man, beneficially affects the host by improving the properties of the indigenous flora. A more recent, but probably not the last definition is "live microorganisms, which when consumed in adequate amounts, confer a health effect on the host" [6]. Probiotics are live, nonpathogenic microorganisms that may interact with gastrointestinal and vaginal microflora. Clinical studies indicate that certain probiotics may be useful in treating some diarrheal disorders, respiratory allergies, and eczema, as well as in controlling inflammation and reducing the risk of Candidal vaginitis and colon cancer. Dietary sources of probiotics are usually found in dairy products such as Yogurt, which contains two groups of intestinal bacterial species of Lactobacilli and Bifidobacteria of probiotic bacteria. Gastric survival rates of probiotics are estimated at 20 to 40 percent, with the main obstacles to survival being gastric acidity and the action of bile salts. Investigations into different modes of administering probiotics may expand their applications in functional foods. It is generally accepted that antibiotics deplete the friendly bacteria in our intestines which perform many important functions. Stress, faulty diet, and poor digestion are also factors in the depletion of these vital factors. Replenishing the flora in our gut on a regular basis, and particularly when undergoing a course of antibiotics, is a simple, important and effective strategy to protect our overall health. Probiotics are friendly and beneficial bacteria. Although there are hundreds of different strains of bacteria that live in the digestive tract, beneficial bacteria are generally placed in two categories: Lactobacillus acidophilus and Bifidobacterium bifidum. These bacteria are normal inhabitants of the large and small intestines. They provide many health benefits.
38
PREBIOTICS A prebiotics substance has been defined as a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and or activity of limited number of bacteria in the colon. Therefore, compared to probiotics which introduce exogenous bacteria into the colonic micro flora, a prebiotic aims at stimulating the growth of one or a limited number of the potentially health promoting indigenous microorganisms thus modulating the composition of the natural ecosystem. VITAL ROLE OF PROBIOTICS Probiotics reduce the levels of harmful bacteria such as E. coli and Salmonella by producing metabolic end-products that inhibit or antagonize them. These compounds include hydrogen peroxide, lactic and acetic acids. Inhibiting levels of microbial pathogens: L. Acidophilus may inhibit pathogens by lowering the pH in the intestines. The production of organic acids effectively lowers intestinal pH to a level that is beneficial to good bacteria and destructive to pathogens. Protecting the immune system. Preventing establishment of harmful fungus and parasites: L. Acidophilus and B. Bifidus aggressively attach themselves to the walls of the colon. In doing so, they may inhibit Candida albicans, bacteria and the parasite Giardia lamblia. Lowering levels of toxic by-products: Harmful bacteria can produce toxins, such as indole, skatole, and methane because of their metabolic reaction to certain foods. Reducing their numbers may lower toxin levels in the colon. Assist in digestion of lactose: Clinical studies have shown that L. Acidophilus assists the bodys natural process of digestion, particularly lactose and dietary carbohydrates. Synthesizing important B vitamins: Probiotics have been found to be beneficial in the synthesis of folic acid, niacin, pantothenic acid, and biotin. IDENTIFICATION OF PROBIOTICS Probiotics are microorganisms---such as bacteria, fungi and yeasts---that can be seen only under a microscope and that are often referred to as "healthy" or "good" bacteria. According to the National Center for Complementary and Alternative Medicine (NCCAM) and defined by the 39
World Health Organization, probiotics are "live microorganisms, which, when administered in adequate amounts, confer a health benefit on the host." The benefits of incorporating probiotics into one's diet have been widely speculated, as little evidence exists to support the long-term health benefits. Despite the lack of formal evidence, the probiotic trend has swept the health and diet industries for their potential cleansing benefits, immune boosting powers and nutritional value. GENETIC MODIFICATION OF PROBIOTIC MICROORGANISMS The perceived desirable qualities of probiotics are many and it is highly unlikely that any one strain will harbor all the qualities or provide the multitude of proposed benefits. There is a numerous of possible probiotic strains, coupled with a highly diverse set of phenotypes and potential benefits. Therefore, screening genetic traits offers considerable promise in attacking the almost insurmountable task of surveying for functional probiotic properties, or building combinations of probiotic strains that can elicit multiple effects. Moreover, genetic modification of probiotic bacteria offers the added developmental potential to annex new beneficial activities (e.g. vaccine presentation) or improve the effectiveness of existing properties (e.g. bacteriocin levels). Consumers in developed countries will accept products manufactured or containing GMOs (genetically modified organisms); if they offer benefits to health and product quality that the consumer can clearly recognize [7]. In this regard, because probiotics by definition, offer health benefits, consumers may willingly accept and use genetically modified probiotics if there are substantial and tangible benefits provided over their traditional counterparts. CHARACTERIZATION OF GOOD PROBIOTIC STRAINS Parameters that are employed for characterizing bacteria for being used in probiotics includes following steps or tests: Accurate taxonomic identification Production of antimicrobial substances, including bacteriocins, hydrogen peroxide, and organic acids Antagonistic toward pathogenic/cariogenic bacteria Resistant to bile 40
Immunostimulatory Amenable to production processing: adequate growth, recovery, concentration, freezing, dehydration, storage, and distribution
Able to compete with the normal microflora, including the same or closely related species; potentially resistant to bacteriocins, acid, and other antimicrobials produced by residing microflora
Lactobacilli and bifidobacteria constitute two extremely important groups of probiotic bacteria. As members of the normal microflora of the gastrointestinal tract of humans, they offer considerable potential as probiotics because of their history of safe use and thegeneral body of evidence that supports their positive roles.
Genetic analysis and manipulation of these bacteria will be paramount to understanding their probiotic roles and maximising their performance in vitro and in vivo.
ROLE OF PROBIOTICS IN VARIOUS DISEASES Health effects of probiotics in pediatrics: In probiotic foods, cultures of beneficial live microorganisms characteristic of the healthy human gut microbiota are administered in order to provide a safe microbial stimulus. The probiotic effects are attributable to the restoration to the normal of increased intestinal permeability and an unbalanced gut micro ecology. They improve the intestines immunological barrier functions, alleviate the intestinal inflammatory response, and reduce generation of proinflammatory cytokines characteristic of local and systemic allergic inflammation [8].Probiotics have been found as beneficial in the treatment of diarrhea, inflammatory bowel diseases and allergic diseases. Effects of probiotics in treatment of diarrhea: The use of probiotic microorganisms for the prevention or therapy of gastrointestinal disorders is an obvious measure and perhaps the most usual application of probiotics because most health effects attributed to them are related directly or indirectly (i.e., mediated by the immune system) to the gastrointestinal tract.
41
The mechanisms and the efficacy of a probiotic effect often depend on interactions with the specific microflora of the host or immunocompetent cells of the intestinal mucosa. The gut or its associated lymphoid system; GALT [9] is the largest immunologically competent organ in the body, and maturation and optimal development of the immune system after birth depend on the development and composition of the indigenous microflora and vice versa. Many strains of probiotic microorganisms have been shown to inhibit growth and metabolic activity as well as the adhesion to intestinal cells of enteropathogenic bacteria (Salmonella, Shigella, E. coli, or Vibrio cholerae) [10] to modulate (temporarily) the intestinal microflora and to have immunostimulatory or -regulatory properties. MECHANISM OF PROBIOTICS Probiotic bacteria have different influences on the host. Different organisms can influence the intestinal luminal environment, epithelial and mucosal barrier function, and the mucosal immune system. They exert their effects on numerous cell types involved in the innate and adaptive immune responses, such as epithelial cells, dendritic cells, monocytes/macrophages, B cells, T cells, including T cells with regulatory properties, and NK cells [11]. Alteration of the intestinal microbiota Probiotic bacteria can antagonize pathogenic bacteria by reducing luminal pH, inhibiting bacterial adherence and translocation, or producing antibacterial substances and defensins. One of the mechanisms by which the gut flora resists colonization by pathogenic bacteria is by the production of a physiologically restrictive environment, with respect to pH, redox potential, and hydrogen sulfide production. Probiotic bacteria decrease the luminal pH; this pH reduction was associated with increased animal survival [12] Augmentation of barrier function In this mechanism probiotics increase mucous production and enhance the barrier integrity.In addition to the inhibition of growth of conventional organisms or potential pathogens, probiotics can influence mucosal cell cell interactions and cellular stability by the enhancement of intestinal barrier function through modulation of cytoskeletal and tight junctional protein phosphorylation [13] Probiotics are already widely used to prepare fermented dairy products that are becoming popular in Europe and Japan. Some of the commercially available probiotic bacteria are listed in 42
table 1. These products favorably influence digestive functions and colonic flora. The most promising health benefits are the prevention of diarrhea and enhancement of the immune system. These 2 effects may be mechanistically related. Combining probiotics with prebiotics could improve the survival of the bacteria crossing the upper part of the gastrointestinal tract, thus enhancing their effects in the large bowel. Immunomodulation: This concept was demonstrated by Lammers et al. on 2002 and Otte and Podolsky on 2004 [14, 15]. Probiotic bacteria affect the various cells which are the key members of innate and adaptive `immune responses as like:
1. Effects on epithelial cells 2. Effects on dendritic cells 3. Effects on monocytes/macrophage 4. Effects on lymphocytes (B lymphocytes, NK cells, T cells, T cell redistribution)
Table 1: commercially available human probiotic microorganisms
Lactobacillus species L. acidophilus L. casei L. fermentum L. gasseri L. johnsonii L. lactis L. paracasei L. plantarum L. reuteri L. rhamnosus L. salivarius
Bifidobacterium species B. bifidum B. breve B. lactis B. longum
Streptococcus species S. thermophilusz
43
REFERENCES 1. Metchnikoff, E. (1907). Lactic acid as inhibiting intestinal putrefaction In: The prolongation of life: optimistic studies. W. Heinemann, London; 161-183. 2. Tissier, H. (1906). Tritement des infections intestinales par la methode de translormation de la flore bacterienne de lintestin. C R Social Biology. 60: 359-361. 3. Lilly, D.M. & Stillwell, R.H.(1965) Probiotics.Growth promoting factors produced by micro-organisms. Science 147, 747-748 4. Fuller, R.: Probiotics in man and animals ((1989)) Journal of Applied Bacteriology 66, 365-378. 5. Havenaar, R. & Huis int Veld, M.J.H.: Probiotics ((1992)) a general view. In: Lactic acid bacteria in health and disease (Ed.: Wood, J.B.J.). Vol 1. Elsevier Applied Science Publishers, Amsterdam. 6. Guarner, F. & Schaafsma, G.J. (1998) F. Probiotics, International Journal of Food Microbiology, 39, 237238. 7. Verrips, C.T. & Berg D.J.C. (1996).Barriers to application of genetically modified lactic acid bacteria. Antonie van Leeuwenhoek 70: 299-316. 8. Isolauri, E., Rautava, S., Kallimki, M, Kirjavainen, P. & Salminen, S. (2002) Role of probiotics in food hypersensitivity. Current Opinion in Allergy and Clinical Immunology, 2: 263-271. 9. Eizaguirre, I., Urkia, N.G., Asensio, A.B., Zubillaga, I., Zubillaga, P., Vidales, C., Garcia-Arenzana, J.M. & Aldazabal, P. (2002) Probiotic supplementation reduces the risk of bacterial translocation in experimental short bowel syndrome. Journal of Pediatric Surgery, 37,699702 10. Coconnier, M.H., Lievin, V., Bernet-Camard, M.F., Hudault, S. & Servin, A.L. (1997). Antibacterial effect of the adhering human Lactobacillus acidophilus strain LB. Antimicrobial Agents Chemotherapy, 41, 104652. 11. Zhang, Z., Hinrichs, D.J. & Lu, H. (2007). the next therapeutic targets for inflammatory bowel disease. International Immunopharmacology 7:409416. 44
12. Venturi, A., Gionchetti,, P. & Rizzello, F. (1999) Impact on the composition of the faecal flora by a new probiotic preparation: preliminary data on maintenance treatment of patients with ulcerative colitis. Alimentary Pharmacology Therapy 13:11031108. 13. Schmitz, H., Barmeyer, C. & Fromm, M. (1999). Altered tight junction structure contributes to the impaired epithelial barrier function in ulcerative colitis.
Gastroenterology. 116:301309. 14. Lammers, K.M., Helwig, U. & Swennen, E. (2002). Effect of probiotic strains on interleukin 8 production by HT29/19A cells. Americal Journal of Gastroenterology, 97, 11821186. 15. Otte, J.M. & Podolsky, D.K. (2004) Functional modulation of enterocytes by Grampositive and Gram-negative microorganisms. American Journal of Physiology, Gastrointestinal and Liver Physiology, 286, G613G626.
45
Chapter III
TISSUE ENGINEERING AND ITS THERAPEUTIC APPLICATION. Atul Kumar Singh1, Chandrabhan Seniya1, Sharad S Lodhi2, Sudhanshu Singh3, G.J. Khan4, Jaykar Jha4*
1
Department of Biotechnology, Madhav Institute of Technology & Science, Gwalior

2 3
Department of Biotechnology, Ministry of Science &Technology, New Delhi
Department of Chemical Engineering, Indian Institute of Technology, New Delhi
Department of Biotechnology, L.N.Mithila University, Darbhanga, Bihar-*Corresponding Author:Dr Jaykar Jha,Professor & Head,Department of Biotechnology,L.N.Mithila University,Darbhanga-846608, Bihar, India,Email:jaykarjha@yahoo.com,
ABSTRACT: Tissue Engineering (TE) is an interdisciplinary field that applies the principles of
engineering and life sciences to develop a fully functional human organ in vitro to cope up with the problem of organ scarcity. TE uses living cells and their extracellular components with polymer based biomaterial scaffolds to develop biological tissues for human body repair, such as organ transplants. Scaffold serves numerous functions critical for the success of tissue regeneration. It allows cells to attach, grow, proliferate, migrate and differentiate. The various types of polymers used for scaffold preparation can be categorized under two broad groups: either natural or synthetic materials. Extensive research is going on in the areas of Cornea TE, Bone TE, Dental TC, Skin TE, Cardiovascular TE, Cartilage TE, Liver TE and Urinary bladder TE. Low level of precision in cell placement, vascularization of thick tissue constructs, incapability of precisely controlling pore size, pore geometry, spatial distribution of pores and construction of internal channels within the scaffold and extremely laborious, slow and costly non automated tissue assembly processes are some of the limitations of traditional tissue engineering. However, cell sheet engineering, organ printing technology and use of bioreactors provide new insights for TE. On the whole, TE appears to be the new frontier of medicine for its impact on regenerative and reconstructive procedures in humans. Each phase in TE must be understood in an integrated manner from the polymer material properties, to the micro- and 46
macro-architecture of scaffold, to the cell, to the tissue-engineered transplant and finally to the host tissue. 1. INTRODUCTION Tissue Engineering (TE) is an interdisciplinary field that applies the principles of engineering and life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function or a whole organ. Tissue engineering has also been defined as understanding the principles of tissue growth, and applying this to produce functional replacement tissue for clinical use. During the last decade, tissue engineering research has made significant progress towards the design of tissue constructs to repair or replace lost morphology and functions in diseased or damaged organs. The ultimate goal of tissue engineering is to develop a fully functional human organ in vitro to cope up with the problem of organ scarcity. Currently the number of donors available is far less than the requirement of organs for transplantation. Several approaches are currently being used to overcome the donor scarcity like artificial mechanical organs, xenotransplantation (using animal organs), tissue engineering and regenerative medicine. The main problem of organ transplantation from donor (human/animal) is the immune response problem in patients body and any disease can be transferred from the donor to the patient. The problem in case of artificial mechanical organ is the very poor integration between the organ and native tissue and also they deteriorate with time in vivo and loose their function. Regenerative medicine is also a promising field in this case but this technique is most effective in the early stage of a disease. At terminal stages or for injured tissue, there is a still need for organ replacement. So, in this case tissue engineered organ is the right answer to overcome the problem of organ shortage. Tissue engineering uses living cells and their extracellular components with polymer based biomaterial scaffolds to develop biological tissues for human body repair, such as organ transplants. 2. Scaffold: Structure and Function Scaffold serves numerous functions critical for the success of tissue regeneration. It allows cells to attach, grow, proliferate, migrate and differentiate. It provides the space for vascularization, neotissue formation and remodeling. Scaffolds also enable the efficient transport of nutrients, growth factors, blood vessels and removal of waste materials. 47
The structural aspect of scaffolds includes pore size, porosity, pore size distribution, pore connectivity and reproducibility of pores. These aspects are vital as they provide optimal spatial and nutritional conditions for the cells. The interconnectivity of pores determines the transport of nutrients and waste and thus influences the success of tissue engineering. Generally, an increase in porosity leads to increase in the permeability, but for this to happen the pores need to be highly interconnected. Therefore, permeability/porosity ratio is an indicator of the percolative efficiency per unit porous volume of a scaffold. The reproducibility of scaffolds is also very important as it determines the dimensional stability of the scaffold and consistency of the tissue formation. 2.1 Characteristics of Scaffolds: The scaffold used in tissue engineering should at least have some important features which include biocompatibility to avoid unwanted host tissue responses to the implant; excellent surface chemistry to allow attachment, migration, proliferation, and differentiation of the cells; interconnected pores with proper pore size to support cell infiltration and vascularization; a controlled biodegradability to aid the formation of new tissue; adequate mechanical properties to maintain the structure and function immediately after implantation and during remodeling of the implants; acquisition of the sufficient mechanical properties to provide better environment for cells, deliver inductive molecules or cells to the repair site and provide cues to control the structure and function of newly formed tissue; support for the formation of extra cellular matrix (ECM) by promoting cellular functions; ability to provide the bimolecular signals to the cells and; ability to mimic in part the structure and biological function of the extracellular matrix [18]. 3. Polymers used in Tissue Engineering: The various scaffold materials investigated to date can be categorized under two broad groups: either natural or synthetic materials. Examples of commonly used polymers include proteins such as albumin, collagen, fibrin, hyaluronic acid derivatives, silk fibroin, polysaccharides such as alginate, chitosan, heparin, and other naturally occurring biodegradable polymers of sugar units. The best natural scaffold into which cells naturally migrate during spontaneous wound repair is the fibrin clot, fibres with fibrin coating, or fibrin fibres. Synthetic resorbable polymers, such as poly(a-hydroxy ester)s, poly(lactide) (PLA), poly(glycolic acid) 48
(PGA), copolyesters of the lactic acid and glycolic acid, poly(lactide-co-glycolide) (PLGA), polyanhydrides, polyorthoesters, polyphosphazens, polycarbonates, polyamides, polyanhydrides, polyamino acids, polyortho esters, polyacetals, degradable polycyanoacrylates and degradable polyurethanes have been developed. PLA, PGA and their copolymers have a long history of use as synthetic biodegradable materials and are approved by U.S. Food and Drug Administration (FDA) for some specific clinical uses. 3.1 Alginate: Alginic acid is 1,4-linked (homopolymeric or heteropolymeric) copolymer of -Dmannuronic acid and -L-guluronic acid. The alginate is usually derived from seaweed source. There is no specific interaction known between mammalian cells and the alginate polysaccharide. Furthermore, alginate carries a negative charge balance, so proteins are not readily adsorbed due to electrostatic repulsion and thus alginate hydrogel and/or fibrous matrix provide somewhat anti-adhesive surface [9]. This anti-adhesive nature of alginate can have dramatic effect for tissue regeneration process, as for cartilage tissue engineering chondrocytes encapsulated within alginate found to proliferate less but produce higher levels of cartilage specific proteins- e.g., glycosaminoglycan (GAG) and collagen than those in monolayer culture on petri-dish [10]. 3.2 Chitosan: Chitin and chitosan attracted attention of tissue engineers mainly due to its cheap availability, biocompatibility, biodegradability and acceleratory effect on wound healing [11]. Chitin, poly[ (14)-2-acetoamido-2-deoxy- D-glucopyranose], is one of the most abundant natural polysaccharide in the world, which is present in crustacean, insects, fungi, yeasts. Deacetylation of chitin by alkali generates chitosan, poly[(14)-2-amino-2-deoxy-Dglucopyranose] . A typical preparation of chitosan fibers can be done by wet spinning method [12]. 3.3 Hyaluronic acid: Hyaluronan is a naturally occurring linear polysaccharide. Hyaluronan consists of basic disaccharide units of D-guluronic acid and D-N-acetylglucosamine, linked together by alternating -1,4 and -1,3 glycosidic bonds. In 1934, Karl Meyer described a procedure for 49
isolating a novel glycosaminoglycan from the vitreous humor of bovine eyes, and named it hyaluronic acid. At physiological pH all carboxy, groups are dissociated, and hence the polysaccharide should be called hyaluronate. The physico-chemical characterisation of hyaluronan was carried out during 1950s and 1960s. The molecular weight could be in the range of several millions, while in solution the chain behaves as an extended random coil, with a diameter of 500 nm. These properties enable hyaluronan to regulate water balance, osmotic pressure and flow resistance, to interact with proteins, and also to act as a lubricant, and to stabilise the structures by electrostatic interactions [13]. 3.4 Silk fibroin: Recently silk is emerging as the most highlighted candidate for designing of biomaterials and tissue-engineering scaffolds with a wide range of mechanical properties. The dragline silk from the orb weaving spider, Nephilia clavipes, has the highest strength of any natural fiber, and challenges the mechanical properties of synthetic high performance fibers. Silk fibrous scaffolds compression resistant; are stable at physiological temperatures and; are insoluble in aqueous and organic solvents. Silk structures have been studied as a model for the study of structure-function relationships of fibrous proteins. Thus, biocompatibility, the ability to engineer the materials with specific and impressive mechanical properties, and a diverse range of surface chemistries for modification or decoration suggests that silk has the potential to be used as tissue engineering (TE) scaffold. 3.5 Collagen: The extracellular matrix (ECM) is the natural scaffold for the cells, acting as a mechanical support and creating a microenvironment to which the cells can respond. Constructing a matrix or scaffold that simulates the ECM environment is therefore desirable and a widely used strategy in tissue engineering. Such a scaffold has the potential to promote cell growth and to restore key functions to damaged tissues and organs. To mimic the high proportion of collagen present in most native tissues, collagen scaffolds are widely used in tissue engineering [14]. Collagens as a source of natural polymers are in particular interesting as scaffolds for bone tissue engineering as they reflect the major source of proteins in bone extracellular matrix, which consists of up to 90% type I collagen among other matrix proteins [15-17]. 50
3.7 Poly-caprolactone (PCL): The repeating molecular structure of PCL homopolymer consists of five nonpolar methylene groups and a single relatively polar ester group. The presence of hydrolytically unstable aliphatic-ester linkage causes the polymer to be biodegradable. Poly (e-caprolactone) (PCL) fibres are produced by wet spinning from solutions in acetone. To induce bone in-growth into the scaffold, a bioactive coating of the pore surface or additional bioactive fibres, i.e. calcium phosphate glass fibres, can be included [18]. 3.8 Polydimethylsiloxane (PDMS): Polydimethylsiloxane (PDMS) belongs to a group of polymeric organosilicon compounds which are commonly referred to as silicones. PDMS is optically clear, and is generally considered to be inert, non-toxic and non-flammable. It is occasionally called dimethicone. PDMS is viscoelastic, meaning that at long flow times (or high temperatures), it acts like a viscous liquid, similar to honey. However at short flow times (or low temperatures) it acts like an elastic solid, similar to rubber. PDMS has been widely used as a biomaterial in ophthalmic and other applications due to its good compatibility, high mechanical strength, excellent oxygen permeability and transparency [19-21]. However, for use as an artificial cornea, contact lens and in other applications, modifications with hydrophilic functional groups or polymers are necessary to improve wettability for tear protein and mucin interactions and to improve glucose permeability for cellular health. 3.9 Poly (N-isopropylacrylamide): Poly (N-isopropylacrylamide) (PIPAAm) exhibits thermo-responsive hydrophobicity changes in aqueous solutions. At temperatures below 32 C PIPAAm molecules are highly hydrated, so the PIPAAm grafted surfaces are hydrophilic. At temperatures above 32 C the surfaces suddenly change to hydrophobic due to extensive dehydration of PIPAAm molecules [22-27]. This property of PIPAAm is well exploited in cornea tissue engineering. 4. Applications of tissue engineering: 4.1 Cornea Tissue Engineering: Tissue engineering has great potential to appease the growing demand of corneal transplantation. Traditional tissue engineering methods have generally focused on one of two 51
strategies: either the injection of isolated cell suspensions or the use of biodegradable scaffolds to support tissue formation, following which production of corneal tissue seems to be an achievable target. However, current strategies for developing tissue engineered corneal construct has few hurdles to cross and hence until now no successful bioengineered keratoplasty strategy has been clinically implemented. Several attempts failed due to presence of synthetic scaffold material, which impairs transparency and causes strong inflammatory responses upon biodegradation. It has been observed that the implantation of nearly all polymer materials, even if non-toxic, causes a nonspecific inflammatory response [28-29]. Cell Sheet Engineering is a promising approach to treat the cornea. Till now the designed cell sheets are of single layer i.e. epithelial cell sheets, endothelial cell sheets and with randomly oriented cells that affects transparency. So for proper transplantation, it is important to have a tissue engineered properly oriented complete corneal construct which suffers no rejection, no dependence on donor and most of all transparent. Tissue engineered cornea is generated by temperature-responsive polymers of N(isopropylacrylamide), and have attached these polymers to various surfaces. This new surface modification technology demonstrates several intelligent functions, including dramatic, reversible surface property alterations with slight temperature changes. Scientists are still facing some problems regarding tissue engineered (TE) cornea. One of the major problems is that these TC corneas have not enough tensile strength to allow surgical manipulation and fixation, and it is hard to form the required surface curvature. 4.2 Bone Tissue Engineering Although bone is a dynamic and well-vascularized tissue with innate healing and remodeling capacities up to 10% of the bony fractures are complicated by non-union, which require additional treatment with bone grafts in order to achieve defect union and healing. In fact, bone grafts have become the second most transplanted tissue in the world after blood. [30] Bone grafts can be categorized into three types: autografts, allografts, and synthetic grafts. Autografts are harvested from a secondary site from the patients own body, commonly the iliac crest. Allografts, bone grafts harvested from another donor (mainly from cadavers), are an alternative with enhanced flexibility of graft size and shapes [31-33]. However, they introduce the possibility of immune rejection and pathogen transmission. Moreover, processing techniques, such as demineralization, strip the tissue of osteoinductive factors necessary for stimulating bone 52
repair, resulting in impeded healing times, as compared to autografts [34]. Synthetic grafts are made from metals or ceramics, which can be fashioned to different shapes and sizes and are nonimmunogenic, but thus far, have been hampered by their poor speed of healing and inability to remodel in tandem with the natural healing process [30]. 4.3 Dental tissue engineering Developing a technique to manipulate follicles. enamel organ epithelial (EOE) cells is a significant advance towards enamel replacement and therefore attempts have been made to develop a strategy to generate enamel based on subcultured EOE cells using tissue engineering technology. Sumita et al. provide a promising step towards a new therapy for reforming enamel but further study is needed on how to combine the newly-generated enamel with the original dental dentin or enamel in the tooth [35-37]. As EOE cells disappear in adult teeth after tooth eruption we also need to discover new cell sources to advance this technology in the clinical setting [38-40]. 4.4 Skin Tissue Engineering: Basically, there are three methods of skin repair and replacements are autografting, cadaver skin and in vitro reconstructed skin. Autografting is a widely used method of treating organ loss. Cadaver skin has offered an alternate pathway to skin replacement for many years. Alternatives to cadaveric skin are represented by native biological substitutes, with or without living cells [41]. In vitro reconstructed skin relies on the hypothesis that tissue can, in principle, be cultured in vitro. During the several preceding decades, there have been breathtaking achievements in medical science in the field of in vitro cell cultivation, tissue engineering and new biopharmaceutical as well as recombinant genetic therapies. The skin has been the first tissue-engineered organ from the lab bench to the patient [41]. 4.5 Cardiovascular Tissue Engineering: Cardiovascular disease remains a major cause of morbidity and mortality worldwide, resulting 16.7 million deaths each year, accounting for 29% of all deaths globally. The major modality of death due to cardiovascular disease remains acute myocardial infarctions (MI) and sudden cardiac death with more than one third of first time MI not making it to the hospital. Cardiovascular tissue engineering is an emerging field with an enormous potential for revolutionizing the next generation of heart failure therapies; we are witnessing the evolution of 53
therapies from pharmaceuticals and device treatments to the era of biological therapies with cell and engineered tissue do mechanisms Cardiovascular tissue engineering is a materials-based approach and involves preformed three-dimensional (3D) scaffolds in the form of mesh, patch, or foam. This inert biocompatible material will then implanted with differentiated cells to form appropriate functional tissue to be transplanted in-vivo. This has been the focus of intense research in the cardiovascular tissue regeneration field over the past decade [42-45]. 4.6 Cartilage Tissue Engineering: Cartilage is a remarkably complex bio-composite material which exhibits outstanding stiffness, strength, resiliency and shock absorption. It is amazing that only few millimetres thick (500 m up to 7 mm) articular cartilage, as an avascular tissue consisting of only one cell type (chondrocytes), represents at first sight a relatively simple example of tissue engineering but still no fully successful tissue engineered cartilage product is commercially available. The reason for this discrepancy lies in the challenging environment the implant will be exposed to in human body, for example the enormous mechanical forces acting on knee articular cartilage. Accordingly, sufficient mechanical properties of the implant, integration of the implant into the host tissue, and prevention of cell dedifferentiation are required. Once damaged, human cartilage has a poor regenerative ability. Significant progress has been made by using fibrous scaffolds for tissue engineering of articular cartilage. 4.7 Liver Tissue Engineering: Orthotopic liver transplantation is currently the only treatment option available for the people suffering from end-stage liver diseases. But technical complexity, high cost, need for immunosuppression to control allograft rejection (which can cause long-term damage to both the liver and kidneys) are highlighting about the need to develop tissue engineered liver. Differentiated hepatocytes are difficult to culture in vitro because they do not proliferate well and quickly lose viability. Function of Liver is challenging enough to simulate in vitro, which includes synthesis (e.g. of clotting factors, plasma proteins), metabolism (of endogenous and exogenous substances) and elimination (bile secretion) of specific substances over prolonged time periods. The success of tissue engineering of other organs such as bone, cartilage, and skin has been comparably easier to achieve because they are not as highly metabolic and do not
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require an extensive vasculature. But the complex physiology of liver tissue demands preestablish a vasculature bed and seed the cells around the network. 4.8 Urinary bladder Tissue Engineering Tissue engineering, using autologous cells for implantation might offer a solution to this problem. Recently, several studies have confirmed feasibility of bladder reconstruction using engineered segments which were formed using biomaterials seeded with autologous cells in vitro [47]. To date, the clinical application of engineered tissues has been hampered by slow vascularization, poor nutrition leading to cell death and consecutive tissue fibrosis [48-49]. For successful bladder reconstruction revascularization of the construct is essential to support short and long term survival. Furthermore, to restore physiological bladder function reinnervation is indispensable. In respect to bladder TE the ideal scaffold material should prrovide structural support for distinct cell layers, including an adequate surface for stable attachment of urothelial cells; give adequate biomechanical support to harbor a high density of smooth muscle cells on the exterior surface without inducing premature collapse of the hollow organ; serve as a barrier between luminal contents and the body cavity and support the formation of unidirectional muscle tissue in defined layers and allow for rapid innervations and vascularisation [50-51]. 5. Limitations of traditional Tissue Engineering The main limitations of the traditional tissue engineered solid scaffold approach are the following: 1. Low level of precision in cell placement, especially when engineering multicellular constructs. i.e., difficult to obtain 3D precision positioning of several cell types. 2. An intrinsic problem with vascularization of thick tissue constructs. 3. Extremely laborious, slow and costly non automated tissue assembly process. 4. Incapable of precisely controlling pore size, pore geometry, spatial distribution of pores and construction of internal channels within the scaffold. For example, scaffolds produced by solvent-casting particulate-leaching cannot guarantee interconnection of pores because this is dependent on whether the adjacent salt particles are in contact. Furthermore, skin layers are formed during evaporation and agglomeration of salt particles makes controlling the pore size difficult. 55
Moreover, only thin scaffold cross-sections can be produced due to difficulty in removing salt particles deep in the matrix. For gas foaming, it has been reported that only 10-30% of the pores gets interconnected. Non-woven fibre meshes have poor mechanical integrity. Excluding gas foaming and melt molding, conventional scaffold fabrication techniques use organic solvents, like chloroform and methylene chloride, to dissolve synthetic polymers at some stage in the process. The presence of residual organic solvent is the most significant problem facing these techniques due to the risks of toxicity and carcinogenicity it poses to cells. 5. Cellular structure plays a major role in determining cellular activity. The cellular membrane structure, the extracellular matrix and basement membrane influences cellular metabolism, via the proteinprotein interactions. But this cellular metabolism gets disturbed in 2D cell culture because the cells isolated directly from higher organisms frequently alter metabolism and alter their gene expression patterns. Thus, the adaptation of cells in case of 2D cell culture requires a lot of significant adjustment of the surviving cell population not only to changes in oxygen, nutrients and extracellular matrix interactions, but also to alter waste disposal. 6. Molecular gradients play a vital role in biological differentiation, determination of cell fate, organ development, signal transduction, neural information transmission and countless other biological processes. In case of traditional scaffold based tissue engineering the cultured cells lack key signaling and hormonal agents supplied in the in vivo situation by the circulatory system. 7. Conventional fabrication techniques produce scaffolds that are foam structures. Cells are then seeded and expected to grow into the scaffold. However, this approach has resulted in the in vitro growth of tissues with cross-sections of less than 500 m from the external surface. This is probably due to the diffusion constraints of the foam. The pioneering cells cannot migrate deep into the scaffold because of the lack of nutrients and oxygen and insufficient removal of waste products; cell colonisation at the scaffold periphery is consuming, or acting as an effective barrier to the diffusion of oxygen and nutrients into the interior of the scaffold. Furthermore, for bone tissue engineering, the high rates of nutrient and oxygen transfer at the surface of the scaffold promote the mineralization of the scaffold surface, further limiting the mass transfer to the interior of the scaffold. Thus cells are only able to survive close to the surface. However, most other 3D tissues require a high oxygen and nutrient concentration. The human body supplies its tissues with adequate concentrations of oxygen and nutrients via blood vessels. 56
8. Growing cells can significantly alter production of their own extracellular matrix protein and often undergo morphological changes (e.g., an increase in spreading). Because of this the receptors on cell surface might not be in correct orientation and clustering and thus this would affect communication between cells. 6. Advances in Tissue Engineering: 6.1 Cell Sheet Engineering: Cell sheet technology enables novel approaches to tissue engineering without the use of biodegradable scaffolds. Cell sheet technology consists of a temperature-responsive culture dish, which enables reversible cell adhesion to and detachment from the dish surface by controllable hydrophobicity of the surface. This allows for a non-invasive harvest of cultured cells as an intact monolayer cell sheet including deposited extra cellular matrices. The monolayer cell sheet can be transplanted to host tissues without using biodegradable scaffolds and sutures. Thick tissue constructs and patterned cell sheets using two or more kinds of cell source are also developed by means of layered cell sheets in vitro. With the injection of single cell suspensions, the injected cells are expected to remain around the damaged host tissues for the maintenance and recovery of native functions. However, in most cases, the injected cells cannot be retained around the target tissue, causing difficulties to control the size, shape and location of the injected cells. To overcome these problems, biodegradable polymer scaffolds have been seeded with cells to be used to fabricate threedimensional tissue-like grafts. Upon polymer degradation, cells are believed to proliferate and migrate to replace the polymer scaffold. However, the places previously occupied by the polymer scaffolds are filled with large amount of extracellular matrix (ECM). For larger constructs, cells in the center of the constructs become necrotic though the cells on the periphery are unimpaired. This is due to the limits on passive diffusion, that is, insufficient delivery of oxygen and nutrients and removal of metabolic waste. Strong inflammatory responses are often observed upon biodegradation of the scaffolds. Macrophages and neutrophils with collagenase and elastase activities migrate to the implant site during the early wound healing response [52]. Therefore, a new method to avoid the use of biodegradable scaffolds is strongly expected for the further development of tissue engineering. The monolayer cell sheet can be collected simply by reducing culture temperature lower than 32C for less than 30 minutes, without any enzymes or 57
chelating agents. By this technology, we can transplant cell sheets to host tissues without using biodegradable scaffolds [53]. Advantages of cell sheet engineering compared with more conventional approaches: Cell sheets harvested from thermoresponsive substrates adhere to the host tissues without the need for sutures [54]. Tissue engineering strategies require the scaffold material to be surgically placed between cultured cells and the injury site, which can complicate or delay integration of the tissue implant [52]. Cell sheet engineering avoids the use of scaffold materials, which can have potential for inflammatory or foreign body reactions or other complications arising from the by-products of scaffold biodegradation [55]. Thermoresponsive substrates avoid the use of deleterious enzymes that typically are used to remove cell monolayers from conventional culture dishes [56]. Control over the spatial distribution of cells within three-dimensional stratified tissues can be achieved by layering cell sheets created from different cell types [57]. Cell sheet engineering offers better control over cell seeding; that is, when cells are seeded at a cell concentration similar to that observed on a confluent monolayer, final tissue-like constructs have greater cell densities and less ECM, reflecting the varying cell densities of the different native tissues [58]. 6.2 Organ Printing Technology: It has always been a question to be answered that how a tissue engineer can closely imitate the developmental morphogenesis for capturing the above. The answer to this has been solved by the same emerging interdisciplinary field of tissue engineering that has integrated developmental biology in a biomimetic approach. This biomimetic approach intends to provide the biological, chemical, and mechanical clues to guide the gradual cell differentiation taking place and also the assembly into a 3D tissue construct with a real-time insight. Current limitations of exogenous scaffolds or extracellular matrix based materials have underlined the need for alternative tissue-engineering solutions. Scaffolds may elicit adverse host responses and interfere with direct cellcell interaction, as well as assembly and alignment of cell-produced ECM. Thus, fabrication techniques for production of scaffold-free engineered 58
tissue constructs have recently emerged. Here we report on a fully biological self-assembly approach, which we implement through a rapid prototyping bioprinting method for scaffold-free small diameter vascular reconstruction. Now the question arises how can this biomimetic approach be utilized in a beneficial way to serve the above purpose? This is done by using the principle of Biomimetic-writing. Biomimetic-writing or organ printing, which is also known as the biomedical application of rapid prototyping is defined as additive layer by layer biomanufacturing. It is an emerging transforming technology that has the potential of surpassing traditional scaffold based tissue engineering. Thus, in a more narrow sense, it can be defined as computer aided, layer by layer deposition of biologically relevant materials. The ultimate goal of this technology is to fabricate 3D vascularised functional living human organs suitable for clinical implantation. The main practical outcomes of this technology are industrial scalable robotic biofabrication of complex human tissues and organs, automated tissue based in vitro assays for clinical diagnostics, drug discovery and drug toxicity, and complex in vitro models of human diseases. Tissue engineering is based on fabrication of porous solid biodegradable scaffolds with sequential cell seeding in bioreactors. It is the ultimate traditional or classic way of making scaffolds but this standard or method itself has lot of limitations that makes a person to think of an alternative for constructing or making scaffolds. The main rationale behind this approach is a need to maintain, atleast initially, the shape and mechanical properties of the tissue engineered construct and to provide a substrate for cell attachment and signals for cell differentiation and tissue development. Thus, organ printing or biomimetic writing or robot fabrication offers an interesting alternative to solid scaffold based tissue engineering. It has become apparent that 3D cell culture offers a more realistic micro- and local-environment where the functional properties of cells can be observed and manipulated that is not possible in 2D culturing. Rapid prototyping has many different variations. The basis for this technique is to produce usable scaffolds in a short time scale (i.e. hours to days). Solid freeform fabrication (SFF) and 3D printing are two of the more popular rapid prototyping techniques that are capable of generating multi-material and multi-cellular anatomical constructs. Most bone TE methods involve seeding of acellular constructs or insertion of acellular implants with the expectation of 59
cellular ingrowth in vivo. Some successful studies include the use of porous coral in the shape of a distal phalanx seeded with periosteal cells for thumb reconstruction, 3D printing brushite implants and a cranial segment using tricalcium phosphate (TCP) and tetracalcium phosphate respectively. Shek et al. used localized gene therapy to increase and localize cellular and tissue ingrowth using an SFF polypropylene fumarate/TCP composite that provided a stable matrix that could be matched to specific patient defect geometry. Work by Sherwood et al. in conjunction with Therics, Inc. (Princeton, NJ, USA) produced osteochondral composites using TCP combined with either PLG or PLA for the chondral surface. The composite structure exhibited region specific mechanical properties and integration between the two biomaterials making it suitable for implantation. Therics, Inc. also has a number of other TCP based therapeutic products that are currently undergoing clinical studies. SFF techniques are able to produce patient specific scaffolds that can be modified to increase and guide cellular in growth through variation of surface roughness, chemically bonded growth factors, and altered scaffold porosity. For more heterogeneous tissues, such as the meniscus, heart valve and liver, control over spatial and temporal differences in cell type/morphology and mechanical properties is necessary. Achieving structures that have the necessary cell distributions and biomechanical properties is a major challenge. Cytoscribing, as termed by Klebe involved alternating deposition of layers of cells and materials to generate 2D and 3D tissues. Klebe established this technique using a variety of different cell types from different species and bound them to substrates using fibronectin that was deposited via Hewlett Packard graphics plotter of ink jet printer. More recently several groups have demonstrated simultaneous co-deposition of cells and materials. An excellent example of this is by Cohen et al. via SFF using alginate and chondrocytes. The work established the ability to print cell seeded alginate using different materials (i.e. two different grades of alginate) and in different structurally sound shapes including a disc, crescent and meniscus based on CT data with printing resolution of 270 m. Rapid prototyping has also been used in the fabrication of 3D hepatic tissues with complex internal microstructure. Constructs were generated using both multi-cell and multi-material as means to improve nutrient transport. Cell printing efforts by Chang et al. have evaluated cell viability of HepG2 cells based on dispensing pressure and nozzle diameter with calcium cross-linked alginate and combined these SFF techniques with lithography methods to generate 3D micro-organs. The micro-organs had vascular networks serving as pharmacokinetic models and were able to replicate consistent prints with 250 m resolution. 60

Power Switch No.1 Power Switch No.2

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Figure 1: Schematic Diagram of Direct Write Assembly Rapid prototying is already well established and includes many technology variants such as stereolithigraphy, selective laser sintering, fused deposition modelling, 3D printing, ballistic particles manufacturing and others. The main challenge of this technology is the adaptation of existing systems for specific biological materials when it is technically possible, as well as development of novel deposition systems specifically designed for biologically relevant materials. 6.3 Bioreactors in Tissue Engineering: A tissue engineering bioreactor can be defined as a device that uses mechanical means to influence biological processes [59]. Bioreactors can be used to aid in the in vitro development of new tissue by providing biochemical and physical regulatory signals to cells and encouraging them to undergo differentiation and/or to produce extracellular matrix prior to in vivo implantation. Bioreactors are devices in which biological or biochemical processes develop under a closely monitored and tightly controlled environment. Cells respond to mechanical stimulation and bioreactors can be used to apply mechanical stimulation to cells. This can encourage cells to produce extracellular matrix (ECM) in a shorter 61
time period and in a more homogeneous manner than would be the case with static culture. For example, in comparisons between ECM protein levels of equine articular chondrocytes cultured on polyglycolic acid scaffolds after 5 weeks in culture, constructs cultured under hydrostatic pressure showed significant improvements over constructs cultured in static medium [60]. A benefit of ECM production is the increase in mechanical stiffness that it provides to the construct. A six-fold increase in equilibrium aggregate modulus (an intrinsic property of cartilage which is a measure of stiffness) was found after 28 days of culture in a compression bioreactor compared to free swelling controls [61]. Another important application of bioreactors is in cellular differentiation. Mechanical stimulation can be used to encourage stem cells down a particular path and hence provide the cell phenotype required. Bioreactors can provide biochemical and physical regulatory signals that guide differentiation [62]. There is great potential for using mesenchymal stem cells and other multipotent cells to generate different cell types and bioreactors can play an important role in this process. As well as providing mechanical stimulation, bioreactors can also be used to improve cellular spatial distribution. A heterogeneous cell distribution is a major obstacle to developing any threedimensional tissue or organ in vitro. Defects requiring tissue engineering solutions are typically many millimeters in size [63]. Scaffolds in such a size range are easily fabricated, however, problems arise when culturing cells on these scaffolds. As the size of the scaffold increases, diffusion of nutrients to the centre of the construct becomes more difficult. Static culture conditions result in scaffolds with few cells in the centre of the construct. It is hypothesized that this is due to limited cell penetration during seeding, cell migration to the scaffold periphery during culture, or cell death in the centre of the scaffold [63]. The only mechanism by which nutrients and waste can move when a scaffold is in static culture is by diffusion. It has been shown that despite homogeneous cell seeding, after long periods in culture, more cells are found on the periphery of constructs [64] leading to peripheral encapsulation which hinders nutrient and waste exchange from the centre, resulting in core degradation of tissue engineered constructs. This is of major concern in the field of tissue engineering, and is a major obstacle in the formation of a viable tissue in vitro. For this reason, for a number of tissue types, the move towards clinical trials has been slow and progress to date in engineering significant quantities of functional tissue in vitro for implantation in humans in vivo has been somewhat disappointing.
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Thus, bioreactors can be used in tissue engineering applications to overcome problems associated with traditional static culture conditions, improve cellular distribution and accelerate construct maturation [65] whilst applying biophysical signals to constructs to improve tissue formation in vitro prior to in vivo implantation. In general, bioreactors are designed to perform at least one of the following five functions to 1. Provide a spatially uniform cell distribution, 2. Maintain the desired concentration of gases, and nutrients in culture medium, 3. Facilitate mass transport to the tissue, 4. Expose the construct to physical stimuli and/or 5. Provide information about the formation of 3D tissue. 7. CONCLUSION: Scaffolds are used to provide sites for cells attachment, proliferation, differentiation and migration by up regulating and down regulating the synthesis of protein and growth factors. They provide mechanical support; deliver inductive molecules or cells to the repair site. Scaffolds also provide clues to control the structure and function of newly formed tissue. Recently decellularized extracellular matrix has been suggested as a scaffold for heart valve tissue engineering or direct implantation. However, cell removal damages the physical and biochemical properties of the valve leaflet structure. Matrix /polymer hybrid scaffold with improved biomechanical characteristics may be advantageous for many tissue engineering aspects such as heart valve tissue engineering. Equilibrium is needed between highly porous scaffolds that allow rapid tissue ingrowths and minimize diffusion limitations and less porous materials that retain both construct shape and the ability to bear mechanical loads in a complex biochemical and mechanical environment. On the whole, tissue engineering appears to be the new frontier of medicine for its impact on regenerative and reconstructive procedures in humans. In fact, its ultimate goal is to develop powerful new therapies, namely biological substitutes, for structural and functional disorders that have been proven to be difficult or impossible to tackle successfully with the current approaches of interventional medicine. In addition, providing cell-to-tissue replacement parts of the human body it can ultimately afford the irremediable shortage of transplantable organs.
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Tissue engineering presents enormous opportunities for materials science from the perspective of both materials design and materials processing. Scaffolds must direct the arrangement of cells in an appropriate three-dimensional configuration and present molecular signals in appropriate spatial and temporal manner so that the individual cells will form the desired tissue structures and do so in a way that can be carried out reproducibly, economically, and on a large scale. Scaffolds are the materials equipped with molecular cues mimicking certain aspects of structure or function of natural extracellular microenvironments. Each phase in tissue engineering must be understood in an integrated manner from the polymer material properties, to the micro- and macro-architecture of scaffold, to the cell, to the tissue-engineered transplant, to the host tissue.
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43. Zimmerman, W. H., Melnychenko, I., Wasmeier, G., Didie, M., Naito, H., Nixdorff, U., Hess. A., Budinsky, L., Brune, K., Michaelis, B., Dhein, S., Schwoerer, A., Ehmke, H. & Eschenhagen, T. (2006). Engineered heart tissue grafts improve systolic and diastolic function in infracted rat hearts. Nature Medicine 12, 452. 44. Zhong, S., Teo, W. E., Zhu, X., Beuerman, R., Ramakrishna, S., Yue, L. & Yung, L. (2005). Formation of collagen-glycosaminoglycan blended nanofibrous scaffolds and their biological properties. Biomacromolecules 6, 2998-3004. 45. Atala, A. & Lanza, R.P. (2002). Methods of Tissue Engineering. Academic Pr. 46. Zhao, X. Y., Li, W., Lv, Z., Liu, L., Tong, M., Hai, T., Hao, J., Guo, C. L., Ma, Q. W., Wang, L., Zeng, F. & Zhou, Q. (2009). iPS cells produce viable mice through tetraploid complementation. Nature, 461, 86. 47. Atala, A. & Bauer, S. B. (2006). Tissue-engineered autologous bladders for patients needing cystoplasty. Lancet, 367, 1241. 48. Yoo, J. J. & Meng, J. (1998). Bladder augmentation using allogenic bladder submucosa seeded with cells. Urology, 51: 221-225. 49. Ko, H. C. & Milthorpe, B. K. (2007). Engineering thick tissues the vascularisation problem. European Cells and Materials, 14, 1. 50. Brittberg, M. & Lindahl, A. (1994). Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. New England Journal of Medicine, 331, 889. 51. Bent, A. E. & Tutrone, R. T. (2001). Treatment of intrinsic sphincter deficiency using autologous ear chondrocytes as a bulking agent. Neurourol Urodyn, 20, 157. 52. Yang, J., Yamato, M., Kohno, C., Nishimoto, A., Sekine, H., Fukai, F. & Okano, T. (2005). Cell sheet engineering: Recreating tissues without biodegradable scaffolds. Biomaterials, 26, 6415. 53. Shimizu, T., Yamato, M., Kikuchi, A. & Okano, T. (2001). Two-dimensional manipulation of cardiac myocyte sheets utilizing temperature-responsive culture dishes augments the pulsatile amplitude. Tissue Engineering, 7, 141. 54. Nishida, K., Yamamoto, M. & Hayashida, Y. (2004). Corneal reconstruction using tissueengineered cell sheets comprising autologous oral mucosal epithelium. New England Journal of Medicine, 351, 1187. 55. Yang, J., Yamato, M., Shimizu, T., Sekine, H., Ohashi, K., Kanzaki, M., Ohki, T., Nishida, K. & Okano, T. (2007). Reconstruction of functional tissues with cell sheet engineering. 68
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Chapter IV
RNA INTERFERENCE: MOLECULAR MECHANISM AND THERAPEUTIC APPLICATIONS G.J.Khan1, Chandrabhan Seniya2, Prabhat N Jha3, Atul K. Singh2, , Jaykar Jha1*
1 2
Department of Biotechnology, L.N. Mithila University, Darbhanga, Bihar-, India
Department of Biotechnology, Madhav Institute of Technology and Science, Gwalior, India

