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The germination percentage of teak seed is generally very poor due to its higher percentage of empty seed and poor seed viability. The viable seeds exhibit protracted germination behaviour due to their inherent seed dormancy and other physiochemical characteristics. Hence establishing a teak nursery for largescale plantation activities is a challenging task. This study was undertaken to study the effect of ageing on in vitro true seed and in vivo drupe germination and its dormancy mechanism in teak. Fresh, one-year and two-year stored drupes were used to represent different levels of ageing. Under in vivo conditions, poor drupe germination was observed in fresh drupes (3%) and germination percentage was increased when the drupes were subjected to ageing for one year (17%) or two years (32%). When true seeds separated from fresh drupes and germinated under in vitro conditions, enhanced germination (58.3%) was observed. Biochemical analysis showed that indole-3- acetic acid, indole butyric acid, abscisic acid and coumarin are not present in fresh, one year and two-year-old true seeds. The gibberellic acid was increased with an increase in ageing, but the GA3 did not influence the germination percent under in vitro conditions. Scanning electron microscope (SEM) image of fresh teak true seed showed that embryo tip was shrivelled, whereas one and two-year-old true seed embryo tip bulged; this was confirmed that one and two-year-old true seed embryos were matured and satisfied the after-ripening requirement. Nursery studies revealed that one and two-year-old drupes recorded the highest germination compared to fresh drupes.

An invasive pest, fall armyworm, Spodoptera frugiperda (J.E.Smith) (Lepidoptera: Noctuidae) attacks maize at every stage of development, from seedling emergence up to cob formation. Early instar larvae were seen mostly on leaves of maize with characteristics pin or shot hole symptoms. Later instar larvae were confined to deep whorls, leaving typically ragged like appearance and fed on the reproductive stage of the crop especially tassels and developing cobs resulting in quality and quantity loss of maize produce. The effect of commercially available Bacillus thuringiensis subsp. kurstaki product, Dipel® against the second instar larvae of Fall Armyworm (FAW )was not promising under laboratory conditions. Hence, an effort was made to add an adjuvant along with B. thuringiensis to increase the virulence of commercially available B. thuringiensis.The Laboratory bioassays with B. thuringiensis and seven chemical additives ( T1- Bt + Boric acid, T2- Bt + Zinc oxide, T3- Bt + Sodium nitrate, T4- Bt + Peptone, T5- Bt + Urea, T6- Bt + EDTA, T7- Bt + Citric acid & T8- Bt alone T9- Control) were tested against second instar larvae of Spodoptera frugiperda larvae. The results showed that B. thuringiensis plus sodium nitrate (T3) promoted maximum mortality 82.2 per cent with a minimum LC50 value of 54.620 mg/l. Sodium nitrate boosted B. thuringiensis activity at a concentration of 0.05 per cent by 2.128-fold than B. thuringiensis alone. Overall, sodium nitrate improved the efficacy of B. thuringiensis spray at the maximum level followed by boric acid, urea, EDTA and peptone.

Background Tilapia (Oreochromis niloticus) cultures are frequently infected by Vibrio vulnificus, causing major economic losses to production units. Previously, tilapia expressing recombinant delta-5 desaturase and delta-6 desaturase (D56) were found to be resistant to V. vulnificus infection. In this report, we profile the D56-mediated molecular changes underlying this resistance in tilapia. A comparative transcriptome analysis was performed on V. vulnificus-infected wild-type and D56-transgenic tilapia using Illumina’s sequencing-by-synthesis approach. Gene enrichment analysis on differentially expressed unigenes was performed, and the expression patterns were validated by real-time PCR. Results Comparative transcriptome analysis was performed on RNA-sequence profiles obtained from wild-type and D56-transgenic tilapia at 0, 6 and 24 h post-infection with V. vulnificaus. GO and KEGG gene enrichment analyses showed that D56 regulates several pathways and genes, including fatty acid (FA) metabolism associated, and inflammatory and immune response. Expression of selected FA metabolism-associated, inflammatory and immune responsive genes was validated by qPCR. The inflammatory and immune responsive genes that are modulated by FA-associated D56 likely contribute to the enhanced resistance against V. vulnificus infection in Tilapia. Conclusions Transcriptome profiling and filtering for two-fold change variation showed that 3795 genes were upregulated and 1839 genes were downregulated in D56-transgenic tilapia. These genes were grouped into pathways, such as FA metabolism, FA elongation, FA biosynthesis, biosynthesis of unsaturated FA, FA degradation, inflammation, immune response, and chemokines. FA-associated genes and immune-related genes were modulated by D56 at 6 h and 24 h post infection with V. vulnificus. The expression patterns of FA-related genes, inflammatory genes, antimicrobial peptide genes and immune responsive genes at 0, 3, 6, 12, 24 and 48 h post-infection suggests these genes are involved in the enhanced resistance of D56 transgenic tilapia to V. vulnificus.

