Antibacterial Efficacy and Mechanisms of Action of a Novel Beta-Defensin from Snakehead Murrel, Channa striata.

Beta-defensins, identified from fishes, constitute a crucial category of antimicrobial peptides important in combating bacterial fish pathogens. The present investigation centers on the molecular and functional characterization of CsDef, a 63-amino acid beta-defensin antimicrobial peptide derived from snakehead murrel (Channa striata). The physicochemical attributes of CsDef align with the distinctive characteristics observed in AMPs. CsDef was recombinantly produced, and the recombinant peptide, rCsDef, exhibited notable antibacterial efficacy against bacterial fish pathogens with an MIC of 16 µM for V. proteolyticus. A. hydrophila exhibited 91% inhibition, E. tarda 92%, and V. harveyi 53% at 32 µM of rCsDef. The rCsDef exhibited a multifaceted mechanism of action against bacteria, i.e., through membrane depolarization, membrane permeabilization, and generation of ROS. The rCsDef was non-hemolytic to hRBCs and non-cytotoxic to normal mammalian cell line CHO-K1. However, it exhibited anticancer properties in MCF-7. rCsDef demonstrated notable stability with respect to pH, temperature, salt, metal ions, and proteases. These findings suggest it is a potential candidate molecule for prospective applications in aquaculture.

A Novel Beta-Defensin Isoform from Malabar Trevally, Carangoides malabaricus (Bloch & Schneider, 1801), an Arsenal Against Fish Bacterial Pathogens: Molecular Characterization, Recombinant Production, and Mechanism of Action.

Antimicrobial peptides (AMPs), including beta-defensin from fish, are a crucial class of peptide medicines. The focus of the current study is the molecular and functional attributes of CmDef, a 63-amino acid beta-defensin AMP from Malabar trevally, Carangoides malabaricus. This peptide demonstrated typical characteristics of AMPs, including hydrophobicity, amphipathic nature, and +2.8 net charge. The CmDef was recombinantly expressed and the recombinant peptide, rCmDef displayed a strong antimicrobial activity against bacterial fish pathogens with an MIC of 8 µM for V. proteolyticus and 32 µM for A. hydrophila. The E. tarda and V. harveyi showed an inhibition of 94% and 54%, respectively, at 32 µM concentration. No activity was observed against V. fluvialis and V. alginolyticus. The rCmDef has a multimode of action that exerts an antibacterial effect by membrane depolarization followed by membrane permeabilization and ROS production. rCmDef also exhibited anti-cancer activities in silico without causing hemolysis. The peptide demonstrated stability under various conditions, including different pH levels, temperatures, salts, and metal ions (KCl and CaCl2), and remained stable in the presence of proteases such as trypsin and proteinase K at concentrations up to 0.2 µg/100 µl. The strong antibacterial efficacy and non-cytotoxic nature suggest that rCmDef is a single-edged sword that can contribute significantly to aquaculture disease management.

Antibacterial activity and modes of action of a novel hepcidin isoform from the shrimp scad, Alepes djedaba (Forsskål, 1775).

Hepcidin, initially identified in human blood ultrafiltrate as cysteine rich Liver Expressed Antimicrobial Peptide (LEAP-1), is a core molecular conduit between iron trafficking and immune response. Though a great share of studies has been focused on the iron regulatory function of hepcidins, investigations on the antimicrobial aspects are relatively less. The present study is aimed at identification of hepcidin from a teleost fish, Alepes djedaba followed by its recombinant expression, testing antibacterial property, stability and evaluation of cytotoxicity. Modes of action on bacterial pathogens were also examined. A novel hepcidin isoform, Ad-Hep belonging to the HAMP1 (Hepcidin antimicrobial peptide 1) group of hepcidins was identified from the shrimp scad, Alepes djedaba. Ad-Hep with 2.9 kDa size was found to be a cysteine rich, cationic peptide (+4) with antiparallel beta sheet conformation, a furin cleavage site (RXXR) and 'ATCUN' motif. It was heterologously expressed in E. coli Rosettagami B(DE3)PLysS cells and the recombinant peptide, rAd-Hep was found to have significant antibacterial activity, especially against Edwardsiella tarda, Vibrio parahaemolyticus and Escherichia coli. Membrane depolarization followed by membrane permeabilization and Reactive Oxygen Species (ROS) production were found to be the modes of action of rAd-Hep on bacterial cells. Ad-Hep was found to be non-haemolytic to hRBC and non-cytotoxic in mammalian cell line. Stability of the peptide at varying temperature, pH and metal salts qualify them for applications in vivo. With significant bactericidal activity coupled with direct killing mechanisms, the rAd-Hep can be a promising drug candidate for therapeutic applications in medicine and fish culture systems.