3
Dept. of Biosciences, Birla Institute of Technology & Science, Pilani, India
*Corresponding Author: Dr Jaykar Jha, Professor & Head, Department of Biotechnology, L.N.Mithila University, Darbhanga-846608, Bihar, India, Phone: +919431627901 Email:jaykarjha@yahoo.com,
ABSTRACTS: RNA interference (RNAi) is a conserved cellular defence mechanism for

controlling the expression of foreign genes in most eukaryotes including humans. It is triggered by double-stranded RNA (dsRNA) and causes sequence-specific mRNA degradation of singlestranded target RNAs homologous in response to dsRNA. The mediators of mRNA degradation are small interfering RNA duplexes (siRNAs), which are produced from long dsRNA by enzymatic cleavage in the cell. siRNAs are approximately twentyne nucleotides in length, and have a base-paired structure characterized by two nucleotide 3'-overhangs. Chemically synthesized siRNAs have become powerful reagents for genome-wide analysis of mammalian gene function in cultured somatic cells. siRNAs hold great potential as gene-specific therapeutic agents.
INTRODUCTION:
RNA interference (RNAi) is a phenomenon leading to post-transcriptional gene silencing (PTGS) after endogenous production or artificial introduction into a cell of small interfering double strand RNA (siRNA) with sequences complementary to the targeted gene [1]. RNAi prevents the translation of the target protein by selective sequence specific degradation of its encoded messenger RNA (mRNA). RNAi is an RNA-dependent gene silencing process that is 70
controlled by the RNA-induced silencing complex (RISC) and is initiated by short doublestranded RNA molecules in a cell's cytoplasm, where they interact with the catalytic RISC component argonaute. It is a novel gene regulatory mechanism that limits the transcript level by either suppressing transcription (TGS) or by activating a sequence Specific RNA degradation process [PTGS/RNA interference (RNAi)] [2]. Two types of small RNA molecules microRNA (miRNA) and small interfering RNA (siRNA) are central to RNA interference. The RNAi pathway is found in many eukaryotes including animals and is initiated by the enzyme Dicer, which cleaves long double-stranded RNA (dsRNA) molecules into short fragments of about 20 nucleotides called siRNA. The siRNA is unwound into two ssRNA, namely the passenger strand and the guide strand. The passenger strand is degraded, and the guide strand is incorporated into the RNA-induced silencing complex (RISC). Posttranscriptional gene silencing occurs when the guide strand base pairs with a complementary sequence of a mRNA molecule and induces cleavage by argonaute, the catalytic component of the RISC complex. The effects of both RNAi and PTGS are potent and systemic in nature. The gene silencing molecules (RNAi) is amplified and the concentration of RNAi increases by many fold. The amplification of the RNAi makes it potent even if the concentration is quite low at initiation stage. This process is known to spread systemically throughout the organism despite initially limited molar concentrations of siRNA. The natural function of RNAi refers to the mechanism involved in cellular defence against viruses, genomic containment of
retrotransposons, and post-transcriptional regulation of gene expression. Primarily, it is involved in the response to exogenous pathogenic and endogenous parasitic nucleic acids [3,4], as well as in the basic cellular functions, such as gene regulation and heterochromatin formation [57]. Currently, RNAi has been widely used not only as an extremely powerful approach for reverse functional genomics [8], but also as an effectively potent strategy for gene silencing-based therapeutics [9,10]. As compared with other gene silencing reagents, such as antisense oligonucleotides (ODNs), ribozymes, and DNAzymes, siRNAs have apparently become the most powerful and widely used gene silencing reagents for manipulating gene activity. RNAi can specifically silence individual genes, creating knockout phenotypes, either in transformants that can produce the required hairpin RNAs, or upon infection with recombinant RNA viruses that carry the target gene (VIGS, viral-induced gene silencing) [11]. The integrity of the RNAi pathway is essential in mammals [12,13], where endogenous RNAi controls different 71
developmental and physiological processes and has been implicated in the development of cancer and other diseases [14,15]. Understanding these functions is of critical importance when exploiting RNAi for different purposes, as exogenous manipulation could undesirably interfere with the function of endogenous RNAi. Knowing the biological processes in which RNAi participates would allow us to anticipate and monitor the appearance of unexpected adverse effects derived from the application of these therapeutic molecules.
IMPORTANT FEATURES OF RNA SILENCING:

RNA silencing has been coined variously as PTGS in plants, RNAi in animals, quelling in fungi, andvirus-induced gene silencing in plants and animals, have converged on a universal paradigm of gene regulation. The common components of the paradigm are: (i) the inducer is the dsRNA; (ii) the target RNA is degraded in a homology dependent fashion; (iii) the degradative machinery requires a set of proteins which are similar in structure and function across most organisms [16]. In most of these processes, certain invariant features are observed, including the formation of small interfering RNA (siRNA) and the organism specific systemic transmission of silencing from its site of initiation.
BRIEF HISTORY OF RNA INTERFERENCE:

Napoli and Jorgensen were the first to report an RNAi type of phenomenon in 1990 [17]. The discovery of RNAi was the unexpected result of attempts to make the colour of petunia blooms more purple by over expressing the transgene chalcone synthetase (CHS)). However, instead of increasing anthocyanin levels, the transgenic model showed apparent silencing of both the endogenous and exogenous CHS genes. The levels of endogenous as well as introduced CHS were 50-fold lower than in wild-type petunias, which led them to hypothesize that the introduced transgene was cosuppressing the endogenous CHS gene. In 1992, Romano and Macino reported a similar phenomenon in Neurospora crassa [18], noting that introduction of homologous RNA sequences caused quelling of the endogenous gene. RNA silencing was first documented in animals by Guo et al., [19] who observed that the introduction of sense or antisense RNA to par-1 RNA resulted in degradation of the par-1 message in Caenorhabditis elegans. At that time, antisense was one of the most attractive means of eliminating gene expression. Antisense was thought to function by hybridization with endogenous mRNAs resulting in double stranded RNA (dsRNA), which either inhibited translation or was targeted for 72
destruction by cellular ribonucleases. Guo et al., produced the loss-of-function or gene-knockout phenotype in the offspring of the injected C. elegans by direct injection of antisense siRNA (par1 gene) and a similar inhibitory effect on par-1 gene function was elicited by the injection of sense siRNA [19]. In 1998, Fire et al., reported that double-strand RNA was at least tenfold potent as a silensing trigger than were antisense or sense RNA alone [20]. Due to the versatile application of RNAi in diverse systems, different groups around the globe are trying to explore the molecular mechanism of its action. It has been established that siRNAs are the key mediators of RNAi [21] and the cloning of the RNAse III-like enzyme Dicer [22], have provided insight into the RNAi pathway. RNAi renders promising result in therapy specially in infectious disease, cancer, and dominantly inherited disorders [23, 24]. RNAi has also shown exceptional promise as a genome-wide screening tool [25-27].
SALIENT FEATURES OF RNA INTERFERENCE

The mechanism by which RNAi inhibits the conversion of mRNA into protein is a potent and robust system which protect the organism from genetic contamination and bears the following features. Double stranded RNA rather than single-stranded antisense RNA is the interfering agent. High degree of specific gene silencing with less effort. Highly potent and effective. Silencing can be introduced in different developmental stages. Systemic silencing. Avoids problems with abnormalities caused by a knocked out gene in early stages Silencing effects passed through generations.
COMPONENTS OF RNAi:
Both genetic and biochemical approaches have been undertaken to understand the basis of silencing. Genetic screens were carried out in the fungus Neurospora crassa, the alga Chlamydomonas reinhardtii, the nematode Caenorhabditis elegans, and the plant Arabidopis thaliana to search for mutants defective in quelling, RNA interference, or PTGS. Analyses of these mutants led to the identification of host-encoded proteins involved in gene silencing and also revealed that a number of essential enzymes or factors are common to these processes. Some 73
of the components identified serve as initiators, while others serve as effectors, amplifiers, and/or transmitters of the gene silencing process. Dicer: Dicer was given its name by Emily Bernstein, who first demonstrated the enzyme's dsRNA "dicing" activity [28]. Dicer is an endoribonuclease in the RNase III family that cleaves double-stranded RNA (dsRNA) into short double-stranded RNA fragments called small interfering RNA (siRNA), about 20-25 nucleotides long usually with a two-base overhang on the 3' end. Dicer contains two RNase III domains and one PAZ domain; the distance between these two regions of the molecule is determined by the length and angle of the connector helix and determines the length of the siRNAs it produces. RNase III family members are among the few nucleases that show specificity for dsRNAs [29] and cleave them with 3 overhangs of 2 to 3 nucleotides and 5 -phosphate and 3-hydroxyl termini [30]. Bernstein et al. [31] identified an RNase III like enzyme in Drosophila extract which was shown to have the ability to produce fragments of 22 nucleotides, similar to the size produced during RNAi. Cleavage by Dicer is thought to be catalyzed by its tandem RNase III domains. Some Dicer proteins, including the one from D. melanogaster, contain an ATP-binding motif along with the DEAD box RNA helicase domain. Dicer homologues from many different sources have been identified; some recombinant Dicers have also been examined in vitro, and phylogenetic analysis of the known Dicer-like proteins indicates a common ancestry of these proteins [32]. Small interfering RNA (siRNA): The key insight in the process of PTGS was provided from the experiments of Baulcombe and Hamilton [33], who identified the product of RNA degradation as a small RNA species (siRNA) of 25 nucleotides of both sense and antisense polarity. siRNAs are formed and accumulate as double-stranded RNA molecules. siRNAs were detected first in plants undergoing either cosuppression or virus-induced gene silencing and were not detectable in control plants that were not silenced. siRNAs were subsequently discovered in Drosophila tissue culture cells in which RNAi was induced by introducing 500-nucleotide long exogenous dsRNA [34]; in Drosophila embryo extracts that were carrying out RNAi in vitro [35] and; also in Drosophila embryos that were injected with dsRNA [36]. Guide RNAs and RNA-Induced Silencing Complex: At the end of 2005, two independent groups, Gregory et al. and Matranga et al., overturned the long-held belief that helicases played a major role in unwinding ds siRNAs by demonstrating that Ago2 is responsible for cleaving the non incorporated (passenger) strand of the siRNA duplex, allowing the other strand to be 74
incorporated into RISC [37, 38]. Evidence that the passenger strand of the siRNA serves as the first target of RISC is derived from detection of the predicted 9-nucleotide 5 cleavage and 12nucleotide 3 cleavage products. How the RISC/Ago2 determines which strand of the doublestranded siRNA to cleave and which strand to incorporate into the RISC complex were a mystery. In 2003, a group led by Jayasena addressed this question by comparing the thermodynamic properties of functional and nonfunctional siRNAs [39]. Their results indicated that the 5 antisense region of the functional siRNAs were less thermodynamically stable than the 5 sense regions, providing a basis for entry into RISC. The siRNA duplex containing ribonucleoprotein particles (RNPs) are subsequently rearranged into the RNA induced silencing complex. The functional RNPs contain only single stranded RNA. The estimated apparent molecular mass ranges from between 130 to 160 kDa in the case of high salt purified RISC from human cells to 500 kDa or up to the 80S range in the case of RISC isolated from D. melanogaster cell lysates. One thing these effector complexes have in common is the presence of an Argonaute protein. Although Ago2 is the enzyme responsible for cleaving the passenger strand siRNA as well as target mRNA, purified Ago2 cannot use double-stranded siRNAs as a substrate for target mRNA cleavage. This observation suggests that additional cofactors are needed for RISC activity. Recent evidence has suggested that Dicer may not only play a role in the initiator phases of the RNAi pathway, but also at later phases [40, 41] The assembly of RISC is ATP dependent [42,43] which probably reflects the requirement for energy driven unwinding of siRNA . RNA-Dependent RNA polymerase: The effect of RNAi is both potent and systemic in nature. This has led to a proposed mechanism in which RNA-dependent RNA polymerases (RdRPs) play a role in both triggering and amplifying the silencing effect. Transgenic and virus-infected plants show an accumulation of aberrant transgenic and viral RNAs. The RdRP enzymes might recognize these aberrant RNAs as templates and synthesize antisense RNAs to form dsRNAs that are finally the targets for sequence-specific RNA degradation [44-47]. Transmembrane Protein (Channel or Receptor): The systemic spread of gene silencing from one tissue to another has been well established in C. elegans and plants. To investigate the mechanism of systemic RNAi, Winston et al. [48] constructed and used a special transgenic strain of C. elegans (HC57). They identified a systemic RNA interference deficient (sid) locus required to transmit the effects of gene silencing between cells with green fluorescent protein 75
(GFP) as a marker protein. Of the 106 sid mutants belonging to three complementation groups (sid1, sid2, and sid3), they isolated and characterized sid1 mutants. The sid1 mutants had no readily detectable mutant phenotype other than failure to show systemic RNAi. Interestingly, these mutants also failed to transmit the effect of RNAi to the progeny. The SID1 polypeptide is predicted to be a 776-amino-acid membrane protein consisting of a signal peptide and 11 putative transmembrane domains. Based on the structure of SID1, it was suggested that it might act as a channel for the import or export of a systemic RNAi signal or might be necessary for endocytosis of the systemic RNAi signal, perhaps functioning as a receptor. No homologue of sid1 was detected in D. melanogaster, which may be consistent with the apparent lack of systemic RNAi in the organism [49, 50]. However, the presence of SID homologues in humans and mice might hint at the systemic characteristics of RNAi in mammals.
MECHANISM OF RNA INTERFERENCE

RNA interference takes place predominantly within the cytoplasm of the cell and is triggered by the introduction of a double stranded oligonucleotide into the cell cytoplasm. The mechanism can be divided into two distinct phases: an initiation and an execution phase. The initiation phase involves processing of dsRNA into small interfering RNA molecule (siRNA). In the execution phase, siRNAs are then incorporated into large ribonucleoprotein complexes. These effector complex interfere with gene expression by using the small RNA strand to identify their complementary mRNA and the same is cleaved and degraded [51]. In the gene silencing pathways initiated by dsRNA precursors (small RNA duplex), Dicer-mediated cleavage yields small dsRNA intermediates, which must dissociate into competent single strands in order to function as guides for RISCs. For each small RNA duplex, the guide strand, is loaded onto a specific Argonaute protein and assembled into the active RISC; other strand, the passenger strand, is destroyed. The key factors in converting the RISC from its precursor form (the preRISC), which contains the small RNA duplex, to its mature form (the holo- RISC), which contains the guide strand, are small RNA strand unwinding and preferential strand selection. [52]. Their results indicated that the 5 antisense region of the functional siRNAs were less thermodynamically stable than the 5 sense regions, providing a basis for entry into RISC. For example, in D. melanogaster, DCR-2 does not simply transfer siRNAs to a distinct RISC but, instead, forms part of the RISC together with the siRNAs, indicating that the role of DCR-2 76
extends beyond the initiation phase. The loading of siRNA duplexes onto AGO2 is facilitated by the RISC loading complex, which contains DCR-2 and its dsRBD-containing partner, R2D2 [53, 54]. The particular strand of the siRNA duplex that is loaded onto AGO2 seems to be determined by the orientation of the DCR-2R2D2 heterodimer on the siRNA duplex [55]. R2D2 is thought to sense the thermodynamic stability of the siRNA duplexes and bind to the more stable end of the siRNA, whereas DCR-2 is recruited to the less stable end. The heterodimer probably recruits AGO2 through an interaction between DCR-2 and AGO2. Previous models have proposed that the transition from a double-stranded silencing trigger to a single-stranded one is mediated by an unidentified ATP-dependent RNA helicase. However, the unwinding of the siRNA duplex and the loading of a single strand into the RISC are facilitated by the slicing of the unincorporated (passenger) strand by AGO2, a process that does not require ATP [5658]. Cleavage in the middle of the passenger strand, as though the passenger strand were an mRNA target, would be expected to reduce the annealing temperature and the free energy of duplex formation, which in turn facilitates the separation of the siRNA strands. These data support a model in which siRNAs are initially loaded as duplexes onto an AGO2-containing pre-RISC. By contrast, in humans, premiRNAs are known to bind to a preformed trimeric complex of AGO2, DICER1 and DICER1s dsRBDcontaining partner, TRBP [59]. This complex can cleave target RNAs using pre-miRNA and can distinguish miRNA from miRNA* in the absence of ATP hydrolysis [59,60], suggesting that DICER1-mediated cleavage and sensing of thermodynamic stability occur in series in the AGO2DICER1TRBP complex. This process by which a pre-RISC is converted to a holoRISC can also occur by a slicer-independent mechanism. Three of the four Argonaute proteins in humans (AGO1, AGO3 and AGO4) lack slicer activity but are nonetheless loaded with singlestranded guide siRNAs [61-63]. Similarly, single-stranded miRNAs are found associated with AGO2 in humans, despite the expectation that mismatches in the unwound pre-miRNA should block the passenger-strand cleavage activity of AGO2. Thus, a cleavage-independent (bypass) mechanism for RISC assembly must exist. RNA helicase A has been identified as a candidate for unwinding the duplex in this process [64]. Amplification Of siRNAs: One of the many intriguing features of RNA interference is the apparently catalytic nature of the phenomenon. A few molecules of dsRNA are sufficient to degrade a continuously transcribed target mRNA for a long period of time. Although the conversion of long dsRNA into many small siRNAs results in some degree of amplification, it is 77
not sufficient to bring about such continuous mRNA degradation. Since mutations in genes encoding RNA-dependent RNA polymerase (RdRP) affect RNAi, it was proposed that this type of polymerase might replicate siRNAs as epigenetic agents, permitting their spread throughout plants and between generations in C. elegans. Recent studies by Lipardi et al. [65] and Sijen et al. [66] provided convincing biochemical and genetic evidence that RdRP indeed plays a critical role in amplifying RNAi effects. Lipardi et al. [65], while investigating the dsRNA-dependent degradation of target mRNA in a Drosophila embryo cell extract system, showed the generation of full-length cognate dsRNAs from labeled siRNAs at early time points. Both single stranded RNAs (equivalent to target mRNA) and dsRNAs served as templates for copying by RdRP. New full-length dsRNAs were formed rapidly and cleaved. They also showed a strict requirement for the 3 hydroxyl group and 5 phosphate group on siRNAs for primer extension in the RdRPmediated reaction [65].
Figure1: Different steps in Gene Silencing: 78

Sijen et al. [66] further revealed the role of RdRP activity in RNAi. In an RNAi reaction, they observed the formation of new siRNA species corresponding to target mRNAs but different from trigger dsRNAs. They named these new siRNAs secondary siRNAs. With a primary trigger dsRNA specific for the lacZ region of the target mRNA that encoded a GFP-LacZ fusion protein, these authors demonstrated the degradation of a separate GFP mRNA target. This kind of RNAi induced by secondary siRNAs was named transitive RNAi. These authors demonstrated the requirement for the rrf1 gene, a C. elegans gene with sequence homology to RdRP, in the generation of secondary siRNAs and transitive RNAi [66]. Amplification of siRNAs might occur at various stages of the RNAi reaction and has been documented in plants, C.elegans, N. crassa, and Dictyostelium discoideum but not in flies and mammals [67]. Though the RdRP activity is present in Drosophila embryo extract, as mentioned earlier, it is surprising that the fly genome does not code for RdRP. Additionally, numerous experiments also suggest that RdRP is not required for RNAi in D. melanogaster [68]. Degradation of mRNA: In the effector step of RNAi, the double-stranded siRNAs bind an RNAi-specific protein complex to form a RISC. This complex undergoes activation in the presence of ATP so that the antisense component of the unwound siRNA becomes exposed and allows the RISC to perform the downstream RNAi reaction. Zamore et al [69] demonstrated that a 250 kDa precursor RISC, found in Drosophila embryo extract, was converted into a 100 kDa complex upon being activated by ATP. This activated complex cleaved the substrate. The size and constitution of the precursor as well as the activated RISC might vary depending on the choice of system [68]. The antisense siRNAs in the activated RISC pair with cognate mRNAs, and the complex cuts this mRNA approximately in the middle of the duplex region. A few independent studies demonstrated the importance of the RISC complex in this part of RNAi reactions. The mRNA cleaving RNA-protein complexes have also been referred to as siRNP (small interfering ribonucleoprotein particles). It is widely believed that this nuclease is probably different from Dicer, judging from the substrate requirements and the nature of the end products. Since the target cleavage site has been mapped to 11 or 12 nucleotides downstream of 5 end of the guide siRNA, a conformational rearrangement or a change in the composition of an siRNP ahead of the cleavage of target mRNA is postulated. Finally, the cleaved mRNAs are perhaps degraded by exoribonucleases [70]. A part of cleaved fragments of mRNA at the end of step 2 might also be converted to the duplex forms by the RdRP-like activity. These forms might have 79
siRNA-like functions and eventually enter the pool of the amplification reaction. Thus, it is likely that amplification of the RNAi reaction takes place at both step 1 and step 2 of RNAi. In another model, it has been proposed that siRNAs do not act as primers for the RdRP-like enzymes, but instead assemble along the length of the target RNA and are then ligated together by an RNA ligase to generate cRNA. The cRNA and target RNA hybrid would then be diced by the DCR protein. All these models were summarized by Schwarz et al. [71]. Most of the steps involved in the mechanism of RNAi have been illustrated schematically in Fig 1.
THERAPEUTIC APPLICATIONS OF RNA INTERFERENCE

RNAi is an excellent therapeutic tool for disease in which selective knockout of a gene would slow or completely halt the disease process in the affected cells. Ideally this would be accomplished with no or tolerable side effects. Although there are candidate gene targets for many different diseases, we will focus on four different types of diseases that are very common and for which RNAi approaches are currently being tested in preclinical studies. A. Cancer: Cancer cells exhibit two main abnormalities. Uncontrolled growth and resistance to cell death (apoptosis) [72]. The ability of siRNAs to silence specific oncogenic variants while sparing the wild-type products of genes has been demonstrated in human pancreatic carcinoma [73]. Similarly, a single point mutation in the tumour suppressor p53 was effectively targeted but not the wild-type product using siRNAs [74]. RNAi technology can be directed against cancer by specifically modulating the expression of oncogenes, promoting apoptosis and regulating cell cycle.The goals for RNAi approaches for cancer therapy are therefore to modulate the expression of apoptotic protein/cell cycle protein in the cancer cells thereby stopping tumour growth and killing the cancer cells. To selectively eliminate cancer cells without damaging normal cells, the RNAi is used to target a gene specifically involved in the growth or survival of the cancer cell. Advantages of RNAi methods for the growth suppression and killing of cancer cells have been shown in different studies. Viral vectors have also been used to express siRNAs and inhibit cancer cell growth and tumorogenicity. For example, a retroviral vector was used to specifically and stably inhibit expression of the oncogenic K-RASV12 allele in human tumor cells [73]. Depletion of K-RASV12 resulted in loss of anchorage-independent growth and tumorigenicity. In addition to blocking the expression of normal genes that are required for cancer cell growth and survival, RNAi can be used to target specific cancer-causing mutations. For example, dsRNA 80
was employed to target the M-BCR/ABL fusion site to kill leukemic cells with such a rearrangement [75]. Leukemic cells without BCR/ABL rearrangement were not killed by MBCR/ABL-dsRNA. Several other studies have demonstrated efficacy of liposome-mediated or viral vector-mediated transfection of cancer cells in suppressing their growth and/or inducing their death [76]. The next step in the development of RNAi technology for cancer therapy will be to establish methods for targeting tumor cells in vivo. Another approach might be to target genes that promote angiogenesis. Tumor cells require a rich supply of blood and achieve this by stimulating the process of angiogenesis; it may therefore be possible to inhibit tumor growth by targeting the vascular endothelial cells involved in angiogenesis. As evidence, it was shown that depletion of the crk adaptor protein using RNAi inhibited the migration of cultured vascular endothelial cells [77]. B. Infectious Diseases: The ability of RNAi to inhibit the replication and cellular uptake of viruses and other infectious agents has been clearly demonstrated in cell culture studies and therefore, holds promise for the treatment of human patients. Transfection of human cells with siRNAs against different genes in the poliovirus genome resulted in resistance of the cells to infection with poliovirus [78]. The ability of siRNAs targeting the gene encoding the death receptor Fas to protect mice from liver failure and fibrosis in two models of autoimmune hepatitis was tested by Song et al. [79]. Intravenous injection of Fas siRNA specifically reduced Fas protein levels in the livers of mice during 10-day period. Fas siRNA treatment abrogated hepatocyte necrosis and inflammatory infiltration and markedly reduced serum concentrations of transaminases demonstrating a clear hepatoprotective effect of the siRNA therapy. Synthetic siRNAs and expressed siRNAs have been used to target several early and late HIV-encoded RNAs in cell lines and in primary haematopoietic cells including the TAR element [80], tat [81, 82], rev [ 81], gag [83, 84], env [84], vif [80], nef [80] and reverse transcriptase [82]. Despite the success of RNAi-mediated inhibition of HIV-encoded RNAs in cell culture, targeting the virus directly represents a substantial challenge for clinical applications because the high viral mutation rate will lead to mutants that can escape being targeted [85]. RNAi-mediated downregulation of the cellular cofactors required for HIV infection is an attractive alternative or complementary approach. Cellular cofactors such as NF-B [82], the HIV receptor CD4 [83] and the co-receptors CXCR4 and CCR5 [86] have been successfully downregulated by RNAi, resulting in the inhibition of HIV replication in numerous human cell lines and in primary cells 81
including T lymphocytes and haematopoietic -stem-cell-derived macrophages [80, 81, 84, 8689]. C. Cardiovascular and Cerebrovascular Diseases: Cardiovascular disease is the leading cause of death in many industrialized countries. It most commonly results from the progressive occlusion of arteries in a process called Atherosclerosis, which can ultimately culminate in a myocardial infarction or stroke. Atherosclerosis involves damage to vascular endothelial cells, local production of inflammatory cytokines and the recruitment of macrophages to the site forming foam cells. In addition, apoptosis of foam cells and vascular smooth muscle cells occurs [90]. The severe irritability that occurs in heart or brain cells during a myocardial infarction or stroke results in the death of cardiac tissue rapidly by necrosis, many other cells die more slowly by apoptosis; data from animal studies suggest that such cardiac myocytes and brain neurons that die by apoptosis can be saved [91, 92]. It may be possible to use RNAi technology to intervene in the process of atherosclerosis or to reduce the damage to heart tissue and brain cells that patients suffer following a myocardial infarction or stroke. A key step in the process of atherosclerosis is the up-regulation of cell adhesion molecules in vascular endothelial cells, which play an essential role in the recruitment of macrophages to the site of endothelial damage. The production of cell adhesion molecules can be selectively suppressed in cultured cells [93]. D. Neurodegenerative Disorders: Recent rapid progress in the application of RNAi offers new approaches to drug target identification and validation. Advances in targeted delivery of RNAiinducing molecules has raised the possibility of using RNAi directly as a therapy for a variety of human genetic and other neural and neuromuscular disorders. Alzheimers disease, Parkinsons disease, Huntingtons disease and Amyotrophic lateral sclerosis (ALS) are examples of relatively common age related neurodegenerative disorders that are increasing as average life expectancy increases. Each disorder is characterized by the dysfunction and death of specific populations of neurons: hippocampal and cortical neurons involved in learning and memory processes in Alzheimers disease, dopamine-producing neurons in the substantia nigra that control body movements in amyotrophic lateral sclerosis. Specific genetic mutations are responsible for a small percentage of cases of Alzheimers and Parkinsons disease and Amyotrophic lateral sclerosis [94], whereas all cases of Huntingtons disease result from mutations (polyglutamine expansions) in the Huntington protein [95]. Studies of patients and of animal and cell culture models of each disease have revealed shared biochemical cascades that result in neuronal death. 82
Those cascades include increased oxidative stress, dysregulation of cellular calcium Parkinsons disease and spinal cord motor neurons in homeostasis and apoptosis [91]. Therefore, there have been two different strategies for preventative and therapeutic interventions in neurodegenerative disorders. One strategy is to block the disease-specific events that are believed to initiate the neurodegenerative process, whereas the second strategy targets downstream events in the neurodegenerative cascade. Recent studies have shown that cultured neurons can be efficiently transfected with siRNAs and that the targeted genes are effectively silenced. In one study, it was shown that cultured neurons can be depleted of the p75 neurotrophin receptor, a protein in the TNF receptor family that has been implicated in neuronal apoptosis in certain settings [96]. Proapoptotic members of the Bcl-2 family [97] and caspases [98] have been effectively targeted and neuronal death have been prevented using RNAi methods. Caplen and colleagues [61] performed studies aimed at determining whether RNAi could be used to target the pathogenic process in inherited neurodegenerative disorders caused by polyglutamine expansions. Thus, now it is possible, at least in cell culture, to selectively silence a transcript associated with an important group of time genetic diseases by RNAi.
CHALLENGES IN RNA INTERFERENCE

Intravascular degradation: Naked siRNA is unstable in circulation owing to serum RNase A-type nucleases and rapid renal clearance, leading to degradation and a short half-life [100]. Some investigators have turned towards the chemical modification of the sugars, the backbone or the bases of oligoribonucleotides for stabilization [101,102]. However, the hydrophobic cell membranes create a challenge for the intracellular delivery of negatively charged polymers. Tissue penetrance and intracellular delivery: For safe and effective delivery of RNAi to the mRNA target of interest, many variables must be negotiated. Although nanoparticles by definition range in size from 1 nm to 1,000 nm in size, it has become clear that intravenously injected particles > 100 nm in diameter are likely to be trapped by the reticuloendothelial system (RES) in the liver, spleen, lung and bone marrow, leading to degradation by activated monocytes and macrophages.[103] The fate of RNAi in biological fluids: Although intracellular barriers to RNAi delivery are being explored in depth, extracellular factors in the microenvironment are less well studied 83
and could be more important. For successful delivery, nanoparticles must extravasate and move through the complex extracellular matrix (ECM) to reach the cancer cells [103]. Immune-mediated toxicities: Unknown or unexpected toxicities that are related to systemic RNAi delivery must be anticipated. The innate immune response to siRNA is highly contextualized and divided into two groups: Toll-like receptor (TlR)-mediated or non-TlRmediated groups. TlR3, TlR7 and TlR8 are activated when engaged with nucleic acids within endosomal and lysosomal compartments. Ligands that activate TlR7 and TRl8 include duplex siRNA, or its corresponding single strand [103].
CONCLUSION
Even at this early stage of understanding the molecular mechanisms of RNAi and in the development of methods for the use of RNAi technology for selective gene silencing, it is clear that RNAi will be a widely used tool for establishing the functions of genes. The ability to selectively deplete a single protein of interest in cultured cells using siRNAs, and plasmids and viral vectors, is now established. Improvements on the currently available protocols for RNAi are being made, and the methods are being applied by thousands of investigators in diverse fields. With the advent of these methods has come an explosion of studies that have employed RNAi.
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Chapter V
BIOTECHNOLOGICAL INTERVENTIONS TO COMBAT ABIOTIC STRESS IN PLANTS

Rajesh Mehrotra1,2, Uthra Balasubramaniyan1, Purva Bhalothia1, Bhavya Ravi1 and Sandhya Mehrotra1
1
Department of Biological sciences, Birla Institute of Technology & Sciences (BITS), Pilani, Rajasthan, India Pin-333031, 2 Louisiana state university, Baton Rouge, Louisiana 70803.
Corresponding Author: Rajesh Mehrotra, E-mail: rajmeh25@hotmail.com ABSTRACT: Molecular programming of plants enables them to withstand diverse environmental stress conditions by dynamic alterations in physiology and cellular composition. An insight into the mechanisms involved in such adaptations will help not only in engineering stress resistance in agronomically important crop varieties but also in understanding patterns of plant development and distribution in response to the environment. This chapter focuses on the various types of stress conditions such as drought, salinity and temperature extremes and their molecular manifestations with an emphasis on signaling cascades involving phosphatases, kinases, nuclear factors and stress responsive genes. The role of cis elements present in promoters and the design of synthetic promoters for achieving inducible stress-specific expression profiles have also been explored. Keywords: Stress, protein engineering, salinity, chilling
INTRODUCTION
Abiotic factors such as water availability and temperature extremes have had a major influence on the evolution of plant systems. Nevertheless, in recent times, drastic environmental disturbances have affected agricultural productivity to a large extent. Abiotic stress has been shown to negatively influence survival, biomass production and accumulation, and grain yield of most crops [1,2]. The perception of varying environmental conditions and the processes of adaptation for survival under these conditions represent a complex interplay between various nuclear factors and signaling molecules. Engineering of stress tolerance therefore demands a comprehensive knowledge of genes and regulatory components activated in response to different 93
kinds of stress. Since abiotic stress responses are multigenic in nature, research has focused on the characterization of abiotic stress related genes, transcription factors and regulatory sequences present in plant promoters. These have interesting implications both in terms of stress tolerance and understanding of plant biochemical and developmental processes. Many recent studies have suggested that genes involved in abiotic stress tolerance are regulated at the level of transcription and hence a brief understanding of transcription regulation becomes essential at this stage.
TRANSCRIPTIONAL REGULATION: AN OVERVIEW

DNA, in isolation, cannot result in protein synthesis. The protein machinery comprising the basal transcription factors, trans-acting elements and the membranous and intercellular proteins involved in signal transduction pathways is indispensable in the events that finally result in transforming the genetic information on DNA into protein products. Even within the nucleus, eukaryotic genes are present as chromatin which is a complex of DNA and histone proteins. A stretch of approximately 160 base pairs of DNA wraps around the central kernel of the histone octamer (H2A, H2B, H3 and H4) in two left-handed super helical turns and the assembly is referred to as a nucleosome, which is the basic unit of chromatin structure [3]. The nucleosomes are attached by short stretches of linker DNA and are wound into tight solenoids stabilized by histone H1 which assembles the nucleosomes into tight arrays thereby preventing the transcription factors and RNA polymerase from gaining access to the genes [4]. This represents the default condition of an inactive gene. Gene activation and regulation therefore, require a change in this chromatin structure. The two major classes of chromatin modifying complexes that have been characterized are histone acetyltransferases and methyltransferases and ATP dependent chromatin remodeling complexes [5]. Histone acetyltransferase (HAT) and deacetylase mediate the addition of negatively charged acetyl groups to the basic residues of histone tail. This reduces the electrostatic interaction between the histones and DNA, thus loosening the histones and making DNA available for transcription. Histone methyltransferases on the other hand transfer methyl groups to histones and can either activate or further repress transcription depending on the amino acid that is methylated and other covalent modifications in the vicinity. [4] In a very elegant study Wada et al have shown association between up regulation of stress responsive genes and hypomethylation of genomic DNA in tobacco plants [6]. 94
ATP dependant chromatin remodeling complexes use the energy of ATP hydrolysis to locally disrupt or alter the association of histones with DNA. They contain an ATPase subunit that belongs to the SNF2 superfamily of proteins. Based on the identity of this subunit, they have been classified into two main groups, the SWI2/SNF2 group and the imitation SWI (ISWI) group [7]. A third class of ATP-dependent complexes contains a Snf2-like ATPase which also displays deacetylase activity. In addition to the open reading frame (ORF) sequences coding for proteins, specific DNA sequences called cis regulatory elements are crucial for the regulation of transcription. Cisregulatory elements are small nucleotide sequences usually arranged in a unique fashion in a promoter regulatory region. The size of cis-regulatory elements varies from 3 to 25 nucleotides and they interact specifically with the transcription factors, thus regulating the expression of gene located in front of them. They are usually located upstream of the transcription start site where the RNA polymerase binds. The most common consensus sequences found in promoters are the CCAAT box (known as the CAT box), GC box and the TATA box (consensus TATAAA sequence). Coupling element 1 and coupling element 3 have been shown to be involved in dehydration stress. These elements have an ACGT core motif to which bzip class of proteins bind. Enhancers are cis-regulatory elements which act on promoters situated on the same chromosome and are situated upstream or downstream relative to the gene, or within the intron sequence. Enhancers are modular in the sense that a gene can have several enhancer elements turning it on in different cells. An enhancer that is important for the expression and regulation of AtHKT1gene involved in salt tolerance has been reported [8]. Trans-acting factors are proteins that bind to the cis-acting sequences like promoters, enhancers and silencers and interact with one another to activate or repress transcription of a gene. Through their DNA binding domain, they bind to the cis-acting sequences and through their trans-activating domain interact with proteins bound with RNA polymerase to activate or suppress the transcription of a gene. For instance, eukaryotic RNA polymerase II which is responsible for transcribing mRNAs cannot directly bind to the DNA sequence of a promoter. It requires other protein factors called basal transcription factors such as TFIID, TFIIA, TFIIB, TFIIE, TFIIF and TFIIH which bind to the promoter in a defined order and interact with RNA polymerase II to efficiently transcribe the mRNA. Multiple families of transcription factors have 95
been characterized and grouped based on structural similarities and their mode of action. For example, proteins like Hox and Pax belong to the homeodomain family and are involved in turning on several developmental genes. Other families include the zinc finger proteins, helixloop-helix family and basic leucine zipper [4]. These factors may be expressed in a spatial or temporal stage inside the cell or upon activation by modifications like phosphorylation or binding of specific ligands to the enhancer sites for the initiation of transcription. The activation of specific transcription factors in cells is achieved through specific signal transduction pathways. These include ligand binding to specific cell surface receptors, phosphorylation and activation of nuclear transcription factors. The signaling machinery is complex and involves secondary messengers (Ca2+, Inositol triphosphate (IP3) and reactive oxygen species (ROS), sensory kinases, phosphatases and nuclear transcription factors [9].
ENVIRONMENTAL STRESS CONDITIONS

TEMPERATURE STRESS The temperature maintained in plant systems relies heavily on the ambient environment. Generally plant systems are maintained 5-8 degrees lower than the surroundings through transpirational cooling. CAM plants, in particular, can generally withstand higher temperatures because they keep their stomata closed during the day and as a result do not lose water by transpiration [10]. Temperature stress resistance in plants can be mainly categorized into: 1. Heat tolerance 2. Chilling tolerance and 3. Freezing tolerance Heat tolerance: Elevated temperature levels have disastrous consequences on cellular organization due to the denaturation of proteins and alterations in membrane fluidity. For instance, exposure of leaf and root tissues of Agrostis stolonifera to high temperatures showed significant changes in membrane lipid composition with high levels of palmitic acid and reduced amounts of linolenic acids [11]. Change in the lipid composition of membranes thus has severe effects on the action of membrane-associated enzymes and electron carrier molecules which are involved in photosynthesis and respiration.
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Therefore, plants have evolved several adaptations to heat stress. The physiological changes include development of leaf hairs and waxes, change in orientation of the leaf and the development of small leaves so as to minimize the boundary layer resistance for heat transfer to the environment. Plants exposed to high temperatures also begin to accumulate osmotic compounds to reduce the water potential of cells. These include proline, glycine betaine, -4amino butyric acid (GABA). Plant hormones such as abscisic acid and ethylene which have roles in normal developmental processes such as seed germination, flowering and seed dormancy are also found to be overproduced in thermo tolerant plants [12]. Molecular responses to heat stress involve the production of heat shock proteins (HSPs), which act as molecular chaperones and assist in proper folding of cellular proteins. Five different classes of HSPs have been found in plants which have been found to be localized to cytosol, mitochondria, chloroplast and endoplasmic reticulum. These include the Hsp70 (DnaK) family, the Hsp90 family, the Hsp100 (Clp) family, the chaperonins (GroEL and Hsp60) and the small Hsp (sHsp) family. Low molecular weight HSPs (15-30 kDa) are found abundantly in plants. Their function is still not clear, but they are found to be mainly expressed during certain developmental stages [13]. A specific transcription factor, HSF, acts on the transcription of the HSP mRNA. The trimeric form of this factor accumulates during heat stress and upon
phosphorylation activates the production of HSP mRNAs [10]. Chilling tolerance: Chilling temperatures generally lead to inhibition of photosynthesis, degradation of cellular proteins and lower respiration rates. This implies an impaired membrane function, particularly the chloroplast and mitochondrial membranes. Chilling sensitive plants have been found to contain a high proportion of saturated fatty acids in their membrane phospholipids which tend to crystallize at lower temperatures. In contrast, membrane lipids of chilling resistant plants have been found to possess a high proportion of unsaturated fatty acids and desaturase enzymes [10]. This lowers the temperature at which phase transition of membrane lipids occur. When plants are exposed to long periods of freezing temperature, ice crystals begin to form outside cells resulting in dehydration of cells. Plant proteins called anti-freeze proteins (AFPs) help in preventing ice crystal formation by binding to ice crystal surfaces and inhibiting their growth [14]. Certain compounds such as sucrose, mannitol, raffinose and sorbitol also help in freezing tolerance by acting as cryoprotectants.
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Freezing tolerance: Freezing tolerance in plants generally refers to prevention of cellular dehydration. This is accompanied by the formation of extracellular ice crystals. Plants employ both colligative and non-colligative mechanisms to combat freezing-dehydration. As the temperature drops below 0 degrees, apoplastic ice formation begins to occur [15]. This freezing is further enhanced by ice nucleators which trigger ice formation. Intracellular ice formation is highly deleterious to the cell as the plasma membrane is ruptured. This severely impairs membrane-associated processes such as ion transport, photosynthesis, metabolite synthesis, signaling pathways which are membrane dependent. Several cryoprotective proteins such as antifreeze proteins are produced by plants against freezing stress [16]. AFPs have been mainly isolated from the apoplastic space where they interact with ice crystals and modify their growth. They result in an overall decrease in the freezing temperature of the plant. The difference between melting and freezing temperature is defined as thermal hysteresis and it forms the basis for the quantitative assay of AFPs [17]. Genes coding for antifreeze proteins have been cloned by several researchers. cDNA encoding an antifreeze protein of 64kDa from the bittersweet nightshade, winter S. dulcamara, has been cloned by using a polyclonal antibody [18]. This protein was found to contain a zinc finger motif and a specific DNA binding ability. The interaction between AFPs and ice crystals has been studied. It is thought that AFPs interact with the relatively flat surfaces of ice crystals through van der waals interactions and hydrogen bonds [19]. Different AFPs interact with different sides of ice crystals and inhibit growth. This is thought to be the reason behind evolution of different AFPs. Apart from inducing freezing tolerance, AFPs are thought to minimize freezing damage by inhibiting recrystallisation and minimizing the size of crystals. Chilling-resistant plants can be produced by acclimation to cold conditions and exogenous application of ABA. Cold-stress induced genes are activated by transcription factors such as C-repeat binding factors (CBF1, CBF2, and CBF3). CBF/DREB1-type transcription factors bind to CRT/DRE elements (C-repeat/dehydration-responsive, ABA-independent sequence elements) in gene promoter sequences. Studies have revealed that CBF1/DREB1 is induced by cold stress whereas DREB2 is induced by dehydration stress [20]. The expression of CBF/DREB1 is controlled by a transcription factor, ICE (Inducer of CBF expression) [21]. Membrane phospholipids: Functions and possible engineering mechanisms: Membrane lipids form the backbone for several cellular functions and are the most susceptible to 98
environmental stress conditions. The lipid components of plastid and cytoplasmic organelle membranes follow separate biosynthesis pathways. While the chloroplast membrane is mainly composed of monogalactosyl diacylglycerol (MGD), digalactosyl diacylglycerol (DGD) and sulfoquinovosyl diacylglycerol, the endoplasmic reticular membrane and other extrachloroplast membranes are mainly composed of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) [22]. Thus it has been observed that every membrane has a specific lipid composition and these lipids in turn have defined fatty acid compositions. For instance, phosphatidic acid produced in the plastids always contains 16:0 at sn-2 position and 18:1 at sn-1 position. This is in contrast to phosphatidic acid produced in the extrachloroplast membranes where 18 carbon chains are found at the sn-2 position and 16:0 fatty acids at the sn-1 position predominantly. The different pathways for lipid biosynthesis go hand in hand for maintaining the defined lipid composition of membranes. This is shown in studies involving act1 mutants which are deficient in chloroplast acyl-ACP sn-glycerol-3-P acyl transferase. It was observed in such mutants that the eukaryotic pathway predominated and compensated for the shortcomings in the prokaryotic pathway thus maintaining a constant lipid composition [23] . All the major glycerolipids present in membranes are initially synthesized with 16:0 and 18:0 acyl groups [22]. Additional polyunsaturation is achieved by the action of membrane associated desaturases. Several desaturases have been characterized by mutational studies of A. thaliana [23]. The engineering of membrane lipids therefore offers exciting possibilities for enhancing stress tolerance. For instance, fab1 mutants of A. thaliana have reduced levels of the enzyme ketoacyl synthase II which is responsible for the elongation of 16:0 fatty acids to 18:0 acyl entities. Fab2 mutants which have a mutated 18:0 ACP desaturase isozyme have also been studied. Both these mutants show enhanced levels of saturated fatty acids compared to wild type. Polyunsaturated fatty acids are very important when considering low temperature membrane behavior. As they are not tightly packed, they dont crystallize easily at low temperatures. They have been studied for roles in membrane stability in relation to cold tolerance. Efforts have also been made to alter the level of polyunsaturated fatty acids (PUFA). fad6 mutants contain reduced levels of 18:3 and 16:3 FAs and elevated levels of 18:1 and 16:1 precursors due to the deficiency of chloroplast desaturase. Studies with fad3-2fad7-2 fad8 mutants have shown that trienoic fatty acids in membrane lipids may have important functions in enhancing the tolerance of the photosynthetic machinery to temperature extremes [24]. The importance of PUFAs for endowing 99
low temperature tolerance is further strengthened by studies with fab2 mutants that are deficient in ER 18:1 desaturases. These mutants show poor phenotypic characteristics at low temperatures which underlines the importance of PUFAs [25]. Several studies have shown that the thylakoid membrane is extremely sensitive to temperature changes. They mainly contain MGD, DGD, SL and PG as component lipids. The level of saturated and unsaturated fatty acids is very important. This is reflected in studies with mutants of Chlamydomonas reinhardtii with reduced levels of unsaturated fatty acids which show high heat tolerance with enhanced activity of PSII [26]. Phospholipase D: Threatening membrane integrity: Phospholipase D cleaves membrane phospholipids such as phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine (PE) to produce phosphatidic acid [27]. Since this enzyme is involved in disruption of membrane integrity, it might be overexpressed during conditions of stress. Studies with low temperature acclimatised grape berries (Vitis vinifera) showed that when exposed to high temperatures, PLD activated during low temperature acclimation induced the production of salicyclic acid and HSP73 which are important mediators of thermo-tolerance [28]. Phosphatidic acid (PA) is an important secondary messenger which is produced with the help of phospholipase D (PLD) and phospholipase C (PLC), and diacylglycerol kinase. It can be further broken down to soluble molecules by phosphatases, acyl hydrolases and lipoxygenases. The model plant A. thaliana has been found to contain 12 distinct PLD families. The main domains that are present are phox homology domain, pleckstrin homology domain and C2 domain. Since PLDs play a major role in membrane lipid hydrolysis, their regulation is very important. They are mainly regulated by Ca2+, PIP2 and alpha subunit of G-proteins [29]. Regulating the levels of PLD in plants would therefore aid immensely in preserving the integrity of cell membranes during conditions of stress. SALINITY STRESS High salinity leads to osmotic stress in plants and is largely dependent on the amount of evaporation and precipitation. Salt stress and drought stress have overlapping consequences as salt concentration and water deficit are closely linked. The major ions involved in salinity stress are Na+, K+, H+ and Ca2+. Excessive quantities of Na+ cause membrane disintegration, toxicity to metabolic pathways and a reduction in photosynthesis. High salinity causes an increase in 100
cytosolic Ca2+ levels. The genes involved in salt stress belong to the SOS (Salt Overly Sensitive) family. SOS3 is a myristoylated calcium binding protein that can change its conformation in response to calcium ion concentration. SOS2/CIPK24 encodes a serine/threonine protein kinase and contains an auto inhibitory domain. SOS3 interacts with SOS2 through this auto inhibitory domain and activates it in a calcium-dependent manner. Many Arabidopsis mutants have been studied to determine their function in salinity stress. AtHKT1 codes for a histidine kinase transporter which mediates Na+ entry into the root cells of Arabidopsis [30]. It has been found that Athkt1 mutants also have deficiency in SOS3 function. SOS3 and SOS2 are thought to down regulate AtHKT1 activity under salt stress. Salt stress mechanisms mainly focus on efflux of Na+ across the plasma membrane and sequestration into intracellular compartments. The transport of solutes such as Na+ and K+ across the plasma membrane is aided by the H+-ATPases that generate the proton motive force required for these processes. For instance, SOS2 has been found to interact with vacuolar Na+/H+ antiporter and H+/Ca2+ antiporter [31]. Salt stress almost invariably leads to cellular dehydration. This effect is compensated by the accumulation of metabolites called as compatible solutes [32]. These include sugars and sugar alcohols such as sucrose, fructose, trehalose, raffinose and mannitol. Osmolytes such as glycine-betaine, proline and ectoine are also accumulated. The presence of such compatible solutes increases the water potential of cells and thereby prevents water loss. Overexpression of genes involved in the biosynthesis of osmolytes has been investigated as a possible mechanism for salt stress induction. Polyamines: Polyamines are aliphatic nitrogen compounds that are positively charged at physiological pH. Arginine decarboxylase (ADC), agmatine iminohydrolase and N-carbamoyl putrescine amidohydrolase and S-adenosine methionine decarboxylase (SAMDC) are the major enzymes involved in the biosynthesis of polyamines. In Arabidopsis, ADC2 has been found to be induced in response to drought, wounding, high salinity and potassium deficiency while ADC1 is responsible to cold stress. SAMDC1 and SAMDC2 have been found to be induced by cold [33]. ABA produced during abiotic stresses also induces genes involved in polyamine synthesis. Mutants in Arabidopsis with reduced ADC activity, spe-1 and spe-2 are found to have reduced salt tolerance [34].
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DROUGHT STRESS Drought is considered as one of the major abiotic stresses in the world. Water stress affects the integrity of the lipid bilayer and leads to increased salt concentration inside cells thereby creating osmotic imbalance. In response to drought stress, the synthesis of LEA (Late Embryogenesis Abundant)/dehydrin-type genes and proteinases is up regulated. Most of the plants respond to drought stress by closing their stomata to avoid transpirational water loss. Hydroactive closure of stomata which is metabolically dependent on ion fluxes is regulated by ABA. Due to a decrease in pH of the xylem sap during water deficit, transport of ABA to the shoots is enhanced. The accumulated ABA causes a depolarization of the guard cell membrane which results in reduced stomatal conductance. But this response may affect the rate of
photosynthesis with the limited availability of CO2. The electron transport chain is affected and reactive oxygen species such as superoxide, hydrogen peroxide and hydroxyl radicals are produced. Several major regulatory elements that are active in response to abiotic stress have been identified in Arabidopsis. Dehydration-responsive element binding protein 1(DREB1)/C-repeat binding factor (CBF) and DREB2 function in an ABA-independent gene expression, whereas the ABA-responsive element (ABRE) binding protein (AREB)/ABRE binding factor (ABF) functions in an ABA-dependent gene expression .In addition to these major pathways, other regulatory factors including the NAC (NAM, ATAF1,2 and CUC) and MYB/MYC (myeloblastosis/ myelocytomatosis) factors are involved in abiotic stress-responsive gene expression [20]. The promoter of rd29A has been found to contain both ABRE as well as DRE/CRT elements. ABA-mediated regulation of gene expression involves cis ABA responsive (ABRE) and coupling (CE) elements, required for ABA responsiveness, which have been shown to be present in the promoters of stress responsive genes [35]. Proteins (ABFs) that bind to ABRE elements containing a basic leucine zipper (bZIP) motif activate ABA stress responsive genes [36]. ABRE motif is somewhat similar to the G-box that functions in regulating plant gene expression in response to light conditions and wounding. On the other hand, dehydration response element (DRE) and DRE binding factors involved in ABA-independent regulation of gene expression during osmotic stress [20, 37, 38, 39], showed that dehydration response gene RD22BP1, and ATMYB2, MYC and MYB related proteins act as transcriptional activators of 102
rd22 (dehydration response) in response to drought and ABA, respectively. Leung et al. [40, 41] isolated and characterized arabidopsis ABI1 and ABI2 homologous (ABA insensitive 1 and 2) loci, which are crucial for plant responsiveness to ABA and negatively regulate ABA signaling. [42]
RECENT ADVANCES IN ABIOTIC STRESS TOLERANCE