The effect of drupe size on in vivo and in vitro germination and its dormancy mechanism in teak have been studied in this paper. Teak drupes were size graded to five categories namely very large with more than 15 mm, large with 13 to

Groundnut is a predominant oilseed crop in India and ranks first among the edible oilseed groups, which produces high nutritive value kernels. A field experiment was conducted involving 55 groundnut accessions to evaluate their performance under sodicity by relative comparison with normal sandy soil during Kharif, 2019. The genotypes VG 18096, VG 15008, TMV 13 and VG 18100 recorded high per se performance for pod yield and kernel yield per plant under sodic soil condition. Higher estimates of phenotypic and genotypic coefficient of variation was recorded for the number of matured pods per plant (32.37 and 27.22), pod yield per plant (29.53 and 23.80) and kernel yield per plant (29.45 and 21.44). Heritability and genetic advance as per cent of the mean was found to be high for the number of matured pods per plant, hundred kernel weight and pod yield per plant, which indicated that these traits were governed by additive gene action and were less influenced by the environment. These traits could be improved through simple phenotypic selection.

Additional file 1: Supplementary Figure S1. Analysis of sample correlation.

Additional file 4: Supplementary Table S2. Comparison of RNA-seq and qRT-PCR.

Additional file 2: Supplementary Figure S2. Melting curve analysis of target genes.

Additional file 3: Supplementary Table S1. List of primers used in qPCR.

The antimicrobial peptide (AMP) piscidin was identified from Epinephelus lanceolatus and demonstrated to possess antimicrobial and immune-related functions. Supplementation of feed with recombinant Epinephelus lanceolatus piscidin (rEP)-expressing yeast pellets may minimize the excessive use of antibiotics and control pathogens in aquaculture or animal husbandry. However, before implementing rEP as a supplement, it is necessary to understand whether it harbors any toxicity. Since toxicological information on the topic is scarce, the present investigation was carried out to test whether rEP exhibits allergenic and/or toxic effects. In an oral acute toxicity test (OECD 425), Sprague Dawley (SD) rats were administered rEP dissolved in reverse osmosis water, yielding an LD50 &gt, 5000 mg/kg (no observed animal death). The compound was therefore classified as non-toxic by oral administration. In an acute respiratory toxicity test (OECD 403), heads and noses of SD rats were exposed to liquid aerosol for 4 h (the highest concentration that could be administered without causing any animal death), and a lethal concentration (LC50) &gt, 0.88 mg/L was obtained. The mass medium aerodynamics diameter (MMAD) of rEP aerosol particles was 8.18 &mu, m and mass medium aerodynamics diameter (GSD) was 3.04, which meant that 25.90% could enter the airway (&lt, 4 &mu, m) of a rat, and 58.06% (&lt, 10 &mu, m) could be inhaled by humans. An ocular irritation test (OECD 405) with rEP powder was performed on New Zealand White (NZW) rabbits. Signs of irritation included conjunctival swelling and diffuse flushing 1 h after administration. The signs were less apparent after 24 h and disappeared after 72 h. The classification assigned to the powder was mild eye irritation. Skin sensitization was performed for a local lymphoproliferative test (OECD 442B) using BALB/c mice, with the highest soluble concentration of the rEP considered to be 100% test substance, formulations were diluted to 50% and 25%, and bromodeoxyuridine (BrdU) incorporation was used to measure the degree of lymphocyte proliferation. The stimulation indexes (SIs) were 1.06 (100%), 0.44 (50%), and 0.77 (25%), all of which were less than the cutoff value for a positive sensitization result (1.6). Negative response was also seen in the bacterial reverse mutation test (OECD 471), and no chromosomal effects on Chinese hamster ovary (CHO)-K1 cells were observed (OECD 487). Based on these six toxicity tests, rEP showed neither acute toxic effects in experimental animals nor mutagenicity. Thus, rEP can be considered safe for use in subsequent research on its application as a feed additive for poultry, cattle, or aquatic animals.