Mangrove-associated endomycota: diversity and functional significance as a source of novel drug leads.

Endophytic fungi are known for their unprecedented ability to produce novel lead compounds of clinical and pharmaceutical importance. This review focuses on the unexplored fungal diversity associated with mangroves, emphasizing their biodiversity, distribution, and methodological approaches targeting isolation, and identification. Also highlights the bioactive compounds reported from the mangrove fungal endophytes. The compounds are categorized according to their reported biological activities including antimicrobial, antioxidant and cytotoxic property. In addition, protein kinase, α-glucosidase, acetylcholinesterase, tyrosinase inhibition, antiangiogenic, DNA-binding affinity, and calcium/potassium channel blocking activity are also reported. Exploration of these endophytes as a source of pharmacologically important compounds will be highly promising in the wake of emerging antibiotic resistance among pathogens. Thus, the aim of this review is to present a detailed report of mangrove derived endophytic fungi and to open an avenue for researchers to discover the possibilities of exploring these hidden mycota in developing novel drug leads.

In vitro propagation of infectious hypodermal hematopoietic necrosis virus (Penaeus stylirostris penstyldensovirus) in PmLyO-Sf9 cells.

Infectious hypodermal hematopoietic necrosis virus (IHHNV/PstDVI) was isolated and propagated in the hybrid shrimp-insect cell line PmLyO-Sf9. A few hours after inoculation with an infected tissue extract or virus suspension, cytopathic changes could be observed in the cell line, including clustering, enlargement, syncytium formation, granulation, vacuole formation, tapering, irregularities in the plasma membrane with extended tails, detachment, cell death, and accumulation of cellular debris. Expression of viral genes, the presence of virions, and cytological changes observed using transmission electron microscopy suggested replication of the virus in these cells. The virus was purified by ultracentrifugation, negatively stained, and examined using an electron microscope, and the purified virus was found to be infectious both in vitro and in vivo. This development opens avenues for the study of the basic molecular mechanism of IHHNV infection, pathogenesis, and replication, which is much needed for developing an antiviral strategy in aquaculture.

A novel anti-lipopolysaccharide factor from blue swimmer crab Portunus pelagicus and its cytotoxic effect on the prokaryotic expression host, E. coli on heterologous expression.

BACKGROUND: Invertebrates like crabs employ their own immune systems to fight against a number of invasive infections. Anti-lipopolysaccharide factors (ALFs) are an important class of antimicrobial peptides (AMPs) exhibiting binding and neutralizing activities against lipopolysaccharides. RESULTS: This study identified and characterized a novel homolog of ALF (Pp-ALF) from the blue swimmer crab Portunus pelagicus. Pp-ALF has a 369bp open-reading frame encoding a protein with 123 amino acids. The deduced protein featured an LPS-binding domain and a signal peptide. The predicted tertiary structure of Pp-ALF contains three α helices packed against four ß sheets. The deduced amino acid sequence of Pp-ALF had a net positive charge of +10.75 and an isoelectric point of 9.8. Phylogenetic analysis revealed that Pp-ALF has a strong ancestral relationship with crab ALFs. CONCLUSION: Antibacterial, antiviral, antifungal, anticancer, and antibiofilm activities of Pp-ALF could be revealed by in silico prediction tools. Recombinant expression of Pp-ALF was unsuccessful in the Escherichia coli Rosetta-gami expression system due to the cytotoxic effect of the peptide to the host. The toxic effect of Pp-ALF to the host was displayed by membrane permeabilization and death of the host cells by fluorescent staining with Syto9-Propidium Iodide and CTC-DAPI- FITC.