ABA is an important plant hormone which has roles in inhibition of growth and stomatal opening, particularly under conditions of stress. It is also involved in the regulation of seed maturation and dormancy. Due to its capability of inducing desiccation tolerance in developing seeds, it is known to have functions in water stress tolerance. Enhanced levels of ABA are often found in plants subjected to stress. Conditions of water scarcity lead to a rise in the pH of the xylem sap which promotes the transport of ABA through the root xylem into the shoot. ABA promotes the efflux of potassium ions from the guard cells which reduces the turgor pressure of the stomata and causes stomata closure and inhibits its opening during stress conditions. [10] At the molecular level, this is achieved by chromatin remodeling through changes in histone acetylation. ERF7 (Ethylene responsive factor 7) from Arabidopsis thaliana is found to interact with atsin3 (arabidopsis thaliana sin3) and hda19 (histone deacetylation 19) to repress transcription of genes. Transgenic Arabidopsis plants have been created which express aterf7 and are sensitive to ABA in guard cells leading to an increase in transpirational water loss [43]. Overexpression of AtHD2C, which is repressed by ABA results in a lower transpiration rate in comparison to the wild type plants. Chromatin remodeling has also been found to be responsible for stomatal closure in plants. [30] The promoters of ABA-inducible genes contain multiple copies of cis elements known as ABA-responsive elements (ABREs; PyACGTGG/TC), or the combination of an ABRE with a coupling element (CE). [44] AREB/ABF bZIP proteins are found to require post-translational modifications for their activation. This is performed by SnRK kinase family members in the presence of ABA. bZIP subfamily members such as AREB1/ABF2, AREB2/ABF4 and ABF3 have been found to be induced in the case of dehydration and ABA treatment in vegetative tissues. [45]
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Regulation of ABA mediated stress responses: ABA signaling involves the following essential components [46]: 1) PYR1/PYL/RCAR1 receptors; 2) Protein phosphatases (PP2C); 3) Protein kinases (SnRK2) and; 4) Downstream targets (ABRE) Phosphatases as negative regulators: Protein phosphatases are broadly classified into two major classes: protein tyrosine phosphatases and protein serine/threonine phosphatases. The protein serine/threonine phosphatases are further classified into the PPP (protein phosphatase P family) and PPM (protein phosphatase magnesium divalent ion dependent family) gene families. The PPP family comprises PP1, PP2A, and PP2B which share sequence homology in their catalytic domains and are sensitive to specific inhibitors like okadaic acid. PPM family includes PP2Cs and these have not been found to share any sequence homology with PPPs [47]. PP2Cs are monomeric enzymes and the largest protein phosphatase family in plants known. For instance, 76 members have been found in Arabidopsis thaliana [48]. Several ABA-insensitive mutants such as ABI1, ABI2, HAB1 (homology to ABI1) HAB2, AHG1/AHG3 (ABAhypersensitive germination I) have been developed which act as negative regulators of ABA. [46] This evidence was supported by developing double or triple PP2C knockout mutants which shows more hypersensitivity to ABA [49, 50]. The first ABA-insensitive mutant that was developed, ABI1, was found to encode protein phosphatase 2C (PP2C). The expression of PP2Cs varies in different tissues. ABI1 is expressed in seeds and guard cells and found in both cytosol and nucleus, whereas AHG1 and AHG3/AtPP2CA are expressed mostly in seeds and localized to the nucleus. [49, 46] During stress ABA directly interacts with its target receptors PYR/PYL/RCAR (pyrabactin resistance 1/pyrabactin resistance like/regulatory component of aba receptor 1). These receptors are known to inhibit the activity of phosphatases especially protein phosphatase 2C (PP2C) [51]. ABA interacts with the lysine residues of the target receptor molecule through a carboxylic acid residue. ABA interacts with the water filled space of the receptor cavity. As soon as ABA binds one of the loops containing -pleated sheet closes like a gate bringing all the amino acids present in the cavity in close vicinity to the bounded ABA [52]. The receptor serine residues (PYL1 S112 and PYL2 S89) that are exposed upon ABA binding, form hydrogen bonds with a conserved tryptophan residue in PP2C and result in inhibition of its phosphorylating activity [46]. The whole mechanism is reversed in the absence of ABA. PP2C interacts with the 104
PYR1/PYL/RCAR1 receptor thereby inhibiting the auto phosphorylation of the kinases. The inactive kinases are unable to phosphorylate AREB which, in turn, cannot bind to the ABAresponsive element. Therefore stress inducible gene expression is hindered. Kinases as Positive Regulators: Kinases mainly act by phosphorylating transcription factors which regulate stress-responsive genes. Kinases lead to auto phosphorylation of a family of
SnRKs (sucrose non-fermenting -related protein kinase). SnRK2 (OSRK1) is capable of auto phosphorylation, and phosphorylates myelin basic protein (MBP) and histone protein and it is found to be induced under conditions of dehydration [53]. It has been reported that PP2C directly dephosphorylates sub class III SnRK2 kinases. It has been observed that the SnRK2 phosphorylation which happens in the presence of ABA is absent in abi1-1 mutants. This shows that PP2C is involved in the regulation of the ABA-responsive activation of SnRK2. Internal phosphorylation by SnRK2 occurs at Ser175 residue in its kinase activation loop. The same sites are dephosphorylated in the absence of the ABA. [46] Several kinases have been characterized in different plants. These include SNF1-related protein kinase 2 SRK2D/SnRK2.2 (SRK2D), SRK2E/SnRK2.6/OST1 (SRK2E) and SRK2I/SnRK2.3 (SRK2I) from Arabidopsis thaliana, PKABA1 from wheat and SAPK8, SAPK9, SAPK10 from rice. SRK2E has been found to function primarily in regulation of the stomatal responses, while SRK2D & SRK21 are involved in seed germination and root growth. It has been determined that three kinases within the Arabidopsis SnRK2 family, subclass III, SRK2D/SnRK2.2, SRK2E/OST1/SnRK2.6 and SRK2I/SnRK2.3 are strongly activated by ABA. It has been reported in soyabean that the SPK1 and SPK2 kinases are stimulated in response to hyperosmotic stress while the activity of SPK3 and SPK4 kinases increases in response to dehydration and high salinity. Among the ten SnRk2 kinases characterized in rice, SAPK 8, SAPK 9 and SAPK 10 kinases were found to be activated by ABA. A maize ZmSPK1 has been reported to be expressed in roots, mature leaves and vessels and present in high quantities in reproductive organs. In the case of tobacco NtOSAK kinase was found to be activated by osmotic stress [46, 53]. PP2C inactivates SnRK2 by dephosphorylation when ABA is not present or plants are not exposed to stress. Once ABA level rises because of adverse environmental conditions or developmental cues, the ABA-bound PYR/PYL/RCAR receptors interact with PP2C and inhibit its phosphatase activity. The negative regulation of SnRK2 by PP2C is eliminated. Therefore, AnRK2 phosphorylates serine /threonine residues at R-X-X-S/T sites in the conserved regions of its 105
target site in the transcription factor AREB/ABF. bZIP transcription factors have been characterized in different crop varieties such as TaAbF from wheat [54] and TRAB1 from rice [55]. The ABA-dependent phosphorylation of AREB/ABF transcription factors is required for the complete activation of stress-inducible promoter cassettes. AREB is known to bind to the ABA-responsive element ( PyACGTGG/TC), a conserved cis-element found upstream of the ABA-inducible gene. This happens in a cyclic manner where PYR/PYL/RCAR negatively regulatesPP2C, and PP2C negatively regulates SnRK2. Helicases: The partial unwinding of the double helical DNA is crucial for life-defining processes such as replication, recombination and transcription. This is achieved by DNA helicases which are proteins capable of disrupting the hydrogen bonds between nucleic acid duplexes. This unwinding of the duplex DNA is NTP/dNTP hydrolysis dependent and all the RNA and DNA helicases have inherent NTPase activity. Helicases have been classified into 3 super families: SF1, SF2 and SF3. Highly conserved domains are mainly involved in ATP-hydrolysis and unwinding of the nucleic acids. Due to the presence of the sequence motif II (DEAD, DEAH or DEAX), the helicase family is also called DEAD-box protein family [56]. As helicases are indispensable in basic cellular processes governing phenotypic expression and physiological adaptation, engineering of helicases has been explored as a possible mechanism for stress tolerance. Genes encoding DEAD-box DNA helicases such as PDH45, PDH47 and LOS4 have been expressed and found to be induced in response to cold, salinity and other stress conditions. PDH45 is thought to exert its role in a coordinated fashion alongwith DNA topoisomerase I which is involved in relieving the tension caused by partial unwinding of DNA [32].
PROMOTER ENGINEERING
The diverse molecular patterns in a cell are a result of a complex interplay between cisand trans-acting factors. Synthetic promoters are created by combining natural or artificially synthesized core-promoters with multiple cis-regulatory elements either derived from plants or other sources. Strategies for the construction of synthetic promoters depend on the cis-elements located upstream of the core or basal promoter. In plants, as many as 469 cis-regulatory elements have been documented in relation to transcriptional regulations according to Plant cis-acting regulatory DNA elements (PLACE) database. Microarray analysis has been performed to 106
identify target stress inducible genes of DREB1A [57]. Full length cDNA micro array and Gene Chip array were employed for exploring downstream transgenes overexpressing DREB1A. More than 40 genes were identified as the DREB1A downstream genes [58]. Two-component gene switches can also be constructed. A gene sequence coding for a transcription factor forms the initial component followed by a regulatory module comprising the core promoter and multiple cis motifs upstream of it. But the number of such regulatory motifs has to be controlled in order to prevent the monopolization of transcriptions factors that may regulate endogenous gene expression. A surplus of synthetic TF binding sites can cause depletion of endogenous TFs and thereby lead to transcriptional inactivation of other essential housekeeping genes [59]. With a prior knowledge of the type of cis elements influencing specific genes, two core promoters can be placed in the opposite orientation with the common cis-regulatory elements placed in between. Such promoters are called bidirectional promoters. An artificial bidirectional promoter for high level expression of transgenes which can carry two genes at a time in different orientations has been designed [60]. The most widely used promoters in generating transgenic plants are constitutively expressed, i.e., they are turned on all the time and throughout the plant life cycle. However, in cases where the gene expression needs to be tailored to a specific organ or a specific time, such constitutive promoters may not be a suitable choice, especially for the stress-induced genes. This is because the constitutive expression of some stress induced genes may have serious deleterious effects on the plant growth and developmental processes. For instance, the DREB1A cDNA has been overexpressed in A. thaliana under the control of CaMV 35S (constitutive) and rd29a (stress induced) promoters [61]. The use of the strong CaMV 35S promoter to drive the expression of DREB1A also resulted in severe growth retardation under normal growing conditions. In contrast, expression of DREB1A under the control of rd29a gene promoter caused minimal negative effects on plant growth, while providing even greater tolerance compared to CaMV 35S promoter. The possibility of inducing gene regulatory units by chemical compounds which do no have any side-effects on normal plant developmental processes such as growth and cell division have directed research strategies towards chemically inducible promoters. The chemicals commonly used for the induction of transgenic plants are benzothiadiazole, tetracycline, dexamethasone and ethanol [62]. For instance, two novel seed specific promoters 107
namely HaFAD2-1 and HaAP10, have been isolated and functionally characterized from sunflower. These promoters contained seed specific motifs like AACA motif, ACGT element, EBoxes, SEF binding sites and GCN4 motif [63]. The basic findings on stress promoters have led to a major shift in the paradigm for genetically engineering stress tolerant crops in recent years. Most of the stress promoters contain an array of stress specific cis-acting elements that are recognized by specific transcription factors [64, 65, 66, 67, 68, 69]. Transcriptional activation of stress-induced genes has been possible in transgenic plants over expressing one or more transcription factors that recognize promoter regulatory elements of these genes. Two families, bZIP and MYB, are involved in ABA signaling and its gene activation. Many ABA inducible genes share the (C/T) ACGTGGC consensus, cis-acting ABA-responsive element (ABRE) in their promoter regions [70, 71]. Heat shock promoters, rd29 (induced by osmotic stress), and adh (induced by anaerobic stress) gene promoters have been subject of intensive research. The levels of rd29 mRNA changes differentially in response to dehydration, low temperature, salt stress or exposure to ABA [66]. The over expression of DREB1A was found to improve drought and low temperature stress tolerance in tobacco [72]. These results highlight the importance of stress inducible rd29A promoter and the DREB1A gene in improving drought, salt and freezing stress tolerance. It will become feasible to employ stress- induced promoters for raising transgenics against low or high temperatures, drought or salinity stresses when promoters responding to these are fully characterized and then cloned in suitable plant transformation vector systems. The factors pertaining to cis-elements taken into consideration are position, orientation, copy number and spacing. Two cis-motifs ACGT and GT which are involved in the regulation of pathogen defense have been studied. These motifs placed 50 nucleotides or 100 nucleotides upstream to TATA box were studied [73]. It was interpreted that a single ACGT motif enhanced promoter expression when placed 100 nucleotides upstream to TATA box while two ACGT motifs separated by 5 nucleotides enhanced promoter expression when placed 50 nucleotides upstream to minimal promoter. Two copies of ACGT when separated by 5 nucleotides allowed promoter activation by salicylic acid and when separated by 25 nucleotides, promoter was induced by abscisic acid but not salicylic acid [74]. The differential induction is expected to 108
involve the recruitment of different bZIP transcription factors [75] and this change in spacing between two copies of a given motif can alter the signal pathway to which a promoter responds. Various cis-acting DNA motifs could function as an activator by itself as well as a synergizing activator in the presence of other neighboring homologous as well as heterologous motifs. This synergistic effect is well illustrated in the work of Sawant et al. where the effect of eight cis-acting motifs on transcription from the basal promoter (Pmec) was studied by placing these upstream of the TATA-box at the -38 position as in plant genes. [76]. Therefore, a comprehensive knowledge about various cis-elements and their corresponding effect on stress-responsive genes will pave way for designing synthetic promoter cassettes for achieving enhanced levels of stress-specific resistance.
CONCLUSION
Poor plant growth and productivity under adverse environmental stress conditions has triggered research to devise novel strategies for the induction of stress resistance. Signaling pathways involved in stress responses have been characterized through mutant studies, mostly of the model plant, A. thaliana. Signal transduction pathways involved in cellular responses to myriad stress conditions seem to be tightly interlinked. Therefore, information about additional signaling components is required to make sense of the complete picture. A common denominator among different types of stress responses seems to be the loss of membrane integrity. This implicates essential roles of membrane lipid composition. Therefore, engineering of membrane lipid composition and activity of membrane-associated enzymes such as PLD also offers exciting possibilities for enhancing stress tolerance. As abiotic stress resistance is polygenic, plant
engineering will require manipulation of complex metabolic or regulatory pathways involving multiple genes. Current research methodologies include manipulation of genes encoding several stress-responsive components to mitigate the effects of stress. However, a better approach would be to control stress responsive genes themselves through cis-element engineering instead of overexpressing components produced in response to stress. Simultaneous transfer of several genes will be the likely next step to achieve practical levels of plant stress tolerance.
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Chapter VI SYNTHESIS AND APPLICATION OF MAGNETIC NANOPARTICLES: A BIOLOGICAL PERSPECTIVE

Arpit Bhargava, Navin Jain and Jitendra Panwar*
Centre for Biotechnology, Department of Biological Sciences Birla Institute of Technology & Science, Pilani-333 031
*
Corresponding author: Jitendra Panwar
E-mail: drjitendrapanwar@yahoo.co.in; jpanwar@bits-pilani.ac.in
ABSTRACT: The fast growing field of nanotechnology presents great potential to influence diverse areas like energy, environment, agriculture, healthcare and consumer goods and therefore building great expectations not only in the academic community but also among investors, governments and industries. This chapter deals with most recent advances in the biogenic synthesis and biological applications of magnetic iron nanoparticles [MINPs]. Owing to the limitations and drawbacks of physiochemical methods of MINP synthesis, considerable interest has been generated in biogenic synthesis methods as the later being ecofriendly and cost effective. Magnetotactic bacteria [MTB] represent a fascinating example of biologically controlled mineralization forming MINPs in the form of magnetosomes. Progress in the biological methods can be realized with the number of reports coming in the past decade on MINP synthesis using bacteria, fungi and plants. The later section of the chapter discusses various biological applications of MINPs viz. in separations of biomolecules and cells, detection and diagnostics, imaging, hyperthermia, drug and therapeutic delivery, nucleic acid delivery and transfer, cell manipulation and tissue engineering and in agriculture. Further, the chapter evaluates the advantages of biosynthesized MINPs over commercially available ones and finally concludes with the possible directions of future research in this field.
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INTRODUCTION
Nanotechnology serves as a prospective tool for overcoming critical issues faced by society. Nanomaterials are at the leading edge of the rapidly developing field of nanotechnology. Recent developments in the fabrication and application of nanostructured materials has delivered solution to various problems like climate change and pollution control [1], clean water technology [2], energy generation [3, 4], information storage [5] and biomedical applications [6]. Over the years the increasing inflow of investment in the field of nanotechnology proves its potential [7, 8]. Nanoparticles are of great scientific interest as they bridge the gap between bulk material and atomic or molecular structures. Presence of nanoparticles is well known in many natural processes, from magnetotactic bacteria that sense the earths magnetic field using nanomagnets, to the facilitated transport of radionuclides in groundwater [9]. Magnetic iron nanoparticles (MINPs), a class of nanostructured material possess amenable properties making them important in view of biological applications. The nanometric size interacts efficiently with biological entities like cell [10-100 m], viruses [20-450nm], protein [5-50nm] and gene [10-100nm] thus leading to exciting opportunities in the field of MRI imaging [10], hyperthermic effect [11], cell tagging and tracking [12], drug and gene delivery [13, 14] and detection of probes [15, 16] as well as a prospective material to be applied in agriculture. This chapter explores new insights in the synthesis and applications of MINPs in biological perspectives. In the later part of the chapter, specific advantages and importance of magnetosomes and biosynthesized MINPs have been discussed. The chapter finally concludes with a brief discussion and propose hypothesis for future trends of research in this field.
BIOSYNTHESIS OF MINPs
Significant application of MINPs at commercial platform makes their synthesis protocols a valued subject for research and innovation. Physicochemical as well as biological methods are employed for the synthesis of MINPs. The physicochemical methods include microemulsion, sol-gel synthesis, hydrothermal reactions, flow injection synthesis, electrochemical synthesis, pyrolysis and chemical co-precipitation method [17]. 118
Although the physicochemical methods are fast, efficient with considerable ease in upgrading to industrial scale production, complexity of these processes owing to colloidal nature of nanoscale iron particles is a major constraint which limits their applications. The major problem is the difficulties in controlling the size and shape of nanoparticles. Other issues surrounding these methods are difficulties in controlling experimental conditions, polydispersity, tedious downstream processing with generation of toxic by-products and yield of particle in nonpolar organic solvent, thus precluding their application [17, 18]. Considering the ill-effects of physicochemical methods, alternate methods for nanoparticle synthesis deserves merit. The increased awareness towards the development of clean, reliable and eco-friendly alternate for synthesis of MINPs has encouraged the researchers to look into newer Greener methods [19, 20]. The green synthesis involves selection of solvent medium, environmentally benign reducing agent and non-toxic substances for the stability of nanoparticles, which must be evaluated based on green chemistry perspective [21]. The landmark discovery of magnetotactic bacteria [MTB] by Richard Blakemore made the path for enormous research in finding biological agents capable of syntheszing MINPs [22]. MTB are the oldest reported microbial factories for MINPs fabrication. As a naturally occurring phenomenon, it involves the formation of intracellular magnetic structure, the magnetosomes, which are nanosized, membrane enclosed crystals of magnetic iron minerals composed of magnetite [Fe3O4] or greigite [Fe3S4]. Magnetosome crystals have species-specific morphologies, sizes and arrangements [Figure 1; 23] and their formation is genetically regulated by a complex set of genes situated mainly at magnetosome island in MTB genome [24, 25]. Magnetosomes are involved in magnetotaxis and direct the bacterial cells to swim towards the growth favouring microoxic zones of the chemically stratified natural water [26]. Almost all the reported MTB are microaerophiles which can form multiple magnetosomes within them through an oxygen sensitive process and are motile by means of flagella. The group is highly diverse in terms of phylogeny, morphology and physiology [27]. Despite of their high abundance and ubiquitous occurrence in marine and fresh water habitats, most MTB are difficult to isolate and cultivate invitro, which is probably due to their adaptation to complex chemical gradients typically encountered in stratified sediments. Only a limited number of MTB strains have been isolated in pure culture [28], out of which species of Magnetosiprillum are extensively studied for elucidation of mechanism for magnetosome formation [Figure 2; 29]. In general, the particles are 119
usually arranged along the axis showing single or multiple chains with particle size ranging between 35 and 120 nm, with single domain magnetism [30]. Surrounding the mineral core is the magnetosome membrane which is a lipid bilayer membrane with composition considerably similar to cytoplasmic membrane [31]. It originates from the cytoplasmic membrane by invagination, represents a distinct subcellular compartment and has a unique biochemical composition. Roughly 20 magnetosome-specific proteins function in the vesicle formation, magnetosomal iron transport and; control the crystallization and intracellular arrangement of MINPs [32]. It has been hypothesized that these membrane proteins may play functional roles like accumulation of supersaturating iron concentration, maintenance of reductive conditions and mineralization of iron either by oxidation or by partial reduction and dehydration of ferrihydrite intermediate to magnetite [23]. Apart from the group of MTB which synthesize MINPs in microaerophillic environment, recent years have observed reports of many other aerobic bacterial species synthesizing iron nanoparticles. Bacteria like acid tolerant Leptospirillum ferriphilum [33] and iron oxidising Leptothrix ochracea [34] were found to produce magnetosome like mineral nanoparticles. Both of these bacterial species belongs to the class of Chemolithotrophs which utilizes iron as an energy source. Bharde et al. [35, 36] have successfully obtained iron oxide (magnetite and maghemite) and iron sulphide (gregite) nanoparticles from an aerobic bacterium, an Actinobacter species using aqueous mixture of potassium ferricyanide/ferrocyanide. Somewhat similar to magnetosomes membrane, the nanoparticles produced by Actinobacter under aerobic condition were found to have a protein coat [36]. The only account of fungi producing iron nanoparticle is by Bharde et al. [37], where Fusarium oxysporum and Verticillium sp. were challenged with mixture of potassium ferricyanide/ferrocyanide at room temperature thus producing magnetite nanoparticles extracellularly. The group also explored the possibility of using mixture of ferric and ferrous chloride but the particles formed were irregular in shape and had poor crystal structure.
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Figure 1: TEM images of magnetotactic bacteria. (a) Magnetospirillum magneticum strain AMB-1 and (b) Desulfovibrio magneticus strain RS-1. BacMPs of (c) the AMB-1 strain and (d) the RS-1 strain. Reproduced from [23] with permission from American chemical Society.
Phytosynthesis of nanoparticles is a recent, rapid, low cost, eco-friendly and a single step method. Various plant parts viz. leaf extract, sun dried leaves, and fruits can be used and many have proved to be successful [38]. Herrera-Becerra et al. [39] successfully demonstrated a green chemistry method for the production of MINPs with size less than 5 nm by exposing a homogeneous suspension of powdered milled alfalfa and FeNH4(SO4)2.12H2O solution. Recently, amorphous iron nanoparticles of various size and morphologies (spherical, platelets and nanorods) have been synthesized using tea polyphenols [40]. In a separate study, soyabean sprouts were used for synthesis of superparamagnetic Fe3O4 nanoparticles [41]. In a very recent examination, iron nanoparticles were synthesized at room temperature using aqueous sorghum bran extract as both the reducing and capping agent [42].
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Figure 2: Model for step-wise assembly of magnetosomes in Magnetospirillum magneticum AMB-1. Steps leading to magnetosome formation are indicated on the left side of the model, and the factors known to play a role in each of these steps are indicated on the right side. Protein names in black indicate factors discovered in previous studies in AMB-1; in orange, proteins whose potential functions were defined in the present study. Asterisks indicate proteins characterized in MSR-1. Black octagons represent growing or mature, euhedral magnetite crystals. Red symbols indicate inner membrane proteins; purple dots indicate magnetosomeassociated proteins; yellow lines represent the MamK cytoskeletal filaments. IM, inner membrane; OM, outer membrane. Reproduced from [29] with permission from Proceedings of the National Academy of Sciences, USA
BIOLOGICAL APPLICATIONS OF MINPs

Nanotechnology is beginning to allow scientists, engineers and physicians to work at the cellular and molecular levels to produce major advances in the life sciences and health care. Interdisciplinary approach combining the field of nanotechnology, material sciences and biology enable us to fully comprehend the potential uses of nanoparticles in biomedical applications.
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MINPs offer attractive possibilities in biomedicine as they can be manipulated and transported to a particular site by using external magnetic field gradient. MINPs with diameter less than 20 nm are said to be superparamagnetic and can have potential biomedical applications, as they do not retain any magnetism after removal of external magnetic field. This phenomenon arises because these nanoparticles consist of a single crystal domain, and thus exhibit a net magnetic moment directed along an anisotrophy axis. This is in contrast to the multi-domain ferromagnetic particles which aggregate after exposure to an external magnetic field [43]. This part of the chapter deals with various biomedical applications of MINPs.
SEPARATION OF BIOLOGICAL MOLECULES AND CELLS

Isolation, separation and purification of biologically active compound, biomolecules and cells raise the cost of downstream processing in many industries. Although variety of techniques are available for the separation process but all suffer from issues like cost, eco-friendliness and complexity [44]. Magnetic separation is based on the simple principle of magnetic attraction of biocompatible MINPs towards a magnet [45] and likely to be a cost effective alternative technique. A series of modification have been performed on the magnetic separation technique but the core principle remains the same. In general, the magnetic separation using biocompatible nanoparticles is a two-step process, involving (i) the tagging or labelling of the desired biological entity with magnetic material, and (ii) separating out these tagged entities via a fluid-based magnetic separation device [46]. Magnetic separation technique has been successfully employed in various applications like cell sorting, protein purification and nucleic acid extraction. In general, separation can be performed by one of the two ways, [i] direct or targeted separation and [ii] indirect separation. In the direct or targeted separation, MINPs coated with high affinity ligand are targeted towards specific biological entity to which these modified MINPs attaches and thus can be eluted out by using magnetic separator. In the indirect approach which can also be said as sandwiched separation, an antibody specific to the molecule need to be separated is introduced first, followed by the introduction of MINPs coated with high affinity ligand against the antibody. Strategies can be made either to separate the desired molecule or cell from the mixture using magnetic separation or removing the contaminants and undesired one [45].
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Conventionally proteins are being separated using chromatography, ultracentrifugation and membrane filtration technique, where each method requires tedious pre-processing of sample to achieve high efficiency and purity. The same task can be done using MINPs coated with protein specific ligand like streptavidin, protein A or antibody, which can easily and quickly separate out target protein from crude cell extract without tedious frills [47]. Kim et al. [48] reported an efficient method for purification of native polyhistidine-tagged proteins using modified MINPs. In this experiment they synthesized superparamagnetic iron oxide [SPIO] nanoparticles coated with bis-nitrilotriacetic acid [NTA] chelate using catechol anchor which when loaded with Ni [II], binded to polyhistidine fusion proteins in their native, folded conformations. In an another study, SPIO nanoparticles were coated with hydrophilic resins prepared by radical polymerization of magnetite [Fe3O4], styrene, divinyl benzene [DVB] and glycidyl methacrylateiminodiacetic acid [GMAIDA] in ethanol/water medium to improve purification of His-tagged proteins [49]. Wei et al. [50] devised a novel strategy based on MINPs coated with zirconium phosphonate for the enrichment of phosphopeptides used for mass spectroscopy. Under optimized experimental conditions, 1 10-9 M phosphopeptides in 50 L tryptic digest of -casein could be enriched and identified successfully using the adopted strategy. Nucleic acid separation had been a time consuming and laborious process based on several extraction and centrifugation steps, often limited by small yields and low purities of the separation products and unsuitable for automation and up-scaling. Although availability of nucleic acid extraction kit has diluted the complexity but it involve increased cost and still the process lacks rapid extraction. Use of MINPs explored the possibility to use them to isolate and purify nucleic acids. During last few years, specifically functionalised magnetic particles have been developed that allow quick and efficient purification directly after extraction from crude cell extracts, eliminating repeated steps of centrifugation and incubation. In addition, the new approach provided an easy automation of entire process and isolation of nucleic acids from larger sample volumes [51]. SPIO nanoparticles [Fe3O4] coated with a multivalent cationic agent, polyethylenimine [PEI] were employed to simplify the purification of plasmid DNA from bacterial cells [52]. Approximately 35 g of high-purity plasmid DNA was isolated using magnetic nanobeads within 10 minutes from 3 ml of overnight bacterial culture [53]. Yet in another study, Park and Chang [54] developed a method for high throughput human DNA 124
purification with the amino-functionalized silica coated MINPs having average particle size of 9 nm and coating thickness of 1040 nm. In advancement to this method, isolation of messenger RNA (mRNA) from mammalian cells and extraction of the supercoiled form of plasmid DNA from agarose gel was achieved using Carboxyl-coated MINPs [55]. These MINPs were attached with 5-NH2-tagged oligo-[dT]25 primer and were used to isolate mRNA from breast cancer cells. The ability to separate living cells is an essential aspect of research in cell biology. The need of cell separation arises during some advanced medical procedures and certain cell replacement therapies [56]. Magnetic cell separation methods are among some of the most efficient methods for bulk cell separation [57]. The background principle exploits the concept of immunogenic interaction between antigen and specific antibody or ligand and receptor which in this case is coated or decorated on MINPs. Magnetic cell separation typically employs the use of antibodies against specific cell surface epitopes to tag cells of interest with magnetic particle conjugates [47]. Under the effect of applied magnetic field, the magnetically tagged cells tend to have higher magnetophoretic mobility enhancing their separation [57]. Inglis et al. [58] have developed a continuous-flow microfluidic device that enables cell by cell separation in which cells were selectively tagged with MINPs. One of the most critical contributions of this technique has been in ART [Assisted Reproductive Technique], where MACS [magnetic activated cell sorting] uses annexin V-conjugated SPIO nanoparticles to separate non-apoptotic spermatozoa based on expression of phosphatidylserine [59]. Qiu et al. [60] used the MACS strategy with anti-CD14 and anti-CD16 antibody beads to deplete macrophages and neutrophils from sputum and to enrich the content of bronchial epithelial cells from 1.1% in the starting population to 40%.
DETECTION AND DIAGNOSTICS

The rapid detection of biomolecules and specific cells in biological samples is of paramount concern in the field of medicine. Biosensing strategies based on MINPs have received considerable attention because they offer unique advantages over the other detection techniques. Biological samples exhibit virtually no magnetic background, and thus highly sensitive measurements can be performed in turbid or otherwise visually obscured samples without further processing [61]. The superb structural and functional properties of MINPs have made them a promising candidate to be used in nanoscale biosensors for detection of biomolecules, cells and 125
other biological entities [62]. Although the detection/ diagnostic or sensing principle may vary in different type of applications but the biological principle is more or less common relating to interaction between target molecule or cell and its specific ligand which in many cases may be monoclonal antibodies. A sensor generally consists of two components: a recognition element for target binding and a transduction element for signalling the binding event [63]. Majorly, three sensory principles are used in development of various biosensors using MINPs [64]. The first one, called magnetic relaxation switches suggests that aggregated and dispersed states of MINPs have different transverse spin-spin relaxation times [values of T2]. When used as targeted contrast agents, surface-modified nanoparticles bind specific molecules producing local inhomogenieties in the applied magnetic field in tissues where molecular targets are present. These inhomogenieties result in decreases in the T2 relaxation time (or increases in 1/T2) which in turn lead to changes in the contrast of magnetic resonance images [65]. The second type of biosensors, called magnetic particle relaxation-based sensors, exploits the change in relaxation of magnetic moments within magnetic particles. In theory, the effective relaxation rate depends on the Brownian relaxation rate and the Neel relaxation rate. Faster relaxation time between these two, governs the effective relaxation process. Target induced aggregation can decrease the rates of the Brownian or Neel relaxations and this is the basis of assays for molecular targets [17]. The third principle, called magnetoresistive sensors, involves the binding of magnetic nanoparticles to a sensor surface. The magnetic fields of the particles alter the magnetic fields of the sensor which result in electrical current changes within the sensor [66]. Two mechanisms have been suggested for binding of magnetic nanoparticles to the sensor surface [67]: [i] direct labeling and [ii] indirect labelling (a sandwich type binding) which has been principally discussed in the previous section of this chapter. Even though there can be different type of detector systems and biosensors particular for enormous range of biological units present, it is beyond reach to discuss the specific application of all the available systems. Mass spectrometry specifically MALDI-TOF MS is one of the standard method for the study of biomolecules. However, this approach is not suitable for detection of small molecules due to the interference from matrix in the low molecular weight region of the mass spectrum. To overcome this limitation, Lin et al. [15] have developed nanoprobe-based affinity mass spectrometry [NBAMS] in combination with direct protein identification by MALDI-TOF MS. It 126
eliminates conventional time-consuming sample purification and enrichment processes and also removes the interference caused by the background matrix as in NBAMS, the surface of MINP acts as a matrix. One of the methods for detection and quantification of DNA has been proposed by Kouassi and Irudayaraj [68], in which DNA can be labelled with appropriate fluorophores decorated on MINPs. Another variant for detection of DNA using MINP is electrochemical detection, which usually involves immobilization of a single-stranded probe nucleic acid to a solid substrate and the production of an electrochemical signal using a redox couple when the desired target hybridizes to the probe. The advantage of this method lie in the fact that since the electrochemical redox couple does not bind to the DNA, the nucleic acid retains its natural state and can be recycled [69]. MINPs are helpful in detection of viral particles as in case of supramolecular assemblies that take advantage of changes in the nanoparticles optical or magnetic properties upon viralinduced assembly. Such structures act as magnetic relaxation switches by causing extensive spinspin relaxation time changes of surrounding water molecules and thus can be used as potential nanosensors for rapid and sensitive detection of clinically relevant viruses. Perez et al. [70] have developed method for detection of Herpes simplex virus [HSV] and Adenovirus [ADV] even at very low concentration (5 viral particles per 10 l). They conjugated the monodispersed
magnetic nanoparticles with virus specific antibodies. In presence of virus these nanoparticles create a super-molecular structure and their presence can be selectively detected by magnetic resonance methods like NMR and MRI. Utilizing the fact that many bacteria use the mammalian cell surface carbohydrates as anchors for attachments which subsequently results in their infection, magnetic glyconanoparticle -based system has been used to detect E. coli within 5 min, as well as remove up to 88% of the target bacteria from the medium [71]. The system is found to be capable of identifying different E. coli strains on the basis of their response patterns to MINPs.
IMAGING
The basis of magnetic resonance imaging [MRI] is the directional magnetic field or moment associated with charged particles in motion. Nuclei containing an odd number of protons and/or neutrons have a characteristic motion or precession. Because nuclei are charged 127
particles, this precession produces a small magnetic moment. Clinical MRI uses the hydrogen protons and its interaction with both a large external magnetic field and radio waves to produce highly detailed images of the human body [73]. Conventional MRI scans uses gadolinium as a contrast agent which sharpens the content by changing the proton behaviours in their proximity. Advances in this technology involve replacement of gadolinium by iron oxides particles as effective contrast agents in imaging [74]. MRI contrast agents act to improve image quality by reducing the relaxation time and hence altering the NMR signal intensity of water in the body tissues containing the agent. SPIO nanoparticles represent an alternative class of NMR contrast agents that are usually referred to as T2 [transversal relaxation time] or T2 contrast agents as opposed to T1 [longitudinal relaxation time] agents such as paramagnetic gadolinium [III] chelates [75, 76]. Gandolinium makes complexes with low molecular weight chelating molecules, such as diethylene triamine pentaacetic acid [DTPA] as well as capsulated in lipsome and micelles. However, interference due to ion exchange with other metals and their complexes in the surrounding limits their application [77]. Advantages of MINPs lie in their inclusivity within tissues enabling a very large signal to be obtained from a MRI scanner. Based on the size of functionalized nanoparticle used they have been classified as supreparamagnetic iron oxides [SPIO] where particles have a size greater than 50 nm [coating included] and ultrasmall superparamagnetic iron oxides [USPIO] where particles are smaller than 50 nm [78]. The size affects their half-life in vivo as well as their bio-distribution and pharmacokinetic properties. Advances in the field of nanoparticle engineering have developed magnetism engineered iron oxide [MEIO] nanoparticle which are discussed to be better contrast agents as compared to traditional SPIO nanoparticle and cross linked iron oxide nanoparticle [CLIO]. It is because MEIO have high and tunable mass magnetization values needed to enhance the relaxation process of the proton nuclear spins for improved imaging [79]. MRI imaging using MINP depends on the effectiveness of the nano-sized contrasting agents to be captured by the cells through the endocytosis pathway so as to insert the agents in the target cells. For efficient imaging, plasma half life of particle plays an important role. In general, mechanical filtration process in spleen and liver eliminates MINP above 200nm where as particles below 10nm are cleared by opsonisation and renal clearance [80]. To ensure prolonged plasma half life, fictionalization of SPIO in terms of coating or encapsulation with amphiphilic coatings are preferred which can drastically improve plasma half life as well as targeting capabilities of the 128
contrast agent [81]. Two of the most common coating is of polysacharride dextran [82] and poly ethylene glycol [PEG] [83]. PEG coated nanoparticles offer advantage of being escaped from macrophages leading to opsonisation thus have increased blood flow time [84, 85]. Application of SPIO in imaging can be conceptualized as passive targeting which involves SPIO probes lacking explicit molecular specificity thus allowing generalized and nontargeted cellular uptake, enhanced retention in tumors, macrophage phagocytosis, as well as accumulation in the liver, spleen, and lymph nodes. Active targeting engages SPIO functionalized against specific bio molecules. The SPIO used for active targeting may or may not have surface coating. Presence of surface coating gives additional reactive moieties for the attachment of a variety of ligand and antibodies [80]. As the trend has shifted towards active targeting using molecular specific probes, some other recent applications using this technique has helped in diagnosis and detection of diseases, tumours, infections and molecular imaging. Huang et al. [86] developed biocompatible poly-vinylpyrrolidone coated SPIO nanoparticles using high temperature process that can be efficiently labelled to Mice islet cells. These biocompatible SPIO can be used to visualize islet cells during transplantation which makes it a non-invasive technique for assessing islet engraftment and the early recognition of graft loss. The usefulness of SPIO as contrast agents in MRI extend to diagnosis of abnormalities in central nervous system [CNS]. It can be used to detect bloodbrain barrier dysfunction due to tumours, neuro-inflammatory pathologies and in vivo cellular tracking in CNS disease or injury [87]. Recently Wang et al. [88] reported that non-pathogenic anaerobic bacteria can anchor and proliferate within solid tumours hypoxia area, making these bacteria a potential representative for cancer diagnosis and therapy. They have developed bio functionalized SPIO nanoparticles tagged on anaerobic bacteria for their detection and evaluation using MRI. Ease of detection and screening of these labelled bacteria have enhanced the development in the field of cancer therapy and diagnosis.
HYPERTHERMIA
Hyperthermia is a therapeutic procedure mainly used in the treatment of cancer alongwith chemotherapy, radiotherapy or surgery. The principle of magnetically mediated hyperthermia [Figure 3b] is based on two facts: (i) Tumour cells are sensitive to temperature above 41C than normal tissue cells [89]. At high temperature range, disruption and modification in the function 129
of many structural and enzymatic proteins occurs within cells resulting in altered growth and differentiation leading to the tumour destruction. (ii) Metallic objects with magnetic properties when placed in an alternating magnetic field induce currents flowing within them. As the metal resists the flow of current, heat generation takes place. The amount of current and thereby heat is proportional to the size of the magnetic field and the size of the object [90]. Exploiting these two facts, hyperthermia has proved to be an effective way of destroying tumours and thus treating cancer. The heating power of the particles is quantified as the specific absorption rate [SAR] and describes the amount of energy converted into heat per unit time and mass. As evaluated by Rosensweig [91], superparamagnetic materials show impressive levels of heating at low magnetic fields as compared to ferromagnetic material which require much higher magnetic field strength for effective heat generation. On the basis of mode of introduction of magnetic agents, hyperthermia can be categorized into: (i) Arterial Embolization Hyperthermia [AEH], which uses the arterial supply to deliver magnetic particles into the tumour tissue, (ii) Direct Injection Hyperthermia [DIH], where the particles are directly injected into the tumour and (iii) Intracellular Hyperthermia [IH], which engages biocompatible magnetic particles facilitating their cellular uptake by the tumour [47]. Recent developments in the field of hyperthermia, has initiated the use of this technology to a targeted tissue by conjugating the MINP with specific antibody or ligand [92]. Balivada et al. [93] evaluated the efficiency of magnetically mediated hyperthermia using bimagnetic core/shell nanoparticles (Fe/Fe3O4) in order to prevent the growth of subcutaneous mouse melanomas. Their results indicated that intra-tumoral administration of surface modified MINPs can attenuate mouse melanoma after a short alternating magnetic field exposure. For practical implementation of this therapy the site of injection, dose of heating agents, property of tumour and tissue as well as temperature susceptibility should be accurately optimized [94]. Apart from being used as an anti-tumour therapy, magnetically mediated hyperthermia can also be employed as antimicrobial treatment. As reported by Thomas et al. [95] thermoablation of the bacterium Staphylococcus aureus is possible using SPIO nanoparticle biofunctionalized with carboxylic acid moiety.
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DRUG AND THERAPEUTICAGENT DELIVERY

The drawback of conventional drug delivery system is non-specificity, uneven distribution of therapeutic agent in the body, drug retention and side effects due to the need of higher drug dose [96]. The magnetic drug targeting although may not be the final solution to the aforesaid mentioned problems however, serves as a healthier system for the targeted delivery of therapeutics [Figure 3a]. Most imperative benefit of MINPs for drug delivery is the capacity to deliver the drug to desired area under external magnetic field [13]. In comparison to paramagnetic nanoparticles, SPIO nanoparticles do not aggregate even after removal of the external magnetic field [97]. Molecular targeting and assisted internalization of nanoparticles can be achieved using specific ligand based delivery system involving use of cell or tissue specific antibodies, proteins, low molecular weight ligands or charged molecules [10]. To increase the blood half life of the delivery molecule and to avoid early detection by Reticulo-endothelial System [RES], particles are generally coated with a variety of hydrophilic polymers like Dextran, PEG or liposomes [98]. The overall size of MINPs must be sufficiently small to evade rapid spleen filtration but large enough to avoid renal clearance. Parameters such as the physicochemical properties of MINPs loaded with the drug, their field strength and geometry, depth of the target tissue, rate of blood flow, and vascular supply play a major role in determining the effectiveness of the drug delivery process [99]. Due to the enhanced permeability and retention [EPR] effect (Figure 4), MINPs are vital in the treatment of cancer and other tumours [100]. EPR is a phenomenon of enhanced extravasation of particles from tumour blood vessels, and their subsequent retention in tumour tissue, which is not observed in normal vasculature. The leaky vascular blood vessels and a discontinuous endothelial cell lining, the matrix metalloproteinases (collagenases) affect disintegration of the matrix tissue surrounding blood vessels and permits the entry of particles that have limited access to normal tissue [101]. Doxorubicin (DOX), a well known anticancer therapeutic drug, coated on MINPs, has been delievered to tumor blood vessels and reported to suppress metastasis [102]. Combining targeted drug delivery with MRI has been the topical inclination in current research. This dual functionality of active MINP have been successfully applied in treatment of notable malady of heart, CNS, tumours etc. Jain et al. [103] reported treatment of breast cancer using DOX and Paclitaxel drugs incorporated on biocompatible MINP 131
as anti-proliferative agents with simultaneous imaging through targeted MRI. Yang et al. [104] exploited the interaction between natural receptor urokinase plasminogen activator receptor (uPAR) and its ligand urokinase plasminogen activator (uPA) by functionalizing the ligand on biocompatible iron oxide nanoparticles containing anti-tumor drug DOX. During an animal trial experiment on rat and rabbit, DOX loaded SPIO nanoparticles showed decrease in relative tumour volume up to two- and four-fold as compared to free-DOX and commercial liposome drug DOXIL along with higher MRI sensitivity comparable to a conventional MRI contrast agent suggesting them as a promising candidate for treating liver cancer and monitoring the progress of cancer using MRI [105]. Therapeutic proteins have revolutionized the treatment of many diseases thus are potential candidate which can be loaded on naoparticles for delivery to target cells. Antioxidant enzymes such as catalase and superoxide dismutase [SOD] which are therapeutically important in disorders related to increased production of reactive oxygen species [ROS] can be delivered to the target site by loading them on MINPs [106]. Success of drug delivery will only rely on the efficiency of internalization of drug molecule inside the cell. Radionuclides as an alternate treatment have been targeted to the tumours in many experiments as their activity is not dependent on molecule internalization [107].
NUCLEIC ACID DELIVERY AND TRANSFER

The success of incorporating drug on magnetic particle for targeted drug delivery has increased the attention of scientific community towards using them for delivery of nucleic acid molecules [DNA & RNA] to target cells [108]. Although similar to drug delivery, considering the fragility of nucleic acid, one of the most important requirements is to protect the bio molecule. Therefore, for a successful delivery system, the carriers must form a condensed complex with biomolecules which aid in its protection, facilitate penetration of the cell membrane after complexation by targeting through external magnetic field by exploiting biological phenomenon of endocytosis and finally should unload their payloads inside of cells at appropriate time which can carefully modulated by condition sensitive interaction between bounded nucleic acid and nanoparticle. Molecules can be attached to MINP surface either by chemical or biological contact or through electrostatic charge in view of the inherent property of DNA being negatively charged [109]. Polyethylene amine [PEI] is the most commonly used 132
surface coating agent which is cationic in nature [110]. The facilitation of nucleic acid delivery using superparamagnetic particle with the help of external magnetic force is termed as Magnetofection [111]. Advanced approach related to this method are attachment of non-viral [112] as well as viral vectors [113] containing gene of interest attached to nanoparticle, which has been found to increase the efficiency of transfection by many folds and also allows possible transfection to defiant cells.
Figure 3: In vivo therapeutic applications of distally controlled MINPs. [a] Magnetic particles [gray spheres] associated to therapeutic molecules [shown as orange spheres] act as vehicles for drug delivery, and after systemic administration they are concentrated to the target organ [O] with the help of a magnet [M]. [b] In magnetically mediated hyperthermia [MMH], systemically administered magnetic particles [gray spheres] accumulate in a tumor [T], either through the enhanced permeability and retention [EPR] effect or upon magnetic- or ligand-based targeting. Particles in the tumor are then heated [illustrated by the change to red spheres] through the external application of an alternating magnetic field [AMF], and this results in the death of the tumor cells. Reproduced from [47] with permission from Elsevier.
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Figure 4: Schematic diagrams showing enhanced permeability and retention (EPR) effect of nanoparticles in tumours.
Gene delivery imaging system for diagnosis of liver diseases has been achieved by using chitosan-linoleic acid coated SPIO nanoparticles targeted to hepatocytes and reported to be efficiently used as a carrier for gene silencing with specific siRNAs [114]. Yet in another experiment, poly-L-lysine-modified iron oxide nanoparticles [IONP-PLL] carrying the NM23H1 gene, the first suppressor gene of cancer metastasis, were successfully delivered to tumour cells in vivo and have significantly extended the survival time of an experimental mouse [115]. Cationic nanoparticle coupled to an integrin v3-targeting ligand were used to deliver Raf gene selectively to angiogenic blood vessels in tumor-bearing mice, as ATP -Raf blocks the endothelial signalling and angiogenesis in response to multiple growth factors and can result in apoptosis of the tumour-associated endothelium [116].
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CELL MANIPULATION AND TISSUE ENGINEERING

Once tagged with MINPs or internalization of the same compels the cell to behave specifically under magnetic field. This principle can be modified in variety of ways to manipulate cell structure, growth pattern and intrinsic property providing a valuable tool for studying cell science. To study structure and morphology of the membrane of submicronic endosomal compartments endosomal internalisation of the MINPs was carried out [117]. The internal magnetization of endosomes allows deforming them along the direction of an applied field. A detailed overview on manipulation of cell by MINPs has been reviewed by Dobson [118]. Technologies for fabricating functional tissue architectures are highly attractive for tissue engineering. At present methods such as microcontact printing and lithography have been developed but issue of complexity like requirement of specialized surfaces delimits their use. MINPs and magnetic force can be resourcefully employed to allocate cells on arbitrary surfaces and in this way, one can generate tissue constructs of various shapes. The success in this field has opened up new opportunities in magnetic-force-based tissue engineering [Figure 5]. For example, magnetite cationic liposomes [MCLs], magnetically label to target cells were allowed to grow on a steel plate placed on a magnet [119]. On a similar note, human mesenchymal stem cells [MSCs] magnetically labelled with MCLs were seeded onto an ultra-low attachment culture surface with a magnet on reverse side which facilitated growth of multilayered sheet-like structures after a 24-h culture period retaining its ability to differentiate into various cell lines [120]. With the collaboration in the fields of cell and molecular biology, material sciences and chemistry, magnetic particle based tissue engineering show great promise for future research.
AGRICULTURAL APPLICATIONS OF MINPs