Hepcidin regulates iron homeostasis and host-defense mechanisms, while the hepcidin-like protein, Tilapia hepcidin (TH)2-3, functions as an antimicrobial peptide (AMP). Since AMP dietary supplements may be used as alternatives to antibiotics in livestock, we tested the effects of recombinant (r)TH2-3 as a dietary supplement in grouper aquaculture. rTH2-3 was produced by a Pichia pastoris expression system and exhibited thermostability and broad-spectrum antimicrobial activity. The feed conversion ratio and feed efficiency were determined in Epinephelus lanceolatus (grouper) fed with rTH2-3-supplemented diet for 28 days. In addition, grouper showed enhanced superoxide dismutase (SOD) activity after rTH2-3 feeding compared to regular-diet-fed fish. Gut microbiota analysis revealed that microbial diversity was enhanced by feeding grouper with 1% rTH2-3. After challenging grouper with Vibrio alginolyticus, differential regulation of immune-related genes in the liver and spleen was observed between the TH2-3 and regular-diet groups, including for genes associated with antimicrobial and pro-inflammatory functions, complement components, and major histocompatibility complex (Mhc). These findings suggest that overall immunity was improved. Thus, our results suggest long-term supplementation with rTH2-3 may be beneficial for aquacultured grouper. The beneficial effects of the supplement are likely based on changes in the commensal microbial community as well as immunomodulation.

Supplementing chicken feed with antibiotics can improve survival and prevent disease outbreaks. However, overuse of antibiotics may promote the development of antibiotic-resistant bacteria. Recently, antimicrobial peptides have been proposed as alternatives to antibiotics in animal husbandry. Here, we evaluate the effects of antimicrobial peptide, Epinephelus lanceolatus piscidin (EP), in Gallus gallus domesticus. The gene encoding EP was isolated, sequenced, codon-optimized and cloned into a Pichia pastoris recombinant protein expression system. The expressed recombinant EP (rEP) was then used as a dietary supplement for G. g. domesticus; overall health, growth performance and immunity were assessed. Supernatant from rEP-expressing yeast showed in vitro antimicrobial activity against Gram-positive and Gram-negative bacteria, according to an inhibition-zone diameter (mm) assay. Moreover, the antimicrobial peptide function of rEP was temperature independent. The fermentation broth yielded a spray-dried powder formulation containing 262.9 μg EP/g powder, and LC-MS/MS (tandem MS) analysis confirmed that rEP had a molecular weight of 4279 Da, as expected for the 34-amino acid peptide; the DNA sequence of the expression vector was also validated. We then evaluated rEP as a feed additive for G. g. domesticus. Treatment groups included control, basal diet and rEP at different doses (0.75, 1.5, 3.0, 6.0 and 12%). Compared to control, rEP supplementation increased G. g. domesticus weight gain, feed efficiency, IL-10 and IFN-γ production. Our results suggest that crude rEP could provide an alternative to traditional antibiotic feed additives for G. g. domesticus, serving to enhance growth and health of the animals.

Incomplete clearance of apoptotic cells and reactive oxygen species (ROS) release are known to trigger inflammasome activation causing severe inflammation in acute lung injury and various metabolic and autoimmune diseases. Moreover, it has been reported that apoptotic cell clearance and ROS-mediated apoptosis critically depend on mitochondrial uncoupling protein-2 (UCP2). However, the relationship between UCP2 and inflammasome activation has not been studied. This report investigates the role of UCP2 in the expression and activation of NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome in human macrophages. We found that UCP2 overexpression significantly enhanced the expression levels of NLRP3. The NLRP3 expression levels were significantly suppressed in THP1 cells treated with genipin, a UCP2 inhibitor, compared to controls. In addition, genipin altered adenosine triphosphate (ATP)- and hydrogen peroxide (H2O2)-mediated interleukin-1 beta (IL-1β) secretion and significantly suppressed caspase-1 activity in inflammasome-activated human macrophages. Taken together, our results suggest that genipin modulates NLRP3 inflammasome activation and ATP- or H2O2-mediated IL-1β release.