Characterisation of a novel crustin isoform from mud crab, Scylla serrata (Forsskål, 1775) and its functional analysis in silico.

A 336-base pair (bp) sized mRNA sequence encoding 111 amino acid size crustin isoform (MC-crustin) was obtained from the gill sample of the green mud crab, Scylla serrata. MC-crustin possessed an N-terminal signal peptide region comprising of 21 amino acid residues, followed by a 90 amino acid mature peptide region having a molecular weight of 10.164 kDa, charge + 4.25 and theoretical pI of 8.27. Sequence alignment and phylogenetic tree analyses revealed the peptide to be a Type I crustin, with four conserved cysteine residues forming the cysteine rich region, followed by WAP domain. MC-crustin was cationic with cysteine/proline rich structure and was predicted with antimicrobial, anti-inflammatory, anti-angiogenic and anti-hypertensive property making it a potential molecule for possible therapeutic applications.

Functional characterization of a histone H2A derived antimicrobial peptide HARRIOTTIN-1 from sicklefin chimaera, Neoharriotta pinnata.

Antimicrobial peptides (AMPs) are gene encoded short peptides which play an important role in the innate immunity of almost all living organisms ranging from bacteria to mammals. Histones play a very important role in defense as precursors to bioactive peptides. The present study is an attempt to decipher the antimicrobial activity of a histone H2A derived peptide, Harriottin-1 from sicklefin chimaera, Neoharriotta pinnata. Analysis in silico predicted the molecule with potent antibacterial and anticancer property. The Harriottin-1 was recombinantly produced and the recombinant peptide rHar-1 demonstrated potent antibacterial activity at 25 µM besides anticancer activity. The study strongly suggests the importance of histone H2A derived peptides as a model for the design and synthesis of potent peptide drugs.

A hepatic antimicrobial peptide, hepcidin from Indian major carp, Catla catla: molecular identification and functional characterization.

BACKGROUND: Increase of antibiotic resistance in pathogenic microbes necessitated novel molecules for curing infection. Antimicrobial peptides (AMPs) are the gene-encoded evolutionarily conserved small molecules with therapeutic value. AMPs are considered as an alternative drug for conventional antibiotics. Hepcidin, the cysteine-rich antimicrobial peptide, is an important component in innate immune response. In this study, we identified and characterized hepcidin gene from the fish, Catla catla (Indian major carp) and termed it as Cc-Hep. RESULTS: Open reading frame of Cc-Hep consists of 261 base pair that encodes 87 amino acids. Cc-Hep is synthesized as a prepropeptide consisting of 24 amino acid signal peptide, 36 amino acid propeptide, and 26 amino acid mature peptide. Sequence analysis revealed that Cc-Hep shared sequence similarity with hepcidin from Sorsogona tuberculata. Phylogenetic analysis indicated that Cc-Hep was grouped with HAMP2 family. Structure analysis of mature Cc-Hep identified two antiparallel beta sheets stabilized by four disulphide bonds and a random coil. The mature peptide region of Cc-Hep has a charge of + 2, isoelectric value 8.23 and molecular weight 2.73 kDa. CONCLUSION: Functional characterization predicted antibacterial, antioxidant, and anticancer potential of Cc-Hep, which can be explored in aquaculture or human health care.

A Novel Approach of Transducing Recombinant Baculovirus into Primary Lymphoid Cells of Penaeus monodon for Developing Continuous Cell Line.