Agriculture has seen overriding revolution with the introduction of hybrid crops and use of plants as expression system. Even nanotechnology extends its relevance in plant world with development of nanoscale technology for improvement in crop yield qualitatively and quantitatively. Even though, the complete functionality of MINPs is yet to be explored in this field but few reports are there suggesting a pivotal futuristic role of MINP in agriculture and plant sciences. Reports showing presence of nanoscale particle of clay, metal and other humic 135
substances in the soil define their role in natural soil systems [121]. The metal nanoparticles most prominently include various oxides, hydroxides, and oxyhydroxides of Al, Fe, and Mn, formed by weathering and bio-mineralization. Iron derivates occur in nearly all soils in the form of Goethite [-FeOOH], hematite [-Fe2O3], magnetite [Fe3O4], maghemite [-Fe2O3], lepidocrocite [-FeOOH] and ferrihydrite [122]. These nanoscale minerals play a vital role in the adsorption and retention of nutrient anions [e.g. phosphate] by electrostatic interactions and ligand exchange and actively promote clay flocculation and soil aggregate stabilization [123]. Zerovalent iron, in the nanoscale dimension can be applied to stabilize several elements in contaminated soils [124]. Zhu et al. [125] have demonstrated the significant uptake of magnetite [Fe3O4] nanoparticles [0.5 g/L] by pumpkin plants and their subsequent translocation and accumulation in various tissues. During the study, they did not observe any toxicological effects to the plants which suggest their use in nanoparticulate delivery system in plants. However, interactions between plants and nanoparticles need in depth investigations such as uptake route, its mechanism, translocation of nanoparticles and their interactions with plants tissues at cellular and molecular level. In a very recent study, MINP delivery in plants has been very well reviewed by Nair et al. [126]. The research group has evaluated the effect of nanoparticles in plant systems and demonstrated an outline for the safe use of agri-nanotechnology. This suggests that new nutrient delivery systems that exploit the nanoscale porous domains on plant surfaces can be developed (127).
ADVANTAGES OF MAGNETOSOMES AND BIOSYNTHESIZED MINPs

Biological methods for nanoparticle synthesis help in circumvent many of the detrimental features observed in chemical and physical synthesis methods by enabling synthesis at mild or neutral pH, pressure and temperature, thus made these nanoparticles more acceptable and compatible in biological systems. The formation of MINPs by biological systems is beneficial in terms of their properties and amenability in bio-medical application due to their uniform size distribution. Biomineralization provides controlled crystals of uniform size without fabrication mechanism of magnetic
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Figure 5: Uses of magnetic particles in tissue engineering. [a] Different cell types, indicated in purple and blue, are separately labeled using magnetite cationic liposomes and sequentially seeded onto an ultra-low attachment plate under which a magnet is placed. This leads to the generation of 3D multilayered heterotypic cell sheets by magnetic-force-based tissue engineering [Mag-TE]. Removal of the magnet allows the recovery of the construct for use. [b] Tubular structures can be generated by folding preformed cell layers, obtained as shown in panel [a], around rod-shaped magnetic models. Such tubular constructs are recovered after removal of the magnet. Reproduced from [47] with permission from Elsevier.
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submission of extreme temperature, pH and pressure which are mostly required in physicochemical process [128]. Biomembrane formed around the mineral core results in better stability, dispersion and thus can be easily handled as compared to synthetic MINPs [129]. Another advantage of natural coating is that it provides an excellent target for modification and functionalization of the particle [130] [Figure 6; 10]. The stoichiometry of membrane proteins can be engineered enhancing the applicability of biogenic MINP [131].
Figure 6: MNP possessing various ligands to enable multifunctionality from a single nanoparticle platform. Reproduced from [10] with permission from Elsevier.
The application of biosynthesized MINPs are presently confined to magnetosomes, which are thus far most extensively, reviewed MINP produced by biological system. Magnetosomes have been reportedly used in various biomedical applications proving them better as compared to commercially available MINPs. Advances in production strategy and bio-engineering of magnetosomes are rapidly escalating their importance. The unique and superior characteristics of magnetosomes as discussed above attracts interest for their use in magnetic separation, diagnostic, detection of analytes, drug and gene delivery and imaging. Table 1 enlist some recent 138
studies showing significant biomedical applications of MINPs. Wacker et al. [138] have demonstrated the concept of magneto immuno-PCR (Figure 7). Functionalization of
Figure 7: Schematic drawing of the Magneto Immuno-PCR [M-IPCR]. [A] HBsAg specific magnetosome-antibody conjugate and DNAantibody conjugate are incubated simultaneously with the serum sample containing HBsAg resulting in a signal-generating detection complex. [B] The detection complex is concentrated using an external magnetic field. Subsequent washing steps permit the removal of unbound materials. [C] After resuspending, a defined volume of the detection complex solution is transferred to a microplate containing the PCR mastermix to enable real-time PCR detection of the immobilized antigen. Reproduced from [138] with permission from Elsevier.
particles for specific application in physiological environment is important and can be performed with ease in case of magnetosome owing to presence of lipid bilayer membrane [132]. Functionalization can be done by biotinylation, charged molecules like PEI and organosilane, polyamidoamine dendrimer as well as by advanced approaches involving genomic and proteomic level manipulation of magetosome membrane [133]. Although the magnetosomes themselves are of low toxicity, the associated bacterial endotoxin lipopolysaccharide [LPS] may 139
pose adverse effect. The LPS can be easily removed as suggested by treatment with detergent retaining selective tightly bound protein on the membrane which can be used as potential anchoring molecules [129]. Arakaki et al. [23] have excellently reviewed the advances and futuristic application of magnetosomes in biomedicine.
Figure 8: Development of nanovaccines: MINP coated with antigen interacting with a B cell.
CONCLUSION
As can be seen in this chapter, MINPs are one of the most extensively commercially used nanomaterial in biomedical world with diversified applications. In the past decade, considerable success has been achieved but the final objectives are still ahead. Shifting the focus towards ecofriendly, cost effective and greener methods of nanoparticle fabrication will attract attention of the scientific community towards development of biocompatible MINPs. Monitoring and understanding the interaction of MINPs with cells and tissues inside the living system will constructively shape the strategies for development of biocompatible nano-magnets. Adapting an interdisciplinary approach can enhance the development futuristic technologies which are more user friendly and close-to-market. Use of MINPs in targeted drug delivery will undoubtedly improve the therapeutic potential of many water insoluble and unstable drugs. However, more efforts are required to find out easy ways to drove these MINPs to internal body organs such as liver, kidney, pancreas etc. We believe that in future, the trends of research will be related to Table 1: Significant biomedical applications of magnetosomes
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Application genre
Key Feature
Reference
Detection and diagnostic; Imaging
System for Streptavidin detection using biotin conjugated to bacterial [134] magnetic particles isolated from magnetic bacteria were used as magnetic markers for magnetic force microscopy [MFM] imaging.
Separation of biological molecule and cell
The efficient and stable display of the immunoglobulin G [IgG]- [135] binding domain of protein A on BMPs using anchor molecule, Mms13, isolated from BMPs was performed. The anchoring properties of Mms13 onto BMPs were evaluated using luciferase fusion studies.
Imaging
Magnetosomes of size 42 nm covalently coupled to the fluorescent [136] dye DY-676 were labelled to murine macrophages J774 and examined by infrared imaging, MRI, confocal laser scanning microscopy and transmission electron microscopy.
Drug delivery
Bacterial magnetosomes were used as the magnetic-targeted drug [137] carrier by loading antitumor drug doxorubicin [DOX]. Loaded magnetosomes were tested for their activity in EMT-6 and HL60 cell lines to evaluate the in vitro and in vivo anti-neoplastic effects on hepatic cancer.
Detection and diagnosis
HBsAg specific magnetosome-antibody conjugate and DNAantibody [138] conjugate are incubated simultaneously with the serum sample containing HBsAg resulting in a signal-generating detection complex which is concentrated using an external magnetic field and subjected to detection by real-time PCR detection of the immobilized antigen.
Gene delivery and transfer
Novel non-viral delivery system based on PEI coated bacterial MINPs [139] transfecting BMPs-PEI bound to the plasmid pCMVbeta encoding beta-galactosidase [pcD-VP1] into mice. Evaluation of efficiency of in vivo delivery of BMPs-PEI bound plasmid pcD-VP1 were determined by MTT assay for T cell proliferation and ELISA for detecting total IgG antibodies.
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Separation of biological molecules and cell
Magnetic separation of monocytes and B-lymphocytes from the [140] peripheral blood was achieved with high purity using magnetosome with membrane expressing protein A to reduce cytotoxicity of innate membrane.
Detection and diagnostic
ER ligand binding domain [ERLBD] was fused to the anchor protein, [141] Mms13, which are used for efficient and stable protein display onto Bacterial MINPs. To evaluate the ligand-dependent binding of GFP coactivator, binding assay of GFP-coactivator to ERLBD Bacterial MINPs was performed.
Detection and diagnostic; Study of cellular mechanism
Expression of GFP in the microaerophilic M. gryphiswaldense at [142] various oxygen levels and analysis of the subcellular localization of the GFP-tagged magnetosome proteins MamC, MamF, and MamG involved in control of the size of growing magnetite crystals. The GFP-modified fluorescent MINPs were purified from bacterial cells, and the stability of expression and fluorescence of MamC-GFPlabeled magnetosomes was studied in vitro under various conditions.
Imaging
Magnetospirillum magneticum AMB-1 were injected in mice with [143] tumour. Bacterial growth conditions were manipulated to produce small magnetite particles, which were observed using transmission electron microscopy. Tumour targeting was confirmed using 64Culabeled bacteria and positron emission tomography and by determination of viable cell counts recovered from different organs and the tumour.
newer functionalization and modification of MINPs to introduce additional functionalities thus designing active sites for incorporation of diverse utility. We thereby hypothesize the development of nanovaccines by antigen [epitope] presentation on nanoparticle surface [Figure 8]. Its specific targeted delivery will boost the commercial importance of MINPs. As far as biosynthesis of MINPs is concerned, apart from MTB other microbial flora must also be evaluated. Sprouting success in finding aerobic organisms capable of producing MINP will strengthen the research in this field. Efforts should be made to cultivate more and more MTB 142
under laboratory condition. In case of MTB, better understanding of the molecular mechanism and specific role of various genes involved in magnetosomes synthesis can improve the pace of research. Attempts can be done in mimicking the magnetosomes synthesis in other expression systems which can be upgraded to commercial scale as well as producing magnetosomes in vitro which will simplify the downstream protocols. New nutrient delivery systems that exploit the use of metal nanoparticles as potential fertilizers for plant can be developed. Though it is challenging to assess the risks of engineered nanomaterials before commercial products are well defined, proactive research is critical to ensure a sustainable nanotechnology industry.
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Chapter VII ADVANCES IN NANOBIOTECHNOLOGY FOR AGRICULTURE

Vinod Saharan
Department of Molecular Biology and Biotechnology, Maharana Pratap University of Agriculture and Technology, Udaipur Corresponding author e-mail: vinodsaharan@gmail.com
ABSTRACT: The successful application of various nanoplatforms in medicine under in vitro conditions has generated interest in agri-nanobiotechnology. Role of nanobiotechnology will be significant in future for agriculture. Nanobiotechnology holds the promise of controlled release and site targeted delivery of agrochemicals (fungicides, herbicides, pesticides, plant hormones etc.). Specifically, the application of nanoparticles for biotic and abiotic stress offer new ways for crop protection. Alongside, plants are become an ideal factory for synthesis of desire size and shape of nanoparticles, which are in great demanded. Nanoparticle mediated plant transformation has the potential for genetic modification of plants for further improvement and replace the old technology. Herein we reviewed in this chapter the delivery of nanoparticulate materials to plants and their ultimate effects which could provide some insights of this novel technology for the improvement of crops. Keywords: Nanoparticles, stress, biotechnology, saponin INTRODUCTION: Nanotechnology is the manipulation or self-assembly of individual atoms, molecules, or molecular clusters into structures to create materials and devices with new or vastly different properties. The definition of nanotechnology is based on the prefix nano which is from the Greek word meaning dwarf. In more technical terms, the word nano means 10-9, or one billionth of something. The word nanotechnology is generally referring to materials with the size of 0.1 to 100 nanometres. Nano size has different properties from bulk (or micrometric and larger) materials as a result of their size. These differences include physical strength, chemical 156
reactivity, electrical conductance, magnetism, and optical effects. Combining nanotechnology with biology evolve nanobiotechnology. Nanobiotechnology has the potential to revolutionize the agricultural and food industry with new tools for treatment of plant diseases, enhancing the bioavailability of agrochemicals and enable plant cells as nanoparticle synthesizer. Improvement in these areas through nanobiotechnology interventions can make agricultural systems more producible and organized. Application of target delivery system for agrochemicals is most imperative field of research in agriculture. Smart Delivery Systems should possess combinations of time controlled, specifically targeted and multifunctional characteristics to avoid biological barriers for successful targeting plant cells. Nanoformulation could increase the efficiency of agrochemicals and, allowing lower doses to be used to achieve desire effects. Thus, nanobiotechnology would protect the environment through control use of agrochemicals. Besides these significant properties, plants provide a biological synthesis route of several metallic nanoparticles which is more ecofriendly and allows a controlled synthesis with well defined size and shape. Development of nanoparticle / nanovesicle mediated genetic transformation also an important area to be exploited. This chapter presents recent advances in nanobiotechnology for agriculture (A) Nano-augmented delivery system for agrochemical: The successful application of various nanoformulations in drug delivery in medicine has raised concern in agriculture. Processes such as nanoformulation /nanoencapsulation/ nanoemultions show the benefit of more efficient use and safer handling of agrochemicals. In the past few decades, the use of adjuvant / surfactant has become common practice to enhance the delivery of agrochemicals to the inner tissue of the plant. The long- term fates of adjuvant/ surfactant in soils and elsewhere in the environment are also largely unknown because of the lack of long-term monitoring data [1]. Therefore, environmental questions have recently been raised about these traditional products. Available methods for delivery of agrochemicals are not specifically targeted; hence, 50-70 % of agrochemicals like fungicides, herbicides, pesticides, plant growth regulators etc. are deposited in environment without reaching the required sites. The activity of agrochemical gets further reduced by UV light, temperature, humidity etc. The agrochemicals loaded into the inner core of nanoparticles would show a typical sustained-release pattern, protection from external environment (UV, temperature, humidity etc.). Hence, such nanocarriers have a promising future in application of agrochemicals. Adjuvant /surfactants are 157
Oil based, Polymers+ lipid based Film-forming materials Inorganic salts Silicone-based nonionic Amine ethoxylate & nonylphenol ethoxylates Adjuvant /surfactants are partially or completely synthetic materials i.e. Raising environmental questions Non-degradable Less efficient and costly Severe necrotic damage to plant cells Toxic to soil & aquatic organisms
Not universal (need to change with different agrochemicals )
(a) Phytosaponin as a delivery system: Saponins are a highly diverse group of glycosides of plant origin. They contain either a steroidal or a tri-terpenoid aglycone to which one or more sugar chains are attached. The sugars are usually glucose, galactose, glucuronic acid, xylose, or rhamnose. The diversity of saponins is a result of the variability in the sugar side chains. The main biological activity ascribed to saponins is their membrane permeabilizing property. Saponins have additionally been reported to exhibit adjuvant-active properties. Based on these observations, an open cage-like immunostimulating complex (ISCOM) of cholesterol, lipid, immunogen, and saponins from the bark of Quillaja saponaria (soap bark tree) has found successful application as an active adjuvant for vaccination [2]. Synthesis of nanosaponin formulation with chitosan and other components for evaluation of drug delivery system for animal system has established successfully. Nanosaponin formulation with chitosantripolyphosphate cross linking found promising for encapsulation of biological active compounds [3,4]. The physico-chemical characteristics of saponins indicate its suitability for delivery of biological active compound. Saponins assemble in micelle structures in aqueous environment. The property of making novel micelles structure enable saponins to encapsulate various biomaterials. Microscopic studies of saponin extract from Quillaja and Balanites plants revealed their self-assembled properties [5,6]. Biological activity of saponins is due to their binding activity with sterols components in the biological membranes and causes partial pour formation [7,8]. Plant based adjuvant; Quillaja saponin has been used abundantly in animal system for delivery of drugs [9]. In this fashion, saponin molecules could play a vital role in delivering agrochemicals in a targeted manner in crop plants. 158
Nanosaponin delivery system Nano size Efficient in encapsulation of target material Biodegradable & non-toxic to soil & aquatic organisms Cheap and easily available UV protection of encapsulating materials Natural binding ability to biological membranes Quillaja and Balanites saponin has been successfully tested to cross cuticle membrane [10, 11]. Study further conclude that saponin at predetermine concentration as ability to bind to lipid molecules and make safe passage for plant growth regulators. Plant growth regulators have been used over the past decades for several purposes in crop production. Plant hormones are involved in controlling the growth, development, metabolism and morphogenesis of higher plants. Foliar and root application of nutrients and plant growth regulators improve yield characteristic. Plant hormones like 2,4-D, BAP, ABA, GA etc. possess a variety of functions in vegetative and reproductive phases of plant life cycle such as seed germination, stem elongation, leaf expansion, flowering, fruit size, grain yield and nutrient use efficiency. Currently more efforts have been concentrated to cope with adverse effect of global warming in agriculture. Agrinanobiotechnology could bring the revolution in this respected by designing target specific delivery system for plant growth regulators. Novel formulation of saponin with plant growth regulator has been evaluated and found excellent for enhancement of in vitro plant growth at lower concentration of plant growth regulators. Studies gave hypothesis that saponin encapsulates the plant growth regulators through ultrasonication and deliver encapsulated material efficiently to the surface/inner part of plant cell (Fig. 1) [11]. (b) Chitosan as a delivery system: Chitosan is a polysaccharide, similar in structure to cellulose. Both are made by linear -(1-4)-linked monosaccharides. However, an important difference to cellulose is that chitosan is composed of 2-amino-2-deoxy--d-glucan combined with glycosidic linkages. The primary amine groups render special properties that make chitosan very useful in carrier/delivery system for animal/plant cells. Compared to many other natural polymers, chitosan has positive charges which render chitosan to bind with biological membranes [12]. Therefore, it is used extensively in drug delivery applications [13]. Chitosan is relatively reactive and can be produced in various forms such as powder, paste, film, fiber, etc [14, 15]. Chitosan is reported to influence the production of substances related to stress
response, such as phytoalexins [16] and chitinases [17, 18]. It is suggested that chitosan can be 159
used commercially for controlling various plant disease under field conditions [19]. Chitosan treatments have plant growth promoting effects, resulting in improved yields and plant health in numerous crops and fruits. The activation of protective mechanisms in plant tissues with chitosan inhibited the growth of taxonomically different pathogens [20]. It has been considered as an alternative to chemical fungicides [21]. Beausejour et al., (2003) [22] reported that combination of S. melanosporofaciens EF-76 and chitosan represents a promising method of biocontrol against common scab in potato crop. Recently, chitosan nanoformulations found promising for encapsulations of various components and applied in animal cells. It could be predicted that nano scale chitosan formulation will act for effectively than normal chitosan on cell membrane either thought proper exposure with receptors or cross easily via membrane pores.
Hydrophilic head Hydrophobic tail
(Saponin molecule)
(Saponin micelle structures)
(Encapsulation of agrochemical into saponin)
Figure 1: Saponin encapsulation of agrochemicals
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(B) Nanobiotechnology as Agronanofactories: Various physical and chemical methods used for synthesis of nanoparticles. Recently the use of biological systems for the synthesis of nanoparticles is gaining interest since it is more ecofriendly compared to existed system [23, 24]. Among various biological systems, plants provide an easy and safe green route for the synthesis of various metals. The formation and growth of gold nanoparticles [Au NPs] inside live alfalfa plants and sesbania seedlings grown in gold enriched media was reported [25]. Microscopic and X-ray absorption studies confirmed the nucleation and growth of nanoparticles inside plants. The uptake of silver ions and their reduction and distribution as Ag NPs within cellular structure of some metallophytic plants has also been investigated [26]. Copper biomineralisation with some wetland plants that transform copper into metallic nanoparticles at soil-root interface with the help of some endomycorrhizal fungi were also reported that could reduce copper toxicity in contaminated soils. The formation of nanoparticles of an alloy of goldsilver-copper using plant was also reported [27]. This opens up a great scope of using plants for the production of mixed metal nanoparticles of specific compositions. The mechanism of formation of nanoparticles; whether they are formed outside in the media and then translocated to plants or whether they are formed by the reduction of metal salts within the plants itself still needs more clarification [28, 29, 30]. The uptake and translocation of nanoparticles across root cells depends on the type of metal ions and plant species. The amount of nanoparticle accumulation in plants also varies with reduction potential of ions and the reducing capacity of plants that depends on the presence of various polyphenols and other heterocyclic compounds present in plants [31, 32]. Different studies regarding the extracellular biological synthesis of nanoparticles using leaf extracts [33, 34, 35, 36, 37, 38] and dead tissues of various plants [39, 40, 41] were also reported. Hence, compared to the chemical synthesis of nanoparticles, this method provides a better and safe means of nanoparticle production with defined size and shape of our interest. (C) Delivering genetic materials into plants: Nano-fibres/nanoparticles/nanovesicles with cationic nature have ability to precipitate negative charged DNA molecules and bring into cell through nonporous of cell membrane. This process has similarity with microinjection method of gene delivery [42, 43, 44]. The application of fluorescent labelled starch-nanoparticles as plant transgenic vehicle was reported in which the nanoparticle biomaterial was designed in such a way that it bind and transport genes across the cell wall of plant cells by inducing instantaneous pore channels in cell wall [45]. It is possible to integrate different genes on the nanoparticle at 161
the same time and the imaging of fluorescent nanoparticle is possible with fluorescence microscope thus understanding the movement of exterior genes along with the expression of transferred genes. Hence successful generation of pores on cell wall and cell membrane by suitable agents help in nanoparticle mediated DNA transfer that might be more successful in regenerative calli and soft tissues. The ability of surface functionalized mesoporous silica nanoparticles (MSNs) to penetrate plant cell walls also opens up new ways to precisely manipulate gene expression at single cell level by delivering DNA and its activators in a controlled fashion [46]. In preliminary experiments protoplasts were incubated with fluorescently labelled MSNs. It was found that surface modification of MSNs with triethylene glycol was necessary to penetrate the cells. Such modification of MSNs also allowed plasmid DNA to adsorb on MSN surface. After entering the protoplasts, the plasmid DNA was released from the MSNs and the green fluorescent protein (GFP) marker encoded in the DNA was expressed in the cell which was detected by microscopy. In this method the minimum amount of DNA that is required to detect marker expression was 1000-fold less than that required for the conventional delivery method. This efficient delivery method has pronounced applications in various protoplast-based gene expression studies. Nowadays gene gun or particle bombardment is one of the popular tools to deliver DNA into intact plant cells [47]. Particles used for bombardment are typically made of gold since they readily adsorb DNA and are non-toxic to cells. Since MSNs are too light, it is difficult for delivering foreign DNA attached on MSNs by gene gun method. This problem was solved by capping MSNs with gold nanoparticles which increased their momentum after acceleration by the gene gun. Experiments showed that the plasmid DNA transferred by gene gun method using gold-capped MSNs was successfully expressed in intact tobacco and maize tissues. The major advantage is the simultaneous delivery of both DNA and effectors molecules to the specific sites that results in site targeted delivery and expression of chemicals and genes respectively. This is how the nanoparticle mediated plant transformation differs from the conventional genetic engineering methods (like electroporation, microinjection, etc). Future scopes include pore enlargement and multifunctionalization of MSNs which provide even better possibilities in target-specific delivery of proteins, nucleotides and chemicals in plant biotechnology.
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CONCLUSION: Nanobiotechnology is heading to the expansion of a range of economical nanotech applications for enhanced plant growth. Nanostructures endow with an efficient means to distribute agrochemicals in a controlled fashion with high site specificity. Designed nanoparticles can be successfully used to deliver agrochemicals or other substances (elicitors, nucleic acids etc.) into localized areas of plant tissues. Plant mediated synthesis of metal nanoparticles
provide a safe synthesis route with better control over morphology of nanoparticles. On other hand nanocarrier would be a leading tools for gene transformation in a variety of plants. REFERENCES 1. Cornish, A., Battersby, N. S. & Watkinson, R. J. (1993). Environmental fate of mineral, vegetable and transesterified vegetable oils. Pestic. Sci., 37, 173-178. 2. Sun, H,X., Xie ,Y. & Ye, Y.P. (2009). Advances in saponin-based adjuvants Vaccine, 27, 1787-1796. 3. Kashappa & Kashappa, G. H. D. & Hyun, J. P. (2005). Preparation and characterization of saponin-loaded chitosan-tripolyphosphate microspheres by spray drying. Drug Development Research, 64, 114-128. 4. Huang, J., Li, Q., Sun, D., Lu,Y., Su,Y. &Yang,X. (2007). Biosynthesis of silver and gold nanoparticles by novel sundried Cinnamomum camphora leaf. Nanotechnology, 18, 5104-5114. 5. Chapagain, B.P, & Wiesman, Z. (2006). Phyto-Saponins as a Natural Adjuvant for Delivery of Agromaterials through Plant Cuticle Membranes. J. Agric. Food Chem., 54, 6277-6285. 6. Walker-Simmons, M., Hadwiger, L. & Ryan, C.A. (1983). Chitosans and pectic polysaccharides both induce the accumulation of the antifungal phytoalexin pisatin in pea pods and antinutrient proteinase inhibitors in tomato leaves. Biochemical and Biophysical Research Communications, 110, 194-199. 7. Morrissey & Morrissey, J. P. & Osbourn A.E. (1999). Fungal Resistance to Plant Antibiotics as a Mechanism of Pathogenesis. Microbiol. and Mol. Biol. Revi, 63, 708724.
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nanoparticles using Diopyros kaki leaf extracts. Bioprocess Biosyst. Eng., 33,159-164. 34. Bar, H. Bhui, D.K. Sahoo, G.P Sarkar, P. Pyne, S. & Misra, A. (2009). Green synthesis of silver nanoparticles using seed extract of Jatropha curcas, Colloids Surf. Physiochem. Eng. Aspects, 348, 212-216. 35. Song, J.Y. Jang, H.K. & Kim, B.S. (2009a). Biological synthesis of gold nanoparticles using Magnolia kobus and Diopyros kaki leaf extracts, Process Biochem., 44, 1133-1138. 36. Song, J.Y. & Kim, B.S. (2009b). Rapid biological synthesis of silver nanoparticles using plant leaf extracts. Bioprocess Biosyst. Eng., 32, 79-84. 37. Narayanan, K.B. & Sakthivel, N. (2008). Coriander leaf mediated synthesis of gold nanoparticles, Mater. Lett., 62, 4588-4590. 38. Song, J.Y. & Kim, B.S. (2008). Biological synthesis of bimetallic Au/Ag nanoparticles using Persimon (Diopyros kaki) leaf extract. Kor. J. Chem. Eng., 25, 808-811. 39. Parsons, J.G., Armendariz, V., Lopez, M.L., Jose-Yacaman, M. & Gardea-Torresdey, J.L. (2010). Kinetics and thermodynamics of the bioreduction of potassium
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Chapter VIII
IN SILICO ANALYSIS OF SEQUENCE VARIATIONS IN CHOLERA TOXIN B SUBUNIT AND THEIR BINDING WITH CARBOHYDRATE LIGANDS MHU Turabe Fazil1,*, Sunil Kumar2,* and Durg V singh1
1 2
Infectious Disease Biology, Institute of Life Sciences, Nalco Square, Bhubaneswar, India, Bioinformatics Centre, Institute of Life Sciences, Nalco Square, Bhubaneswar, India. *Corresponding Authors: MHU Turabe Fazil, Email: turabe_fazil@yahoo.com Sunil Kumar, Bioinformatics Email: skybiotech@gmail.com
ABSTRACT: Vibrio cholerae is the causative agent of the deadly diarrheal disease cholera.
This bacterium after passing the stomach acid barrier adheres to and colonizes in the intestinal epithelial cells leading to production of cholera toxin. Cholera toxin is a hexameric A-B5 type toxin and encoding genes lies on CTX, a filamentous bacteriophage. The toxicity is attributed by enzymatic activity of the A-subunit whereas B subunit stimulates mucosal antibody responses. The mechanism of CTB binding to the GM1 receptor of epithelial cells has not been fully defined. Efficient binding to GM1 could potentially raise the uptake of antigen across the mucosa and lead to an improved presentation of conjugated molecule to the immune system. Although the action of CT is conserved among classical and El Tor strains, the CTB gene sequences differs among the two biotypes. Based on non-random base variations, three types of ctxB genes due to change in the deduced amino acid sequence positions at 39, 46 and 68, have been described. New ctxB variants showing additional polymorphism at amino acid positions 28 and 34 have recently been described in V. cholerae O139 strains. In this chapter, we discussed the possibility of variable binding efficiency of CTB variants to constituent carbohydrates of GM1 ganglioside through molecular and computational approaches. We found subtle variations in hydrogen bond binding abilities of variants of CTB with the carbohydrates ligands constituent of GM1 receptor. These differences can be employed in designing paratopes that can determine specificity of either a precise biotype or a standard anti-cholera toxin monoclonal antibody and could be exploited for designing drugs or vaccines. 168
INTRODUCTION
Food security and increase in average life expectancy can be attributed to biotechnological developments in the last century. The advent of modern molecular techniques, like new generation sequencers, microarrays, crystallographic structure elucidation methods, has helped us to understand natural science and added tremendous inputs to existing knowledge of biology. Since introduction of computers to investigate biological problems, the temporal inhibitions of understanding miniscule proceedings of molecular machinery have been dampened to a good extent. Infectious disease processes, which had elucidated the imagination of biologists, have been unravelled to an understandable level owing to biotechnology. Understanding pathogenesis is not only important to find immediate therapy but also for gaining knowledge on the evolution in a species. In this chapter, we describe the use of gene sequencing and computational protein modelling to address questions on pathobiology of Vibrio cholerae. 1. Cholera- the disease V. cholerae is the causative agent of the deadly diarrheal disease cholera. Cholera can be transmitted after consuming contaminated water and food. The most distinctive and salient features of cholera is epidemiologic behaviour that tend to cause explosive outbreaks, often with several foci of onset, resulting in epidemics and subsequently pandemics. Cholera gravis caused by V. cholerae have been reported infecting populations throughout the world but to specific endemic regions including Asia and Africa. About 35 million cholera cases have been reported per annum with a mortality rate of 20-30% that could increase if not treated in time (http://www.who.int/mediacentre/factsheets/fs107/en/index.html). The severity of the infection depends on several factors like local intestinal immunity arose from previous bacterial infection or vaccination, the size of the inoculums, adequacy of the stomach gastric-acid barrier and the patients blood group. The most distinctive feature of the cholera is the painless purging of voluminous stools resembling rice-water and fishy odour. In case of severe cholera, the rate of purging may quickly reach up to 5001000 mL/h leading to acute dehydration. The fluid loss may be so rapid that the patient will be at risk of death within a few hours of onset of the disease. V. cholerae is classified into more than 200 serogroups of which only O1 and O139 serogroups have the potential to cause epidemic or pandemic cholera. These bacteria after passing the stomach acid barrier, the surviving bacteria reaches to intestine, adhere to and 169
colonize in the intestinal epithelial cells leading to production of cholera toxin (CT) and causing symptoms of cholera. 2. Genetics of CTX The genes encoding for cholera toxin lies on CTX, a filamentous bacteriophage, is one of the principal virulence factors of the diarrhoeal pathogen V. cholerae [1]. Acquisition of CTX is a key event in the emergence of V. cholerae as a pathogen. Virulence factors are frequently encoded within mobile genetic elements such as phages and plasmids. However, CTX phage was shown to horizontally transmit a virulence factor that results in lysogenic conversion of a bacterium to toxigenicity. This single stranded DNA phage, related to coliphage M13, infects host bacteria by adsorbing to a toxin-coregulated pilus (TCP). The gene for the pilus protein is expressed under control of the ToxR regulatory system that also regulates the transcription of the cholera toxin genes. The CTX, a ~6.9 Kb element, can integrate into V. cholerae chromosome, often as an array of tandemly repeated copies, and can replicate and transmit vertically as a plasmid. The CTX is integrated in the large chromosome of toxigenic V. cholerae El Tor and O139 strains whereas O1 classical biotype isolates had prophages in both large and small chromosome. Basically three distinct CTX variants have been described, namely CTXET derived from V. cholerae O1 El Tor, CTXclass derived from classical V. cholerae, CTXCalc derived from O139 V. cholerae [2]. The CTX prophage is often flanked by a related genetic element known as the RS1. The RS1 element is very similar to the RS2 but contains an additional gene, rstC which acts as antirepressor. The RS1 element is the genome of a satellite phage of ~2.7-kb, capable of both autonomous replication and chromosomal integration. Since RS1 lacks the genes for its morphogenesis, it cannot independently packaged into the genome for transmission. RS1 replicates by the mechanism similar to CTX, utilizing the CTX morphogenesis genes to produce RS1 particles [3]. With the exception of the toxin genes (ctxAB), the organization of the CTX core region is similar to other filamentous phage genomes that infect E. coli and V. cholerae. The ctxAB genes do not have any role to play in the CTX life cycle. The GC content of ctxAB also differs from the other CTX genes that led to the hypothesis that ctxAB are acquired after the evolution of a pre-CTX that lacked these genes [4]. This model proposes the existence of a common pre170
CTX ancestor for the currently known CTX variants [5]. Like several other toxin-encoding phages, ctxAB is adjacent to the CTX attachment site indicating that an imprecise excision of the pre-CTX prophage can generate a new phage by acquisition of ctxAB sequences by yet uncharacterized mechanism. 3. Cholera toxin Cholera toxin is a hexameric A-B5 type toxin. The toxicity is attributed by enzymatic activity of the A-subunit that catalyzes ADP ribosylation of the -subunit of GTP binding protein, GS, leading to activation of adenylate cyclase and a concomitant elevation of cAMP levels causing hyper-secretion of Cl- ion and water ultimately leading to profuse diarrhea. Cholera toxin A subunit (CTA) consists of two polypeptide chains, CTA1 and CTA2. CTA1 confers CTmediated toxicity, whereas CTA2 acts as a linker between CTA1 and cholera toxin B subunit (CTB). The five B-subunits of the toxin bind principally to monosialoganglioside (GM1) receptor present on the surface of the intestinal epithelial cells. The B subunit is considered as molecular recognition unit and delivery vehicle for A-subunit [6]. Cholera toxin B subunit is used as an oral cholera vaccine candidate. Dukoral (R) is the only vaccine recommended and registered by WHO for vaccination of populations at risk of cholera epidemic region. This vaccine consists of killed cells of V. cholerae of both toxigenic biotypes and cholera toxin B subunit, given in two doses within 16 weeks, would stimulate antibacterial and antitoxic immunity. The other lyophilized oral vaccine, Orochol an avirulent mutant of V. cholerae strain CVD103HgR, given as a single-dose, has also been found providing antibacterial and antitoxic immunity. Cholera toxin B subunit (CTB) and the related Escherichia coli heat-labile enterotoxin B subunit (LTB) are competent carrier molecules for the stimulation of mucosal antibody responses and oral tolerance. The mechanism behind CTB's efficiency as a mucosal carrier molecule has not been fully defined but believed to be associated with binding to the GM1 receptor present on the epithelial cells. Efficient binding to GM1 could potentially raise the uptake of antigen across the mucosa and lead to an improved presentation of conjugated molecule to the immune system. It has also been shown that CTB induces major histocompatibility complex (MHC) class II expression on B cells and enhances antigen presentation by macrophages in the absence of increased MHC II expression. CTB triggers the polyclonal activation of B cells and selective 171
apoptosis of CD8 cells from both the Peyers patches and intraepithelial lymphocytes. The therapeutic applications of CTB-mediated oral tolerance include immunoglobulin E (IgE)mediated allergic reactions, the treatment of T-cell-mediated autoimmune diseases and infection related pathological inflammatory conditions [7,8]. 4. CTB sequence variation and binding with carbohydrate ligands Although the action of CT is conserved among classical and El Tor strains, the CTB gene sequence differs among the two biotypes formed the basis for ctxB genotyping. Based on nonrandom base variations, three types of ctxB genes due to change in the deduced amino acid sequence positions at 39, 46 and 68, have been described. Genotype 1 is found in classical biotype strains worldwide and in U.S. Gulf Coast strains, genotype 2 in El Tor biotype strains from Australia, and genotype 3 in El Tor biotype strains from the seventh pandemic and the Latin American epidemic. New ctxB variants showing additional polymorphism at amino acid positions 28 and 34 have recently been described in V. cholerae O139 strains [9, 10]. Previous studies indicated that amino acid sequence variations at positions, not involved in receptor binding, can be a reason for epitope variation of CTB and, therefore, need be considered for development of synthetic and natural vaccine against cholera. Mutation analysis of CTB revealed variations in immunoreactivity, haemolysis and GM1 binding ability for the toxin subunit [11]. However, the role of natural variations in CTB and their advantage if any, in survival or pathogenesis is not yet explored. We carried out a gene sequencing, molecular modelling, dynamics simulations and docking studies for all natural CTB variants. Monosaccharide libraries had been earlier used for exploration of binding sites to develop simple and easily synthesizable molecules against E. coli heat labile toxin, a close homolog of CT. In this regard, monosachharide components of GM1 receptor namely galactose, sialic acid, N-acetyl galactosamine and glucose were employed individually for docking simulations. The information generated would be useful in establishing enterotoxic potential of the CTB variants, if any, and in determination of relationships between variants of CTB and severity of disease apart from defining the requisite binding sites of drug like molecules. The total gene sequence of the cholera toxin B sub-unit is comprised of 375 bases. Because of presence of variants of CTX in V. cholerae strains, we employed a phage specific amplification of cholera toxin B subunit [12]. The DNA sequence between rstR gene and the 172
ctxB were selectively amplied using forward primers of rstRET or rstRCalc and reverse primer of ctxB that yielded amplicons of ~6.9 kb. This PCR product was then used as template in nested PCR to amplify ctxB gene of 449 bases, comprising complete coding sequence. The sequence generated was used in obtaining translated amino acid sequence of all the possible CTB variants. The classical type of CTB, designated as genotype 1, was produced by the V. cholerae harbouring classical type of CTX prophages. El Tor type of CTX prophages produces El Tor type of cholera toxin that includes two variants of CTB, designated genotypes 2 and 3. The O139 V. cholerae possesses both El Tor and Calcutta type of CTX prophages includes genotypic variants 4, 5 and 6 (Table 1). Sequencing data indicates that a majority of V. cholerae O139 El Tor phages belong to genotype 4, whereas the Calcutta type phages comprise mostly of genotype 5. However, both variants of CTB produce classical type of cholera toxin B. The experimentally generated crystal structures for all known variants in V. cholerae are not yet available. Therefore, we built molecular models for all the reported variants of ctxB gene (Table 1) by means of comparative modelling using MODELLER software. Crystal structure of the cholera toxin B-pentamer complexed with GM1 pentasachharide (PDB code: 2CHB) was selected as template for all modelling procedures. All calculations were performed by using ACCELRYS DS Modeling 2.0 software suite (Accelrys Inc. San Diego, CA 92121, USA). In order to assess the stereo-chemical qualities of the three dimensional models, PROCHECK, VERIFY 3D and ERRAT analysis was performed. All atom MD simulations of CTB protein in explicit water was carried out using GROMACS 4.0.6 program and the GROMOS96 force field for a time scale of 1ns. The primary model generated was that of classical CTB, as the crystal structure 2CHB represented classical cholera toxin. The in silico mutations performed using the mutate _model command of MODELLER was used to generate the rest of the variants. Amino acid variations affecting the stability, structure and function of the protein were calculated by SDM server (http://mordred.bioc.cam.ac.uk/~sdm/sdm.php). The chemical structures of monosaccharides were extracted from pubchem (http://pubchem.ncbi.nlm.nih.gov). Structures of all four carbohydrate ligands (glucose, galactose, N-acetyl neuraminic acid, N-acetyl galactosamine) included in binding to CTB were retrieved in two dimensional MDL/SDF format. Three dimensional co-ordinates for these molecules were generated using CORINA program. The standard default settings, consisting of population size-100, selection pressure-1.1, niche size-2, migrate-10, cross over-95, number of operations-1, 00,000, number of docking 10, were 173
adopted for GOLD docking. The ligand showing maximum interactions with the protein were plotted using the program LIGPLOT. Table 2 indicates respective variations in percentage solvent accessibility at mutation position and differential free energy of unfolding for all modeled CTB variants, generated by SDM software in comparison to crystal structure of Genotype 1 (classical). Docking simulations studies suggested that the ability of the type1 CTB to interact with galactose and N-acetyl galactosamine were considerably superior to its counterpart type 3 found in V. cholerae biotype El Tor. Among the CTB variants V. cholerae O139, the most recent known type 6, shows most impressive bonding characteristics with galactose, sialic acid and N-acetyl galactosamine in comparison to all other known CTB genotypes (Fig. 1). Table 1. V. cholerae O1 and O139 strains showing amino acids variations of ctxB and their respective genotypes Strains ctxB Genotypes Amino Acid Position Variation 28 34 39 46 68 Classical, 569B Type 1 D H H F T El Tor, Australia Type 2 D H H L T El Tor, N16961 Type 3 D H Y F I V. cholerae O139, (2005) Type 4 D H Y F T V. cholerae O139, (2002) Type 5 A H H F T V. cholerae O139, (2005) Type 6 D P Y F T
V. cholerae O1 biotype El Tor, causative organism of the ongoing seventh pandemic, produces CT of the genotype 3, while the classical biotype produces CT belongs of genotype 1. The CT genotype of the El Tor strains currently associated with cholera in the Indian subcontinent has shifted from genotype 3 to genotype 1. Thus, the current circulating El Tor strains causing cholera have characteristics of El Tor biotype but possess classical CTB. Considering the chronology of evolution in ctxB genotypes, type1 of CTB was the first reported genotype in classical V. cholerae. The phage that encoded this CTB genotype was found integrated in host chromosomes but unable to replicate independent of the host, while the El Tor type phage replicated in the host to produce functional phage. V. cholerae belonging to classical biotype are known to cause more severe cholera, while El Tor biotype strains survive better in aquatic environments. The results of this study thus indicated that there is decrease in binding efficiency of El Tor CTB compared to the classical cholera toxin, among various carbohydrate ligands. The data generated suggests that new ctxB genotypes of V. cholerae O139, similar to 174
classical CTB were more profound in potential to bind carbohydrate ligands. Recent variations in CTB employed by the microbe indicate that V. cholerae produce a potent toxin more proficient in hydrogen bonding, and thus in forming stable complexes with respect to seventh pandemic clone of biotype El Tor. This could be a reason to worry because current strains of V. cholerae O1 and O139 are host to CTX prophages that had acquired capabilities of independent replication and infection and can lead to rapid spread of severe cholera. Table 2. Results obtained using SDM software for CTB variants in comparison to crystal structure of type 1 CTB. Genotypes Position variations % age solvent accessibi-lity Overall differentat difference at position of tial free energy of mutation unfolding 2 46 (Phe - Leu) + 0.7 0.786 Kcal/mol 3 39 (His - Tyr), 68 (Thr - Ile) 39 by - 7.9 and 68 by + 28.9 3.040 Kcal/mol 4 39 (His - Tyr) - 4.7 1.82 Kcal/mol 5 28 (Asp Ala) - 9.1 2.055 Kcal/mol 6 34 (His Pro, 39 (His Tyr) 34 by + 21.8 and 39 by 4.7 3.429 Kcal/mol
Figure 1: Molecular interactions of CTB variants with lignd galactose
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CONCLUSION
The rate of change in the genetic profile of toxigenic V. cholerae has been a reason for concern due to their ability to cause epidemic and pandemic cholera. Of the various markers used to monitor genetic changes, variations in CTB have been studied of late. In this study, we explored the possibility of variable binding efficiency of CTB variants to constituent carbohydrates of GM1 ganglioside, an important step in causing severe cholera through molecular and computational approaches. The results of this study indicate subtle variations in the binding abilities among variants of CTB with the carbohydrates ligands constituent of GM1 receptor, with respect to competence in hydrogen bonding. Though there had been no phenotypic differences among strains harbouring CTB variants, the data provides clues to potential molecular discrepancies in binding of CTB to its receptor. These differences can be employed in designing paratopes that can determine specificity of either a precise biotype or a standard anticholera toxin monoclonal antibody. As CTB is an attractive drug candidate for autoimmune diseases as well as differentiation therapy, the data presented could be exploited for designing drugs or vaccines.
ACKNOWLEDGEMENTS
This work in part was supported by the funds contributed by the Department of Biotechnology, New Delhi, to the Institute of Life Sciences. Senior Research Fellowships awarded by the Indian Council of Medical Research, New Delhi, India and Institute of Life Sciences, Bhubaneswar, to M.H.U. Turabe Fazil are gratefully acknowledged.
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1. Waldor, M.K., Mekalanos, J.J. (1996). Lysogenic conversion by a filamentous phage encoding cholera toxin. Science, 272:1910-1914. 2. Nusrin, S., Khan, G.Y., Bhuiyan, N.A., Ansaruzzaman, M., Hossain, M.A., Safa, A., Khan, R., Faruque, S.M., Sack, D.A., Hamabata, T., Takeda, Y., Nair, G.B. (2004). Diverse CTX phages among toxigenic Vibrio cholerae O1 and O139 strains isolated between 1994 and 2002 in an area where cholera is endemic in Bangladesh. Journal of Clinical Microbiology, 42,5854-5856.
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Davis, B.M., Moyer, K.E., Boyd, E.F., Waldor, M.K. (2000). CTX prophages in classical biotype Vibrio cholerae: functional phage genes but dysfunctional phage genomes. Journal of Bacteriology, 182, 6992-6998.
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Boyd, E.F., Heilpern, A.J., Waldor, M.K. (2000). Molecular analyses of a putative CTX precursor and evidence for independent acquisition of distinct CTXs by toxigenic Vibrio cholerae. Journal o f Bacteriology, 182, 55305538.
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Ledon, T., Campos, J., Suzarte, E., Rodriguez, B., Marrero, K., Fando, R. (2008). El Tor and Calcutta CTX precursors coexisting with intact CTX copies in Vibrio cholerae O139 isolates. Research in Microbiology, 159, 81-87.
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Spangler, B.D. (1992). Structure and function of cholera toxin and the related Escherichia coli heat-labile enterotoxin. Microbiology Review 56,622-647.
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Jertborn, M., Nordstrom, I., Kilander, A., Czerkinsky, C., Holmgren, J. (2001). Local and systemic immune responses to rectal administration of recombinant cholera toxin B subunit in humans. Infection Immunity, 69, 4125-4128.
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Bhuiyan, N.A., Nusrin, S., Alam, M., Morita, M., Watanabe, H., Ramamurthy, T., Cravioto, A., Nair, G.B. (2009). Changing genotypes of cholera toxin (CT) of Vibrio cholerae O139 in Bangladesh and description of three new CT genotypes. FEMS Immunology and Medical Microbiology, 57,136-141.
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Turabe Fazil, M.H.U., Bhanumathi, R., Pandey, H.P., Singh, D.V. (2011). Characterization of Vibrio cholerae O139 belonging to multiple ribotypes and isolated from diarrhoeal patients in Kerala, southern India. Infection Genetics and Evolution, 11, 454-459
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Jobling, M.G., Holmes, R.K. (2002). Mutational analysis of ganglioside GM (1)-binding ability, pentamer formation, and epitopes of cholera toxin B (CTB) subunits and CTB/heatlabile enterotoxin B subunit chimeras. Infection and Immunity, 70, 1260-1271.
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Mantri, C.K., Mohapatra, S.S., Singh, D.V. (2010). Effect of storage and sodium chloride on excision of CTX or pre- CTX and CTX from Vibrio cholerae O139 strains. Infection Genetics and Evolution, 10, 925-930.
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Chapter IX STRUCTURE-BASED DRUG DESIGN APPROACHES IN DRUG DISCOVERY Pramod K. Yadav1, Satendra Singh1, Chandrabhan Seniya2, Atul K. Singh2, G. J. Khan3
1 2
Department of Computational Biology & Bioinformatics, SHIATS, Allahabad-211007, India.

3
Department of Biotechnology, Madhav Institute of Technology & Science Gwalior, M.P., India Department of Biotechnology, L.N.Mithila University, Darbhanga-846008, Bihar, India Corresponding Author: Pramod K. Yadav, E-mail:
ABSTRACT: The traditional method of discovering drugs has always been a tedious process, consuming several years, huge man power and millions of dollars in order to come up with a single effective novel drug. Computer-aided drug design (CADD) represents more recent applications of computers as tools in the drug design process. CADD uses a variety of computational methods to identify novel compounds, design compounds for selectivity, efficacy and safety, and develop compounds into clinical trial candidates. Most common strategies used in drug designing are structure or target based drug designing (SBDD) and ligand or analogue based drug designing (LBDD). The approach used in CADD is dependent upon the amount of information that is available about the ligand and receptor. In structure based drug design approach one should have reliable 3-dimensional structural information for the receptor or the ligand-receptor complex which is available, as from X-ray diffraction, NMR or homology modeling techniques. The ligand-based approach is applicable when the structure of the receptor site is unknown, but when a series of compounds have been identified that produce the activity of interest. Structure-based ligand or inhibitor design aims to identify chemical compounds that bind strongly to key regions of biologically relevant molecules, e.g. enzymes or receptors and should be able to inhibit or stimulate the biological activity of their target molecules. The current methods for structure-based drug design are virtual screening and de novo ligand designing. Virtual screening is performed by docking method in which ligand databases are screened for a target. In de novo ligand design method, new ligand molecules are built up within the constraint of active site of the target. SBDD is a powerful method and it is an integral part of drug discovery today.
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1. INTRODUCTION Evidences for the use of medicines and drugs can be traced back to as far as the first Egyptian dynasty during 3100 B.C. Majority of the time when drugs have been used; their discovery has always been a relied on trial-and-error method testing of chemical substances on cultured cells or animals, and matching the apparent effects to treatments. It was not until the 1960's that some understanding began to develop about the quantitative relationship between the structure of a drug and its biological activity. The understanding about this quantitative relationship heralded in a new era in drug discovery process in the form of computer aided drug design. During the early 1980s, the ability to rationally design drugs using protein structures was an unrealized goal for many structural biologists. The first projects were underway in the mid80s, and by the early 1990s the success stories started pouring in. Today, even though there is still quite a bit of fine-tuning necessary to perfect the process, structure-based drug design is an integral part of most industrial drug discovery programs and is the major subject of research for many academic laboratories. The conventional way of synthesizing drugs has always been a tedious process, consuming several years, huge man power and millions of dollars in order to come up with a single effective novel drug. However, with the advent of information technology, it can be done efficiently in silico, thereby saving huge funds and man power. The pipeline of drug discovery from idea to market consists of seven basic steps: disease selection, target selection, lead compound identification, lead optimization, preclinical trials, clinical trial testing and pharmacogenomic optimization [1]. (Figure 1). 2. COMPUTER AIDED DRUG DESIGN Computer-aided drug design (CADD), also called computer-aided molecular design (CAMD), and represents more recent applications of computers as tools in the drug design process. Sometimes CADD is interchangeably used as rational drug design (RDD). It is a process used in the pharmaceutical industry to discover and develop new drug compounds. RDD uses a variety of computational methods to identify novel compounds, design compounds for selectivity, efficacy and safety, and develop compounds into clinical trial candidates. These methods fall into several categories structure-based drug design, ligand-based drug design, de novo design and homology modeling depending on how much information is available about drug targets and potential drug compounds. CADD approaches have contributed to the 179
successful discovery of novel numerous enzyme inhibitors, including inhibitors of thymidylate synthase, HIV-1 Protease and purine nucleoside phosphorylase inhibitors. In each case, CADD was used to predict the binding affinity of an inhibitor designed from a lead compound prior to synthesis [2]. Disease selection
Target selection
Lead compound identification
Lead optimization
Pre-clinical trials
Clinical trial testing
Pharmacogenomic optimization Figure 1: Overview of drug discovery process
In most current applications of CADD, attempts are made to find a ligand (the putative drug) that will interact favorably with a receptor that represents the target site. Binding of ligand 180
to the receptor may include hydrophobic, electrostatic, and hydrogen-bonding interactions. This approach to CADD optimizes the fit of a ligand in a receptor site. To be used most effectively, one should have structurally similar compounds with high activity, with no activity, and with a range of intermediate activities. In recognition site mapping, an attempt is made to identify a pharmacophore, which is a template derived from the structures of these compounds. It is represented as a collection of functional groups in three-dimensional space that is complementary to the geometry of the receptor site. In applying this approach, conformational analysis will be required, the extent of which will be dependent on the flexibility of the compounds under investigation. One strategy is to find the lowest energy conformers of the most rigid compounds and superimpose them. Conformational searching on the more flexible compounds is then done while applying distance constraints derived from the structures of the more rigid compounds. Ultimately, all of the structures are superimposed to generate the pharmacophore. This template may then be used to develop new compounds with functional groups in the desired positions. In applying this strategy, one must recognize that one is assuming that it is the minimum energy conformers that will bind most favorably in the receptor site. In fact, there is no a priori reason to exclude higher energy conformers as the source of activity. The approach used in CADD is dependent upon the amount of information that is available about the ligand and receptor. Ideally, one would have 3-dimensional structural information for the receptor and the ligand-receptor complex from X-ray diffraction or NMR. The ideal is seldom realized. In the opposite extreme, one may have no experimental data to assist in building models of the ligand and receptor, in which case computational methods must be applied without the constraints that the experimental data would provide. Based on the information that is available, one can apply either ligand-based or receptor-based molecular design methods [3]. The ligand-based approach is applicable when the structure of the receptor site is unknown, but when a series of compounds have been identified that produce the activity of interest. The receptor-based approach to CADD applies when a reliable model of the receptor site is available, as from X-ray diffraction, NMR, or homology modeling. With the availability of the receptor site, the problem is to design ligands that will interact favorably at the active site, which is accomplished by docking procedure [4].
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Drugs can be designed computationally by using two strategies: 1. Ligand or analogue based drug designing (LBDD). 2. Structure or target based drug designing (SBDD). 2.1 Analogue or Ligand based drug designing (LBDD): Many receptors are not readily amenable to receptor-based drug design. For example, many important receptors are membranebound proteins (transmembrane proteins), which are very difficult to crystallize. In such cases, a lead compound or active ligand must be found, and then the structure of the ligand guides the ligand design process. Ligand-based design techniques use information about one or several known active compounds (ligands) as a basis for the designing of lead compounds. Once we have an active compound, we can begin to refine the biological activity. The methods of this approach are applied when the three dimensional structure of macromolecule-target is unknown, and there is difficult to design its reliable model. These methods are based on analysis of sets of ligands with known biological activity [5]. They include: design of pharmacophore models, analysis of quantitative structure-activity relationship (classic QSAR) [6] and its modification 3D-QSAR (which takes into account spatial structure of compounds), quantitative structure properties relationship (QSPR) etc. The classical QSAR is effective for development of close analogues of known compounds [7]. Pharmacophore model represents a set of points in space with the certain properties and distances between them, which define binding of given group of ligands with target.
2.2 Structure or target based drug designing (SBDD): Structure-based ligand or inhibitor
design aims to identify chemical compounds or peptides that bind strongly to key regions of biologically relevant molecules, e.g. enzymes or receptors, for which three-dimensional structures are known. Designed compounds should be able to inhibit or stimulate the biological activity of their target molecules. The rapid progress of the human genome project is providing an ever-increasing number of potential protein drug targets. Together with advances in structural determination techniques such as nuclear magnetic resonance, crystallography and even homology modeling, structure-based design of ligands or inhibitors has emerged as an important tool in drug discovery and pharmaceutical research [8]. We will focus on structure-based drug design in this chapter and describe a few of its salient features.
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3. The Process of Structure-Based Drug Design: The process of structure-based drug design is an iterative one and often proceeds through multiple cycles before an optimized lead goes into phase I clinical trials. The first cycle includes the cloning, purification and structure determination of the target protein or nucleic acid by one of three principal methods: X-ray crystallography, NMR, or homology modeling. Using computer algorithms, compounds or fragments of compounds from a database are positioned into a selected region of the structure. These compounds are scored and ranked based on their steric and electrostatic interactions with the target site and the best compounds are tested with biochemical assays. In the second cycle, structure determination of the target in complex with a promising lead from the first cycle, one with at least a micromolar inhibition in vitro, reveals sites on the compound that can be optimized to increase potency. Additional cycles include synthesis of the optimized lead, structure determination of the new target-lead complex, and further optimization of the lead compound. After several cycles of the drug design process, the optimized compounds usually show marked improvement in binding and, often, specificity for the target. (i) Selection of a drug target The choice of a drug target is primarily made on a pharmacological and biochemical basis. The ideal target macromolecule for structure-based drug design is one that is closely linked to human disease and binds a small molecule in order to carry out a physiological function. The target molecule usually has a well-defined binding pocket or active site. (ii) Evaluating a target for structure-based drug design Once a target has been identified, it is necessary to obtain accurate structural information. There are three primary methods for structure determination that are useful for drug design: Xray crystallography, NMR, and homology modeling. Crystal structures are the most common source of structural information for drug design, since structures determined to high resolution may be available, and the method is useful for proteins that range in size from a few amino acids to 998 kDa. Typically, crystal structures determined with data extending to beyond 2.5 are acceptable for drug design purposes [9]. (iii) Active site identification in the target Structure-based design begins with the identification of a potential ligand binding site (active site) on the target molecule. Ideally, the target site is a pocket or protuberance with a 183
variety of potential hydrogen bond donors and acceptors, hydrophobic characteristics, and sizes of molecular surfaces. The ligand binding site can be the active site, as in an enzyme, an assembly site with another macromolecule, or a communication site necessary in the mechanism of the molecule. The basic inputs for this step are the 3D structure of the protein and a pre-docked ligand in PDB format, as well as their atomic properties. Both ligand and protein atoms need to be classified and their atomic properties should be defined, basically, into four atomic types: (a) hydrophobic atom: all carbons in hydrocarbon chains or in aromatic groups. (b) H-bond donor: Oxygen and nitrogen atoms bonded to hydrogen atom(s). (c) H-bond acceptor: Oxygen and sp2 or sp hybridized nitrogen atoms with lone electron pair(s). (d) Polar atom: Oxygen and nitrogen atoms that are neither H-bond donor nor H-bond acceptor, sulfur, phosphorus, halogen, metal and carbon atoms bonded to heteroatom(s). All aspects thought to be responsible for binding affinity and selectivity is collected. This knowledge is then exploited to create new ideas on ways to improve existing ligands or to develop new alternative bonding skeletons for a target [10]. (iv) Drug Design Methods Once the three dimensional structure of the target is evaluated and the active site in the structure is identified, there are several paths to developing a good lead based on structure of the target. Current methods for structure-based drug design can be divided roughly into two categories. 1. Virtual Screening 2. De novo ligand designing The first category is about finding ligands for a given receptor, which is usually referred as database searching [11]. In this case, a large number of molecules are screened to find those fitting the binding pocket (active site) of the receptor. Some researchers call this virtual screening in analogy to the bioassay screening procedure employed in the traditional drug discovery process. The earliest programs for performing 3D database searching are DOCK [12], AUTODOCK [13] which are freely available for non-commercial use to researchers. 184
Another category of structure-based drug design methods is about building ligands, which is usually referred as de novo design. In this case, ligand molecules are built up within the constraints of the binding pocket by assembling small pieces in a stepwise manner. These pieces can be either atoms or fragments. The earliest de novo design method began with GRID [14] and many groups have been actively involved in this field since then. Current popular de novo design programs include GROW [14], LUDI [15], LIGBUILDER [16], LEAPFROG [17]. SPROUT [18], PROLIGAND [19] etc. These techniques are raising much enthusiasm to the drug design community. 3.1 Docking: Molecular docking is a study of how two or more molecular structures, for example drug and enzyme or receptor, fit or interact together in 3D space (Figure 2). In the first steps of a drug design project the goal is to find a so-called lead structure, a small molecule which binds to a given target protein and can be further developed to a drug. If the threedimensional structure of the target protein is known, docking algorithm can be applied to virtually search for potential leads. The two main questions arising are what the complex between a protein and a potential leads looks like and how strong the binding affinity of the lead is with respect to other candidates [20]. Searching and scoring are the two components that typically make up a computational docking. Usually a docking method can be classified by the way it handles the receptor and ligand and by the type of search algorithm that is applied [21]. Searching refers to the fact that any docking method must explore the accessible configuration space of the interacting molecules. Goal is to find the orientation and conformation of the binding partners corresponding to the minimum of free energy of binding. Full unrestricted degrees of freedom of translation and rotation are not permissible because they present a high number of potential solutions resulting in need of giant computational resources. Two search approaches can be classified: geometric or combinatorial and stochastic or energy driven
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Active site of receptor
Ligand
Figure 2: A ligand (WR99210) docked in the active site of receptor (PFDHFR-TS)
All configurations generated during a search process need to be evaluated and ranked. Scoring [22] functions achieve this, which ranks the searching results and selects out the best binding geometry based on the energies of the of the complexes or, more theoretical value, Gbind , the binding free energy difference between the bound and unbound states of the ligand and protein [23]. Preferred would be the actual free energy as a score. This is not readily available for computation, so scoring functions must approximate binding free energies with sufficient accuracy. Usually knowledge of interaction energies and forces flows into an assigned score. Scoring functions can be classified as either force field based or empirical based on a weighted sum of interactions. Structure-based drug design attempts to use the structure of proteins as a basis for designing new ligands by applying accepted principles of molecular recognition. The basic assumption underlying structure-based drug design is that a good ligand molecule should bind tightly to its target. Thus, one of the most important principles for designing or obtaining potential new ligands is to predict the binding affinity of a certain ligand to its target and use it as
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a criterion for selection. A historical research was done to develop a general-purposed empirical function in order to describe the binding energy.
The concept of the Master Equation was raised. The basic idea is that the overall binding free energy can be decomposed into independent components which are known to be important for the binding process. Each component reflects a certain kind of free energy alteration during the binding process between a ligand and its target receptor. The Master Equation is the linear combination of these components. According to Gibbs free energy equation, the relation between dissociation equilibrium constant, Kd and the components of free energy alternation was built. The sub models of empirical functions differ due to the consideration of researchers. It has long been a scientific challenge to design the sub models. Depending on the modification of them, the empirical scoring function is improved and continuously consummated [24, 25 and 26]. Docking method may be of three types: protein-ligand docking, protein-protein docking and protein-DNA docking which exclusively depend upon the types of macromolecules or small molecules chosen for the study of molecular interaction. Two approaches are often used for the docking studies. 3.1.1 Rigid body docking In rigid body docking, the bond angles, bond lengths and torsion angles of the components are not modified at any stage of complex generation. In other words flexibility is not provided to the interacting molecules, it is also called as lock and key model. A subject of speculation is whether or not rigid-body docking is sufficiently good for most docking. With respect to computational costs, full rigid docking is invincible. The target can be represented via a pre-computed grid, while the ligand is regarded as a rigid body, i.e. the ligand has only 6 degrees of freedom. However, sampling all orientations of the ligand versus the target with
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exhaustive search algorithms demands considerable computational effort. This approach is particularly useful in the case of virtual screening of large number of compounds to a target. 3.1.2 Flexible docking Docking procedure in which conformational change or flexibility allowed in interacting molecules is called as flexible docking, it is also called as induced-fit model. However, scoring all possible conformational changes is computationally expensive and procedures must intelligently select small subset of possible conformational changes for consideration. It has been shown that protein and ligand flexibility is crucial for binding. The induced-fit mechanism proposes that the protein has to undergo usually minor but however considerable changes to bind with the ligand. The same is applied for the ligand. Allowing flexibility in both the receptor and ligand molecules in the computational model virtually increases the conformational search space. This approach of docking gives more reliable docked complexes and is widely employed by majority of docking programs. 3.1.3 Tools for docking Variety of tools are available for docking studies which are given below: i. AutoDock: AutoDock is a suite of automated docking tools. It is designed to predict how small molecules, such as substrates or drug candidates, bind to a receptor of known 3D structure. ii. DOCK: The DOCK suite of programs is designed to find favorable orientations of a ligand in a receptor. It uses matching algorithm for rigid body docking and an algorithm for flexible ligand docking, have been added over the years. iii. GOLD: GOLD is a program for calculating the docking modes of small molecules in protein sites and is provided as part of the GOLD suite. It uses genetic algorithm (GA) for protein-ligand docking and full ligand flexibility is provided. iv. FlexX: FlexX is a fast computer program for predicting protein-ligand interactions two main applications: complex prediction (create and rank a series of possible protein-ligand complexes) and virtual screening. v. HEX: HEX is a protein docking and molecular superposition program. It uses spherical polar Fourier correlations to accelerate docking calculations.
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vi. FT Dock (Fourier Transform Dock): FTDock performs rigid-body docking on two biomolecules in order to predict their correct binding geometry outputs multiple predictions that can be screened using biochemical information. vii. GLIDE: GLIDE is a high-throughput ligand-receptor docking program for fast library screening. Program identifies the best binding mode through Monte Carlo sampling and provides an accurate scoring function for ranking of binding affinities. viii. FlexiDock: FlexiDock is a simple, flexible docking of ligands into binding sites on proteins. It uses fast genetic algorithm for generation of configurations. ix. PatchDock: PatchDock is an automatic server used for molecular docking. Its algorithm is based on shape complementarity principles. The input is two molecules of any type: proteins, DNA, peptides, drugs. The output is a list of potential complexes sorted by shape complementarity criteria. 3.2 Virtual Screening: Virtual screening uses computer-based methods to discover new ligands on the basis of biological structures [11]. Virtual (database) screening (VS) is an increasingly important component of the computer-based search for novel lead compounds. There are, fundamentally, two approaches to the general problem: VS by docking, which requires knowledge of the 3D structure of the target protein binding site to prioritize compounds by their likelihood to bind to the protein; and similarity-based VS, where no information on the protein is necessary instead, one or more compounds that are known to bind to the protein are used as a structural query (Figure 3). The screening procedure extracts compounds from the database according to an appropriate similarity criterion. In order for the screening procedure to be effective, this criterion should regard molecules that bind tightly to the same proteins as similar [27]. Drug design using molecular modeling techniques involve screening a very large number of ligand records or molecules of compounds in a chemical database (CDB) to identify those that are potential drugs. This process is called molecular docking. It helps researchers to predict how small molecules, such as substrates or drug candidates, bind to an enzyme or a protein receptor of known threedimensional (3D) structure. Docking each molecule in the target chemical database is both a compute and data intensive task [28].
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Modelled structure
XRay/NMR Structure
Known active sites
Rank Hits
Commercial databases
Virtual screening
In-house database
In silico Visual selection of Ligands Figure 3: Overview of virtual screening process 3.2.1 ZINC - Database of Commercially Available Compounds for Virtual Screening: ZINC is a library of very huge number molecules, each with 3D structure, using catalogs of compounds from vendors (the size of this library continues to grow). The molecules have been assigned biologically relevant protonation states and are annotated with properties such as molecular weight, calculated LogP, and number of rotatable bonds. Each molecule in the library contains vendor and purchasing information and is ready for docking. Within certain limits, the molecules are prepared in multiple protonation states and multiple tautomeric forms. In one format, multiple conformations are available for the molecules. This database is available for free download (http://zinc.docking.org) in several common file formats including SMILES, mol2, 3D SDF, and DOCK flexibase format. A Web-based query tool incorporating a molecular drawing interface enables the database to be searched and browsed and subsets to be created. Users can process their own molecules by uploading them to a server [29]. Acquire and screen
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3.3 De novo drug Designing: In de novo drug design method, ligand molecules are built up within the constraints of the binding pocket by assembling small pieces in a stepwise manner. These pieces can be either atoms or fragments. The key advantage of such a method is that novel structures, not contained in any database, can be suggested.
De novo Ligand design Methods
Active Site Analysis Methods
Whole molecule Methods
Connection Methods
Sequential Build-up Methods
Fragment Connectio n Methods
Site Point Method
Random connectio n Methods
Figure 4: Overview of De novo Ligand Design Methods
As shown in Figure 4, de novo ligand design methods fall into a few different categories, depending on the approach used: 1. Site-point connection methods: In this method desirable locations of individual atoms which play crucial role in the active site of the target (site points) are determined and then suitable fragments at those locations are placed. 2. Fragment connection methods: This is started with the previously positioned fragments and then Linkers or scaffolds are determined to connect those fragments without moving them. 3. Sequential buildup methods: In this method a new ligand is constructed within the constraint of active site using the atom-by-atom or fragment-by-fragment buildup approach. The set of building block is generally small, and the construction process may be random.
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4. Random connection methods: A special class of techniques combining some of the features of site point, fragment connection, and sequential buildup methods, along with bond-disconnection strategies and other methods to introduce randomness. Among all four methods described above, sequential buildup and fragment connection methods are frequently used in de novo ligand designing. There are two strategies to build up ligand molecules: one is growing strategy while the other is linking strategy. GROW and LINK is designed to carry out them, respectively. The concept of these two strategies is illustrated in Figure 5. With the growing strategy, the building-up process starts from a seed structure that has been pre-placed in the binding pocket. The user can assign certaingrowing sites on the seed structure and then the program will try to replace each growing site with a candidate fragment. The newly formed structure will serve as the seed structure for the next growing cycle. With the linking strategy, the building-up process also starts from a pre-placed seed structure. However, in this case the seed structure consists of several separated pieces that have been positioned to maximize the interactions with the target protein. The growing of fragments happens simultaneously on each piece and the program will always try to link these pieces in an acceptable way. This process continues until all the pieces have been integrated into one molecule [16]. Future Prospects of Structure-based drug design Structure-based drug design is a powerful method, especially when used as a tool within an armamentarium, for discovering new drug leads against important targets. After a target and a structure of that target are chosen, new leads can be designed from chemical principles or chosen from a subset of small molecules that scored well when docked in silico against the target. After a preliminary assessment of bioavailability, the candidate leads continue in an iterative process of re-entering structural determination and reevaluation for optimization. Focused libraries of synthesized compounds based on the structure-based lead can create a very small promising lead which can continue to phase-I clinical trials. As structural genomics, bioinformatics, and computational power continue to explode with new advances, further successes in structurebased drug design are likely to follow. Each year, new targets are being identified, structures of those targets are being determined at an amazing rate, and our capability to capture a quantitative picture of the interactions between macromolecules and ligands is accelerating.
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Structure-based drug design is an integral part of drug discovery today. While it can't be said that the approach is full proof and sufficient to getting a drug to market, no single technology can claim that ability. The reason behind this is that SBDD is in fact a collection of technologies that includes molecular biology, computational chemistry and bioinformatics. After designing the new drug molecules, ultimately we will have to go to wet laboratory experiments to verify the pharmacological properties of designed drugs. Above all, the approach hinges on people working together in interdisciplinary teams to attack a disease target through customdesign of a new drug. It is clear that targeted strategies to find new drugs are a successful approach that is saving lives every moment. As more drugs derived from structure-based drug design come to market, many more success stories will be waiting to be told.
Figure 5: Growing strategy (the left) and linking strategy (the right) for building up ligands
REFERENCES
1. Augen, J. (2004). Bioinformatics in the Post-Genomic Era: Genome, Transcriptome, Proteome, and Information-Based Medicine. Addison-Wesley Edition: 1st edition, 388pp. 2. Reddy, M.R. and Erion, M.D. (2005). Computer Aided Drug Design: strategies Used in the Discovery of Fructose 1, 6-Bisphosphatase Inhibitors, Current Pharmaceutical Design, 11, 283-294. 193
3. Anderson, A.C. (2003). The process of structure-based drug design. Chemistry & Biology, 10, 787797. 4. Bevan, D.R. (1997). QSAR and drug design, Network Science.
http://www.netsci.org/Science/Compchem/feature12.html.
5. Veselovsky, A.V. and Ivanov, A.S. (2003). Strategy of Computer-Aided Drug Design, Current Drug Targets - Infectious Disorders, 3, 33-40. 6. Kubinyi, H. (1994). Variable Selection in QSAR Studies I. An Evolutionary Algorithm, Quantitative Structure-Activity Relationships, 13, 285-294. 7. Fujita, T. (1997). Recent success stories leading to commercializable bioactive compounds with the aid of traditional QSAR procedures. Quantitative Structure-Activity Relationships, 16, 107-112. 8. Zeng, Jun. (2000). Computational Structure-Based Design of Inhibitors that Target Protein Surfaces, Combinatorial Chemistry and High Throughput Screening, 3, 355-362. 9. Nissen, P., Hansen, J., Ban, N., Moore, P. and Steitz, T. (2000). The structural basis of ribosome activity in peptide bond synthesis. Science, 289, 920930. 10. Gerhard, Klebe (2000). Recent developments in structure-based drug design, Journal of Molecular Medicine, 78,269281. 11. Meng, E.C., Shoichet, B.K. and Kuntz, I.D. (1992). Automated Docking with Grid-Based Energy Evaluation, Journal of Computational Chemistry, 13, 505-524. 12. Schoichet, B. K. and Kuntz, I. D. (1993). Matching chemistry and shape in molecular docking, Protein Engineering, 6, 723-732. 13. Morris, G.M., David, S.G., Robert, S.H., Ruth, H., William, E.H., Richard, K.B., Arthur, J.O. (1998). Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function, Journal of Computational Chemistry, 19, 16391662. 14. Goodford, P.J. (1985). A computational procedure for determining energe- tically favorable binding sites on biologically important macromolecules, Journal of Medical Chemistry, 28, 849- 857. 15. Bohm, H. J. 1994). "The development of a simple empirical scoring function to estimate the binding constant for a protein-ligand complex of known three-dimensional
structure". Journal of Computer Aided Molecular Design, 8, 24356. 16. Wang, R., Gao, Y. and Lai, L. (2000). LigBuilder: A Multi-Purpose Program Based Drug Design, Journal of Molecular Modeling 6, 498 516. 194
for Structure-
17. Cramer, R. D., DePriest, S. (1996). Implemented in the SYBYL program, Tripos Associates, St.Louis, MO, USA. 18. Gillet, V. J., Newell, W., Mata, P. (1994). Sprout: recent developments in the de novo design of molecules Journal of. Chemical Information and Computer Science, 34, 207-217. 19. lark, D. E., Frenkel, D., Levy, S. A. (1995). PROLIGAND: an approach to de novo molecular design. Apllication to the design of organic molecules. Journal of .Computer-Aided Molecular Design, 9, 13-32. 20. Rarey, Matthias (2002). Protein- ligand docking in drug design. In: Thomas Lengauer (ed.) Bioinformatics- from genomes to drugs, Vol I (Wiley-VCH) 7: 315-318. 21. Wilke, Christoph (2004). Molecular conformation and ligand- receptor docking, Seminar: Algorithms in drug design, WSI Computer Science Department. 22. Kuntz, I.D., Meng, E.C. and Shoichet, B.K. (1994). Receptor-Based Molecular Design, Accounts of Chemical Research, 27, 117-123. 23. Tao, P. and Lai, L. (2001). Protein ligand docking based on empirical method for binding affinity estimation, Journal of computer-aided molecular design, 15, 429-426. 24. Gohlke, H., Hendlich, M., Klebe, G. (2000). Knowledge-based scoring function to predict protein-ligand interactions. Journal of Molecular Biology, 295, 33756. 25. Clark, R.D., Strizhev, A., Leonard, J.M., Blake, J.F., Matthew, J.B. (2002). Consensus scoring for ligand/protein interactions. Journal of Molecular Graphics and Modeling, 20, 28195. 26. Wang, R., Lai, L., Wang, S. (2002). Further development and validation of empirical scoring functions for structure-based binding affinity prediction. Journal of Computer Aided Molecular Design, 16, 1126. 27. Lengauer, T., Lemmen, C., Rarey, M. and Zimmermann, M. (2004). Reviews, Novel technologies for virtual screening, Drug discovery today, 9,1- 27. 28. Buyya, R., Branson, K., Giddy, J. and Abramson, D. (2003). The Virtual Laboratory: a toolset to enable distributed molecular modeling for drug design on the World-Wide Grid, Concurrency and Computation: Practice and Experience, 15,125. 29. Irwin, J.J. and Shoichet, B.K. (2005). ZINC A Free Database of commercially available Compounds for Virtual Screening, Journal of Chemical Information and Modeling, 45, 177182. 195
Chapter X
NITROGEN ACQUISITION IN CYANOBACTERIA

1
Pradeep kumar L and 2Vani B*
1,2
Birla Institute of Technology and Science, Pilani, Rajasthan (India)
Corresponding Author: Vani B., Assistant Professor, E.mail: bvani70@gmail.com
ABSTRACT: Cyanobacteria are photoautotrophic prokaryotes and constitute a large taxonomic