Cell line development from shrimp is not a novel venture as researchers across the globe have been trying to have crustacean cell lines over 30 years. The reason for not attaining a crustacean or precisely a shrimp cell line is believed to be the replicative senescence and the inability to maintain telomere length in vitro. Moreover, spontaneous in vitro transformations do not happen in shrimp cells. Oncogenic induction in primary cell culture is one of the ways to attain in vitro transformation by way of disrupting the mechanisms which involve cellular senescence. In this context, a recombinant baculovirus with shrimp viral promoter IHHNV-P2 was used for the transduction aimed at immortalization. An oncogene, H-ras, was successfully amplified and cloned in to the baculoviral vector, downstream to shrimp viral promoter IHHNV-P2 and upstream to GFP. Recombinant baculovirus with H-ras was generated and used for transduction into shrimp lymphoid cells during early dividing stage. Accordingly, fibroblast-like primary cell culture got developed, and H-ras and GFP expression could be confirmed. The study suggests that the simple method of incubating recombinant baculovirus with minced tissue enables in vitro transduction during early dividing stage of the cells, and the transduction efficiency gets enhanced by adding 5 mM sodium butyrate to the culture medium.

ß-Defensins from common goby (Pomatoschistus microps) and silver trevally (Pseudocaranx georgianus): Molecular characterization and phylogenetic analysis.

Antimicrobial peptides (AMPs) are biologically active molecules involved in host defense present in a variety of organisms. They are an integral component of innate immunity, forming a front line of defense against potential pathogens, including antibiotic-resistant ones. Fishes are proven to be a prospective source of AMPs as they are constantly being challenged by a variety of pathogens and the AMPs are reported to play an inevitable role in fish immunity. Among them, ß-defensins form one of the most studied multifunctional peptides with early evolutionary history and recently being considered as host defense peptides. The present study highlights the first-ever report on ß-defensin AMP sequences from common goby (Pomatoschistus microps) and silver trevally (Pseudocaranx georgianus). A 192 bp cDNA fragment with an open reading frame encoding 63 amino acids (aa) comprising a 20 aa signal peptide region at the N-terminal was obtained from the mRNA of gill tissue of both P. microps and P. georgianus by RT-PCR. These peptide sequences when characterized in silico at the molecular level revealed a 43 aa cationic mature peptide with the signature intra-molecular disulphide bonded cysteine residue pattern ascertaining its ß-defensin identity, further confirmed by phylogenetic analysis. The data collected will pave the way for further research on varied facets of the peptide-like, tissue level expressions, antimicrobial activities on commonly encountered pathogens, and its feasibility as a therapeutant in the aquaculture scenario.

Molecular characterization of a novel ß-defensin isoform from the red-toothed trigger fish, Odonus niger (Ruppel, 1836).

BACKGROUND: The concern regarding a post-antibiotic era with increasing drug resistance by pathogens imposes the need to discover alternatives for existing antibiotics. Antimicrobial peptides (AMPs) with their versatile therapeutic properties are a group of promising molecules with curative potentials. These evolutionarily conserved molecules play important roles in the innate immune system of several organisms. The ß-defensins are a group of cysteine rich cationic antimicrobial peptides that play an important role in the innate immune system by their antimicrobial activity against the invading pathogens. The present study deals with a novel ß-defensin isoform from the red-toothed trigger fish, Odonus niger. Total RNA was isolated from the gills, cDNA was synthesized and the ß-defensin isoform obtained by polymerase chain reaction was cloned and subjected to structural and functional characterization in silico. RESULTS: A ß-defensin isoform could be detected from the gill mRNA of red-toothed trigger fish, Odonus niger. The cDNA encoded a 63 amino acid peptide, ß-defensin, with a 20 amino acid signal sequence followed by 43 amino acid cationic mature peptide (On-Def) having a molecular weight of 5.214 kDa and theoretical pI of 8.89. On-Def possessed six highly conserved cysteine residues forming disulfide bonds between C1-C5, C2-C4, and C3-C6, typical of ß-defensins. An anionic pro-region was observed prior to the ß-defensin domain within the mature peptide. Clustal alignment and phylogenetic analyses revealed On-Def as a group 2 ß-defensin. Furthermore, it shared some structural similarities and functional motifs with ß-defensins from other organisms. On-Def was predicted to be non-hemolytic with anti-bacterial, anti-viral, anti-fungal, anti-cancer, and immunomodulatory potential. CONCLUSION: On-Def is the first report of a ß-defensin from the red-toothed trigger fish, Odonus niger. The antimicrobial profile showed the potential for further studies as a suitable candidate for antimicrobial peptide therapeutics.