group within the domain of eubacteria. Different species of cyanobacteria utilize / assimilate nitrogen available to them in different forms. Based on their nitrogen acquisition ability, they are divided as N2-fixing and non N2-fixing. Some of the filamentous species are subdivided into those with and without a heterocyst which is a differentiation from vegetative cells for fixing nitrogen. The aim of this chapter is to highlight facts and ideas on the physiology of nitrogen utilization, which have not received much attention in the past. Keywords: Nitrogen fixation, Cyanobacteria, nif
INTRODUCTION
Cyanobacteria belong to the oldest group of organisms on earth [1]. They are photosynthetic prokaryotes that resemble eukaryotic green algae, making them classified as "blue green algae". They are found in very diverse habitat, from oceans to fresh water to bare rock to soil. They also provide an extraordinarily wide-ranging input to human daily life [2] and are of economic importance [3]. Further, they are important primary producers and their nutritive value is very high. They contribute globally to soil and water fertility [4]. The optimal growth of a cyanobacterial species depends on the availability of nitrogen. Nitrogen (N) can represent as much as about 11% of the dry weight of a cyanobacterial cell [5]. Cyanobacteria use numerous nitrogen containing compounds as sources of nitrogen. Nitrogen fixing cyanobacteria fixes atmospheric nitrogen to cope up with nitrogen depletion. The non N2-fixing group can use nitrate, nitrite, ammonium, organic compounds like urea, amino acids, and some nitrogencontaining bases as nitrogen source (Fig. 1). Cyanobacteria shows preference over some nitrogen sources than other, such that more easily assimilated source of nitrogen is available to the cells. 196
This phenomenon was called nitrogen control [6]. Nitrogen acquisition in all these organisms consume photosynthetically generated ATP and reducing power (ferredoxin) [6].
Figure 1: Core pathways of nitrogen assimilation [6]. Nitrogenase - protein chemistry Cyanobacterial (di) nitrogen fixation is catalyzed by the enzyme complex nitrogenase. They are the best studied organism on the subject, because several of them, can be easily genetically modified by molecular techniques. The ammonium production in the process is defined with the equation [7]:
Cyanobacterial nitrogenase consists of two components, an iron protein (Fe-protein) and an iron-molybdenum protein (MoFe-protein). The Fe-protein homodimer is composed of a single Fe4S4 cluster bound between identical 3240 kDa subunits. The Fe4S4 cluster is redox-active
[8], capable of obtaining more than two oxidative states [9] and transfers electrons to the MoFeprotein [10]. The MoFe-protein is a 22 heterotetramer of 250 kDa. Each 22 dimer of the larger nitrogenase protein binds one FeMo cofactor and one P cluster. The P cluster may have an 197
N2 fixation specific role forcing reversibly bound N2 to the irreversible reduction pathway [11]. The FeMo cofactor consists of 1 Mo atom, 7 Fe atoms, 9 S atoms, and homocitrate, plus an asyet-unidentified light atom (or ion) at its center [7]. The FeMo cofactor is the site of substrate binding and reduction [11]. Nitrogenases are more susceptible to O2, as O2 reversibly damages Fe4S4 cluster and P cluster. In cyanobacteria, the O2 problem is enhanced by the photosynthetic evolution of this gas [7].
Figure 2: Schematics representation of different adaptations enabling nitrogen fixation in cyanobacteria and its light and dark regulation [17]. Reproduced with permission of Elsevier Adaptation in N2 fixation: heterocyst differentiation Some of the cyanobacterial genera undergo cell differentiation, called the heterocysts (Fig. 2), as a means to protect their nitrogenases against atmospheric O2 [12,13]. During aerobic growth conditions, their vegetative cells perform photosynthetic O2 evolution, whereas nitrogenase resides in heterocyst. These differentiate from vegetative cells by cell division and extensive metabolic changes, including Photosystem II (PSII) degradation, so that they cannot perform the photosynthesis [14,15]. In turn, heterocysts provide nitrogen as glutamine formed via N2 fixation and glutamine synthetase/glutamate synthase (GS/GOGAT) [16]. Residual O2, if reaches the inside of the heterocysts, gets immediately consumed by high respiratory activity and
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also other reactions in the heterocysts. Thus heterocysts provide an anaerobic environment, perfect enough for nitrogenase to function. . In Anabaena species heterocyst formation takes about 24 h of deprivation. Heterocyst formation is complex process controlled by many genes [15]. HetR, a serine-type protease, and NtcA, a transcription factor, are found to be the key regulators of the process [18, 19, 20]. Nitrogen deprivation upregulates the expression of hetR and the expression is regulated by NtcA is the global nitrogen regulator [21]. NtcA belongs to the catabolite activator protein (CAP) family. In cyanobacteria, it activates the transcription of heterocyst formation genes in response to nitrogen step-down [22]. NtcA comprises of a helix-turn-helix motif at its C-terminal end, which binds to NtcA-dependent promoter. NtcA is the main 2-oxoglutarate (2-OG) sensor [22], controlling heterocyst formation. 2-OG senses C-N balances in cyanobacteria. The reason for it is explained by the fact that, unlike higher plants, cyanobacteria lacks 2-oxoglutarate dehydrogenase [24, 25] leading to NADP1-isocitrate dehydrogenase catalyzed formation and accumulation of 2-OG [26, 27]. 2-OG thus formed, is used by GS-GOGAT pathway as the carbon skeleton for the incorporation of ammonium [28, 20, 29]. The expression of the icd gene encoding isocitrate dehydrogenase was found to be highest under nitrogen stress in Synechocystis sp. strain PCC 6803 [30]. Its principal role as a carbon skeleton for ammonium incorporation would make 2-OG, an ideal sensor of the C-N balances [31]. Other than NtcA, signal protein PII is also involved in regulation of heterocyst formation[32]. NtcA and PII mediated regulation in nitrogen acquisition is detailed in the later part of this chapter. N2 fixation through temporal separation Without cell differentiation, organisms like Plectonema boryanum has evolved to function in low oxygen environments to cope up with O2 problem in nitrogen fixation (Fig. 2). The organism has nitrogenase in all cells in equal amounts [33]. Plectonema is unable to fix nitrogen when grown anaerobically. However, they need oxygen for respiratory processes that are a crucial for ATP and reductant [34].Thus the organism fix nitrogen under microaerobic conditions [35]. Under these conditions, Plectonema temporally separates oxygenic photosynthesis and nitrogen fixation [34]. They temporally separate transcriptional regulation 199
nitrogenase and photosynthetic genes [36]. Phormidium, and some species of Pseudoanabaena also undergo such temporal separation as that of Plectonema [9]. Nevertheless, some of the non-heterocystous cyanobacteria can undergo temporal separation without the need for a microaerobic environment. Symploca, a filamentous cyanobacteria [37] and the unicellular Gloeothece and Cyanothece belongs to the same category of N2-fixers [38, 39]. They fix nitrogen at night and nitrogenase is typically found in all cells. In these organisms high nitrogenase activity coincides with high respiration rates, maintaining a phase difference of 12 h from the peak of photosynthetic activity. Such pattern is also reflected at the transcriptional level, and observed under either continuous light or darkness. Thus, these organism have adapted a circadian control to cope up with C:N balance [40, 41]. Further, some non-heterocystous species of cyanobacteria can fix nitrogen during the day time (Fig. 2). The mechanism of protection from photosynthetically evolved oxygen in Trichodesmium, involve compartmentalization of nitrogenase in a fraction of the cells. These cells are arranged consecutively along the trichome [42] with active photosynthetic components [43, 44]. Thus, spatial aggregation of nitrogenase into these cells is not sufficient to protect against photosynthetic oxygen evolution. Hence, a significant fraction of nitrogenase would be inhibited by O2. Different research groups have recently explained this complex phenomenon. In Trichodesmium, protection against oxygen was found to involve a complex interaction between spatial and temporal segregation [42, 43]. A circadian clock controls the transcription of nitrogenase and the expression of photosynthetic genes essential to the activation of photosystems I and II (PSII), with a temporal phase difference of ~6 h [45]. This enables the cells to turn photosynthetic activity on or off, without damaging the photosynthetic machinery [17]. Genetic structure of nif gene In general nitrogen fixation genes have two basic structural components a) genes encoding for nitrogenase reductase (nifH), alpha and beta subunits of nitrogenase (nifDK) and b) genes encoding the two subunits of the NifNE protein complex involved in biosynthesis of the FeMo-cofactor [46, 47]. Non-heterocystous cyanobacteria (both unicellular and filamentous) have a contiguous nifHDK operon [48, 49]. On the contrary, in heterocystous species the 200
structural nif operon of most is interrupted by a large transposon [51]. This transposon gets removed during the heterocyst formation. This process is well established in Anabaena. In vegetative cells of Anabaena, the nifD gene is interrupted by an 11 000 base pair DNA element. During heterocyst differentiation of this element is excised by site-specific recombination. The excision leads to the restoration of the nifD coding sequence and of the entire nifHDK transcription unit [52]. The products of nifN and nifE function as a 200-kDa 22 tetramer (NIFNE) that has been purified, partially characterized, and found to contain what appears to be a single 4Fe-4S center [53]. Interestingly, these genes nifN and nifE exhibit striking similarity to nifK and nifD [54]. Nitrogen acquisition from other sources of nitrogen The loss of cyanobacterial nif genes was also an adaptation strategy existed early, where some organisms were able to adapt to an oxic world, while others were not (Fig. 2) (e.g., Oscillatoria). Nitrate assimilation is nitrogen acquisition for most of these non-nitrogen fixing cyanobacteria [55] It enters the cells by an active transport system and gets sequentially reduced to ammonium by the action of nitrate reductase (NarB) and nitrite reductase (NirA) [56]. Fig. 3 represents the nitrate assimilation system as found in some of these fresh-water cyanobacteria. Unlike N2-fixing group, nitrate uptake and reduction to ammonia in these organisms, uses the photosynthetically generated assimilatory power namely, ATP and reduced ferredoxin, required for nitrate reduction [57, 58]. The structural elements of nitrate uptake and assimilation include [58]: ABC-type permease Ferredoxinnitrate reductase (FdNar) Ferredoxinnitrite reductase (FdNir) ATP-binding cassette (ABC)-type uptake transporter Combined nitrogen sources are taken up through permeases, gets metabolized to ammonium, and incorporated into carbon skeletons through the GOGAT pathway. The cyanobacterial ATPbinding cassette (ABC)-type permeases are involved in nitrate uptake [6]. They are composed of a periplasmic substrate-binding protein that is anchored to the membrane, two transmembrane subunits and two ATPase subunits that are located in the cytoplasmic side of the membrane. Two 201
cytoplasmic subunits of ABC-type uptake transporter powers the transport reaction, which are of very high conservation in sequence throughout cyanobacterial genera [58]. They were first identified in Synechococcus elongatus as an ammonium-repressible protein of about 48 kDa, abundant in the cytoplasmic membranes [59-62]. Spirulina platensis genes encoding elements of ABC-type uptake permease are arranged in a operon (NrtA-B-C-D) (Fig. 3) [63, 64]. They couple ATP hydrolysis to the transport of solutes across cell membranes. ATP binding and hydrolysis induce conformational changes in the membrane spanning domains of the permease, which further mediate the transport process [65]. NrtA is a periplasmic substrate-binding lipoprotein that can bind both nitrate and nitrite. NrtA is of about 440 amino acids that is anchored to the cytoplasmic membrane [66]. NrtB bears six transmembrane segments, which is highly hydrophobic protein of about 280 amino acids [67]. NrtD, a protein of about 275 amino acids, represents a conserved component of these permeases [58].
Figure 3: The nitrate assimilation system as found in fresh-water cyanobacteria. Ferredoxinnitrate reductase Once inside the cell, nirate is reduced to nitrite by nitrate reductase (NR). Cyanobacterial nitrate reductases catalyzes the 2-electron reduction of nitrate to nitrite, which is the first step in the nitrate assimilation. They are monomer products of narB gene of 80 kDa, containing a [4Fe 4S] cluster and a Mo-cofactor [68; 69]. They are partially associated with the thylakoid membrane and uses photosynthetically reduced ferredoxin as physiological electron donor [70 202
71]. The complex between NR and negatively charged residues on ferredoxin, is electrostatically stabilized by the interaction of lysine and arginine residues of NR [72]. Ferredoxinnitrite reductase Cyanobacterial nitrite reductases participate in 6-electrons reduction of nitrite to ammonium. Their molecular weight is between 5256 kDa and are monomers products of nir gene, with prosthetic groups including a [4Fe4S] cluster and a siroheme [58]. The electron donors for the enzyme are the photosynthetically reduced ferredoxin or flavodoxin [71]. The interaction of nitrite reductase with ferredoxin seems to be stabilized by electrostatic forces. Two regions in nitrite reductase that are rich in positively-charged amino acid residues are actively involved in regulating its interaction with ferredoxin [73].
Figure 4: Cycle involving ammonium incorporation using 2-OG carbon skeleton, through the GOGAT cycle. Glutamine synthetase-glutamate synthase: The final step completing nitrate assimilation is incorporation resulting ammonium into amino acids by the GS/GOGAT pathway. The process consumes ATP and reducing power and 2-OG, is used up as the final N acceptor (Fig. 4) [74]. However, cyanobacteria can take ammonium from external medium as well. The glnA gene encoding a typical type I glutamine synthetase have been cloned and characterized from numerous cyanobacteria [75]. From Plectonema, genes encoding a ferredoxin-dependent (glsF) and an NADH-dependent (gltB and gltD) glutamate synthase were cloned [76]. Further, gene inactivation studies have revealed that Fd-GOGAT directly utilizes photoreducing power for the catalytic reaction, thus necessary for maintaining nitrogen and carbon equlibrium [76]. Incidentally, any hindrance in the generation of reductant via the photolysis of water and the reduction of the plastoquinone pool or imbalance 203
in the utilization of such reductants for carbon assimilation or by nitrate assimilation causes physiological stress in vivo. Gene cluster involved in nitrate assimilation In both nitrogen fixing and non-nitrogen fixing cyanobacteria, nitrate assimilation genes are commonly found in an operon [77]. The Synechocystis sp. nitrate assimilation operon bears the following genes: nir encoding nitrite reductase [78, 79]; nrtA, nrtB, nrtC and nrtD, encoding a nitrate/nitrite transporter (Fig. 5) [64, 80, 81]; and narB, encoding nitrate reductase (Fig. 5) [64- 65]. Anabaena sp. strain PCC 7120 also posses a similar gene cluster for nitrate assimilation operon [83-84]. This gene cluster is conserved throughout cyanobacteria genera. It ensures production of a balanced amount of the different proteins of the nitrate assimilation system [84]. The expression of the operon is induced in the absence of ammonia [79, 85, 86]. When the cells perceive a high C to N ratio, this operon is transcribed from a complex promoter that includes binding sites for NtcA, a global nitrogen-control regulator [22].
Figure 5: Map of the nrt operon of the genome of Synechocystis sp. NtcA and PII meditaed regulation of gene expression NtcA is a dimer of two monomeric unit (~222 amino-acid) present in almost all cyanobacteria [77, 87]. The recently determined crystal structure of NtcA has given an insight into its mode of action [87]. The transcriptional activity of NtcA is regulated by binding of an effector molecule (2-OG) to the N-terminal effector binding domain (EBD). The 2-OG binds to the EBD at a pocket similar to that used by cAMP in catabolite activator protein, but with a different pattern [87]. When 2-OG bind to EBD, the binding affinity of NtcA towards NtcA promoter increases [22]. Structural analysis have revealed that a tighter coiled-coil conformation of the two C-helices of NtcA, induced by 2-OG maintains the proper distance between the two Fhelices for DNA recognition [87]. NtcA activates expression of the nir operon, as well as of other genes involved in nitrogen assimilation [88 -90]. NtcA mediated regulation of nitrogen control depend on modifications of both enzyme activity and gene expression [77].
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The next level of control in nitrogen assimilation in cyanobacteria is mediated by signal transduction protein, PII [91]. They are yet another central molecule for perception and signalling of the cellular nitrogen status, recognizing ATP and 2-OG [91-94]. ATP and 2-OG controls the reactivity of PII towards various targets [95-96]. Phosphorylation at ser49 in response to the cellular nitrogen and carbon supply is the key factor determining its activity (Fig. 6). Elevation in 2-OG levels (discussed earlier in this chapter) signals this phosphorylation [97-98]. Dephosphorylation of PII-P depends on a protein phosphatase, PphA, which is highly sensitive to 2-OG (even in submillimolar range)[92, 99]. Presence of ammonium initiates dephosphorylation, (Fig. 6) whereas, presence of a nitrogen source induce medial PII phosphorylation, which are modulated by the inorganic carbon supply to the cells. Nitrogen starvation induces highest levels of PII phosphorylation (Fig. 6) [92-100].
Figure 6: PII phosphorylation cycle in response to cellular 2-oxoglutarate levels [92]. Reproduced with permission of John Willey & Sons. PII signalling mediates the NtcA-activated gene expression under conditions of nitrogen starvation [101-102]. However, other direct targets of interaction with PII are still to be revelaed. N-acetyl-L-glutamate kinase (NAGK) was recently identified as one of the target of PII signalling [91, 103]. NAGK, the key enzyme in arginine biosynthesis, forms a tight complex with nonphosphorylated PII, enhancing the catalytic activity of this enzyme [103-104].
205
CONCLUSION
Cyanobacteria are important primary producers and their nutritive value is very high. They contribute globally to soil and water fertility. Nitrogen (N) can represent as much as about 11% of the dry weight of a cyanobacterial cell. Cyanobacteria use numerous nitrogen containing compounds as sources of nitrogen. Nitrogen fixing cyanobacteria fixes atmospheric nitrogen to cope up with nitrogen depletion The non N2-fixing group can use nitrate, nitrite, ammonium, organic compounds like urea, amino acids, and some nitrogen-containing bases as nitrogen source. N2 fixation is catalyzed by the enzyme complex nitrogenase. Some of the cyanobacterial genera undergo cell differentiation, called the heterocysts, as a means to protect their nitrogenases against atmospheric O2 Heterocyst formation is complex process controlled by many genes. HetR, a serine-type protease, and NtcA, a transcription factor are found to be the key regulators of the process. 2-OG senses C-N balances in cyanobacteria. Its principal role as a carbon skeleton for ammonium incorporation makes 2-OG, an ideal sensor of the C-N balances. Other than NtcA, signal protein PII is also involved in regulation of heterocyst formation Some cyanobacteria temporally separate transcriptional regulation of nitrogenase and photosynthetic genes Unlike N2-fixing group, nitrate uptake and reduction to ammonia in these organisms, uses the photosynthetically generated assimilatory power namely, ATP and reduced ferredoxin, required for nitrate reduction . 2-OG, is used up as the final N acceptor. NtcA activates expression of the nir operon, as well as of other genes involved in nitrogen assimilation. NtcA mediated regulation of nitrogen control depend on modifications of both enzyme activity and gene expression PII signalling mediates the NtcA-activated gene expression under conditions of nitrogen starvation. However, other direct targets of interaction with PII are still to be revelaed. Nacetyl-L-glutamate kinase (NAGK) was recently identified as one of the target of PII signalling . NAGK, the key enzyme in arginine biosynthesis, forms a tight complex with nonphosphorylated PII, enhancing the catalytic activity of this enzyme. We therefore conclude with the confidence that nitrogen acquisition in cyanobacteria as a research topic is still alive and well, and promises to yield new insights for many years to come.
ACKNOWLEDGEMENT: The work done in the authors laboratory was supported by grant
from the Department of Science and Technology (DST), New Delhi, India. LPK thanks UGC for a fellowship. 206
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Chapter XI
PLASTIDS IN Plasmodium SIGNIFICANCE IN MALARIA TREATMENT *Vishal Saxena and Shilpi Garg Center for Biotechnology, Department of Biological Sciences, Birla Institute of Technology & Science, Pilani Corresponding author: Vishal Saxena, Email: vishalsaxena12@yahoo.com
ABSTRACT :
Malaria remains one of the most serious infectious diseases in the world,
inflicting acute illness on more than 300 million people and leading to more than two million deaths annually. Human malaria caused by Plasmodium falciparum and P. vivax accounts for most (upto 85%) of the malaria infections all over the world including Sub-Saharan Africa, Southeast Asia and Latin America. Over 108 countries are affected by this disease over decades but there is no permanent cure available for the disease. The situation has become more complicated with the increasing reports on drug resistance in the parasite. A plastid like organelle present in the Plasmodium species found to be indispensable for the survival of the parasite is being looked upon as a putative drug target. The organelle harbors a circular genome similar in constitution to that of chloroplast DNA. However, the functional characterization of the genes identified in the circular genome of plastid still remains hypothetical. Four metabolic pathways specific to prokaryotes are also reported in this organelle and are considered important for the survival of the parasite. The enzymes functional in these pathways are encoded by the nuclear genome of the parasite and are considered as putative drug targets. Thus, a detailed insight is required to understand and decipher the functions of the circular genome and the metabolic pathways. This chapter thus focuses on the origin of this organelle in the Plasmodium species and related genus, its importance in the survival of the parasite, the circular genome and the metabolic pathways of the plastid.
INTRODUCTION
Malaria is probably one of the oldest diseases known to mankind. Deadly fever-probably malaria has been recorded since the beginning of the written word (6000-5500 B.C.). The symptoms of 217
malaria were described in ancient Chinese medical writings. In 2700 B.C., several characteristic symptoms of what would later be named as malaria were described in the Nei Ching, The Canon of Medicine. Details of this disease can be found even in the ancient Indian medical literature like Charaka Samhita and Sushrutha, which even associated these fevers with the bites of the Mosquitoes. However, it was only in the 5th century B.C., that malaria was clinically recognized and described by Hippocrates [1]. He classified the fever types as quotidian (daily), tertian (alternate days) and quartan (fever three days a part). It was only at the turn of this century; Laveran and Ross discovered that the disease was caused by a protozoan parasite transmitted by anopheline mosquitoes. Significant control of the disease was accomplished in the 1930s and 1940s due to the advent of synthetic antimalarial drugs and modern insecticides. The rise in incidence of drug resistant parasites and insecticide resistant vectors, respectively, has however been responsible for resurgence in malaria cases. Prevalence of Malaria Malaria is caused by species of the apicomplexan parasites Plasmodium. Four species of this protozoan parasite are known to cause human malaria viz., P. falciparum, P. vivax, P. malariae and P ovale. Recent reports detail few cases of human malaria by P. knowlesi (a primate parasite). The disease is transmitted to humans by species of female Anopheles mosquito. Malaria is one of the worlds most common and serious tropical diseases. Half of the worlds population is at risk for malaria, which is endemic (where a constant, measurable number of new cases and natural transmission occurs over time) in over 108 countries. Children are at particular risk, accounting for most malaria deaths globally. Although preventable and treatable, malaria causes significant morbidity and mortality, particularly in resource-poor regions. Sub-Saharan Africa is the hardest hit region in the world, and parts of Asia and Latin America also face significant malaria epidemics [2]. Among the four species, P. falciparum and P.vivax are observed worldwide in tropical and some temperate zones. The P.ovale is mainly localized in tropical West Africa where as P. malariae has a very patchy distribution worldwide. P.vivax accounts for over half of all malaria infections outside Africa and an estimated 75 million acute episodes per year in Africa [3] and has been traditionally considered to be the dominant species in India. However, data from intensive studies undertaken in 1930 [4] show an almost equal prevalence of P. vivax and P. falciparum on the basis of microscopic examination
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of blood smears. After the control and eradication programmes of the 1950s and 1960s the number of cases of both species went down considerably. P. falciparum is known to cause most dangerous of malaria and has the highest rates of complications and mortality. As of 2006, it accounted for almost 90% of the deaths. It is more prevalent in sub-Saharan Africa than in other regions of the world; in most African countries, more than 75% of cases were due to P. falciparum. It is well known in causing the varying degrees of severity of malaria with the most complicated form of the disease often termed Severe Malaria. Severe manifestations including organ dysfunction are well documented in P. falciparum malaria. However, recent reports have also clearly established the occurrence of severe manifestations in P. vivax malaria. The clinical data from the patients infected with P. vivax in the studies done in Bikaner, India and some other parts of the world has strongly indicated that P. vivax can cause both sequestration-related and non sequestration related complications of severe malaria, including cerebral malaria, renal failure, circulatory collapse, severe anemia, hemoglobinurea, abnormal bleeding, acute respiratory distress syndrome (ARDS), and jaundice, all of which are commonly associated with P. falciparum infections [57]. The manifestations of severe malaria include (World Health Organisation, 2010): Cerebral malaria, with abnormal behavior, impairment of consciousness, seizures, coma, or other neurological abnormalities Clinical jaundice plus evidence of other vital organ dysfunction Hemoglobinuria (hemoglobin in the urine) due to hemolysis Pulmonary edema (fluid buildup in the lungs) or Acute Respiratory Distress Syndrome, which may occur even after the parasite counts have decreased in response to treatment Abnormalities in blood coagulation and thrombocytopenia (decrease in blood platelets) Cardiovascular collapse and shock
Other manifestations that should raise concern are: Acute kidney failure
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Hyperparasitemia, where more than 5% of the red blood cells are infected by malaria parasites
Metabolic acidosis (excessive acidity in the blood and tissue fluids i.e plasma bicarbonate <15mmol/l ), often in association with hypoglycemia
Hypoglycemia (blood glucose < 2.2mmol/l or <40mg/dl). Hypoglycaemia may also occur in pregnant women with uncomplicated malaria, or after treatment with quinine.
Severe malaria occurs most often in persons who have no immunity to malaria or whose immunity has decreased. These include all residents of areas with low or no malaria transmission, and young children and pregnant women in areas with high transmission. Life Cycle of Plasmodium The life cycle of Plasmodium is carried in two stages in different organisms, one in humans acting as host for asexual stage and second in mosquito acting as vector and site for sexual stage (Figure 1). Various species of anopheline mosquitoes form definitive hosts for the malaria parasites. A female mosquito may ingest male and female gametocytes along with the blood meal. The male gametocyte matures inside the mosquito resulting in the production of a number of slender spindle shaped microgametes. Simultaneously, the female gametocyte matures to form a macrogamete, which on fertilization forms a zygote. The zygote elongates and becomes active forming an ookinete, which penetrates through the stomach wall and forms an oocyst. The oocyst after further maturation produces a large number of slender threads like sporozoites. Some of these enter the mosquito salivary glands and may be inoculated into the blood stream of the intermediate host (man) during the next meal. The sporozoites injected into the blood stream leave the blood vascular system and invade the parenchymal cells of the liver within a very short time. In all the Plasmodium sp. infecting man, asexual multiplication occurs in the liver, resulting in the production of thousands of merozoites. To elaborate further, there are two sub-stages of parasite division during its asexual life cycle, viz. exo-erythrocytic (when sporozoites infect liver cells, develop into schizonts and further into merozoites which infect red blood cells) and erythrocytic stages (includes (a) ring (the earliest stage after initial infection of red blood cells), (b) tropohozoite, (c) schizont (the stage at which the parasite begins to segment into multiple daughter parasites) and 220
(d) merozoite (4-36, formed with in 48-72 hrs and travel through the plasma from the spent blood cell and invades a new host erythrocyte) stage. These are released into the circulation and infect the erythrocytes in the blood. A few of these merozoites may subsequently form male and female gametocytes. Rupture of the RBC releases products of metabolism of the parasites as well as the cells into circulation. It is believed that if a large number of infected cells rupture simultaneously, the volume of toxic material thrown into the blood stream may be sufficient to bring about a malarial paroxysm.
Figure 1: Schematic representation of the life cycle of malaria parasite Chemotherapy for Malaria The fight against malaria began some 100 years back. During this period, various drug molecules have been formulated in way of trials to combat malaria. Antimalarials containing quinoline components are the most effective drugs for malaria chemotherapy. This group of compounds 221
has evolved from the structural modification of quinine (originally extracted from Cinchona tree bark but now also chemically synthesized, was discovered as first anti malarial in 17th century but in use from very old times) and includes 4-aminoquinoline compounds such as chloroquine and mefloquine of which former is more effective, cheap, safe and commonly available drug. The second line drugs include dihydrofolate reductase inhibitors like proguanil, chloroproguanil, pyrimethamine and trimethoprime and sulfa drugs like dapsone, sulfalene, sulfamethoxazole and sulfadoxine. These drugs are normally used in combinations. Tetracycline and its derivatives such as doxycycline are very potent antimalarials and are used for both treatment and prophylaxis. The other useful antimalarials are Artemisinin compounds synthesized from the plant Artemisia annua. These compounds (artesunate, artemether, arteether) are most effective antimalarials and seem to have effect on protein synthesis by the malaria parasite. These are used in combination therapy for the treatment of severe malaria and have shown very rapid parasite clearance in comparison to quinine compounds [8]. However, sadly the cheapest and most effective first line chemotherapies like chloroquine, pyrimethamine, etc. used to fight malaria for decades are now losing efficacy due to emergence of drug resistance in the parasite population. This has led to the resurgence of the disease, with malaria mortality rates redoubling in many areas. Even continuous trials to develop a successful vaccine against malaria have been impeded due to the antigenic variations shown by the parasite. Clearly, there is a need for new antimalarials or identification and exploitation of new therapeutic targets. Many of the more exciting new targets to be revealed by the P. falciparum genome project are enzymes from the Apicoplast a plastid (or chloroplast) that is a legacy of the malaria parasites distant non-photosynthetic ancestry. This cyanobacterial heritage of the apicoplast means that many of its bacteria-like enzymes are fundamentally different from the mammalian host equivalents, making them potential drug targets [9]. Apicoplast A three four membrane organelle was reported some 40 years back in the genus Plasmodium, a member of Apicomplexan family of parasites. The organelle was termed Apicoplast (apicoplast), due to its presence in apicomplexans and similarity to the plastids as indicated by various workers in the past based upon presence of plastidic genome and various enzymes characteristic of plastids. The plastid in Plasmodium is believed to have a red algal origin and is postulated to have appeared in the parasites due to secondary endosymbiosis (Figure 2) [10,11]. 222
The latest hypothesis on the origin of Apicoplast has proposed that a single endocytobiotic event approximately 1300 million years ago, initiated the formation of monophyletic group of chromalveolate protists including the cryptophytes, haptophytes, heterokonts and apicomplexan parasites. This generally well-supported argument proposes that a red algal cell engulfed by an ancestral alveolate protist was enslaved after the transfer of many of its genes to the host cell nucleus and their subsequent inheritance. Following these ancient secondary endosymbiotic events in the red and green lineages, various algal structures and functions became incorporated into chimeric meta-algal cells that underwent considerable diversification as they evolved. A nucleomorph, the remnant of a red algal nucleus still persists in cryptophytes whereas chlorarachniophytes have a green algal version (Figure 3). In apicomplexans, the visible remains of the secondarily acquired algal cell are restricted apparently to a multi-membraned plastid organelle. This is no longer photosynthetic and is bereft of much of its former genic content. Some apicomplexans (Cryptosporidia) may even have lost the plastid organelle altogether, although a store of laterally transferred genes from the algal symbiont has yet to be discounted in such cases [11].
Figure 2: Hypothetical evolutionary scheme outlining sequential endocytobioses carrying primary and secondary plastids. 223
Figure 3: Schematic representation of various stages of hypothetical endosymbiotic events leading to formation of apicomplexans. The discovery of apicoplast is related with the discovery of its circular DNA. In the initial findings, the plastid-like DNA (Figure 4) was first noted in P. knowlesi as a low-density minor satellite in CsCl gradients. Similar low density satellites were also reported in two rodent malaria parasites, P. berghei and P. chabaudi [12]. Kilejian was the first person to see the plastid DNA circles in extracts of isolated mitochondria of the avian parasite P. lophurae. They had a contour length of 10.3 mm (31 kb) and a buoyant density linking them to the previously noted satellites. She understandably assumed them to be of mitochondrial origin [13]. Some similar studies following this in P. berghei and Toxoplasma gondii (another Apicomplexan) stated this circular DNA to be of mitochondrial origin but also raised concern on the large cruciform structures seen in the electron micrographs of the DNA [12, 14]. The circular molecules were first isolated in usable quantity from the simian parasite P. knowlesi [15]. They were 11.5 mm in contour length and showed a cruciform structure just like that of Toxoplasma. Gardner and co workers isolated the counterpart from the human parasite P. falciparum with an estimated size of 35 kb and substantiated the organellar origin of the molecules by obtaining a sequence corresponding to a fragment of a prokaryotic small-subunit (SSU) ribosomal RNA gene [16-18]. Various genes and open reading frames similar to the genes of red and blue-green algae were found in the 35 kb circular DNA. These genes helped 224
researchers to relate the prokaryotic origin of the organelle and pointed to its plastid like provenance [19]. The Plastid DNA Plastid genomes are similar to those of their bacterial progenitors in that they are super coiled DNA circles. At 2735 kb, the apicoplast genome is the smallest known plastid genome, but it appears to have retained a circular, supercoiled architecture. The identity and arrangement of the genes on the 35-kb circle overwhelmingly favor its evolution from a plastid genome. There are about eight to fifteen copies of 35-kb circles per cell in P. falciparum. In other apicomplexans like Toxoplasma gondii, the DNA molecule number goes upto 25 per cell. The P. falciparum plastid DNA circle is packed with over 60 genes. About onethird of the circle includes inverted repeat regions, IRA and IRB. These include tRNA and ribosomal RNA (both small and large) genes. The remaining twothird circle is a single copy region which includes ribosomal protein genes, 7 unidentified ORFs and RNA polymerase genes. There are some specific genes encoding elongation factor EfTu, caseinolytic protease ClpC, and SufB proteins. The genes are usually separated by only a few nucleotides and in some cases even have small overlaps. As compared to plastid DNA from their counterparts in prokaryotes, the Plasmodium plastid DNA has high A+T content (~82-86%) [20,12]. An outline of genes associated to the circular plastid genome in P. falciparum is depicted in Figure 4. Inverted Repeats The inverted repeat (IR) has a total length of approximately 10.5 kb and encodes duplicated genes for the small subunit (SSU) [17] and large-subunit (LSU) rRNAs [18] and nine different tRNAs [21, 22]. Both forms of the rRNA genes are flanked by tRNA genes for met [M], arg [R], val [V], arg [R], leu [L], asp [N], ala [A], Iso [I] and Thr [T]. The order of this IR is conserved in Toxoplasma gondii (another apicomplexan) while it differs from other known plastids. It is thus suggested that it may be an ancestral apicomplexan character derived from numerous rearrangements and deletions in an ancient progenitor of the apicomplexan clade [12].
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Figure 4: An outline of the gene map of the circular DNA of Plasmodium falciparum. The two halves (A and B) of the inverted repeat (IR) are indicated. sufB, clpC and tufA genes are denoted. The details of inverted repeats, RNA polymerase genes and ribosomal protein genes are given in the text. 1,2,3,4 denote 7 unidentified ORFs of varying lengths [20]. The Inverted Repeat regions can be divided into two parts IR-A and IR-B based upon the gene positions. Immediately downstream of the IR-A region encoded on the same DNA strand as the LSU rRNA and tRNA lay ORF470/ SufB which is understood to be a homologue gene encoding SufB protein essential for the growth of cyanobacteria Synechocystis species. It is proposed that the gene might be functioning as an aid in iron metabolism in the apicoplast [23]. Downstream to SufB/ORF470 are two unassigned ORFs (ORF101 and ORF51). The above three ORFs are followed by three larger genes of an RNA polymerase similar to that found in cyanobacteria and chloroplasts and typically not in mitochondria. They are designated as rpoB, rpoC1, and rpoC2, which encode subunits , , and , respectively. Their recognition was one of the first clues to the plastid ancestry of the circle [19, 24]. Another plastid-like feature of the diminutive malarial circular genome is the subdivision of the E. coli rpoC-like gene into two genes, rpoC1 and rpoC2. The rpoB gene is the closest to the plastid sequence of the protist Euglena gracilis while based on some phylogenetic analysis it seems to have diverged considerably from other plastid sequences [12]. 226
Immediately downstream to the IR-B region on the same DNA strand as of rRNA and tRNA genes is present a ribosomal protein gene rps4, which shares the same first three codons as ORF470/ SufB at the other end of the IR-A. These three codons mark the end of repeat regions on both sides. This is followed by a cluster of 10 tRNA genes (his [H], cys [C], leu [L], met [M], tyr [Y], ser [S], asp [D], lys [K], glu [E] and pro [P]). The leucine gene holds the only intron so far recognized on the circle, located, as with other plastid homologs, within the anticodon. Similar to other plastid genomes, a series of ORFs for ribosomal proteins are present downstream to the tRNA genes. The ribosomal proteins are followed by a tufA gene, which encodes the elongation factor Tu (EF-Tu), a G-protein crucial in the elongation step of protein synthesis, similar to other plastid genomes. It is notable that tufA occurs on the plastid genome of many algae but not on that of higher plants. Downstream of tufA, another tRNA genes tRNA[F] lies distinctly on the complementary strand. This is followed by three tRNA genes, gln (Q), gly (G), and trp (W), a short tentative ORF, ORF129, and finally a identified as clpC, a member of the hsp100 gene family; these genes encode ubiquitous heat shock or stress proteins that act as molecular chaperones with diverse functions ( For more details on this genome readers are requested to refer to [12, 20, 23]). Apicoplast Division The division of apicoplast occurs in coordination with the division stages of the parasite. This process has been extensively detailed in apicomplexans like T. gondii and P. falciparum. There appears an intimate linkage between the P. falciparum nuclear division and segregation of parasitic stages and the plastid division. The single apicoplast is crescent shaped during the ring stage (as discussed before in life cycle) of parasites, rounds up into a sphere in early trophozoites and grows in size. During schizont stage, the apicoplast starts forming a reticulate, branched structure, which divides into as many plastids as there are daughter cells in the maturing schizont. After red blood cell rupture, every merozoite contains one copy of slightly elongated plastid ([25]. Thus, as a thumb rule like all the other organelles, one plastid is present in each parasite daughter cell. The apicoplast follows maternal inheritance whereby only those merozoites which develop into female gametocytes carry the copy of apicoplast with them. The readers are suggested to study the reports by Waller et al., 2000 where they have depicted the morphology of plastid division with the help of Green fluorescent protein targeted to different stages of 227
apicoplast division. It has also been observed that the division of apicoplast is immediately followed by division of nucleus and throughout its life cycle in parasite the plastid remains mysteriously attached to the mitochondria. This has been proven by the recent plastid isolation studies whereby it was shown that plastid alone can not be isolated from the parasite but is always accompanied by the mitochondria [26-28]. Apicoplast Functions The importance of the apicoplast has been an ongoing issue of debate since its discovery. The organelle is considered indispensable for the survival of the parasite. The fact has been proven by various experimental data. Parasites die after treatment with drugs that interrupt apicoplast genome replication, transcription or translation [29,30]. Moreover, mutant parasites lacking an apicoplast are not viable [31]. Immediately after apicoplast disruption (either pharmacological or genetic), parasites continue to grow normally in the host cell, but subsequently arrest and die after infecting a new host cell. Presumably, whatever the apicoplast provides for the parasite is crucial for a viable infection process. This could be a component of the parasitophorous vacuole, which surrounds parasites in the host cell, or perhaps a resource that is usually replenished at the time of host-cell invasion [32-34, 9]. The functions of the genes present in the apicoplast genome have been only detailed based upon homology of P. falciparum sequences to orthologues identified from different bacterial databases. The plastid genome sequences from other Plasmodium species are only countable and have been studied to understand the evolutionary position of the parasites. The only gene that has been studied in detail from another human malaria parasite P. vivax is of tufA gene [35] where the scientists have detailed the structural and functional aspects of the Ef-Tu A protein encoded by tufA. Apicoplast is the site for metabolic pathways that are specific to the parasite and are not found in its vertebrate host. All the pathways are of prokaryotic origin, due to this fact the plastid is also considered as putative drug target. A number of enzymes present in these pathways are proven to have drug binding sites in the equivalent enzymes present in plant, algae and bacterial systems. Some of these drugs have also been shown to effect the parasite survival (Table 1) [36]. The enzymes involved in these pathways are encoded by the nuclear genome and are targeted
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from the nucleus to the apicoplast. This transport of proteins takes place by a twofold mechanism and includes a secretory pathway [9]. The apicoplast targeted proteins are tagged at the N terminal with a bipartite leader sequence. The N-terminus starts with a typical hydrophobic signal peptide, while the remainder of the N-terminal extension represents a plastid transit peptide. Deletion of just the transit peptide causes proteins, that now only contained an N-terminal signal peptide, to be secreted from the cell into the parasitophorous vacuole [25, 37] while removal of the signal peptide alone lead to accumulation of the protein in the cytosol [25].The signal peptides are believed to mediate traffic across the outermost membrane of the apicoplast, while the transit peptides mediate traffic across the inner two membranes. One of the four major metabolic pathways for which the apicoplast has been convincingly shown as the site is the de novo Type II fatty acid biosynthesis (FASII) pathway. The discovery of the pathway may be attributed to the identification of nuclear genes which encode for enzymes of this pathway and are targeted to apicoplast. These include b-ketoacyl-ACP synthase I/II (FabB/F) and b-hydroxyacyl-ACP dehydratase (FabZ) enoyl-ACP reductase (FabI). The
pathway includes conversion of acetyl-CoA to malonyl-CoA by acetyl CoA carboxylase (ACCase) which includes addition of malonyl moiety to the acyl chain in repeated rounds of condensation, reduction, dehydration and reduction reactions to form a 10 16 or upto 18 carbon chain long compounds. Initial drug inhibition studies indicated to the presence of this FASII pathway in the blood stage parasites. However, recent reports suggest that FASII pathway is only essential in the late liver stages of the parasite [25, 38 43]. Apart from the much studied FASII pathway, the parasitic plastid also houses isoprenoid biosynthesis [44, 45] pathway. Isoprenoids are diversified group of natural products such as sterols, carotenoids and terpenoids. Isopentenyl diphosphate (IPP), the precursor of isoprenoids can be synthesized in cells by two different types of pathways i.e. Mevalonate dependent pathway and the Non-mevalonate or Deoxyxylulose phosphate (DOXP) pathway. The former is characteristic in fungi, animals, certain bacteria and protozoa while the later one is usually found in plants, algae (within their plastids) and many bacteria. The antibiotic/ herbicide fosmidomycin (and its derivatives) have been shown to block the DOXP pathway by targeting DOXP reductoisomerase in certain rodent Plasmodium species and in P.falciparum as well.
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Table 1: Drugs acting on plastid targets and affecting Plasmodium parasite growth and survival Drug/ herbicide Putative target in plastid Ciprofloxacin Plastid DNA type II topoisomerase Rifampicin Plastid RNA polymerase bsubunit Clindamycin, Erythromycin, Azithromycin, Thiostrpton, Micrococcin, Chloramphenicol Doxycyclin, Tetracyclin Thiolactomycin b-ketoacyl-ACPsynthase III (fabH) Fatty acid biosynthesis Plastid 16S rRNA Protein translation Plastid 23S rRNA Protein translation RNA transcription Metabolic Activity affected DNA Replication
Apicoplast also appears to harbour a ferredoxin-based electron transport system [46] which is characteristic of plant plastids, both photosynthetically active and inactive. As per analogy, the electrons derived from splitting of water are transferred back to ferrodoxin by cofactor NADPH, in non-photosynthetic plastids. The reduced ferrodoxin may serve as reductant for various plastid specific processes. This complete ferrodoxin based electron transport system is yet to be proved in P. falciparum. The presence of genes, viz. sufB (in plastid genome) and sufA, sufC, sufD, and sufS (in the nuclear genome) which are member of the Suf family indicates to some functionality of this pathway in the parasite apicoplast. The Suf system is well known to be involved in the bacterial ferrodoxin based electron transport. This whole system and this complete pathway still remain to be proven in the malaria parasite. The fourth pathway, i.e., the Heme Biosynthesis pathway [47, 48] is required by the P. falciparum for synthesizing heme which may be acting as a precursor in synthesis of certain 230
proteins. One crucial precursor for heme biosynthesis is delta-aminolevulinate (ALA), which is synthesized by two different biosynthetic pathways. The Shemin pathway (characteristic to animals and fungi & located in mitochondria) synthesizes ALA by action of deltaaminolevulinate synthetase (ALAS) on glycine and succinyl CoA. The second pathway C5 or Glutamate pathway synthesizes ALA from glutmate and is found mainly in plastids of algae and plants and in some eubacteria. The enzyme delta-aminolevulinate dehydratase (ALAD) leads to condensation of two ALA from the above two pathways running simultaneously in respective compartments, to yield porphobilinogen. It is thought that Plasmodium uses ALAS and the Shemin pathway in its mitochondria [20, 49]. ALAD has also been found in these parasites. While some evidence indicates that large amounts of the mammalian ALAD enzyme are imported from the red blood cell into the parasite cytoplasm [50 52], other authors have found that the Plasmodium genome encodes its own ALAD. It has been speculated that the malarial parasites synthesize ALA in the mitochondria and continue the biosynthetic pathway of heme with ALAD in the apicoplast, the close contact between these two organelles observed in various life stages supporting this scenario [47, 48]. However, a number of other enzymes involved in the complete heme biosynthesis pathway have now been deduced and have been found to be localized in the apicoplast. Still some more enzymes remain to be identified in apicoplast to completely prove this pathway.
CONCLUSIONS
Apicoplast, the plastid organelle in apicomplexan parasite Plasmodium has paved the way for the identification of novel drug targets in our fight against malaria. The enzymes functional in the metabolic pathways specific to this organelle have been identified as potential targets to various drugs and chemotherapeutic agents. There arises a need to get insight into the reported metabolic pathways and their products as whole that might be helping the parasite to cause infective of varying grades in its vertebrate host. With the availability of the complete nuclear genome data of P. falciparum and plastid genome, the task has become much easier and new studies can be designed to get the details on the function of cluster of genes present in the plastid genome and on function of as yet uncharacterized hypothetical proteins targeted to apicoplast.
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24. Gardner, M.J., Goldman, N., Barnett, P., Moore, P.W., Rangachari, K., Strath, M., Whyte, A., Williamson, D.H. & Wilson, R.J.M., (1994). Phylogenetic analysis of the rpoB gene from the plastid-like DNA of Plasmodium falciparum. Molecular and Biochemical Parasitology, 66, 221-231. 25. Waller, R.F., Reed, M.B., Cowman, A.F. & McFadden, G.I., (2000). Protein trafficking to the plastid of Plasmodium falciparum is via the secretory pathway. EMBO Journal, 19, 17941802. 26. van Dooren, G.G., Marti, M., Tonkin, C.J., Stimmler, L.M., Cowman, A.F., & McFadden, G.I., (2005). Development of the endoplasmic reticulum, mitochondrion and apicoplast during the asexual life cycle of Plasmodium falciparum. Molecular Microbiology, 57, 405419. 27. Kobayashi, T., Sato, S., Takamiya, S., Komaki-Yasuda, K., Yano, K., Hirata, A., Onitsuka, I., Hata, M., Mi-ichi, F. Tanaka, T., Hase, T., Miyajima, A., Kawazu, S., Watanabe, Y. & Kita, K., (2007). Mitochondria and apicoplast of Plasmodium falciparum: Behaviour on subcellular fractionation and the implication. Mitochondrion, 7, 125132. 28. Lim, L. & McFadden, G.I., (2010). The evolution, metabolism and functions of the apicoplast. Philosophical Transactions of the Royal Society: Biological Sciences, 365, 749763. 29. Ralph, S.A., D'Ombrain, M.C. & McFadden, G.I., (2001). The apicoplast as an antimalarial drug target. Drug Resistance Updates, 4, 145151. 30. Fichera, M.E. & Roos, D.S., (1997). A plastid organelle as a drug target in apicomplexan parasites. Nature, 390, 407409. 31. He, C.Y., Shaw, M.K., Pletcher, C.H., Striepen, B., Tilney, L.G. & Roos, D.S., (2001). A plastid segregation defect in the protozoan parasite Toxoplasma gondii. EMBO Journal, 20, 330339. 32. Roos, D.S., Crawford, M.J., Donald, R.G., Kissinger, J.C., Klimczak, L.J. & Striepen, B., (1999). Origin, targeting and function of the Apicomplexan plastid. Current Opinion in Microbiology, 2, 426432. 33. Sullivan, M., Li, J., Kumar, S., Rogers, M.J. & McCutchan, T.F., (2000). Effects of interruption of apicoplast function on malaria infection, development, and transmission Molecular and Biochemical Parasitology, 109, 1723. 234
34. McKean, P., (2002). New hope for the neglected diseases. Microbiology Today, 29, 129131. 35. Saxena, V., Garg, S., Ranjan, S., Kochar, D.K., Ranjan, A., Das, A., (2007). Analysis of elongation factor Tu (tuf A) of apicoplast from Indian Plasmodium vivax isolates. Infection, Genetics and Evolution, 7, 618626. 36. McFadden, G.I. & Roos, D.S., (1997). Apicomplexan plastids as drug targets. Trends in Microbiology, 7, 328333. 37. DeRocher, A., Hagen, C.B., Froehlich, J.E., Feagin, J.E. & Parsons, M., (2000). Analysis of targeting sequences demonstrates that trafficking to the Toxoplasma gondii plastid branches off the secretory system. Journal Cell Science, 113, 3969-3977. 38. Waller, R.F., Keeling, P.J., Donald, R.G., Striepen, B., Handman, E., Lang-Unnasch, N., Cowman, A.F., Besra, G.S., Roos, D.S. & McFadden, G.I., (1998). Nuclear-encoded proteins target to the plastid in Toxoplasma gondii and Plasmodium falciparum. Proceedings of National Academy of Science, USA, 95, 1235212357. 39. Jelenska, J., Crawford, M.J., Harb, O.S., Zuther, E., Haselkorn, R., Roos, D.S. & Gornicki, P., (2001). Subcellular localization of acetyl-CoA carboxylase in the apicomplexan parasite Toxoplasma gondii. Proceedings of National Academy of Science, USA, 98, 2723-2728. 40. McLeod, R., Muench, S.P., Rafferty, J.B., Kyle, D.E., Mui, E.J., Kirisits, M.J., Mack, D.G., Roberts, C.W., Samuel, B.U., Lyons, R.E., Dorris, M., Milhous, W.K. & Rice, D.W., (2001). Triclosan inhibits the growth of Plasmodium falciparum and Toxoplasma gondii by inhibition of Apicomplexan Fab I. International Journal of Parasitology, 31, 109-113. 41. Surolia, N. & Surolia, A., (2001). Triclosan offers protection against blood stages of malaria by inhibiting enoyl-ACP reductase of Plasmodium falciparum. Nature Medicine, 7, 167173. 42. Yu, M., Kumar, T.R.S., Nkrumah, L.J., Coppi, A., Retzlaff, S., Li, C.D., Kelly, B.J., et al., (2008). The fatty acid biosynthesis enzyme FabI plays a key role in the development of liver-stage malarial parasites. Cell Host Microbe, 4, 567578. 43. Vaughan, A.M., ONeill, M.T., Tarun, A.S., Camargo, N., Phuong, T.M., Aly, A.S., Cowman, A.F. & Kappe, S.H., (2009). Type II fatty acid synthesis is essential only for malaria parasite late liver stage development. Cell Microbiology, 11, 506520. 235
44. Jomaa, H., Wiesner, J., Sanderbrand, S., Altincicek, B., Weidemeyer, C., Hintz, M., Turbachova, I., Eberl, M., Zeidler, J., Lichtenthaler, H.K., Soldati, D. & Beck, E., (1999). Inhibitors of the non-mevalonate pathway of isoprenoid biosynthesis as antimalarial drugs. Science, 285, 15731575. 45. Wiesner, J., Hintz, M., Altincicek, B., Sanderbrand, S., Weidemeyer, C., Beck, E. & Jomaa, H., (2000). Plasmodium falciparum: detection of the deoxyxylulose 5-phosphate reductoisomerase activity. Experimental Parasitology, 96, 182-186. 46. Vollmer, M., Thomsen, N., Wiek, S., & Seeber, F., (2001). Apicomplexan parasites possess distinct nuclear-encoded, but apicoplast-localized, plant-type ferredoxinNADP+ reductase and ferredoxin. Journal of Biological Chemistry, 276, 5483-5490. 47. Sato, S. & Wilson, R.J.M. (2002). The genome of Plasmodium falciparum encodes an active deltaaminolevulinic acid dehydratase. Current Genetics, 40, 391-398. 48. van Dooren, G.G., Su, V., DiOmbrain, M.C. & McFadden, G.I., (2002). Processing of an Apicoplast leader sequence in Plasmodium falciparum, and the identification of a putative leader cleavage enzyme. Journal of Biological Chemistry, 277, 2361223619. 49. Surolia, N. & Padmanaban, G., (1992). de novo biosynthesis of heme offers a new chemotherapeutic target in the human malarial parasite. Biochemical and Biophysical Research Communication, 187, 744-750. 50. Bonday, Z.Q., Taketani, S., Gupta, P.D. & Padmanaban, G., (1997). Heme biosynthesis by the malarial parasite. Import of delta-aminolevulinate dehydrase from the host red cell. Journal of Biological Chemistry, 272, 21839-21846. 51. Bonday, Z.Q., Dhanasekaran, S., Rangarajan, P.N. & Padmanaban, G., (2000). Import of host delta-aminolevulinate dehydratase into the malarial parasite: identification of a new drug target. Nature Medicine, 6, 898903. 52. Padmanaban, G. & Rangarajan, P.N., (2000). Heme metabolism of Plasmodium is a major antimalarial target. Biochemical and Biophysical Research Communications, 268, 665-668.
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Chapter XII
THE ENIGMA OF Helicobacter pylori INFECTION AND GASTROINTESTINAL DESEASES

1
Sushil Kumar, 1Shailesh K. Shahi, *1Ashok Kumar and 2V.K. Dixit

1
School of Biotechnology, Banaras Hindu University, Varanasi, India
Department of Gastroenterology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India
*
Coressponding author: Dr. Ashok Kumar, Professor of Biotechnology, School of Biotechnology, Banaras Hindu University, Varanasi, India E.mail: kasokbt@rediffmail.com
ABSTRACT: Helicobacter pylori, a Gram-negative, microaerophilic, spiral-shaped bacterium,

colonizes the human stomach and is estimated to inhabit at least half of the worlds human population. It is a highly successful bacterial pathogen that persistently colonizes the mucosa of the human stomach and infection occurs mostly in early childhood, frequently leading to persistent infection lifelong. The bacterium has been recognized as the causative agent of chronic gastric inflammation, which can progress to a variety of other gastroduodenal diseases, such as peptic ulcers, mucosa associated lymphoid tissue lymphoma, or even gastric cancers. Notably, only a fraction of colonized individuals develop peptic ulcer and gastric cancer, perhaps depending on factors such as host characteristics that are governed by genetic polymorphisms, differentially represented bacterial determinants and/or specific interactions between a particular strain and its host that occur during decades of coexistence.
INTRODUCTION:
About 25 years ago, Barry Marshall and Robin Warren described the successful isolation and culture of a spiral bacterial species from the human stomach [1]. During that period (198283) the conventional thinking was that no bacterium can survive in the human stomach as the 237
stomach produced excessive amounts of acid which was similar in strength to the acid found in a car-battery. The organism was initially named Campylobacter-like organism, gastric Campylobacter-like organism, Campylobacter pyloridis, and Campylobacter pylori but is now named Helicobacter pylori in recognition of the fact that this organism is distinct from the members of the genus Campylobacter [2]. Self-ingestion of H. pylori experiments by Marshall et al., [3] and Morris and Nicholson [4] and later experiments with volunteers (5), demonstrated that these bacteria can colonize the human stomach, thereby inducing inflammation of the gastric mucosa. Marshall developed a transient gastritis after ingestion of H. pylori; the case described by Morris developed into a more persistent gastritis, which was resolved after sequential therapy, first with doxycycline and then bismuth subsalicylate. These initial data strongly stimulated further research, which showed that gastric colonization with H. pylori can lead to variety of upper gastrointestinal disorders, such as chronic gastritis, peptic ulcer, gastric mucosa associated lymphoid tissue (MALT) lymphoma, and gastric cancer. This knowledge had a major clinical impact with regard to the management of these diseases. In addition, the persistence of a pathogen in an environment long thought to be sterile also resulted in insights into the pathogenesis of chronic diseases. This discovery resulted in the award of the 2005 Nobel Prize in Physiology and Medicine to Robin Warren and Barry Marshall for their discovery of the bacterium Helicobacter pylori and its role in gastritis and peptic ulcer disease. Although the prevalence of H. pylori in the Western world is decreasing, gastric colonization by H. pylori remains widespread in the developing world. Infection with H. pylori can be diagnosed by a variety of tests and can often be successfully treated with antibiotics. Unfortunately, the increase in antibiotic resistance is hindering the efficacy of treatment, and, in spite of the pathogenicity of H. pylori, preventive vaccination strategies still do not exist. A better understanding of H. pylori persistence and pathogenesis is thus mandatory to aid the development of novel intervention and prevention strategies. This paper deals with the basic biology of H. pylori together with various types of gastrointestinal diseases caused by its infection and factors involved in disease appearance in the host. 1. MICROBIOLOGY (i) Genus description and phylogeny of Helicobacter - The genus Helicobacter belongs to the subdivision of the Proteobacteria, order Campylobacterales, family Helicobacteraceae. The most important stage in the development of the taxonomy was proposed by Goodwin and 238
colleague [2] to establish a new genus called Helicobacter. They proposed that C. pylori should be transferred to the genus H. pylori. The creation of the new genus was argued mainly on the basis of the 16S rRNA (small subunit) sequence data [6]. To date, the genus Helicobacter consists of over 20 recognized species but many of them is awaiting formal recognition [7]. Members of Helicobacter genus are microaerophilic, majority of them are catalase and oxidase positive, and many but not all species are urease positive. (ii) Morphology - H. pylori is genetically described as a gram-negative bacterium, measuring 2 to 4 m in length and 0.5 to 1 m in width. Although usually spiral-shaped or curved rod with one to three turns when observed in vivo, the coccoid shapes appear after prolonged in vitro culture or antibiotic treatment [8]. These coccoids loose their ability to be cultured in vitro and are thought to represent dead cells [8], although it has been suggested that coccoid forms may represent a viable, non-culturable state. The bacterium has 2 to 6 unipolar (lophotrichate) sheathed flagella of approximately 3 m in length, which often carry a distinctive bulb at the end of flagellum. Bacterial cells are mostly actively motile in viscous solutions such as the mucus layer overlying the gastric epithelial cells. (iii) Genome, plasmids and strain diversity - The genome (chromosomal) DNA of H. pylori is a single circular molecule. The size of the five sequenced H. pylori (Shi 470, HPAG1, J99, 26695 and G27) genome is approximately 1.7 Mbp, with a G+C content of 35 to 40%. The H. pylori strain 26695 genome includes 1,587 genes, whereas the genome of strain J99 includes only 1,491 genes. Genomes of J99 and 26695 contain two copies of the 16S, 23S, and 5S rRNA genes. Many strains (ca 45%) carry one or more cryptic plasmids, which do not seem to carry genes responsible for virulence or antibiotic resistance [9]. Generally bacterial pathogens are highly clonal (such as Shigella dysenteriae and Mycobacterium tuberculosis), in contrast, H. pylori is genetically heterogeneous, suggesting a lack of clonality. This results in every H. pylori-positive subject carrying a genetically distinct strain [10] although differences within relatives may be small. The reasons of genetic heterogeneity are possibly an adaptation of H. pylori to the harsh gastric conditions of its host, as well as to the distinct patterns of the hostmediated immune response to H. pylori infection [11]. Genetic diversity is thought to occur via several methods of DNA rearrangement and the introduction and deletion of foreign sequences [12]. The latter usually have an aberrant G+C content and often carry genes involved in virulence. A striking example of this is the cytotoxic associated gene, Pathogenicity Island (cag 239
PAI), but other plasticity regions have also been suggested to play a major role in the pathogenesis of H. pylori infection [13]. At the molecular level, the diversity in genome may be seen via several mechanisms, including transcriptional and translational phase variation and mutation. Often phase variation occurs via reversible slipped-strand mispairing in homopolymeric G or C tracts, resulting in a shift in translation of the affected gene, thereby causing phase variation via a single mutation. This event shows a reversible phenotypic diversity with only minor genetic variation. Several virulence genes, namely sabA, sabB, hopZ, and oipA (outer membrane protein-encoding genes), display such phenotypic variation, as do lipopolysaccharide (LPS) biosynthetic enzymes [10]. 2. EPIDEMIOLOGY The prevalence of H. pylori shows wide geographical variations in different parts of the world. More than 80% of the population of various developing countries are H. pylori positive [10]. In the industrialized countries, H. pylori prevalence generally remains under 40% of the population. Prevalence of H. pylori is considerably lower in children and adolescents than in adults and elderly people [14]. Within particular geographical areas, the prevalence of H. pylori is inversely proportional with socioeconomic status, this is due to living standard during childhood [15]. H. pylori infection rates rise rapidly in the first 5 years of life in developing countries and remain constantly high thereafter, indicating that H. pylori is acquired early in childhood. However, H. pylori prevalence is low in early childhood and slowly rises with increasing age in industrialized countries. Overall, new H. pylori infection is more common in childhood and persists for life long unless specifically treated. 3. DISEASES ASSOCIATED WITH H. pylori INFECTION (i) Gastritis - It has been elegantly demonstrated that infection with H. pylori always causes chronic active gastritis. Gastritis commonly refers to inflammation of the lining of the stomach. Infection with the H. pylori provokes invasion of the mucosa by lymphocytes/plasma cells (a marker for the grade of gastritis), and neutrophils (a marker for the activity of gastritis). The association between gastrointestinal (GI) symptoms and chronic superficial gastritis [16]) is uncertain. However, it frequently develops into chronic atrophic gastritis with loss of epithelial glands over a period of decades.
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(ii) Peptic ulcer - Gastric or duodenal ulcers (peptic ulcer) is defined as a defect in the gastric mucosa of at least 0.5-cm in diameter and penetrating through the muscularis mucosa, with or without bleeding and perforation [17]. Peptic ulcer (PU) disease is strongly associated to H. pylori infection. The discovery of H. pylori infection has dramatically changed the understanding of the pathogenesis of ulcer disease and also its treatment. H. pylori infection is diagnosed in 95% of duodenal ulcer (DU) where as 85% of gastric ulcer occurred in the presence of the infection of H. pylori worldwide [18, 17]. The remaining DUs and PUs, especially in elderly, are due to drugs like NSAID use and other causes (rarely) such as Zollinger-Ellison syndrome and Crohns disease [18]. Occurrence of most ulcers is at sites where mucosal inflammation is most severe. The corresponding prevalence of infection in the case of gastric ulcer patients has been more complicated to establish since biopsy based diagnosis generally shows false-negative results due to association between gastric ulcers and atrophic changes of the mucosa. Serological confirmation of H. pylori infection has, however, verified an H. pylori seroprevalence of 60100% in gastric ulcers cases [17]. Seroepidemiological evidence from different populations revealed that the lifetime risk for peptic ulcer disease among H. pylori infected individuals is 620%, which is at least 3-4 times higher than for uninfected individuals [19, 17]. Ulcer development in the presence of H. pylori is influenced by variety of host, bacterial and environmental factors. (iii) Nonulcer dyspepsia - Dyspepsia is simply, a term which includes a group of symptoms that come from a problem in upper gastrointestinal tract. Non-ulcer or functional dyspepsia is defined as the presence of symptom of upper gastrointestinal distress without any identifiable structural abnormality during diagnostic work-up, in particular upper gastrointestinal tract. The main symptom of dyspepsia is usually pain or discomfort in the upper abdomen, in addition, may be, heartburn, bloating, belching, quickly feeling 'full' after eating, feeling sick (nausea) or vomiting. Symptoms are often related to eating. 30 to 60% of patients with nonulcer dyspepsia carry H. pylori, but this prevalence is not much different from that in the unaffected population [10]. Two randomised double-blind placebo controlled studies in adults with nonulcer dyspepsia did not find association between H. pylori eradication and relief of symptoms [20]. However, a third well-designed trial reported a benefit of eradication treatment in a small subgroup of patients [21].
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(iv) Gastric cancer - Gastric adenocarcinoma is the second most common cause of cancer related death world wide and 14th cause of overall death [22]. Gastric adenocarcinoma is histopathologically sub-divided into intestinal-type and diffuse type. Intestinal-type gastric adenocarcinoma, which occurs in older people, is more common than diffuse type. A small subset of diffuse type adenocarcinoma is of familial origin, caused by mutations in the Ecadherin gene [23]. Epidemiological studies as well as infection studies using animal models have indicated that H. pylori plays a critical but major role in the development of both types of gastric adenocarcinoma [24]. A large-scale prospective study also revealed that the risk of development of gastric carcinoma was much greater in the population infected by H. pylori than in the H. pylori-uninfected population. Eradication of H. pylori significantly decreased the rate of gastric carcinoma development in H. pylori-infected patients without precancerous lesions, providing evidence for the causative role of H. pylori in gastric carcinogenesis [25]. The bacterium colonizes over half of the population worldwide. Infection is usually acquired in childhood and in the absence of antibiotic therapy and therefore persists for the lifelong of the host. Although most infected patients will develop a chronic active gastritis, the majority are asymptotic. Though the risk varies with age, geographical location, diet and ethnicity, overall 1520% of infected patients will develop gastric or duodenal ulcer disease and only a fraction of all H. pylori infected cases (1-2%) is likely to develop gastric carcinoma. A multitude of host and environmental factors, including interaction with other infectious agents and dietary factors, affect the immune response to H. pylori and may impact disease presentation over the life of the host [26].
(v) Gastroesophageal reflux disease - Gastroesophageal reflux disease (GERD) is defined

as chronic symptoms of mucosal damage produced by the abnormal reflux of gastric contents into the esophagus [27]. Even though GERD is primarily a motility disorder, other pathophysiological disturbances seem to play a role in its pathogenesis. Spectrum of the esophagus injury includes esophagitis, stricture, the development of columnar metaplasia in place of the normal squamous epithelium (Barretts esophagus) and adenocarcinoma [28]. GERD has long been considered to occur independently of H. pylori infection, i.e., to occur with the same frequency and severity in H. pylori-positive and H. pylori negative subjects [10]. However, further studies suggested that the curing of H. pylori infection in DU patients may provoke reflux esophagitis. A 2-year follow-up, based on 276 DU patients randomised to either eradication 242
therapy or long-term omeprazole treatment, revealed that both regimens were equally effective in controlling dyspeptic symptoms and GERD in patients with a healed DU [29]. Furthermore, the healing of ulcers was not found to increase the risk of GERD. (vi) Extragastric manifestations - Till date there is a general belief that H. pylori is linked to a variety of extragastric diseases, thereby opening the new extragastric manifestations of H. pylori infection field [30]. These include coronary heart disease, dermatological disorder such as rosacea and idiopathic urticaria, autoimmune thyroid disease and thromophenomenon, scleroderma, migraine, iron deficiency anemia and Guillain-Barre syndrome [10, 31]. Sidropenic anaemia has been reported to be associated with H. pylori infection and the reversal of iron deficiency anaemia after eradication of H. pylori has been observed in many patients [10]. The observations might be due to subclinical bleedings from the GI tract but are also likely to be due to the genetic factors indicating that H. pylori scavenge iron, presumably from its host. In some paediatric studies, association of H. pylori infection with short stature has been suggested. However, confirmation of these observations would need further studies taking into account various factors such as genetic and socio-economic/nutritional conditions. Among children in developing country, a positive association has been noted between H. pylori and Vibrio cholerae. A higher H. pylori prevalence in children with chronic diarrhoea has been observed. In contrast, a population-based study of German pre-school children found an inverse association between H. pylori and diarrhoea [32].
4. DIAGNOSES Invasive and non-invasive diagnostic methods - The invasive tests for H. pylori infections
are based on gastric specimen for culture, histology, rapid urease assays and polymerase chain reaction (PCR) which by amplifying segments of DNA from H. pylori can detect very small numbers of bacteria and characterize the various genes and this being direct methods. Since these methods require upper endoscopy, they are generally not considered suitable for paediatric epidemiological investigations, for ethical reasons and for the cost. The non-invasive methods based on peripheral samples such as blood, breath samples, stools, urine or saliva for detection of antibodies, bacterial antigens or urease activity all being indirect methods are preferred. The choice of a specific test for an individual patient depends on severity of disease, local experience,
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clinical setting and cost. In hospital-based care, many patients undergo endoscopy, which is then combined with an invasive test of H. pylori.
(i) Culture - Culturing of bacterial colonies from gastric biopsies is regarded as a definitive
proof of H. pylori infection. However, H. pylori is fastidious and microaerophilic organism, its culture is not always successful in many laboratory. The ability to culture the organism, and thus the sensitivity of the test, may vary between laboratories since the method is most technically demanding. The organisms are sensitive to temperature, oxygen and medium which create problem during transportation and the culture itself requires special conditions and 3-7 days of incubation. The main advantages of culture are the excellent specificity and the opportunity to test for susceptibility to antibiotics in patient management [33]. In addition, the genotype of the isolate with regard to cytotoxin associated antigen gene (cagA) and the vacuolating cytotoxin antigen gene (vacA) and other pathogenic gene status can be determined, although this investigation is limited to selected research centres. Genetic diversity study can also be easily made by culture of bacteria.
(ii) Histology - H. pylori is one of the microorganisms that can be confidently recognised by
histology. Also, the histopathologists are uniquely placed both to identify infection and to assess the disease process associated with it, i.e., gastritis and in adults premalignant (dysplastic) changes or neoplastic lesions such as carcinoma, lymphoma or carcinoid. Even though the sensitivity of histology is generally high, this method may be affected by observer related factors and topographical changes in the stomach may introduce sampling errors. Although a single biopsy from the lesser curvature (23 cm from the pylorus) will yield a positive result in over 90% of infected stomachs [34], sensitivity can be improved by examining additional samples from the antrum and corpus. Poor orientation or small biopsy samples will lower sensitivity. The most important factor affecting sensitivity is the experience and consciousness of the observer. Specificity is generally not a big problem. Difficulties arise when patients use anti-Helicobacter treatment where the bacterial count may be much reduced but infection not completely eradicated. If H. pylori adopts a coccoid form then confident identification becomes impossible. In this situation immunohistochemical staining [35] and other technique such as in situ hybridization, [36] or PCR [37] may resolve the issue. (iii) Rapid urease test - This test uses the high concentration of pre-formed urease enzyme activity in H. pylori infected gastric biopsy samples. The increasing alkalinity resulting from the 244
production of ammonium ions (NH4+) is detected by a colour change in the pH indicator and urea containing medium. The test can be carried out using small quantities of standard urea broth for in-house methods but commercial kits are also available. The sensitivity of the test depends on the number of bacteria present in the sample. It has been calculated that 104 microbes are required for a positive result, and a proportion of patients will harbor densities lower than this threshold [33]. Low levels of colonisation are to be expected if patients used anti-Helicobacter treatment where the bacterial count may be much reduced but infection not completely eradicated. Increased sensitivity can be achieved by using two biopsy specimens and incubation at 37C. The specificity of this rapid urease test is very good when the test is read at one hour, but declines with the length of the incubation. Thus, at 24 hours false positive results may be obtained by other urease producing bacteria [38]. Recently many urease positive microbes other than H. pylori in human gastric juice and mucosa have been discovered [39]. In this situation this test will give false positive for H. pylori. (iv) 13C-Urea breath test - The urea breath test (UBT) is one of the most important non-invasive methods for detecting H. pylori infection. The test exploits the rapid hydrolysis of orally administered labelled urea by the enzyme urease, which is produced by H pylori in large quantities. Urea is hydrolysed to ammonia and carbon dioxide, which diffuses into the blood through mucus layer of the gastric mucosa via the systemic circulation and is excreted in the expired air through lungs.
13
C-labeled urea is becoming increasingly popular because this non-
radioactive isotope is innocuous and can be safely used in children and pregnant women. The test gives a direct measure of an active gastric infection [40], also reflecting the bacterial load and grade of histological gastritis [41]. The test is easy to perform and can be repeated as often as required in the same patient. The UBT is a very sensitive and specific method, thus providing a gold standard against which the accuracy of other diagnostic tests (both invasive and noninvasive) can be validated [42]. However, it may not be reliable in assessing patients who have had gastric surgery. One of the most important advantages of the UBT is that it samples the whole stomach and there is no chance of sampling error unlike biopsy based tests (invasive test), which can be influenced by the patchy distribution of H. pylori infection within the gastric mucosa. Occasionally, oral urease producing bacteria may cause false positive results [43]. Additionally, many urease positive microbes other than H. pylori present in human gastric juice and mucosa may give false results [39]. Also, antibiotics, bismuth salts and proton pump 245
inhibitor (PPI), which reduce the extent of H. pylori infection, may introduce false negative results. The cost of producing
13
C-urea is high, but it may be possible to reduce the dosage
further by administering it in capsule. Another major disadvantage is the high initial cost for the test, caused by the need for expensive mass spectrometric equipment. Recommendation of UBT is for monitoring the effect of eradication treatments and it is also a suitable test for epidemiological study, particularly in children. However, reservations have been made since the test is less reliable in children <5 years and it is not yet evaluated among the youngest (<2 years) [44]. (iv) Serology - Serological test is also one of the non-invasive tests. These tests are used for the detection of H. pylori infection and have been helpful in epidemiological studies of prevalence, allowing for the development of preventive infection early in life [45]. Serology is the easiest way to identify H. pylori passive infection in individuals not undergoing gastroendoscopy and this test is based on the principle of detecting the circulating antibodies against H. pylori. Common designs are based on antibody detection tests which include the Western Blot (WB) and enzyme-linked immunosorbent assay (ELISA) technique. The ELISA format has the advantage that many serum samples can be tested in parallel and the process can be completely automated [45]. This is the most widely used method due to its simplicity and cost effective features, speed, and minimal patient discomfort thereby making it suitable also for large-scale screening [46]. Advantage of this test is that the result is not affected by sampling errors like other invasive or non-invasive tests. However, one of the major drawbacks of this test is that a positive test does not necessarily indicate a current (active) infection however similar to the UBT, the test is not affected by sampling errors. It has been demonstrated that initially lower specificity of the method was due to false positive results mainly due to nonspecific crossreaction with other microorganisms, such as Campylobacter sp. [47]. Many different types of antigen preparations can be used in the ELISA and the upper limit of normal values, i.e., the cutoff level, needs to be evaluated for each assay system and in the population being investigated. Although ELISA has proven to be highly accurate for the diagnosis of H. pylori infection in adults, in children it has shown variable sensitivity and specificity [48, 49]. It has been suggested that the cut-off value needs to be corrected specially in the case of children since they produce lower antibody responses than adults, not only due to delayed and low antibody production, but also due to the occurrence of maternal antibodies up to the age of 3-7 months. It has shown 246
limited sensitivity (80%) and
specificity (70%) of H. pylori-specific IgA and IgG antibodies
detection in saliva from general population, however, H. pylori-specific IgG in urine has shown more sensitivity (95.5%) and specificity (83%) than saliva based assay. Most IgG diagnostic tests are serum-based. (vi) Polymerase chain reaction (PCR) (a) Standard PCR assay for the detection of H. pylori- Soon after the discovery of PCR, this was quickly applied to the detection of H. pylori. This method revolutionized the study of DNA, especially after the introduction of a thermostable DNA polymerase obtained from Thermophilus aquaticus (Taq polymerase). Its application in the field of H. pylori concerns not only the detection of the bacterium but also its genome sequencing, quantification and detection of specific genes relevant to pathogenesis such as cagA, vacA, iceA, babA etc, as well as in diversity study based on RAPD, AFLP, ERIC-PCR etc and specific mutations associated with antimicrobial resistance. Target choice is the most important factor in the primers designing which must be specific for H. pylori but conserved in all strains of the species. Therefore, it is necessary to know the DNA sequence of the target in as many strains of H. pylori and as many strains of other bacterial species as possible. In the case of direct detection of H. pylori from specimen it is also necessary to know host DNA sequence. The first target of H. pylori gene used was the genes of the urease operon: ureA [50] and glmM, formerly named ureC [51], and the 16S rRNA gene [52]. 16S rRNA gene is highly conserved in bacteria and exhibits sequences which are specific for different species. Among the drawbacks of PCR are; (i) existence of possible Taq and other themostable polymerase inhibitors which can decrease the sensitivity of the reaction, and (ii) the specimen could be contaminated by exogenous H. pylori DNA or organism, which alters the specificity. To date several methods have been proposed to improve sensitivity and specificity of PCR. To improve DNA extraction, phenol extraction, CTAB mini prep method or the use of many commercial kits for DNA extraction and purification to eliminate the possible PCR inhibitors in target DNA have been employed. Recently, it has been demonstrated that use of an internal control helps in the test of possible PCR inhibitors, it is also advantageous in checking false negative. If the target is too low, use of a nested or seminested PCR has been suggested to escape false positive as well as false negative result [10]. However, the use of nested or heminested PCR increases the risk of airborne contamination by amplicons and must be discouraged. Methods such as the reverse dot blot line probe assay (LiPA) [53] and liquid 247
phase (DNA-enzyme immunoassay) have also been proposed. Several authors have reported the use of reverse transcription-PCR (RT-PCR) assay. This method is based on mRNA, it determines the viability of the bacteria present, but no improvement in sensitivity has been shown in this method. A significant improvement in sensitivity of PCR was obtained with the introduction of real-time PCR method. As is always the case when a new method is more sensitive than the reference method, it is necessary to prove that the results of new methods are not giving false positive results. The specificity of PCR is not usually a problem when dealing with gastric biopsy specimens. However, the possibility of false positives arises if grinding apparatus in the lab, endoscopes [54] or biopsy forceps are not carefully cleaned. (b) PCR assay for the detection of pathogenicity genes of H. pylori - An important application of standard PCR is the genotyping and detection of specific pathogenic factors of H. pylori. There are two most important pathogenic factors i.e., the cag PAI and the polymorphism of the vacA gene. Other genes involved in pathogenicity (oipA, dupA, iceA) and adherence (babA2, sabA) can also be detected by PCR. When detection for both cagA and vacA is performed, a strong association is seen between the presence of cagA and vacA s1, corresponding to strains with the highest production of cytotoxin [55] as well as more severe pathology and disease. Usefulness of PCR for detection of H. pylori in different types of biological samples for invasive as well noninvasive tests, such as gastric juice, saliva, dental plaque and faeces has been emphasized. Although sensitivity and specificity of this test have high practical consideration but due to cost it has limited use. Some of the investigators developed strip- based detection by which H. pylori genes are amplified with a biotin-labelled PCR primer and subsequently analyzed by a single-step reverse hybridization on the strip [53]. However, recently developed method based on multiplex PCR assay seems very useful [37]. This method does not require cultivation of bacteria, extraction of genomic DNA, boiling of biopsy samples or frequent steps of centrifugation. Template for amplification is obtained simply by vortexing the biopsy samples. Using this assay the prevalence of type I and/ or type II strains, vacA signal sequence (s1 and s2) and mid-region (m1 and m2) alleles as well as the presence or absence of a cagA gene can be detected in a single reaction, directly from gastric biopsy samples. This method is cost effective, simple and fast [37]. (c) Real-time PCR - More recently, the development of real-time PCR has facilitated more rapid, specific and quantitative estimation of gene targets as they are amplified in real-time [56]. 248
Advantage of this technique is not only detection of H. pylori in a quick and precise manner but also its quantification and the detection of point mutations associated with antibiotic resistance. The principle of this method is quantification of the amplicons formed in real time. This has been done by using an intercalating agent, e.g., SYBR green I dye, or the fluorescence resonance energy transfer (FRET) principle [57].
(d) PCR for quantification - PCR is also used for the quantitation of H. pylori initially by
using a competitive PCR assay, followed by real-time PCR which has considerably facilitated the process. However, the exponential nature of DNA amplification is prone to burden the experimental data with significant standard error due to the tube-to-tube inherent variations also known as tube effect in amplification efficiency. Therefore it necessitates the introduction of a variable amount of internal standard (PCR mimic) of different sizes for the reliable quantitation of DNA or RNA templates by PCR and co-amplification of a constant amount of target DNA sequence with the same set of primers. A visual comparison allows the detection of bands with the same intensity indicating the amount of target DNA present [58], however, a detection by colorimetric method is also possible. The standard curve is constructed on the basis of band intensity as well as colorimetric data of internal standard templates (ordinate) against the log of the ratios of the PCR products amplified from the known amounts of wild-type template and standards (abscissa). The amount of unknown amount of wild template can be calculated on standard curve. The validation of this method i.e., quantity of H. pylori in gastric mucus was correlated with other invasive tests as well as with the urea breath test (UBT) in a study made by Furuta et al. [59]. However, they also used this method for post-eradicated patient follow-up with a high predictive value [59]. 5. H. pylori infection parameters Available reports suggest that the causes of different outcomes of H. pylori infection are governed by host and environmental factors as well as differences in bacterial virulence factors [60]. Several different characteristics and products of H. pylori have been suggested as virulence factors. Infection scenario of H. pylori is very important to distinguish between colonization and virulence factors. The colonization factors are those that are necessary for the survival of H. pylori in the human stomach at the very first step/acute infection when the H. pylori initially
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enters the stomach. The virulence factors are those, which aid in persistence of the infection of H. pylori such as toxins and factors causing tissue inflammation in the host.
(i) H. pylori colonization factors (a) Urease - The first and most crucial requirement for the pathogen such as H. pylori is to
enter the stomach and survive in the acidic milieu. To colonize the stomach, H. pylori is equipped with the urease enzyme and expression of this enzyme is a universal feature among all H. pylori strains. This enzyme is a 550 kDa protein complex, the cytosolic urease enzyme hydrolyses urea into ammonia and carbon dioxide, which neutralizes gastric acidity [10]. The ammonia increases the pH in periplasmic space and protects the bacteria from the acidic environment. H. pylori is well equipped to survive in strong acid but it is not an acidophile and needs to leave the gastric lumen, also to avoid discharge of the urease in the intestine gastric epithelium as it needs neutral pH environment. Activity of urease enzyme is tightly controlled by a pH-gated urea channel, UreI, which is open at low pH and closed at neutral pH conditions, allowing the bacterium a precise level of control over its pH environment. A urease mutant was shown to be deficient in colonizing gnotobiotic piglets as compared to the wild type [61] emphasizing the importance of this enzyme for survival and successful colonization. (b) Flagella - Motility is also important for colonization, to move through the viscous gastric mucus layer. H. pylori uses the polar flagella so that it can encounter the surface of the gastric epithelium. H. pylori has a unipolar set of 4-6 flagella which makes this bacterium highly motile [62]. These consist of two structural subunits: a major 53 kDa FlaA and a minor 54 kDa FlaB encoded by genes located at the H. pylori chromosome [63]. Movement of flagella is guided by chemotactic factors, which include bicarbonate ions and urea. H. pylori has been shown to possess the enzymatic ability to disrupt the oligomeric structure of mucin, which may help the pathogen to move freely in the mucus layer. Most probably protection of the flagella from acidinduced damage is by the flagellar sheath, which is an extension of the outer membrane. Ottemann and Lowenthal [64] showed that motility of bacterium is required for colonization of H. pylori in gastric mucosa of mice. It has been demonstrated in mouse animal model that mutant H. pylori strain which has non-motile flagella was less virulent in comparison to its wild type strain in the infectious dose. Motility is not only essential for bacteria to escape the acidic surrounding and reach the gastric mucus, but is also important for persistence of the H. pylori 250
infection. The mucus layer always secretes mucus toward the lumen for protecting gastric epithelium from acid since motility is required for movement of the bacteria into the newly formed mucus. (c) Adhesion - A final step in colonization of the stomach is the adhesion of H. pylori to the gastric surface epithelium. It is prerequisite that the bacteria strongly adhere to the gastric tissue to accomplish colonization against the peristalsis movement of the gastric wall combined with the fast turnover rate of the epithelial cells. Adhesion of the bacteria to the epithelial layer of host is mediated by a large family of 32 related outer membrane proteins (Hop proteins) that include the adhesions [65]. BabA is a 75-kDa adhesion protein molecule that mediates the attachment of H. pylori to Lewis b (-1, 3/4-difucosylated) blood group antigens on human gastric epithelial cells. Three bab alleles namely babA1, babA2, and babB have been identified [66]. However, babA1 and babA2 are identical alleles except that babA1 has a 10-bp deletion of the signal peptide sequence that leads to the elimination of the translational initiation codon. The babA2 and babB alleles, which encode homologous proteins, have polymorphic mid region sequences but have conserved sequences in the 5 and 3 regions [10, 66]. Only the babA2 gene product is necessary for Lewis b binding activity [66]. Bacterial strains which possess the babA2 gene adhere more tightly to epithelial cells and promote a more aggressive phenotype. It has been reported that BabA2 is associated with a higher incidence of gastric adenocarcinoma. One of the most common epitopes on the surface of gastro-intestinal epithelium and expressed in soluble glycol-conjugates in saliva and tears of 80% of the population is the histo-blood group antigen Lewis b. The relation between development of gastrointestinal disease and the blood groups have been demonstrated half a century ago, where it has been reported that the incidence of peptic ulcer is higher among individuals of blood group O phenotype. Moreover, the frequency of incidence of peptic ulcer is more in Lewis b antigen secretor than non-secretor individuals. Based on the conditions of the host tissue, H. pylori can bind to a variety of different receptors on the host tissue and possibly adopts the properties of adhesin. When H. pylori colonizing the mucus, approaches the epithelial lining and adheres to the Lewis b antigen on the surface of the epithelial cells, adhesion and interaction with host cells induce inflammation and finally sialylation of tissue cells takes place. The bacteria also adapt to the new environmental conditions in the tissue and express a new adhesin allowing it to bind to sialylated structures for
251
further enhanced adhesion [67]. Such precise and programmed adaptations increase the phenotypic fitness of H. pylori towards successful persistent infection. (ii) H. pylori virulence factors - Several pathogenic factors contribute to the outcome of disease, but the most important ones are the virulence factors produced by any given strain. On the basis of the cagA and vacA genotypes, two types of H. pylori have been defined. Type I strains contain cytotoxin-associated gene A, cagA, and express a functional vacuolating cytoxin A, vacA, and these genes are associated with chronic gastritis, peptic ulcers and gastric carcinoma. Type II strains do not contain the cagA gene, and do not produce functional VacA cytotoxin. Type II strains are generally not associated with gastric pathology [37]. (a) Cytotoxin-associated gene A (cagA) - CagA, a 120145-kDa protein with a carboxyterminal variable region [68, 69] is the most important pathogenic factor of H. pylori that was first described as a virulence factor associated with peptic ulcers [68, 70]. The cagA gene is localized at one end of the cag pathogenicity island (cag PAI), a 40 kb DNA segment containing 31 putative genes. It is presumed that cagA gene was most likely incorporated into the H. pylori genome by a process of horizontal transfer. The cag PAI -DNA segment is comparable to encoding components of a bacterial type IV secretion system, which is a molecular syringe through which macromolecules are delivered from inside of the bacteria to the eukaryotic cells. Clinically, infection with the cagA-positive H. pylori strains are associated with higher grades of gastric inflammation and severe atrophic gastritis and has been suggested to play an important role in the development of gastric carcinoma [71]. Depending on the presence of virulence genes of the type IV secretion system, translocation of CagA into host cells occurs. Once injected into the gastric epithelial cells by the type IV secretion system, CagA localizes to the inner surface of plasma membrane and undergoes tyrosine phosphorylation by several members of the SRC family of kinases (SFK) such as SRC, FYN, LYN and YES [72, 73]. Tyrosine phosphorylation of EPIYA motif of CagA by SFK occurs in the absence of any stimuli, the resulting SFK are constitutively activated in gastric epithelial cells. The above interaction indicates that the CagA might disrupt key signaltransduction pathways, contributing to cell transformation. Tyrosine phosphorylation of CagA occurs at a unique 5-amino-acid sequence- Glu-Pro-Ile-Tyr-Alu (EPIYA) motif that is present in multiple numbers in the carboxy-terminal variable region of the protein [74]. On the basis of 252
flanking sequence of EPIYA motif, EPIYA motif is part of four distinct EPIYA sites i.e., EPIYA-A, B C and D, each contains a single EPIYA motif which surrounds the amino-acid sequence. The CagA proteins from H. pylori strains of Western countries (Western CagA) such as Europe, North America and Australia, possess EPIYA-A and EPIYA-B sites, followed by the EPIYA-C site (A-B-C type EPIYA), which is placed within a 32, 40 and 34 amino acids respectively. The EPIYA-C segment, a 34 amino acid stretch is mostly repeated one to three times in different Western CagA species as a result of homozygous recombination or misaligned replication of a 102 bp cagA gene segment. The tyrosine residue of EPIYA-C segment is the major site of tyrosine phosphorylation by SFK in the gastric epithelial cells in Western CagA, whereas those present within the EPIYA-A and EPIYA-B segment are also phosphorylated at tyrosine residues but less frequently than EPIYA-C segment [74, 73]. CagA proteins of H. pylori strains isolated in East-Asian countries such as Japan, Korea and China are known as EastAsian CagA, and possess the EPIYA-A, EPIYAB and EPIYA-D site (A-B-D type EPIYA) and lack repeatable EPIYA-C segment. The EPIYA-D site of East-Asian CagA represents the major tyrosine phosphorylation site. Once tyrosine phosphorylation by SFK takes place, CagA acquires the ability to bind specially to cytoplasmic protein (SRC homology 2) SHP-2 domain containing protein tyrosine phosphatase (PTP) called SHP-2. SHP-2 has two tandemly repeated SH2 domain termed as N-SH2 and C-SH2 protein tyrosine phosphatase domain. The physical complex formed between SHP-2 and CagA is detectable in cell infected with CagA positive H. pylori in vitro as well as in vivo. The CagA-activated SHP-2 potentiates ERK MAP kinase activity and regulates both cell morphology and cell growth. SHP-2 also dephosphorylates a cellular substrate and decreases the level of tyrosine phosphorylated cortactine, which is involved in the induction of hummingbird phenotype that is associated with elevated cell mortality. (b) Vacuolating cytotoxin A (vacA) - Vacuolating cytotoxin, VacA is a major virulence factor of H. pylori and causes cytoplasmic vacuolization in gastric epithelial cells. This protein plays an important role in the pathogenesis of both peptic ulceration and gastric cancer [10]. The effect of the toxin was first observed on monolayer cell culture of eukaryote when vacuolization was induced by a cell-free supernatant of H. pylori culture. The activities of VacA toxin include membrane channel formation, effects on integrine receptor-induced cell signalling, disruption of endosomal and lysosomal activity, interference with cytoskeleton dependent cell function, 253
induction of apoptosis and immune modulation [10]. A chromosomal gene of H. pylori encodes the VacA responsible for the above effects. The VacA protein is produced as a 140 kDa protoxin that is cleaved into the 88-kDa mature form when secreted into the extracellular space as soluble protein, but can also remain localized on the surface of H. pylori [75]. Although nearly all H. pylori secrete VacA toxin, there is considerable variation in vacuolating activities among strains. This is due to the sequence variability within the signal region (s) and the middle region (m) of vacA gene [49]. The N terminal known as s of the gene encodes the signal peptide, and occurs as either an s1 or s2 type, whereas the C terminal known as m contains the p58 cell binding domain and exists as an m1 or m2 type. Vacuolating activity is high in s1/m1 genotype than s1/m2 genotype and there is no activity in s2/m2 genotype. Due to high activity in s1/m1 genotype it is more frequently associated with peptic ulceration and gastric cancer. Recently, a new vacA polymorphic site, the intermediate (i) region, has been reported where vacuolation assays showed that i-type determined vacuolating activity among s1/m2 strains [76]. In the mature toxin, the p33 domain near the N-terminus of VacA contains only one strongly hydrophobic region. Three tandem GXXXG motifs (defined by glycine residues at positions 14, 18, 22 and 26) [75] are contained in this region that is characteristic of transmembrane dimerization sequences. VacA has a role in membrane channel formation in epithelial cell and is required for VacA-induced cell vacuolation thereby inducing the release of urea and anion from the host cells. Transcellular permeability is also increased which leads to the release of nutrients and cation. Interestingly, a significant part of toxin remains associated with the outer membrane of H. pylori and is not released into the environment [77]. These toxin clusters are transferred to the host cell surface and exert their toxic action when bacteria contact with host cells. The involvement of specific receptors that mediate the bacterium-cell contact [77] has been suggested by this contactdependent direct delivery mechanism. 6. DIVERSITY OF GENOME Compared to other bacterial species H. pylori genome, displays an unusual degree of diversity. The variability between strains was initially reported by the restriction endonuclease digest analysis of genomic DNA. Using the same technique several researchers have subsequently substantiated a high level of diversity in a number of isolates world wide [6]. A 254
high degree of diversity between strains of the H. pylori has also been confirmed by analyses of the genome by a variety of other molecular techniques. Prominent among these are profiling of rRNA gene [6, 52], fingerprinting based on polymerase chain reaction (PCR) and macro restriction digest profiling by pulsed field gel electrophoresis [78]. Data obtained have clearly indicated the apparent colonization by their own particular genomic variant of H. pylori in most individuals. It has been demonstrated that strains undergo genome rearrangements after they infect a new human host however, the reasons for the genetic diversity among H. pylori strains remain unexplained. The stresses associated with colonization and adaptation to a new environment may be responsible for diversity [78]. A number of factors may be involved in this variation such as the movement of short repetitive DNA sequences, uptake of DNA by natural transformation or silent mutations, which is combined with a high rate of recombination events. The degree of change observed within almost every part of the genome so far analysed cannot be attributed to a single factor and yet the genome size, overall inter-strain DNA sequence homologies, base composition and phenotype appear to be conserved. A high rate of genetic variation due to mutation such as silent point mutation may be the mechanism that enables persistent infection of H. pylori for many years in a hostile environment. Till date no studies have been made for monitoring the evolution of a H. pylori strain over a period of several years in situ in the gastric mucosa. However, evidence suggests that the same genotype was maintained in one strain in an individual who had been subjected to endoscopy on four separate occasions over a period of 9 month. This occurred between and after two different treatment regimens. Laboratory maintained strains show evidence on the stability of the genome even on being subjected to the stress of lyophilization and frequent sub-culturing under laboratory conditions. 7. CONTRIBUTION OF HOST GENETICS It has been reported that not only the characteristics of the pathogen and environmental factors but also host genetics play an important role in determining susceptibility and severity of H. pylori infection. It is known that an array of pro- and anti-inflammatory cytokines regulate this inflammation. Genetic polymorphisms directly influence inter individual variation in the magnitude of cytokine response, the expression levels of gene products by generation or deletion of transcription factor sites or by affecting RNA splicing and subsequent translation. It is speculated that the most relevant candidate genes would be ones whose products were involved in handling the H pylori infection and resulting inflammation. These candidate genes would be 255
prohibitively extensive; the initial search focuses on genes that are most relevant to physiology of gastric and, in particular, gastric acid secretion. Three main gastric phenotypes have been observed, and each is associated with a set of pathophysiologic abnormalities. The most common phenotype by far, is known as simple or benign gastritis phenotype and characterized by mild pangastritis with little disruption of gastric acid secretion [79]. This phenotype is observed in subjects who have no symptoms (asymptomatic) and they develop no serious gastrointestinal (GI) disease. The second phenotype is duodenal ulcer and this phenotype is characterized by an antral-predominant pattern of gastritis with relatively limited acid producing corpus mucosa. This phenotypic subject has high antral inflammation, high gastrin, very high acid secretion and relatively healthy corpus mucosa, all these pathophysiologic changes lead to development of peptic ulcer [80]. The third phenotype is the gastric cancer phenotype, and is the most serious phenotype which is characterized by a multifocal gastric atrophy, corpus-predominant pattern of gastritis and achlorhydria or hypochlorhydria [81]. Interesting part of above observation is that subjects who develop duodenal ulcers are protected from gastric cancer development, suggesting that the 2 outcomes are mutually exclusive [82]. Thus, it is clear that in the presence of H pylori, an endogenous agent is up regulated and has a profound proinflammatory effect and is also acting as an acid inhibitor. Interleukin 1B (IL-1) fits this profile perfectly because it is not only most important proinflammatory cytokines in the context of H pylori infection but also the most powerful acid inhibitor so far known [81]. (i) Role of IL-1 gene cluster polymorphisms in H. pylori-induced gastric damage - The expression levels of gene products directly affect genetic polymorphisms by addition or deletion of transcription factor sites or by affecting RNA splicing and subsequent translation. The metabolism of certain compounds is directly influenced or the expression of immune mediators downstream of the gene is affected indirectly with specific polymorphism. Previous studies laid stress on the role of certain mutations in the gene that encodes cytochrome P450, which affects the metabolism of protein pump inhibitors (PPIs) and antibiotics thus affecting the efficacy of eradication treatment of H. pylori. Secondly the effects of mutations in genes encoding antioxidative proteins such as glutathione S-transferase (GST) have also been reported [83]. However, colonization of H. pylori is not directly affected by these mutations. The complex interplay of proinflammatory and anti-inflammatory mediators control and maintain the chronic active inflammation which is responsible for the pathogenic effects of H. pylori infection [84]. 256
Independent studies conducted in recent years have shown the role of H. pylori in gastric disorders and the genetic polymorphisms that affect the expression levels of these inflammatory mediators. Thus it can be concluded that IL-1 gene is related to increased risk of developing gastric cancer which is caused by proinflammatory genetic polymorphisms. A gene cluster which contains the polymorphic IL-1B (encoding the IL-1 cytokine) and IL-RN (encoding the IL-1 receptor antagonist) genes encode the IL-1 cytokine. The most powerful known inhibitor of acid secretion is IL-1 which is also a powerful proinflammatory cytokine. High-level expression of IL-1 is enabled by the IL-1 gene cluster that contains several polymorphisms, such as IL-1B31C/C, IL-1B-511T/T, and IL-1RN 2/2. This eventually contributes to decline in acid production, which is related to corpus-predominant colonization by H. pylori, resulting the formation of atrophic gastritis, pangastritis, and increased risk of gastric cancer [85, 81, 86, 87]. Polymorphisms in other inflammation-associated genes e.g., the genes encoding tumour necrosis factor alpha (TNF-) and IL-10 show similar effects. TNF- is a proinflammatory cytokine, and TNF-A gene shows several polymorphisms. (ii) Other cytokines - Increased TNF- production, is associated with the TNF-A* 308A genotype which along with IL-1, influences the production of gastrin and consequentially affects acid production by gastric parietal cells [88]. Thus the association of TNF-A*308A genotype with H. pylori gives rise to infection and increases the risk of gastric cancer [86,89]. Likewise, the IL-10 gene haplotypes affect the expression of the anti-inflammatory cytokine IL-10. Higher expression level of IL-10 is related to the GCC haplotype and hence gives rise to antiinflammatory response, whereas decreased level of IL-10 is related to the ATA haplotype which favours a shift toward a proinflammatory response [86,89]. The GCC haplotype favours colonization with more-virulent H. pylori strains, whereas increased risk of gastric cancer is related to the ATA haplotype [86, 89]. Several independent studies have established that vulnerability to development of gastric cancer is increased only two-to three fold by single polymorphisms while the presence of multiple proinflammatory genotypes increases it substantially [86,89]. NFB, a master regulator of pro-inflammatory cytokines and anti-apoptotic signalling molecules, is one of the most well-studied transcription factors activated by H. pylori infection [90]. Overall, the increased risk of development of gastric cancer cannot be attributed to a specific polymorphism, as much depends on the vulnerability to H. pylori. Eventually the disease outcome is jointly contributed to factors like the interaction between the different pro257
and anti-inflammatory polymorphisms, the immune status of the host, and the characteristics of the colonizing H. pylori strain.
CONCLUSION:
Helicobacter pylori is usually acquired in childhood and develops acute infection leading to chronic gastritis in virtually all persistently colonized persons. Eighty to ninety percent infected persons never show any symptoms. Clinical outcome is highly variable and depends on bacterial and host factors. Several methods have been developed and are extensively used for screening and testing the prevalence of H. pylori in patients suffering from various types of gastrointestinal diseases. Excellent methods mostly based on PCR have been very useful in genotypic analysis of various H. pylori strains. Higher acid secretor patients, due to infection and host genetic factor, are likely to develop antral-predominant gastritis, which leads to the appearance of duodenal ulcers. Lower acid secretor patients are more likely to have gastritis in the body of the stomach, which leads to develop gastric ulcer and can initiate a sequence of events that, in rare cases, finally develop in gastric carcinoma.
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Chapter XIII
Toxicity of Silver Nanoparticles: The Flip Side of the Coin

1 1,2,3
Madhu Yashpal, 2Brigesh Shahare, 3Gajendra Singh
Electron Microscope Facility, Department of Anatomy, Institute of Medical Sciences, Banaras Hindu University, Varanasi Corresponding Author: Madhu Yashpal, E.mail: madhuyashpal@gmail.com
ABSTRACT: Silver nanoparticles, which have recently been found to be established

antimicrobial agent, are being used increasingly as vehicles in drug delivery systems, food packaging, therapeutics, wound dressing, detergents or antimicrobial coatings, biosensors, cosmetics etc., are rapidly becoming a part of our daily life in different forms. With the strong growth in commercial applications, it is most likely that AgNPs enter the environment in the course of their production, use or disposal and thus, more and more apprehensions have been raised regarding their potential toxicity and jeopardy to the environment. Regardless of the prevalent use of AgNPs, relatively few studies have been carried out to determine the detrimental consequences of AgNPs exposure. Assessing the risks of AgNPs in human health as well as environment requires an understanding of their mobility, reactivity, ecotoxicity and persistency. The chapter reflects on the sources, exposure, and impacts of AgNPs on the environment in order to generate information appropriate to attempt a risk assessment. Keywords: Nanoparticles, silver, toxicity
INTRODUCTION
Nanotechnology, is the science involved in the design, synthesis, characterization and application of molecules in the nanoscale (i.e., 10-9 m or one-billionth of a meter) size range. [1]. The conceptual keystone of nanotechnology arose in 1959 by the physicist Richard Feynman [2] in his lecture, Theres plenty of room at the bottom. He explored the likelihood of maneuvering material at the scale of individual atoms and molecules, envisaging the whole of the Encyclopedia Britannica written on the head of a pin and foreseeing increasing ability to examine and control the matter at nanoscale.
268
The dramatic expansion of this emerging multidisciplinary science has led to the potential application of nanopartilces (NPs) in various fields e.g., aerospace engineering, nanoelectronics, photonics, catalysis, magnetics, cosmetics, pharmaceutics, environmental remediation and medicines [3]. NPs, by definition, are the structures that have dimension in the 1-100nm range [4]. Compared to their bulk materials, due to their special chemical, electrical, magnetic, mechanical, and optical properties [5], NPs are proven to be the quintessence of the nanoscience. In 2006, the estimated global market was worth US $ 10.5 billion and because of their widespread application, the commercial nanotechnology industry is expected to increase significantly to $3 trillion by 2015 [6]. With the escalating charisma of NPs in a broad array of commercial products, more and more apprehensions have been raised regarding their potential toxicity and jeopardy to the environment [7]. Owing to the specific physiochemical properties of NPs, including elevated reactivity due to a large surface to volume ratio [8], the use of NPs raise concerns about possible detrimental effects. Because little is known about the toxicity profile of NPs, no benchmarks or safe levels have been set for the concern of human health. Also, inadequate availability of toxicological information on NPs renders people to endure a high risk of using these novel materials, especially in biological and medical applications. Silver, a naturally occurring precious metal, known to be used in a wide variety of applications, has some special properties like high electrical and thermal conductivity [9]. Ancient civilizations used this precious metal in medicine, eating utensils, ornaments, money (coins), clothes, building materials and as a disinfectant for water and human wounds [10]. Over the last decade, silver nanoparticles (AgNPs) have received substantial recognition due to their attractive broad continuum of antibacterial activity [11]. At present, it is estimated that of all the NPs in consumer products, AgNPs applications have the highest degree of commercialization [12]. AgNPs are deployed in a wide range of medical and consumer products ranging from cardiovascular implants, central venous catheters, neurosurgical catheters, bone cement or wound dressings [13,14], fabrics, home appliances to water treatment [15,16]. Moreover, the characteristic plasmon-resonance optical scattering properties of AgNPs make them available to be used in bio-sensing [17] and imaging applications [18]. 269
Despite the upcoming prevalent use of AgNPs, comprehensive biological and toxicological information is lacking. In addition, exposure and allied jeopardy to human and environmental health have not been explored systematically. In this chapter, we endeavor to emphasize on the adverse effects of the AgNPs. The aim is to give an overview and scrutinize the various toxic effects of AgNPs. EXPOSURE ROUTES OF SILVER NANOPARTICLES In this booming era of nanotechnology, exposure to AgNPs is becoming more and more complex, consequential in an escalating entre to tissues, cells and biological molecules within the body through various portals e.g. inhalation into the respiratory tract, the gastrointestinal tract (GIT), the skin and the female genital tract [13]. It has been revealed that AgNPs used in our household and industrial merchandises find its way through the waste disposal routes into the wastewater treatment facilities and end up in wastewater sludge [19]. Further escape of these NPs into the effluent pollute the aquatic and soil environment results in direct exposure to humans via skin contact, inhalation of aerosols, contaminated drinking water or indirect exposure from ingestion of sea food such as fish. However, accidental or involuntary contact of AgNPs is most likely to occur via the lungs. The inhaled AgNPs can reach systemic circulation in the body and thus can be disseminated to a number of different target organs including brain, olfactory bulb, liver, kidney, immunological system, and blood vessel walls [20,21]. Ji et al. [20] proposed three feasible routes of their translocation to the blood from tracheobronchial region by a mucociliary escalator, with subsequent ingestion in to the alimentary tract; into lymph nodes & lymphatics and via alveolar epithelial cells to the circulatory system. MECHANISM OF SILVER NANOPARTICLE INDUCED TOXICITY Copious research studies have examined the toxicity of AgNPs on an array of organisms, mammalian, and human cells [17,22-25]. Even though, it is well known that these versatile particles cause detrimental effects by their oxidative and inflammatory [26] nature, subsequently inducing genotoxic [27] and cytotoxic [28] outcomes, the precise toxicity mechanisms are still ambiguous. Toxic effects of AgNP possibly stems from their small size [29] as the tiny size of the NPs makes them highly motile both in the body and the environment [30] and smaller particles, in general, exhibit the greatest toxic effects [31-34]. In addition, shape, concentration, surface 270
chemistry, capping agent and propensity to aggregate are also important factors that may influence the AgNP induced toxicity [35-37]. Another possible means for the AgNPs to cause toxicity is the production of silver ions (Ag+) [24,25,38-40], as they move into the cells leading to the production of superoxide radicals [41] and disruption in the ion efflux system [42]. In addition, other imperative factors accountable for AgNP toxicity include association of AgNPs with cell membranes that might cause physical damage (e.g., pitting) or the subsequent penetration resulting in cell malfunction including cell wall destruction [43-45], stimulation of reactive oxygen species (ROS) via a reaction with oxygen [46,47] that damages cellular constituents causing disruption of cell functions and interaction with specific proteins and/or enzymes inhibiting their activities [48,49]. The production of ROS, whether inside or outside of the cell has been considered as a key factor resulting in toxicity induced by NPs [50]. In contrast to their micro-sized counterparts, NPs are distinguished by a higher surface reactivity and can produce comparatively more ROS than larger particles [51,52]. Augmentation of ROS generation triggers the apoptotic cascade by activating oxidant sensitive signaling pathways, and stimulating inflammation, cytotoxic and genotoxic responses, eventually culminating in cell death [36,37, 53-60]. IMPACTS OF SILVER NANOPARTICLES ON BIOLOGICAL SYSTEMS AgNPs, when in contact with biological systems, can educe a spectrum of impacts on tissues and the novel properties, which make them so attractive in numerous applications, could prove to be deleterious and contribute to their toxicological profile. Inside a cell, these NPs can get metabolized and/or altered and go through a series of processes like binding and reacting with proteins, get phagocytosed, deposited, cleared or translocated ensuing inflammatory, oxidative, genotoxic, and cytotoxic effects. However, there is an inadequate understanding into the prejudicial upshots of AgNP exposure. Cytotoxicity Studies reveal that AgNPs exert perilous cytotoxic effects, most likely through decreasing cell viability and/or increasing apoptosis, in a variety of cells, including peripheral blood mononuclear cells [61] and human cervical cancer HeLa [62,63], human acute monocytic leukemia THP-1 [64], MLO-Y4 osteocytic [63], mouse peritoneal macrophage RAW264.7 [60], human lung carcinoma cell line, A549 [64] and Drosophila melanogaster [65] cell lines. 271
Moreover, AgNPs were also found to be notably cytotoxic on different cultures such as in a monolayer cell culture, a tissue explants culture model and mouse expurgated wound model [66]. More recently, the cytotoxic effects of AgNPs are reported to pilot the impairment of the developmental potential of embryo. Using mouse blastocysts as the assay model, Li et al. [67] reported that AgNPs repress embryonic cell proliferation during the blastocyst stage predominantly via inducing apoptosis in the inner cell mass and trophectoderm. Wei et al. [68] reported that AgNPs induce cytotoxicity in mouses fibroblast cells (L929) with manifestation of severe morphological deformities. Park et al. [60] reported cytotoxicity of AgNPs to mouse peritoneal macrophages derived RAW264.7 cell lines. They described that AgNPs were ionized in the cells to cause cytotoxicity by a Trojan-horse type mechanism by increasing sub G1 fraction indicating cellular apoptosis. AgNPs decreased intracellular glutathione level, increased NO secretion, increased TNF- in protein and gene levels, and increased gene expression of matrix metalloproteinases (MMP-3, MMP-11, and MMP-19). Genotoxicity Health risk assessment of NPs is a sprouting field, genotoxicity being an imperative endpoint to be tested. Despite increasing use of NPs, very little has been done with respect to examine the genotoxicological safety of AgNPs. Recent studies have reported that the AgNPs result in DNA damages such as double strand breaks and disrepair of strand breaks in DNA, resulting in chromosome reorganization and chromosomal aberrations thus disturbing genomic stability and integrity. These NPs were found to induce genotoxic effects in human glioblastoma and fibroblast cells [27], human hepatoma derived cell line HepG2 [23] and in mouse embryonic stem cells and fibroblasts [12]. Moreover, AgNPs are reported to exhibit its genotoxic activity mainly via induction of somatic recombination in the wing imaginal disk cells of Drosophila melanogaster [69]. More recently, Hackenberg et al. [70] reported AgNPs induced inhibitory effect on tissue regeneration by suppressing stem cell migration in mesenchymal stem cells derived from human adipose tissue at high exposure concentrations. In addition, few studies as well revealed that AgNPs treatment induced DNA damaging effects on aquatic [59,71,72] and plant [73,74] cells with impairment of cell-division and causing different effects such as chromatin bridges, stickiness, disturbed metaphases and multiple chromosome breaks.
272
Neurotoxicity Owing to their ability to cross the blood brain barrier (BBB) via systemic distribution [10,75,76] and pass through biological membranes, NPs could progress inside the brain and induce detrimental effects [3,77-79]. NPs can also access the brain by sensory nerve endings embedded either in airway epithelia [80] or in olfactory bulb [3,78], followed by axonal translocation to CNS structures. In addition, NPs may also be taken up directly into the brain by trans-synaptic transport [81]. AgNPs entering via the BBB can accrue in different regions of the brain [82], which may pose adverse effects [83,84], as it has been reported that NPs exposure can induce impairments of normal neurons [85], microglia [86] and even aggravate the process of brain pathology [87]. Recently, AgNPs were found to be toxic by ensuing cell shrinkage and irregular membrane borders and reducing the level of dopamine in a neuro-endocrine cell line (PC-12 cells) [88,89]. Mitochondrial toxicity: The foremost toxicological apprehension is the fact that some manufactured NPs are transported across cell membranes, especially into mitochondria [90,91]. The key event in NP induced cytotoxicity is the mitochondrial perturbation that has significant biological effects together with the commencement of apoptosis and diminished ATP production [92,93]. Several reports have demonstrated a clear deleterious effect of AgNPs on mitochondrial function in the liver, lung, nervous system and kidney [12,31,94,95]. However, how exactly AgNPs gain access to and induce mitochondrial damage is unknown. AgNPs can induce alterations in mitochondrial membrane permeability [96,97] consequential in an uncoupling effect on the oxidative phosphorylation system [98]. Another possibility is that these NPs gain access to mitochondria because of their small sizes or ROS generated at its exterior and might then release redox cycling chemicals that damage the inner membrane [91] or by disrupting the mitochondrial respiratory chain, ultimately resulting in the production of ROS and disruption of ATP synthesis, which in turn cause DNA damage [27,33,68]. AgNPs induced ROS generation cause release of cytochrome c into the cytosol of mitochondria [33,99], which commences a cascade of signaling mechanisms that leads to the activation of caspase 3 and caspase 9 [100,101] and thus apoptosis. Pulmonary Toxicity 273
The NPs are allied with adverse pulmonary and cardiovascular effects following inhalation in humans [102,103]. Airborne NPs can deposit in all regions of the respiratory tract, be taken up by cells, and translocate to sensitive organs via the blood or lymph [52]. Once NPs enter the interstitial air spaces of the lungs, they are quickly taken up by alveolar cells and are likely to induce oxidative stress, DNA damage and inflammation leading to fibrosis and pneumoconiosis, and the underlying mechanisms causing pulmonary toxicity [104]. Unfortunately, there is a lack of information regarding the adverse consequences associated with pulmonary exposure to AgNPs. Prolonged exposure to AgNPs is reported to elicit an inflammatory response within the alveoli and induced alterations in lung function [20,105,106]. Both inhalation and instillation experiments have revealed that AgNPs are taken up by alveolar macrophages and persevere there at least for up to 7 days [21]. These aggregated AgNPs are reported to be cytotoxic to alveolar macrophage cells as well as epithelial lung cells [107]. Scarcely available studies on inhalation toxicity of AgNPs demonstrate that these particles may translocate from their site of exposure, so that they have the potential to accumulate within a number of secondary targets, including the liver, spleen, and brain, following pulmonary exposure, which necessitates their transfer into blood or transport within neurons [36]. Reproductive toxicity: Despite the fact that the NPs might pose potential reproductive harm, there has been little substantive evidence to support or refute these concerns. Review of literature has shown that NPs can translocate systemically from primary sites of access across the blood testes barrier, and get deposited and bioaccumulated in gonads [108-111]. In aquatic species it has been shown that NPs can translocate in the testes after gill absorption of contaminated water [112]. AgNP can induce toxicity to germ line stem cells via reduction in mitochondrial function and induction of membrane leakage and apoptosis [113]. Moreover, AgNPs can also induce DNA damage response in embryonic stem cells and embryonic fibroblasts [12]. Recently, Braydich-Stolle et al. [113] reported that AgNPs can induce a significant decline in mouse spermatogonial stem cells proliferation. Dermal toxicity: The potential of NPs to breach the skin and to diffuse into underlying structures raises a considerable health and safety issue for their topical use. AgNPs are reported to pervade through 274
the intact skin and appreciably through the damaged skin [114] and the intradermal NPs could gain access to systemic distribution through subcutaneous lymphatics [115]. The applications of AgNPs in cosmetics, topical products, surgical prosthesis and anti-microbially active smart textiles" [15,116] have substantially increased the potential for human skin exposure. Review of literature reveals that depending on the quantity of silver coating, fabric quality, pH and sweat formulation, silver could be released from antibacterial fabric products into sweat [117]. Commercial wound dressings containing AgNPs are cytotoxic to keratinocytes and fibroblasts [118-120]. Arora et al. [121] found that AgNP induced cell death and oxidative stress in human fibrosarcoma and skin carcinoma cells. In subsequent studies, it has been demonstrated that AgNPs could enter the fibroblasts and liver cells and cause DNA damage and apoptosis [122]. Gastrointestinal tract toxicity Probably, gastrointestinal ingestion is the most common voluntary route of exposure for AgNPs, because these are employed in widely peddled products for water disinfection and food stabilisation [123] and health maintainers or immuno-boosters. After ingestion, the kinetic mechanism of AgNP translocation is unclear, but it has been demonstrated that the uptake can take place trans-cellularly via normal enterocytes and through paracellular pathways [124,125] or through the intestinal lymphatic tissue [126]. Oral exposure of AgNPs can also cause
noteworthy changes in serum alkaline phosphatase and cholesterol concentration, bile duct hyperplasia with infiltration of inflammatory cells, including eosinophils [94] and other hepatoxic consequences including lymphocytic infiltration and expression of genes related to apoptosis and inflammation [32].
CONCLUSION
AgNPs have emerged as noteworthy class of NPs for a wide range of industrial and medical applications. Despite the promising applications of AgNPs, there are still doubts regarding their safety. These are reported to induce toxicity in a range of organs including the lung, liver, brain, vascular system and reproductive organs. Probable mechanisms of AgNPs induced toxicity include generation of ROS, oxidative stress, DNA damage and apoptosis. These initial findings assure that AgNPs do pose a certain degree of potential hazards to environmental and human health. The preliminary results necessitate further comprehensive studies to clearly 275
elucidate the mechanisms of AgNP induced toxicity. Furthermore, the field of AgNPs toxicology, besides investigations on their adverse effects, also necessitates uninterrupted monitoring and risk assessment.
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Chapter XIV SUSPENSION ARRAY TECHNOLOGY: A MULTIPLEXING APPROACH IN IMMUNODIAGNOSTICS

*1 1
Manoj Kumar and 1Subhash V. Kapre
Research and Development Department, Serum Institute of India Research Foundation 212/2, Hadapsar, Pune, Maharashtra - 411028 INDIA Corresponding author: Manoj Kumar, MVSc, PhD In-Charge, Immunobiology and Molecular Research Laboratory Email: manoj.kumar@seruminstitute.com
*
ABSTRACT: Analytical assays are one of the important part of biomedical research,
diagnostics and prophylactics. Various qualitative and quantitative assays have been developed till date having respective advantages and disadvantages. A useful assay should be simple, specific, sensitive, fast, repeatable and cost effective. In recent times there is a focus on multiplexing assays due to an extensive increase in the number of analytes being required to be tested from the same sample e.g. cytokine profiles, serum antibody titers, detection of pathogens, gene expressions and single nucleotide polymorphism etc. The present chapter focuses on suspension array technology for multiple analyte profiling in a single assay with a major focus on immunodiagnostics. The basics of technology, its applications in immunobiology and benefits of the technology are discussed as compared to other analytical tools.
INTRODUCTION:
During the last couple of decades, there has been major advances in the field of the biomedical science especially biotechnology and microbiology, various diseases have been targeted for prophylaxis and control, human genome sequence has been completed and there has been upsurge in newer diagnostic tools. Keeping in view the huge increase in the demand of diagnostics and the new vaccine development, the analytical methods have improved significantly during this time. The conventional assays had disadvantages of being less sensitive, time consuming and laborious which have been mostly replaced by advanced and better assay systems e.g. Enzyme-linked immunosorbant assays (ELISAs), fluorescent antibody assays, radio 288
immunoassays, real time quantitative Polymerase chain reactions (qPCR) assays. With the advent of technologies, several multiplexing assays have been developed, one of which are microsphere based flowcytometric assays. Various manufacturers have come up with multiplexed bead based flowcytometric assays for applications in diagnostics, biopharmaceutical industry and medical research. A suspension array multiple analyte profiling (MAP) technology developed by Luminex Corporation is one of the emerging technology in this field. The technology has several advantages over the conventional and competitor techniques and is described in the following sections. Luminex x-MAP (Suspension array) technology is built on proven existing technologies viz., flow cytometry, microspheres, lasers, digital signal processing and traditional chemistry that have been combined in a unique way. The technique has been highly useful in dealing with the issues related with conventional assays in terms of speed, economy and sensitivity. The technique started as FlowMetrix System [1] and several advancements have been seen in the technology over several years. In this technique Luminex 100/200 or FLEXMAP-3D system (or similar) controlled by a software is used in conjunction with the Luminex microspheres of 5.6 or 6.5 micron diameter. The basic mechanism used is, microspheres which are composed of 100 (500 in case of FLEXMAP-3D) different combinations of 2-3 different fluorochromes (Figure 1, 2), each of which is coated with a particular analyte (antigen/antibody/Nucleic acid fragment etc.; Figure 3). Various chemistries have been used for coupling of the analytes to the carboxylated microspheres [2-8], the most common being an EDC (1-ethyl-3-(3After reaction with the
dimethylaminopropyl)-carbodiimide hydrochloride) based reaction.
specific antibody and secondary antibody or other combination of reactants, these microspheres are made to flow through the needle of the instrument. Samples from microplate wells are aspirated by a sampling probe and transported to the flow cytometer through a flexible sampling line. An auto sampler moves the probe from well to well while a continuously running peristaltic pump causes the alternating uptake of fluid samples and air. A rotating cell suspension system periodically rotates microplate to keep sample particles (microspheres) in suspension during any required pre-analysis incubation steps. The microspheres flow through the fluidics system to the cytometer where the laser falls on the beads and the emitted fluorescence is measured by the detector. The instrument is equipped with two types of lasers, red laser and green laser (Figure 4). A 635 nm red diode laser excites the two fluorochromes contained within the microspheres 289
and a 532-nm, yttrium aluminum garnet (YAG) laser excites the reporter fluorochrome (Rphycoerythrin, Alexa 532, or Cy3) bound to the microsphere surface. When the red laser falls on the beads it emits a fluorescence which is detected by the detector system and helps to segregate different types of beads. Microspheres size, determined by 90-degree light scatter, is used to eliminate microsphere aggregates from the analysis. Then the beads are subjected to green laser beam which gives the Median Fluorescence Intensity (MFI) when the emitted florescence from the reporter dye attached to the secondary antibody/primer tag falls on the detector. MFI is proportional to concentration of analyte in the sample. The dyes on the microspheres and reporter dye e.g. Phycoerythrin (PE) emits different wavelengths so there is no chance of overlapping of fluorescence. High-speed digital signal processing classifies the microsphere based on its spectral address and quantifies the reaction on the surface. The software controls every activities of the hardware [9-12].
Figure 1: Luminex microspheres (With permission from Luminex Corporation)
Latest addition to the series of Luminex instruments is MagPix instrument which provides LED/Image-Based Analysis. The sample is aspirated into the instrument, magnetic beads enter magnetic chamber to form a monolayer for imaging. The red LED (635 nm) excites 290
internal bead dyes and determines which analyte is being measured, whereas, green LED (525 nm) excites detection fluorophores and measures the expression. The fluorescence is identified and quantified with CCD imager.
Figure 2: Typical Luminex 200 Bead map: Bead Region Classifications showing 100 different identities (With permission from Luminex Corporation) Luminex x-MAP microspheres: The Luminex 100/200 (Luminex Corporation) or similar system performs multiplexed analysis of up to 100 (500 in case of FLEXMAP 3D) different reactions simultaneously by using a flow cytometer and digital signal processor to perform realtime analysis of multiple microsphere-based assays. The three major components of the system are a bench-top flow cytometer, microspheres and computer hardware and software. Microspheres serve as the vehicles for molecular reactions. The microspheres are 5.6 or 6.5 m polystyrene microspheres that bear carboxyl functional groups on the surface. Microspheres of this size provide sufficient surface area for covalent coupling of 12 x106 target molecules per microsphere. In addition, the small size allows the microspheres to remain in suspension for several hours, which is more than sufficient for assay setup and analysis, and also provides near291
fluid-phase reaction kinetics. The microspheres are available in 100 (500 in case of FLEXMAP 3D) distinct sets that are classified by the flow cytometer by virtue of the unique orange/red emission profile of each set. The carboxylated microspheres can be covalently coupled to virtually any amine-containing target molecule through surface carboxyl groups (Figure 3). Various types of microspheres are available based on the application and type of analyte required to be coupled to the microsphere. They are MicroPlex microspheres, MagPlex Microspheres, xTAG Microspheres, SeroMAP Microspheres and LumAvidin Microspheres
(http://www.luminexcorp.com/applications).
Figure3: Coupling of an oligonucleotide to Luminex Microspheres (With permission from Luminex Corporation) Advantages of x-MAP technology: In order to be a useful assay, an analytical method should have maximum of the following characteristics. Specificity is very important parameter in most protein-based assays. Higher sensitivity facilitates earlier diagnosis and helps in reducing the sample requirement. The simpler the assay, the less will be the chances to go wrong which is important for clinical diagnostic laboratories. Assays that eliminate the need for washing steps are particularly advantageous. Reliability and reproducibility are of significant importance. The ability of an assay to measure multiple analytes at the same time has become a major consideration in the analyses where one need to quantify multiple analytes from the same sample e.g. cytokine estimations. A rapid and cost effective assay is always a preferred choice for the diagnostic facility.
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The x-MAP technology has several benefits over the similar techniques in terms of the following: 1. Cost and labor is reduced by multiplexing 2. Flexible multiplexing in the range of 1-500 analytes 3. Favorable reaction kinetics of liquid bead array approach with high dynamic range 4. Smaller sample requirement due to multiplexing 5. Liquid reaction kinetics gives faster, more reproducible results than with solid, planar assays
PMT 532 nm
635 nm
Figure 4: Optics of Luminex 200 instrument (Detection of identity of microsphere and fluorescence intensity by red and green laser beams). (With permission from Luminex Corporation)
APPLICATIONS OF x-MAP TECHNOLOGY