ß-Defensin from the Asian Sea Bass, Lates calcarifer: Molecular Prediction and Phylogenetic Analysis.

Antimicrobial peptides (AMPs) are an important element of the innate immune system of all living organisms and serve as a barrier that safeguards the organisms against a wide range of pathogens. Fishes are proven to be a prospective source of AMPs, and ß-defensins form an important family of AMPs with potent antimicrobial, chemotactic and immunomodulatory activities. The present study reports a ß-defensin AMP sequence (Lc-BD) from the Asian sea bass, Lates calcarifer, a commercially important fish species in tropical and subtropical regions of Asia and the Pacific. A 202-bp cDNA fragment with an open reading frame encoding 63 amino acids (aa) was obtained from the mRNA of gill tissue by RT-PCR. The deduced aa sequence of Lc-BD possessed a signal and a mature peptide region with 20 and 43 aa residues, respectively. Lc-BD was characterized at the molecular level, and a molecular weight of 5.24 kDa and a net charge of +4.5 was predicted for the mature peptide. The molecular characterization of Lc-BD revealed the presence of three intramolecular disulphide bonds involving the six conserved cysteine residues in the sequence, and the phylogenetic analysis of Lc-BD showed a close relationship with ß-defensins from fishes like Siniperca chuatsi, Argyrosomus regius, Trachinotus ovatus and Oplegnathus fasciatus.

Anti-lipopolysaccharide Factor from Crucifix Crab Charybdis feriatus, Cf-ALF2: Molecular Cloning and Functional Characterization of the Recombinant Peptide.

Antilipopolysaccharide factors (ALFs) are important effectors of innate immunity in crustaceans with broad spectrum antimicrobial activity. Present study deals with the molecular and functional characterization of a 98-amino acid ALF isoform from, crucifix crab, Charybdis feriatus termed as Cf-ALF2. The ALF isoform Cf-ALF2 exhibits characteristic features of an AMP including a cationic net charge of + 9 and a total hydrophobic ratio of 34%. Recombinant peptide rCf-ALF2 showed remarkable antimicrobial activity against Gram-negative and Gram-positive bacteria especially against Staphylococcus aureus (minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of 5 µM) and Escherichia coli (MIC 10 µM and MBC 20 µM). Using scanning electron microscopy, bacterial membrane blebbing, disruption, and cell content leakage were observed in peptide treated E. coli. The recombinant peptide was found to be non-hemolytic and non-cytotoxic in NCI-H460 cell line at the highest tested concentration (20 µM). Thus, this study identified a novel isoform of ALF from C. feriatus and revealed the potent antimicrobial property of the recombinant peptide Cf-ALF2 and the future prospects of using the peptide for therapeutic applications in the future.

A histone H2A derived antimicrobial peptide, Fi-Histin from the Indian White shrimp, Fenneropenaeus indicus: Molecular and functional characterization.