Keeping in view the various benefits of the x-MAP technology, it has been utilized for several application successfully over several years in various fields of biomedical research including drug-discovery, diagnostics and basic research [8-9, 13-24]. Table 1 shows in brief the applications of the Luminex technology. The details of various major applications are described in the following sections.
293
x-MAP IN IMMUNODIAGNOSTICS Various types of immunoassays are required in the vaccine development, its evaluation, biomedical research and diagnostics. Briefly, vaccines are the antigenic preparations, which stimulate bodys immune system to produce antibody against the antigen injected. Vaccination is the administration of a vaccine to stimulate a protective immune response that will prevent occurrence of disease in the vaccinated person, if contact with the corresponding infectious agent occurs. Presently, there are number of vaccines available in the market. We can divide the major vaccine preventable disease in two parts, that are bacterial vaccine preventable disease, for example diphtheria, tetanus, pertussis, pneumonia, meningitis, cholera, influenza, and viral vaccine preventable diseases such as poliomyelitis, influenza, hepatitis-B, rabies, measles, mumps, rubella etc. Few of the vaccines for protozoal, parasitic and mycotic diseases are also developed or are under development. With the increase in the potential vaccine candidates, there is a trend towards development of multivalent vaccines e.g. Trivalent, quadruvalent, Pentavalent and hexavalent etc. Further, there are vaccines against several serotypes/strains of the same pathogen e.g. Bacterial pneumonia, rotaviruses etc. With this approach, multiplexing technique has become an essential requirement for vaccine development and evaluation. The assays can be categorized in to antibody and antigen screening assays. Screening antibodies: Efficacy studies of a vaccine are highly required to evaluate its potency in protecting from the disease. Normally with response to the different antigens/serotypes different type of IgG are formed by the immune system of the affected host. To measure the concentration of IgG, different types of antibody titration methods are available. Among them few of the conventional methods are gel diffusion method, immunoelectrophoresis etc. where diffusion lines are formed due to the antigen (Ag)-antibody (Ab) reaction [12]. RIA (Radioimmunoassay) is also a sensitive method to estimate antibody, in which the antigen labeled with radio isotopes and the precipitation formed by Ag-Ab reaction is estimated by autoradiography but it has potential health hazards. Enzyme Linked Immunosorbant Assay (ELISA) is another method for quantitative estimation of antibody, in which the microtiter plate is coated with Ag followed by reaction with antibodies in serum and a secondary Ab conjugated with an enzyme, and the substrate. The reaction in terms of optical density is then measured with an ELISA reader.
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Conventional Ab titration methods have a lot of disadvantages. 1. Low sensitivity 2. Require large volume of sample 3. Huge time consumption 4. Laborious and tedious method 5. A number of critical steps 6. A number of washing steps 7. If the assay is done for multi-serotype vaccine then it is not cost effective 8. Safety issues Multiplexed immunoassays have been a focus for development and evaluation of multivalent vaccine which help get rid of the disadvantages of the conventional monoplex assays. Using x-MAP technology, multiple immunoassays can be performed simultaneously on an array of microspheres as long as the conditions of the assays (incubation times, sequence of reagent additions, washes) are the same or can be combined. A basic format of IgG estimation assay is presented in the figure 5. The technology has been applied by various authors for development of assays for simultaneous identification and quantitation of antibodies in sera, plasma, culture supernatants etc. A multiplexed indirect immune-fluorescence assay was developed for antibodies to
Haemophilus influenza type b (Hib) polysaccharide and the toxoids of Clostridium tetani (Tet) and Corynebacterium diphtheriae (Dip). The multiplexed Luminex assay was compared to individual ELISAs for the same analytes. The correlations (r2) between ELISA and Luminex assay of the 81 samples were 0.96, 0.96, and 0.91 for Tet, Dip, and Hib, respectively (Pickering et al., 2002a). A fluorescent covalent microsphere immunoassay (FCMIA) method was
developed for the simultaneous (multiplexed) measurement of immunoglobulin G (IgG) antibodies to 23 pneumococcal capsular polysaccharide (PnPS) serotypes i.e., PnPSs 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F. The minimum detectable concentrations for the 24 multiplexed (PnPS and C-PS) FCMIAs ranged from 20 pg/ml for PnPS 3 to 600 pg/ml for PnPS 14 [4].
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Table 1: Various fields of application of x-MAP technology
Sl. No.
Field of application
Targets (Examples) Detection and diagnosis of Infectious diseases (Bordetella pertussis, M. tuberculosis, Clostridium spp., Epstein Barr viruses, Influenza viruses, Rota viruses, Toxoplasma infections etc.) Vaccine testing (Vaccine efficacy surveillance, immunology research etc.) studies, immunization
Immunodiagnostics
Allergy testing (Alternaria, Asp f 1, Bermuda Grass, Bla g 1, Der f 1, mountain cedar, wheat, timothy grass etc.) Autoimmune diseases (Chromatin, Histone, Gliadin, SSA etc.) Human Leukocyte Antigen testing (Donor specific antibody testing, HLA class I or II testing) Newborn screening Gene expression profiling (BCL2, IFN-gamma, IL-10, NKFB1, VEGF genes etc.)
Genomic research
Genotyping (HPV genotyping, Signet genotyping panel etc.) Cystic fibrosis (Testing for various mutations associated with CF) Cytochromes Cytokines, Chemokines and growth factors (ILs, MCPs, MIPs, EGFR, IFNs, TNF, V-CAM etc) Cardiac markers (Adiponectin, Cystatin C, ILs, SGOT, VCAM-1, Fibrinogen etc.)
Protein expression Cancer markers (AFP, CAs, CEA, f-PSA etc.) profiling Isotyping (IgA and IgG isotyping) Metabolic markers (Apolipoproteins, Collagens etc.) Cellular signaling (Akt, CRK, Erk, EGFR etc.) Neurobiology Testing of bio-warfare pathogens and bio-threat agents (Bacillus anthracis, Yersinia pestis etc.)
Biodefence/ Environmental
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A rapid and simple method for the simultaneous quantitation of serum IgG antibodies specific for Neisseria meningitidis serogroups A, C, Y, and W-135 was developed and evaluated. Four bead sets were generated, each conjugated with one of the meningococcal capsular polysaccharides (A, C, Y, or W-135) and serologically assessed by the use of anti-meningococcal international reference sera. Cross-reactivity studies demonstrated no inhibition between monoplex and multiplex assays, and the assay was linear over a 24-fold serum dilution range. Inhibition studies demonstrated that the assay is specific, with <25% heterologous inhibition occurring [25]. Lal et al. [26] described a rapid and simple method for the simultaneous quantitation of IgG to nine pneumococcal serotypes (1, 4, 5, 6B, 9V, 14, 18C, 19F, 23F). Comparison of monoplex and nonaplex assays revealed no evidence of microsphere interference. The nonaplex assay was shown to be specific with <30% heterologous inhibition occurring and assay sensitivity was high with the Limit of detection ranging between 32.3 and 109.7 pg/ml. The assay was shown to be repeatable and conjugated microspheres were shown to remain stable over 12 months. There was a good correlation of the nonaplex assay with the ELISA. Finally, four bead sets coated with meningococcal polysaccharides (serogroups A, C, Y and W135) were added into the nonaplex assay, with no effect on the pneumococcal or meningococcal IgG levels generated. A multiplex system was developed for measuring antibodies against 9 malarial vaccine-candidate antigens, including recombinant proteins from 2 variants of merozoitesurface protein 142 (MSP-142), 2 variants of apical merozoite antigen-1 (AMA-1), erythrocyte binding antigen-175 (EBA-175), merozoite surface protein-3 (MSP-3), and peptides from the circumsporozoite protein (CSP), ring erythrocyte surface antigen
(RESA) and liver-stage antigen-1 (LSA-1). Plasma samples were screened by SAT in a mono- and multiplex format and by ELISA. Optimal amounts of protein required to perform the SAT assay were 10-100-fold less than that needed for ELISA. Excellent agreement was found between the single or multiplex formats (r2 >0.96), even when two variants of the same antigen were used. The multiplex assay was rapid, reproducible, required less than 1 l of plasma and had a good correlation with ELISA [27]. A 13-plex assay for quantitation of pneumococcal antibodies was optimized which showed satisfactory correlation with ELISA [20]. The multiplexed assay has been used for 297
screening the human sera for antibodies against 19 of the pneumococcal serotypes in the Indian population [18]. Further, the technique has been successfully utilized for screening of the culture supernatants from hybridoma cultures during development of various monoclonal antibodies [19]. Various multiplexed pneumococcal IgG quantitation kits are being explored for their use in vaccine efficacy studies [8]. In addition to antibody detection in vaccine development, IgG levels against 6 extractable nuclear antigens associated with collagen vascular diseases were measured by a multiplexed assay in various samples. The results of multiplexed assay corroborated well with those from ELISAs and the results could be used for diagnosis and prognosis of the disease [15].
Standard
Unknown Sample
Phycoerythrin Secondary Antibody
Known antibody Unknown antibody Antigen Polystyrene Bead
Figure 5: Schematic diagram for identification and quantitation of antibody in a serum sample 298
Screening antigens: In addition to the antibody estimation for determining efficacy of various vaccines, vaccine manufacturing process requires highly sensitive analytical tools for antigen estimation also for process development e.g. Fermenter harvest yields, purification process yields and antigen identification and quantitation in the final vaccine vials. Further, the antigen identification and titration is of significant importance in the clinical sample sent to the diagnostic laboratories. Similar to the antibody assays, the multiplexed assays impart big advantage over conventional time consuming and less sensitive assays for antigen identification and quantitation/titration.
Figure 6: Typical standard curve during identification and quantitation of antibody in a serum sample There are two most common formats of antigen estimation assays viz., sandwich assay and competitive inhibition assay. In a typical sandwich assay, an antigen is captured by the antibody on surface of microsphere and detected by a secondary antibody conjugated with a fluorochrome (Figure 7). On the other hand, in a competitive inhibition assay, an unknown antigen is pre-adsorbed with the known antibody and the reduction the signal of antibody due to pre-adsorption with homologous antigen is determined by putting the mixture in an antibody quantitation assay. Lower the fluorescence signal (MFI) indicates higher the content of antigen in a sample (Figure 8). For quantitative 299
sandwich immunoassays, standard curves are optimized one analyte at a time, with the capture antibody coupled to the microsphere and the detection antibody bound to the reporter. Often microsphere-based assays can be performed with no wash steps if the background and potential interfering substances in the samples are minimal. Since each microsphere population is distinguished by size and/or spectral address, the reporter intensity relates to the quantity of analyte bound and thus establishes the relative concentration of that analyte. Commercial antibody pairs optimal for ELISA development may not always be optimal for multiplexed microsphere immunoassays. Antibodies should be tested to find the best pairs, be matched for affinity, and not be reactive with the same epitome. Once optimized, the several individual assays can be combined into the reaction volumes of one assay. The fluorescent intensities of each fluorochrome are measured and, if quantitative, the assay data are transformed with analytical software. A measure of viral load was developed by covalently coupling adenovirus antibodies to microspheres and incubating with DNA specific fluorophores. The MFI was measured from a standard curve of fluorescent intensity versus viral concentration. Various groups have reported bacterial serotyping of multiple strains of Streptococcus pneumoniae. A multiplexed assay has been used by Health protection agency of United Kingdom for detection of 14 pneumococcal serotypes by a competitive inhibition assay format utilizing a polyclonal serum. The assay was found to be simple, robust and repeatable as compared to conventional techniques. Validation of the assay was performed with a selection of serotype specific monoclonal antibodies and the assay was found to be specific [28]. Similar assays are being developed for identification serotype specific antigens at Serum Institute of India .Fluorosphere-based analyses would be applicable for disease surveillance, vaccine development and surveillance, screening of donated blood, and other high throughput screening procedures [29]. Quantitation of Cytokines: Cytokines, chemokines, and growth factors (hereafter referred to collectively as cytokines) play an important role in a wide range of physiological processes including immune response, inflammation, hematopoiesis, and carcinogenesis. Changes in levels of cytokines in the body fluids have been linked to various ailments making them important from diagnostic point of view. Cytokines are 300
excellent candidates for multiplexed analysis because of their complex inter-relationships in immunologic and hematologic functions. Fluctuations in the level of one cytokine often induce changes in others. High levels of circulating proinflamatory cytokines which may be predictive of congestive heart failure and myocardial infarction have been associated with chronic heart failures [30]. A more realistic indication of the complexity of the cellular interactions would include measurements of multiple cytokines at any time point. Thus, the rates of secretion of these analytes in a variety of physiological conditions are best measured simultaneously in identical reaction formats. The selection of monoclonals for multiplexed assays has been focused to get the comparable results with the conventional ELISAs [31]. The advantages and protocol for multiplexed cytokine estimation in human serum and culture supernatants have been described since old [32, 13, 14]. The assays setup is similar to the sandwich assay format shown in Figure 8. Multiplexed microsphere arrays for cytokines developed by commercial vendors or independent laboratories are primarily two-site immunoassays. Relatively high-affinity matched pairs of antibodies for ELISAs for at least 50 human cytokines and related molecules are available from commercial vendors. The limit of sensitivity of these commercial antibody pairs falls in the low picogram range. Multiplexed arrays for various number of cytokines are available based on the experimental requirements e.g. Determining type of T-helper cell response (Th1 or Th2) etc. Simultaneous evaluation of multiple immune mediators was performed by Luminex MAP technology with advantages of higher throughput, smaller sample volume, and lower cost. Ninety-six specimens from women over the course of their respective pregnancies were evaluated for cytokine concentrations using commercially available ELISA kits and commercially available Luminex MAP kits according to the manufacturers directions. Correlations between data sets were evaluated using Pearsons correlation coefficient (r). Excellent correlations were demonstrated for IL-1, IL-4, IL-5, IL-6, IL-10, IFN , and TNF , in contrast to IL-12 p70 and IL-13 [33]. The cytokine levels were estimated in the acute wound fluid by a 27-plex assay and IL-1 was found to be the most potent inducer of signal molecule production [34].
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HLA Testing: Flow cytometry has significantly impacted HLA testing. Microspheres coated with HLA class I and class II antigens have been used to test for HLA antibodies prior to transplant. This technology has recently been implemented on the Luminex platform and has the potential for DNA-based HLA typing, as well as testing for antibodies to specific HLA antigens [35]. Autoimmune testing: The first microsphere-based multiplexed immunoassay application that has received FDA approval was a kit for autoimmune disease testing. IgG antibodies to the markers SSA, SSB, Jo-1, histone, Sm, RNP, Centromere B, and Scl-70 are included in the kit [5].
Phycoerythrin Detection antibody
Unknown Antigen
Capture Antibody Polystyrene Bead
Figure 7: Identification and quantitation of antigen by sandwich assay in a sample Protein phosphorylation assay: Traditional methods for measuring phosphorylated proteins include Western blot analysis with immunologic or isotopic identification of the 302
modified proteins. Both procedures can involve several days of sample processing. Micro-particle based capture and detection of phosphorylated proteins is a rapid, quantifiable flow cytometric procedure. Phosphorylation of the signaling protein ERK-2 has been identified with antibodies specific for the phosphorylated protein [36,5]. Diagnosis of Cystic Fibrosis: A suspension array hybridization assay was developed for detection of 31 mutations and polymorphisms in the cystic trans-membrane conductance regulator gene using x-MAP technology. The oligonucleotide capture probes and PCR amplification primers were designed and the probes were coupled to carboxylated microspheres followed by hybridization of coupled microspheres to oligonucleotide targets and detection of the multiplexed-PCR amplified targets by direct hybridization (Figure 9) to probe coupled microspheres [37].
Phycoerythrin Secondary Antibody Primary antibody Free Primary Antibody
Antigen Polystyrene Bead Unknown Antigen
Figure 8: Identification and quantitation of antigen by competitive inhibition assay in a sample
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Antigen identification by hybridization assays: In addition to the antibody based assays, the antigenic load has been quantitatively estimated for Salmonella and other bacterial pathogens by a hybridization of sample DNA PCR product to the microspheres coupled to the specific oligonucleotide targets [38]. A universal primer-multiplex PCR system was developed and applied for simultaneous detection of Escherichia coli, Listeria monocytogenes and Salmonella spp. [17]. Similarly low levels of various pathogens including E. coli, Bacillus cereus, Salmonella enterica and Staphylococcus aureus have been detected by x-MAP technology [22]. The xTAG respiratory viral Panel developed by Luminex corporation has been used in the characterization of pandemic influenza A (H1N1) specimens [39,24] Single Nucleotide Polymorphism (SNP) Genotyping: SNP genotyping requires methods that are accurate, high throughput, and cost effective. Typical SNP genotyping technologies included solid supports (gels, chips, glass slides), as well as mobile supports, such as mass spectrometry and capillary electrophoresis. Flow metric analysis of fluorescent microspheres has been identified as a novel technology platform for SNP genotype determination. Its advantages include rapid data acquisition (up to thousands of data points per second), excellent sensitivity, and multi-parametric fluorescence analysis that permits multiplexed analysis [40]. One approach to microsphere-based SNP genotyping is enzymatic allele discrimination by oligoligation assay (OLA) or single base chain extension (SBCE). In these methods, a fluorescent reporter molecule is enzymatically added to a capture probe to determine the SNP genotype. The three steps in the genotyping process are 1) allele discrimination by thermal cycling of PCRamplified genomic DNA in a solution-based enzymatic reaction, 2) capture of the enzymatic reaction products by hybridization of DNA sequences (ZipCodes) on the capture probe with complementary sequences on the surface of the microsphere, and 3) flow cytometric analysis of the microspheres to identify both the microsphere population and the fluorescent signal associated with a genotype [41]. The ZipCode concept permits use of a defined set of optimally cZipCode-coupled microspheres for different uses of SNPs. The same ZipCode sequence can be incorporated into the design of new capture probes as new SNPs are added to the assay. Other approaches to SNP genotyping have included direct or competitive hybridization of PCR-amplified products to 304
complementary sequences coupled to microspheres. These approaches require a unique oligonucleotide-coupled microsphere population to match each SNP allele and can require an elevated temperature during the flow metric analysis [5]. The advantages of microsphere-based SNP genotyping include greater flexibility than static platforms such as the chip, excellent accuracy at a reasonable cost, and increased throughput by multiplexing.
TARGET
AMPLIFY
DENATURE
HYBRIDIZE
DETECT
Figure 9: Schematic Microsphere based direct hybridization assay format (With permission from Luminex Corporation) Several other applications and different markers have been targeted using this technology. For more information refer http://www.luminexcorp.com/applications.
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CONCLUSIONS:
The suspension array based x-MAP technology is one of the emerging multiplexing techniques in the field of biomedical sciences. There has been a series of advancements in the technology over several years i.e. shifting from low throughput FlowMetrix to Luminex 100, Luminex 200, FLEXMAP 3D and most recently LED/CCD based MagPix system. The technique has got several advantages over the conventional techniques for various applications such as immunoassays, diagnostic assays and gene profiling studies etc. in terms of speed, sensitivity, flexibility, accuracy, specificity and high throughput. As with any high-throughput method that generates data, the challenges include integrating a robust information-management system to handle the deluge of data and extract reliable information. The technique has huge potential to the biomedical research, diagnostics and vaccine industry in the years to come. REFERENCES: 1. Fulton, R.J., McDade, R.L., Smith, P. L., Kienker, L.J., & Kettman, J. R. Jr. (1997). Advanced multiplexed analysis with the FlowMetrix Chemistry, 43(9), 1749-1756. 2. Pickering, J.W., Martins, T.B., Schroder, M.C. & Hill H.R. (2002a). Comparison of a multiplex flow cytometric assay with Enzyme-Linked Immunosorbant Assay for quantitation of antibodies to Tetanus, Diphtheria, and Haemophilus influenzae Type b. Clinical and Diagnostics Laboratory Immunology, 9(4), 872-876. 3. Pickering, J.W., Martins, T.B., Greer, R.W., Schroder, M.C., Astil, M.E., Litwin, C.M., Hildreth, S.W. & Hill, H.R. (2002b). A Multiplex fluorescent microsphere immunoassay for antibodies to pneumococcal capsular polysaccharide. J. Clin. Pathol., 117, 589-596. 4. Biagini, R.E., Schlottmann, S.A., Sammons, D.L., Smith, J.P., Snawder, J.C., Striley, C.A.F., MacKenzie, B.A. & Weissman D. N. (2003). Method of simultaneous measurement of antibodies to 23 pneumococcal capsular polysaccharides. Clinical and Diagnostic Laboratory Immunology, 10 (5), 744750.
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5. Kellar, K.L. & Iannone, M.A. (2002). Multiplexed microsphere-based flow cytometric assays. Exp. Haematol., 30, 1227-1237. 6. Lal, G., Balmer, P., Stanford, E., Martin, S., Warrington, R. & Borrow, R. (2005). Development and validation of a nonaplex assay for the simultaneous quantitation of antibodies to nine Streptococcus pneumoniae serotypes. Journal of Immunological Methods, 296, 135-147. 7. Schlottmann, S.A., Jain, N., Chirmule, N. & Esser, M.T. (2006). A novel chemistry for conjugating pneumococcal polysaccharides to luminex
microspheres. Journal of Immunological Methods, 309, 75-85. 8. Whaley, M.J., Rose, C., Martinez, J., Laher, G., Sammons, D.L., Smith, J.P., Snawder, J.E., Borrow, R., Biagini, R.E., Plikaytis, B., Carlone, G. M. & RomeroSteiner, S. (2010). Interlaboratory Comparison of three multiplexed bead-based Immunoassay for measuring serum antibodies to pneumococcal polysaccharides. Clin. Vaccine Immunol., 17(5), 862-869. 9. Chen, R., Lowe, L., Wilson, J.D., Crowther, E., Tzeggai, K., Bishop, J.E. & Varro, R. (1999). Simultaneous quantification of six human cytokines in a single sample using microparticle based flow cytometric technology. Clin. Chem., 45,1693-1694. 10. Dunbar, S. A. (2006). Applications of Luminex xMAP technology for rapid, highthroughput multiplexed nucleic acid detection. Clinica Chimica Acta, 363, 71-82. 11. Mohamed, F.E. & McCoy, J.P. (2006). Multiplex bead array assays: Performance evaluation and comparison of sensitivity to ELISA. Methods, 38(4), 317-323. 12. Lim, C.T. & Zhang, Y. (2007). Bead-based microfluidic immunoassays: The next generation. Biosensors and Bioelectronics, 22, 1197-1204. 13. Vignali, D.A.A. (2000). Multiplexed particle-based flow cytometric assays. Journal of Immunological Methods, 243, 243-255. 14. Kellar, K.L. & Douglass, J.P. (2003). Multiplex microsphere-based flow cytometric immunoassays for human cytokines. Journal of Immunological Methods, 279, 277-285. 15. Martins, T.B., Burlingame, R., von Muhlen C.A., Jakowski, T.D., Litwin, C.M. & Hill, H.R. (2004). Evaluation of Multiplexed Fluorescent microshpere 307
immunoassay for detection of autoantibodies to nuclear antigens. Clinical and Diagnostics Laboratory Immunology, 11 (6), 1054-1059. 16. Pickering, W.J., Hoopes, J. D., Groll, M. C., Romero, H. K., wall, D., Sant, H., Astill, M.E., and Hill, H.R. (2007). A 22-plex chemiluminescent Microarray for Pneumococcal antibodies. Am. J. Clin. Pathol., 128, 23-31. 17. Yuan, Y., Xu, W., Zhai, Z., Shi, H., Luo, Y., Chen, Z. & Huang, K. (2009). Universal primer-multiplex PCR approach for simultaneous detection of Escherichia coli, Listeria monocytogens, and Salmonella spp. in food samples. Journal of Food Science, 74(8), M446-52. 18. Kumar, M., Samantray, S., Joshi, A., Kulkarni, P., Raut, S. & Kapre, S. (2010a). Seroprevalence of antibodies against pneumococcal serotypes in healthy Indian population. Proc. of 7th international symposium on pneumococci and pneumococcal diseases, Tel Aviv, Israel, March 14-18, 2010. 19. Kumar, M., Patni, S., Khokale, P. Joshi, A. & Kapre, S. (2010b). Rapid hybridoma screening by suspension array technology. Proc. of 8th Planet xMAP Europe Symposium, Vienna, Austria, 20-21st October, 2010.
20. Elberse, K.E.M., Tcherniaeva, I., Berbers, G.A.M, & Schouls, L.M. (2010). Optimization and application of a multiplex bead-based assay to quantify serotype specific IgG against Streptococcus pneumoniae polysaccharides: Response to the booster vaccine after immunization with pneumococcal vaccine. Clinical and Vaccine Immunology, 17 (4), 674-682. 21. Vistnes, M., Christensen, G., & Omland, T. (2010). Multiple cytokine biomarkers in heart failure. Expert Rev. Mol. Diagn., 10 (2), 147-157. 22. Battaglia, A., Schweighardt, A.J., & Wallace, M.M. (2011). Pathogen detection using a Liquid Array Technology. J. Forensic Sci. .(e-publication). 23. Zivcec, M., Safronetz, D., Haddock, E., Feldmann, H. & Ebihara, H. (2011). Validation of assay to monitor immune response in the Syrian golden hamster (Mesocricetus auratus). Journal of Immunologicals Methods, (e-publication). 24. McCloskey, C.B., Kraft, C.S., Ingersoll, J.M., Hill, C.E., Burd, E.M. & Caliendo, A.M. (2011). Characterization of 2009 pandemic influenza A (H1N1) specimens 308
7-valent conjugate
with a positive hamagglutinin1 (H1) signal in the Luminex xTAG(R) respiratory viral panel (RPV) assay. J. Clin. Microbiol. .(e-publication).
25. Lal, G., Balmer, P., Joseph, H., Dawson, M. & Borrow, R. (2004). Development and evaluation of a tetraplex flow cytometric assay for quantitation of serum antibodies to Neisseria meningitidis serogroups A, C, Y, and W-135. Clinical and Diagnostic Laboratory Immunology, 11 (2), 272-279. 26. Lal, G., Balmer, P., Stanford, E., Martin, S., Warrington, R. & Borrow, R. (2005). Development and validation of a nonaplex assay for the simultaneous quantitation of antibodies to nine Streptococcus pneumoniae serotypes. Journal of Immunological Methods, 296, 135-147. 27. Fouda, G.G., Leke, R.F.G., Long, C., Druilhe, P., Zhou, A., Taylor, D.W. & Johnson, A.H. (2006). Multiplex assay for simultaneous measurement of antibodies to multiple Plasmodium falciparum antigens. Clinical and Vaccine Immunology,13(12), 1307-1313. 28. Findlow, H., Laher, G., Balmer, P., Broughton, C., Carrol, E.D. & Borrow, R. (2009). Competitive inhibition flow analysis assay for the non-culture- based detection and serotyping of pneumococcal capsular polysaccharide. Clinical and Vaccine Immunology, 16 (2), 222-229. 29. Park, M.K., Briles, D.E. & Nahm, M.H. (2000). A latex Bead-based flow cytometric immunoassay capable of simultaneous typing of multiple
pneumococcal serotypes. Clinical and Diagnostics Laboratory Immunology, 7 (3), 486-489. 30. Vistnes, M., Christensen, G., & Omland, T. (2010). Multiple cytokine biomarkers in heart failure. Expert Rev. Mol. Diagn., 10 (2), 147-157. 31. Elshal, M.F. & McCoy, J.P. (2006). Multiplex bead array assays: Performance evaluation and comparison of sensitivity of ELISA. Science Direct Methods, 38, 317-323. 32. Carson, R.T. & Vignali, D.A.A. (1999). Simultaneous quantitation of 15 cytokines using a multiplexed flow cytometric assay. Journal of Immunological Methods, 227, 41-52. 309
33. duPont, N.C., Wang, K., Wadhwa, P. D., Culhane, J. F., & Nelson, E.L. (2005). Validation and comparison of Luminex multiplex cytokine analysis kit with ELISA: Determinations of a panel of nine cytokines in clinical sample culture supernatants. Journal of Reproductive Immunology, 66, 175-191. 34. Grimstad, O., Sandanger, O., Ryan, L., Otterdal, K., Damass, J.K., Pukstad, B. & Espevik, T. (2011). Cellular sources and inducers of cytokines present in acute wound fluid. Wound Repair Regen.(e-publication). 35. Bray, R.A. (2001). Flow cytometry in human leukocyte antigen testing. Semin. Hematol., 38,194-200. 36. Lund-Johansen, F., Davis K., Bishop J. & deWaal M. R. (2000). Flow cytometric analysis of immunoprecipitates: high-throughput analysis of protein
phosphorylation and protein-protein interactions. Cytometry, 39, 250-259. 37. Dunbar, S.A. & Jacobsan, J.W. (2005). Rapid screening for 31 mutation and polymorphism in cystic fibrosis transmembrane conductance regulator gene by Luminex xMAP suspension array. Methods Mol. Med., 114, 147-171. 38. Dunbar, S.A. & Jacobson, J.W. (2007). Quantitative, multiplexed detection of Salmonella and other pathogens by Luminex xMAP suspension array. Methods Mol. Biol., 394, 1-19. 39. Pabbaraju, K., Wong, S., Lee, B., Tellier, R., Fonseca, K., Louie, M. & Drews, S.J. (2010). Comparison of a singleplex real-time RT-PCR assay and multiplex respiratory viral panel assay for detection of Influenza A in respiratory specimens. Influenza other Respi. Viruses (e-publication). 40. Landegren, U., Nilsson, M. & Kwok, P.Y. (1998). Reading bits of genetic information: methods for single-nucleotide polymorphism analysis. Genome. Res., 8, 769-776. 41. Kwok, P.Y. (2001). Methods for genotyping single nucleotide polymorphisms. Annu. Rev. Genomics Hum. Genet., 2, 235-258.
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Chapter XV
CYANOTOXINS: PHARMACOLOGICAL AND ECOLOGICAL EFFECTS R. Ashwin Kumar, S.K. Verma Center for Biotechnology, Biological Science Group, Birla Institute of Technology and Sciences, Pilani, Rajasthan, India. Correspondin Author: E.mail: S.K. Verma, E-mail: skverma@bits-pilani.ac.in, rajuashwinkumar@gmail.com
ABSTRACT:The Cyanotoxins produced toxic cyanobacteria are usually found in

eutrophicated waters such as lakes, ponds, rivers and estuaries and it is a constant problem for water authorities. The world health organization has shown great concern to public health over prolong exposure of low concentration of cyanotoxins. The majorities of cyanotoxins are of nonribosomal origin and can be grouped into classes with conserved structural properties. The biochemical aspects of Cyanotoxins have been extensively studied throughout the world during last two decades but very little information available about its regulatory factors, mechanism of action of its variants and their toxicity profile. In this chapter, we discuss about the major Cyanotoxins along with its variants, their mode of actions and their potential health risk to animals and humans.
INTRODUCTION
Cyanobacteria are photoautotrophic prokaryotes which comprise of a large species of widespread occurrence with diverse morphological, physiological and biochemical properties. They play an important role in the functioning of aquatic ecosystems, because they are involved in much of the primary production, and form the basis of tropic networks [1, 2]. Biochemical processes in cyanobacterial cells are of three types: basal (providing energy and raw materials for cell functions), synthetic (replication and vegetative growth) and secondary metabolites production (utilization of substrate to give a variety of products often specific for given organism). Among these processes the maximum chemical diversity is observed in the secondary metabolites such as 311
cyanotoxins which provide heterogeneity as well as inter-/intra species level markers for organisms. A heavy water bloom formed under adequate light and temperature can also serve a rich source of secondary metabolites of novel chemical and molecular structure [3, 4]. During 1990s, had begun to screen extracts of cyanobacteria on Nostac, Anabaena, Lyngbya, Synechocystis, Spirulina, and Nodularia for various biological activities in fresh water and marine cyanobacteria, which led to high incidence of novel biologically active compounds such as antibiotics, algicides, toxins, pharmaceutical active compounds and plant growth regulators. The species such as Spirulina and Nostac have been used as a source of protein and vitamins for humans and animals [2, 4]. However, these microorganisms can also be harmful, because many species are able to produce toxins that threaten both animal and human health [5]. The products of cyanobacteria contain 40.2% of lipopeptides, 5.6% of pure amino acid composition, 4.2% fatty acids, 4.2% macrolide and 9.4% are amides. Lipopeptides are a potent bioactive group of compounds with approximately 85% being active of which are toxins 41%, anticancer13%, antibiotics12%, enzyme inhibitors 8%, antiviral 4% and antifungal 18%. The remaining activities are as tumor promoter, herbicide, antimycotic, antimalarial, antimicroalgal, cell-differentiation promoter, cardio active compounds and sunscreen pigment [1-4]. The major focus of this chapter is therefore to compile and analyze information of cyanotoxins and discuss their ecological significance.
TOXINS OF CYANOBACTERIA
The toxins produced by planktonic species of cyanobacteria have been particularly well studied. They include Anabaena, Nostoc, Aphanizomenon, Cylindrospermopsis, Microcystis, Nodularia, Oscillatoria, Lyngbya, Scytonema and Tolypothrix [3, 6, 7]. Eutrophication has been a constant problem for water authorities due to its association with the formation of cyanobacterial blooms [5]. Moreover use of chemicals for purification of water like chlorine, copper sulphate (algicides) can lyse the cyanobacterial cells to release the toxins into the surrounding water body. Cyanotoxins in drinking water can disrupt aquatic ecosystems and cause serious public health problems. Numerous poisoning events that have resulted from the consumption of contaminated water through chronic ingestion, inhalation of droplets or contact with nasal mucous membranes,
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dermal contacts through bathing or recreational activities such as wading, swimming, skiing and canoeing [5,8]. To create a comprehensive cyanotoxin contingency plan, it is necessary to be familiar with the physical and chemical properties of the toxin, the nature of the cyanotoxin, i.e., intracellular or extracellular, growth and diurnal cyanobacteria bloom patterns, and effective treatment processes [9]. Based on the chemical structure they are mainly divided into cyclic peptides, alkaloids and lipopolysaccharides. Hepatotoxins Hepatotoxins are the most common offenders worldwide in case of waterborne disease caused by toxins of cyanobacteria [10]. The cyanobacterial hepatotoxins include cyclic peptides (Microcystins and nodularins) and cyclic guanidine alkaloid
(cylindrospermopsin). Cyclic peptides are comparatively large natural products with molecular weight of 800-1,100. They contain either five (nodularins) or seven (microcystins) amino acids, with the two terminal amino acids of the linear peptide being condensed (joined) to form a cyclic compound [9, 11, 12]. Microcystins: Microcystins have been characterised from Microcystis, Anabaena, Oscillatoria, Nostoc, and Anabaenopsis species. About 75 structural variants of microcystins have been characterized so far from cyanobacteria. According to the world health organization, the guide line value for total microcystin (free and intracellular) is 1 g/L [10, 11, 12]. The aminoacid Adda, (2S, 3S, 8S, 9S)-3-amino-9-methoxy-2, 6, 8trimethyl-10-phenyldeca-4, 6-dienoic acid, is the most unusual structure in this group of cyanobacterial toxins with two protein amino acids (X&Z) and five non-protein amino acid. The nomenclature of microcystin variants has denoted by the amino acids present at the X and Z position (fig1). The most common and potent variant is microcystin- LR containing amino acid leucine (L) and arginine (R) at X and Z position [10,12]. Other variants differ in methyl group and amino acid in the ring, resulting in marked differences in toxicity and in hydrophilic and hydrophobic property. The Adda of microcystin provides the molecule with a characteristic wavelength absorbance at 238nm [4, 6, 12]. The Adda moiety is also required for toxicity and is important in the binding of the toxins to the protein phosphatases. The stereochemistry of the dienes in Adda group and level of
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methylation in various structures of cyclic peptides influences the level of toxicity and site of action [12,14].
Figure 1: General Structure of Microcystin The site of action of microcystins is by disrupting the cytoskeleton of hepatocytes, leading to massive hepatic bleeding which may lead to death or chronic intoxication by promoting hepatic tumors. The tumour-promoting activity of microcystin is likely to arise from its ability to potently inhibit PP2A which regulates several mitogen-activated protein kinases (MAPK). The cell cycle is regulated by a delicate balance between phosphorylation and dephosphorylation of key control proteins. The specific dephosphorylation of Ser/Thr residues plays an essential role in control of the cell, with more than 97% of protein phosphates occurring at Ser/Thr residues which suggests that the toxic effects of MC results in so-called activation of protein kinases. Both PP1 and PP2A covalently bind MCLR at cysteine-273 for PP1 and cysteine-266 for PP2A. Therefore, by inhibiting both PP1 and PP2A with a variety of molecular targets, resulting in an overall increase in phosphorylation of regulatory proteins (Fig. 2) [15]. These enzymatic inhibition causes morphologic damage to the tissue; Microcystins in the liver can induce hepatocytes to produce arachidonic acid metabolites such as prostacyclin (6-Keto F1 alpha) and thrombaxane (TXB2) by stimulation of the cyclooxygenase pathway [15].
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Figure 2: The proposed scheme illustrated by Gehringer dipicting cellular events upon MCLR exposure. The MAPK pathway is activated by receptor tyrosine kinases activated through G-linked receptors bycleavage of Phosphatidyllinositol 4,5-biphosphate (PIP) to diacylglycerol (DAG). IP3 triggers the release of Ca2+ from the endoplasmic reticulum (ER), but this does not occur for MCLR. PKC was not found to play a role in MCLRinduced kinase activation in-vivo. PP2A regulates the phosphorylation of both MAPKK and MAPK so inhibition of this PP by MCLR would allow for the continual activation of both these kinases which activates transcription factors. MCLR increases phosphorylation of p53 and formation of reactive oxygen species (ROS) which inturn triggers mitochondrial permeability, however the effect on p21WAF, role of caspases and role of JNK remains to be elucidated [15].
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Microcystin is biosynthesized via a large multifunctional enzyme complex consisting of non-ribosomal peptide synthetase (NPKS) and polyketide synthase (PKS) modules collectively making cluster of microcystin genes (mcy S 55kb) [16]. Peptide synthetases have a modular construction: the order and number of modules at the gene level determines the structure of product. The individual peptide synthetase catalyzes amino/hydroxyl acid activation and thioester formation reaction, the rate of reaction is determined by the growing heptapeptide chain. The production of the fatty acid side chain of the amino acid Adda has been responsible for the function of polyketide synthase. Two operons, namely mcy ABC and mcy DEFGHI form a large gene cluster for the enzyme complex (Fig.3) They are transcribed in opposite direction with a central sequences of approximaterly 900bp containing the promoter and putative regulatory cis element. Certain gene cluster encoding microcystin synthetase has been cloned and sequenced [13, 14, 16, 17].
Figure 3: Microcystin synthetase of Microcystis aeruginosa, 55kb (mcy S) The NRPS module of the second hybrid PKS/NRPS enzyme, McyE, is thought to be involved in the activation and condensation of D-Glu with Adda. McyE converts the polyketide to a -amino acid in the final step of Adda biosynthesis [16, 17]. The mcyF ORF was originally predicted to encode a glutamate racemase, responsible for the epimerization of the L-Glu residue of microcystin and later it founded with evidence that McyF acts exclusively as an Asp racemase and D-Glu residue is provided by L-Glu racemase residing outside the mcyS gene cluster [18]. McyH is in fact responsible for the active transport of microcystin (ABC transporter) to, elicit their toxic effect [17]. Thus, exchange and rearrangement of peptide synthetase modules offer the opportunity to produce novel products. Nodularin:Nodularin is produced predominantly by brackish water cyanobacteria. The first report of nodularins was in 1988 when bloom infested lake in Australia containing Nodularia spumigena was shown to be the cause of stock death. It is a cyclic pentapeptide with a similar structure to microcystin, consisting of Adda, D-glutamic acid 316
(D-Glu), N-methyldehydrobutyrine (MeDhb), D-erythro--methylaspartic acid (DMeAsp), and L-arginine (L-Arg) (Figure 4) [19, 20, 21]. Seven naturally-occurring isoforms of nodularin have been reported to date. Two of these isoforms, have variations within the Adda residue, which reduces or abolishes the toxicity of the compound [16]. The D-Glu residue is essential for toxicity of nodularin, as esterification of its free carboxyl abolishes toxicity; however, substitution at position 1 has little effect on toxicity. The other two isoforms, nodularin-Har and motuporin, are variable at position 2 [22]. Nodularin-Har is produced by the strain N. harveyana PCC7804, with the L-Arg, replaced with L-Homoarginine (L-Har) [23]. Motuporin has been isolated from the Papua New Guinea sponge Theonella swinhoei, and may be synthesized by an associated cyanobacterium. The L-Arg residue of nodularin is replaced by L-Val in motuporin. The L-Val residue is responsible for additional cytotoxicity of motuporin against cancer cell lines [24].
N N OH O NH O N H O COOH O NH COOH O NH H2N

Figure 4: Structure of Nodularin The mechanisms of toxicity are relatively same as that of microcystin by inhibiting pp1 and pp2A with IC50 of 0.1nm. The hydrophobic C20 - amino acids Adda, present in both toxins, blocks protein phosphatases enzyme activity by interacting with the hydrophobic groove and obstructing substrate access to the active site cleft. MeDhb binds to Cys273 of PP2A in a similar fashion to the MeDha residue in microcystin which
NH
317
binds to Cys273 of PP1 and Cys266 of PP2A. However, binding of the toxin does not occur covalently and may be the reason for its additional carcinogenic properties [25].
Figure 5: Nodularin synthetase of Nodularia spumigena, 48kb (nda S) [16] The nodularin biosynthesis gene cluster ndaS, from Nodularia spumigena NSOR10, was sequenced and characterized in 2004. The 48 kb region of the genome consists of nine ORFs (ndaA-I) transcribed from a bidirectional regulatory promoter region (Figure 5). The Adda side-chain is produced via a mixed NRPS/PKS pathway from a phenylacetate starter unit and several malonyl-CoA extensions (NdaC, D and F) (Figure 5). The NRPS module of the hybrid NRPS/PKS, NdaF, subsequently adds D-Glu to the growing chain [16, 26]. Two NRPS enzymes, NdaA and B, complete the cyclic pentapeptide by adding the final amino acid residues, L-Thr, D-MeAsp and L-Arg. The NRPS modules responsible for the activation of D-Ala and D-Leu in mcyS (McyA and B) are absent from ndaS as nodularin lacks these moieties. The NRPS and PKS proteins require posttranslational modification by a phosphopantetheinyl transferase (PPT) protein. The PPT required for activation of the Nda proteins is not clustered with the other nda genes. Recently,degenerate PCR and subsequent functional enzymatic characterization, identified the PPT required for nodularin biosynthesis in N. spumigena NSOR10 [16, 27 28]. Cylindrospermopsins: Cylindrospermopsins a cyclic guanidine hepatotoxic alkaloid first isolated from Cylindrospermopsis raciborski. Later it was also reported from Aphanizomenon ovalisporum, in Israel, Aphanizomenon flos-aquae in Germany and Umezakis natan in Japan. The toxin is a stable tricyclic alkaloid containing a guanido group linked at C7 to hydroxymethyl uracil (Fig.6) [30, 31, 32]. At the hydroxyl bridge, there are two possible epimers, cylindrospermopsin and 7-epicylindrospermopsin. Because of the negatively charged sulfate group and the positively charged guanido group, the molecule is a zwitterion and very water soluble. Both naturally occurring epimers are equally toxic [33]. The acute toxicity studies with crude extract with LD50 of 200 g/kg injected intraperitonally blocks protein synthesis of cells in kidney, liver, lungs, 318
adrenal glands and intestine. The effect pure toxin has been identified in an outbreak of acute heptoenteritis and renal damage among the aboriginal population in Australia [33,34].
O O S O O
H N
OH O NH O
NH HN NH
Figure 6: Structure of Cylindrospermopsins Cylindrospermopsin has also been found to be genotoxic in a number of in-vitro assay systems. Using the micronucleus assay, cylindrospermopsin caused DNA fragmentation and loss of whole chromosomes in a human white blood cell line and in mice liver [35]. These ndings could not be conrmed in CHO K1 cells, suggesting that metabolism of the toxin to an active product was required for genotoxic action. This hypothesis was conrmed in primary mouse hepatocytes where cylindrospermopsininduced DNA fragmentation was detected by the COMET assay, an effect that could be eliminated by application of CYP450 inhibitors [36]. Recent data from primary
hepatocytes show two routes of toxic action, a rapid route probably through toxicity of a CYP450 oxidation product of the toxin and a slower mechanism through the welldocumented inhibition of protein synthesis, which does not require toxin metabolism. Uptake of the toxin is relatively rapid since complete and irreversible block of protein synthesis occurs after a 1 hr exposure in-vitro. Inhibition of protein synthesis occurs at the ribosome during the peptide chain elongation step. Glutathione synthesis is also reduced via a CYP450-dependant mechanism, but this does not lead to an increase in oxidative stress in the cell [33, 34, 35, 36]. Neurotoxins Aphantoxins, anatoxins-a, saxitoxins, neosaxitoxin are known neurotoxins reported from the strains of Anabaena, Aphanizomenon, Cylindrospermum and Oscillatoria
319
respectively. They are generally alkaloids which are well known for its neurotoxicity with low molecular weight [3, 4]. Anatoxins and its variants inhibit transmission at the neuromuscular junction by molecular mimicry of the neurotransmitter acetylcholine and inhibition of acetyl cholinesterase activity [37]. The enzyme acetylcholinesterase catalyzes the hydrolysis of the neurotransmitter acetylcholine and it has been an attractive target for the treatment of Alzheimers disease and other possible therapeutic application in the treatment of Parkinsons disease and Myasthenic gravis [3, 37]. These compounds are potent postsynaptic depolarizing neuromuscular blocking agent that causes hypersalivation and death within minutes to hours in animals. They are only known naturally occurring organophosphate [6]. Anatoxin-a is the structural analogue of cocaine and binds to nicotinic acetylcholine receptors which leads to respiratory failure within few minutes. It can induce apoptosis in non-neuronal cells. Toxin-treated thymocytes showed all the typical morphological and biochemical features of apoptosis including DNA fragmentation, generation of ROS and caspase activation, which are more pronounced than in Vero cells. Exact mechanisms of anatoxin induced apoptosis are not known [38]. Homoanatoxins-a is a potent neuromuscularblocking agents that causes severe paralysis convulsion and death by respiratory arrest. Jamaicamide from Lyngbya majuscule also shares the same site of action as compared to other neurotoxins stated above [39].
H N
H2 O N
Structure of Anatoxins-A
N N H2N N O O P O CH3 O CH3 CH3
Structure of Homoanatoxins-A
Structure of Anatoxins-A (S) 320

Saxitoxins comprises different isoforms and with varied toxicities. They are well known as paralytic shellsh poisoning (PSP) toxins due to their accidental consumption in contaminated seafood. Saxitoxins (STX) and its analogue compounds (neosaxitoxin, aphantoxins) have been reported to occur naturally in marine dinoagellates, lamentous cyanobacteria and in certain heterotrophic bacteria. In freshwater environments, STX toxins are almost exclusively associated with cyanobacteria, and the occurrence of STX producing neurotoxic cyanobacterial blooms has been increasingly reported [3, 4, 16, 41, 42]. PSP toxins selectively block voltage-gated Na+ channels in excitable cells, thereby affecting the impulse generation in animals which can lead to death. STX binds to nerve membranes with high afnity (dissociation constant Kd=2 nM). PSP toxins block resting, active or inactive Na+ channels equally well, through a receptor site named as site 1, occupied by the so-called pore-blockingtoxins. These toxins act from the extracellular side of the plasma membrane by occluding the entry of the Na+ channel pore. The positively charged guanidinium group of PSP toxins interacts with negatively charged carboxyl groups at the mouth of the channel [42, 43, 44].
Br OCH3
O O O
OH O O OH
OH
R4 O H N NH2 N N R5 OH R2 R3
R 1N H2 N
Debromoaplysiatoxins:General Structure Saxitoxins
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Dermatotoxins Most of the lipopolysaccharides of cyanobacteria cause severe dermatitis, oral and gastrointestinal inflammation. The lipopolysaccharide (LPS) endotoxins of cyanobacteria are generally found in the outer membrane of the cell wall where they form complexes with proteins and phospholipids [4, 45]. Lipopolysaccarides, as the name implies, are condensed products of a sugar, usually a hexose and a lipid, normally a hydroxy C14-C18 fatty acid. The many structural variants of LPS are generally phylogenetically conserved (similar fatty acid and sugar components). It has been proved that the fatty acid component of the LPS molecule elicits allergenic response (irritant) in humans and mammals [3]. In 1970 the first LPS was reported from the cyanobacterium Anacytis nidulans, which exhibits both pyrogenic and toxic effects. Both unlike Gram- negative bacteria and cyanobacterial LPS lacks any phosphate in the lipid-A core. LPS of cyanobacterium are found to be 10fold less toxic than other bacterial LPS. The considerable diversity of cyanobacteria in their chemical composition is largely related to the phylogeny [4]. Mechanisms of vomiting and its relationship to LPS activity Nausea and vomiting are normal physiological responses to the ingestion of toxic substances; they are essential defences because they are the end result of the actions of sensory motor systems that operate to identify and rapidly expel hazardous substances from the upper G-I tract. The two main sensory systems that direct the emetic response are local, associated with the gut mucosa (pre-absorptive response) and central, specifically the chemoreceptive trigger zone of the area postrema, located in the dorsal surface of the medulla oblongata (post-absorptive response). Stimulation of chemoreceptors in the stomach, jejunum and ileum by irritant chemicals such as hypertonic saline, copper sulfate or mustard, or by bacterial enterotoxins, leads to the activation of vagal sensory afferent nerves to the brain. Vagal efferent processing through the enteric nervous system stimulates enteric motor neurons to affect emesis. Emetic chemoreceptors are also found in the vascular system; activation of these chemoreceptors will also initiate nausea and vomiting. Endogenous mediators of emesis such as dopamine, acetylcholine and enkephalin are reported. Prostaglandins have well-known emetic actions. Some of the other dermatotoxins which are will reported are Debromoaplysiatoxin from Lynbya gracilis [45, 46]. 322
Cytotoxins Cryptophycin 114 was first isolated from Nostac sp ATCC 53789 and Nostac sp GSV 224, which exhibited potent cytotoxicity against human tumor cell lines and good activity against a broad spectrum of drug-sensitive and drug resistant murine and human solid tumors. Nevertheless, C-14 again appeared to be too toxic to become a clinical candidate. This led to a detailed structure function study and resulted in the isolation of Cryptophycin 815 a semi synthetic analogue, which proved to have a greater therapeutic efficiency and lower toxicity. Cyanovirin 16, a 101 amino acid protein has recently been placed on an accelerated track for clinical development for use as a virucidal agent. Cyanovirin-N (CV-N) was extracted from Nostac ellipsosporum, which prevent the surface receptor of HIV from undergoing the conformational changes that enable the viral and cell membranes to fuse, allowing the transfer of the viral genetic material into the host. CV-N was found to have potent activity against all immunodeficiency viruses HIV1, HIV-2, SIV (simian) and FIV (feline). The use of CV-N, Cryptophycin 114 and its analogue 815 lead to an entirely new class of antiHIV, antitumor drugs [47,48]. Some of sulfolipid of Lyngbya lagerheimi, Dolastatin 3, G, 11, 12 and Serinol-derived malyngamide 1-2 from Lyngbya majuscule also show anti-HIV activity [49]. Some of the other cytotoxins reported are diarrhetic toxin {lipopeptide) from Microcystis species, Anabaena species and Nodularia species, -butyrolactone, +-(s)-butyramidedo-ybutyrolactone, butyrolactone 3-y and hermitamides B from Lyngbya majuscule, 30methyloscillatoxin D, 31-neroscillatoxin and oscillatoxin from Oscillatoria species, hormothamnin and hormothamnione from Hormothamnion enteromoyphoides,
kalipyrone from Tolypothrix species, borphycin and nostocyclophane from Nostoc ellipsosporum, micromide and guamamide from Symploca and tenue cyclamide from Nostoc spongiaeforme [3,4].
CONCLUSION
A number of areas of cyanotoxins toxicology require investigation before the human health risk can be fully assessed. The level of mechanisms of action is not yet known by which route toxic metabolites are formed to produce rapid toxicity and more importantly, genotoxicity. There is considerable evidence for involvement of other variants in toxicity 323
but the involvement of specic isoforms have yet to be identied. Without this information, it is difcult to make accurate predictions about the applicability of animal data to human risk assessment, because it cannot show whether these mechanisms are likely to occur in humans. Detailed toxicokinetics are still lacking. More work needs to be done to determine rates of uptake, tissue distribution and excretion from an oral dose. Effects of cylindrospermopsin on nonhepatic tissues have not been well investigated. Renal effects seemed to be important after chronic low dose exposure of cyanotoxins and thrombotic effects have been described by a number of workers. Long-term oral toxicity studies are needed in both sexes of animal and so far only male mice have been investigated in detail. Reproductive and teratogenic toxicity assessments are also required for all cyanotoxins. Finally, and most importantly, in-vivo carcinogenicity trials need to be undertaken to assess the potential carcinogenicity of cylindrospermopsin, isomers of microcystin, nodularins and other neuro toxins. REFERENCES 1. Shimizu Y. (2003). Micro algal metabolites. Current opinion in microbiology, 6, 236243. 2. Burja A.M., Banaigs B., Abou-Mansour E., Burgess G.J. & Wright P.C. (2001). Marine cyanobacteria-a prolific source of natural products. Tetrahedron, 57, 93479377. 3. Harada K. (2004) Production of secondary Metabolites by freshwater cyanobacteria. Chemical and Pharmaceutical bulletin, 52, 889-899. 4. Pranita J., Pawan K.S. & Radha P. (2008). Cyanobacterial bioactive molecules-an overview of their toxic properties. Canadian Journal of Microbiology, 54, 701-717. 5. Westrick J.A., Szlag D.C., Southwell B.J. & James S. (2010). A review of cyanobacteria and cyanotoxins removal/inactivation in drinking water treatment. Analytical bioanaytical Chemistry, 397, 1705-1714 6. Codd G., Bell S. & Kaya K. Ward C., Beattie K. & Metcalf J. (1999). Cyanobacterial toxins, exposure routes and human health. Euopean Journal. phycology, 34, 405-415 7. Carmichael WW. (1994). The toxins of cyanobacteria. Scientific American, 270, 6472.
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