Antimicrobial peptides (AMPs) derived from histone proteins form an important category of peptide antibiotics. Present study deals with the molecular and functional characterization of a 27-amino acid histone H2A derived AMP from the Indian White shrimp, Fenneropenaeus indicus designated as Fi-Histin. This peptide displayed distinctive features of AMPs such as amphiphilic alpha helical structure and a net charge of +6. The synthetic peptide exhibited significant antimicrobial activity against Gram-negative and Gram-positive bacteria especially against V. vulnificus, P. aeruginosa, V. parahaemolyticus, V. cholera and S. aureus. Disruption of cell membrane and cell content leakage were observed in peptide treated V. vulnificus using scanning electron microscopy. The synthetic peptide Fi-His1-21 exhibited DNA binding activity and found to be non-haemolytic at the tested concentrations. Peptide was also found to possess anticancer activity against NCI-H460 and HEp-2 cell lines with an IC50 of 22.670 ±â€¯13.939 µM and 31.274 ±â€¯24.531 µM respectively. This is the first report of a histone H2A derived peptide from F. indicus with a specific antimicrobial activity and anticancer activity, which could be a new candidate for future applications in aquaculture and medicine.

Molecular Characterisation of a Novel Isoform of Hepatic Antimicrobial Peptide, Hepcidin (Le-Hepc), from Leiognathus equulus and Analysis of Its Functional Properties In Silico.

Hepcidin represents a family of cysteine-rich antimicrobial peptides that are mainly expressed in the liver of living organisms. In this study, we have identified and characterised a novel isoform of hepcidin from the common pony fish, Leiognathus equulus (Le-Hepc). A 261-bp fragment cDNA coding for 86 amino acids was obtained. Homologous analysis showed that Le-Hepc belongs to the hepcidin super family and shares sequence identity with other known fish pre-propeptide hepcidin sequences. The ORF encodes for a 24-amino acid (aa) signal peptide coupled to a 36-aa prodomain followed by a 26-aa mature peptide. The mature peptide region has a calculated molecular weight of 2.73 kDa, a net positive charge of +2 and a theoretical pI of 8.23. Phylogenetic analysis of Le-Hepc showed a strong relationship with other fish hepcidin sequences and clustered into HAMP2 group hepcidins. Secondary structural analysis indicated that Le-Hepc mature peptide contains two antiparallel ß-sheets strengthened by four disulphide bonds formed by eight conserved cysteine residues. The physicochemical properties of the peptide and its structural parameters are in agreement with characteristic features of an antimicrobial peptide. This is the first report of an antimicrobial peptide from the common pony fish, L. equulus.

Marine Algicolous Endophytic Fungi - A Promising Drug Resource of the Era.

Endophytic fungi have currently been acknowledged as the most promising source of bioactive compounds for drug discovery, and considerable progress has been made in exploring their diversity, species richness, and bioprospecting. Fungal endophytes from unique environmental settings offer a pool of potentially useful medicinal entities. Owing to the constant stresses imposed on macroalgae by marine environments, it is believed that algae and their associated endophytic symbionts represent a good source of structurally diverse bioactive secondary metabolites. Despite the proven significance of active metabolites of algal endophytes, little have been exploited. This review highlights the latest discoveries in algicolous endophytic research, with particular focus on the bioactive metabolites from algal endophytes. Compounds are classified according to their reported biological activities, like anticancer, antibacterial, antifungal, and antioxidant properties. Present experimental evidence suggests that a majority of the bioactive metabolites were reported from Phaeophyceae followed by Rhodophyceae and Chlorophyceae. An intensive search for newer and more effective bioactive metabolites has generated a treasure trove of publications, and this review partially covers the literature published up to 2016.

Impaired telomerase activity hinders proliferation and in vitro transformation of Penaeus monodon lymphoid cells.

Retaining terminal transferase activity of telomerase, the ribonucleoprotein enzyme which add telomeric repeats on chromosome end is thought to be required to prevent cellular ageing. Additionally, telomerase considered as a marker for cell proliferation and immortalization in eukaryotes. We examined telomerase activity in tissues and lymphoid cell culture of Penaeus monodon. Along with telomerase activity, telomere repeats and an attempt on identification of telomerase reverse transcriptase (PmTERT) were made. Telomeric repeat amplification protocol revealed that telomerase-dependent telomeric lengthening has been taking place in P. monodon and the adult tissues were retaining this capacity throughout their lifespan with the highest activity in ovary, testis and lymphoid organ. However, telomerase activity could not be detected in lymphoid cells in culture. The canonical telomeric repeats added by telomerase of lymphoid tissue extract were identified as TTAGG, but pentameric repeats GGTTA and AGGTT were also added by the telomerase. PmTERT protein sequence (partial) shared 100 % identity with the TERT sequence of Daphnia pulex, 27 % sequence identity with Purple sea urchin and 24-25 % with Zebra fish. Undetectable telomerase activity in lymphoid cell culture supports the hypothesis that the inadequate telomerase activity or gene expression may be a reason that prevents neoplastic transformation and spontaneous immortalization of the cells in vitro. Thus, it is envisaged that telomerase activation in lymphoid cells may surmount cellular ageing for in vitro transformation and cell line establishment.

Molecular Characterization of a Newly Identified Subfamily Member of Penaeidin from two Penaeid Shrimps, Fenneropenaeus indicus and Metapenaeus monoceros.

Penaeidins are a major group of antimicrobial peptides found in penaeid shrimps. This study reports a new isoform of penaeidin from the hemocytes of Indian white shrimp, Fenneropenaeus indicus (Fi-PEN, JX657680), and the pink shrimp, Metapenaeus monoceros (Mm-PEN, KF275674). Mm-PEN is also the first antimicrobial peptide to be identified from M. monoceros. The complete coding sequences of the newly identified Fi-PEN and Mm-PEN consisted of an ORF of 338 bp encoding 71 amino acids with a predicted molecular weight of 5.66 kDa and a pI of 9.38. The penaeidins had its characteristic signal peptide region (19 amino acids), which was followed by a mature peptide with a proline-rich domain (24 amino acids) at the N-terminal region and a cysteine-rich domain (28 amino acids) at the C-terminal region, designating it to penaeidin-3 subgroup. Structural analysis revealed an alpha-helix in its secondary structure and an extended structure at the proline-rich domain. The newly identified penaeidin isoform showed maximum similarity of 63 % to a penaeidin-3 isoform of P. monodon, which further proves it to be a new isoform. Phylogenetic analysis showed that it possessed similar evolutionary status like other penaeidins, which has subsequently diverged at different phases of evolution. The wide distribution of penaeidins in penaeid shrimps indicates the importance of these AMPs in the innate immunity.

Attempts on producing lymphoid cell line from Penaeus monodon by induction with SV40-T and 12S EIA oncogenes.

In an attempt of in vitro transformation, transfection mediated expression of Simian virus-40 (T) antigen (SV40-T) and transduction mediated expression of Adenovirus type 12 early region 1A (12S E1A) oncogene were performed in Penaeus monodon lymphoid cells. pSV3-neo vector encoding SV40-T oncogene and a recombinant baculovirus BacP2-12S E1A-GFP encoding 12S E1A oncogene under the control of hybrid promoters were used. Electroporation and lipofection mediated transformation of SV40-T in lymphoid cells confirmed the transgene expression by phenotypic variation and the expression of GFP in co-transfection experiment. The cells transfected by lipofection (≥ 5%) survived for 14 days with lower toxicity (30%), whilst on electroporation, most of the cells succumbed to death (60%) and survived cells lived up to 7 days. Transduction efficiency in primary lymphoid cells was more than 80% within 14 days of post-transduction, however, an incubation period of 7 days post-transduction was observed without detectable expression of 12S E1A. High level of oncogenic 12S E1A expression were observed after 14 day post-transduction and the proliferating cells survived for more than 90 days with GFP expression, however, without in vitro transformation and immortalization. The study put forth the requirement of transduction mediated 'specific' oncogene expression along with telomerase activation and epigenetic induction for the immortalization and establishment of shrimp cell line.