Spotted pod borer, Maruca vitrata is one of the most serious lepidopteran pests in pulses which occurs from seedling to pod formation stage. The occurrence of M. vitrata depends upon various biophysical and biochemical characters of host plants. Plant characters viz., plant height, number of branches, number of pods per cluster, pod length, pod width, angle between pods, number of seeds per pod, seed size, trichome density and trichome length in pods were studied to observe the association of different morphological traits with resistance or susceptibility to M. vitrata in nine blackgram varieties. Among the tested nine varieties, CO 5 and MDU 1 were considered susceptible having highest pod damage per cent of 14.38 and 13.70, respectively while VBN (Bg) 5 and VBN 6 have lowest pod damage per cent of 5.79 and 6.15, respectively. It was observed that highly susceptible variety CO 5 has less number of trichomes (2.13/mm2) and trichome length (3.95 mm) while the resistant variety VBN 6 has dense trichomes (4.13/mm2) and trichome length (5.01 mm) which is considered to be the main factor to confer resistance in plants.

Rice is the major staple food crop in Asia and it feeds more than 70 per cent population of India. A study was carried out in rice using F2 segregating population of three cross combinations viz., ASD 16 × CSR 23, ASD 16 × Anna (R) 4 and MDU 6 × Ezhome1 in a non-randomized experimental design under sodic soil condition. Observations were recorded on 12 morphometric traits viz., days to flowering, plant height, flag leaf length, flag leaf width, number of productive tillers, panicle length, number of grains per panicle, spikelet fertility percentage, chlorophyll content, thousand grain weight, grain length breadth ratio and single plant yield. Genetic variability parameters and correlation varied from cross to cross and the traits number of productive tillers and number of grains per panicle showed high PCV and GCV. These characters also showed high positive correlation towards single plant yield with high direct effects in path analysis. Hence, these traits could be relied upon as selection indices under sodic soil conditions in rice.

The marine antimicrobial peptide (AMP) GE33, also known as pardaxin, possesses antimicrobial and anticancer properties, and modulates host signaling. GE33 has cytotoxic effects on murine bladder carcinoma (MBT-2) cells. Here, we investigated the potential of GE33 combined with inactivated MBT-2 as a cancer vaccine. The presence of up to 12.5 μg of GE33 did not inhibit the proliferation or endogenous nitrous oxide (NO) levels of RAW264.7 cells. However, the secretion of MCP-1, IL-6, and IL-12 by RAW264.7 cells was affected by GE33. We proceeded to test the effectiveness of the vaccine by immunizing mice at 7, 14, and 21 days of age, and injecting live MBT-2 cells on the 28th day. Tumor growth by the 58th day was attenuated in mice treated with the vaccine, as compared to the control group. Induction of MBT-2 specific-tumor antigens was increased in mice immunized with our vaccine. Furthermore, activation of T-cell receptors, cytotoxic T-cells, and NK cells was enhanced, and these showed high specificity for targeting tumor cells. Finally, immunization controlled excess recruitment of monocytes, lymphocytes, T-helper cells, and NK cells, and decreased the expression of VEGF. This report provides empirical evidence that our GE33-based vaccine enhances antitumor immunity in mice.

Current efforts to improve the effectiveness of vaccines include incorporating antimicrobial peptides mixed with a virus. The antimicrobial peptide, epinecidin (Epi)-1, was reported to have an antiviral function, and an Epi-1-based inactivated vaccine was postulated as a model and discussed. In this report, we demonstrated modulation of immune responses by Epi-1 and an Epi-1-based Japanese encephalitis virus (JEV)-inactivated vaccine against JEV infection in mice. Under in vitro conditions, Epi-1 prevented JEV infection-mediated loss of cell viability in BHK-21 cells. When Epi-1 and JEV were co-injected into mice and mice were re-challenged with JEV after 14 days, all mice survived. In addition, Epi-1 modulated the expressions of immune-responsive genes like interleukin (IL)-6, IL-10, MCP-1, tumor necrosis factor-α, interferon-γ and IL-12, and elevated the levels of anti-JEV-neutralizing antibodies in the serum. The presence of Epi-1 suppressed the multiplication of JEV in brain sections at 4 days after an injection. Mice immunized with the developed vaccine showed complete survival against JEV infection, and it was superior to the traditional formalin-based JEV-inactivated vaccine. This study demonstrates the use of Epi-1 to develop an inactivated vaccine can provide guidelines for the future design of Epi-1-virus formulations for various in vivo applications.

The antimicrobial peptide, tilapia hepcidin (TH) 2-3, belongs to the hepcidin family, and its antibacterial function has been reported. Here, we examined the TH2-3-mediated regulation of proinflammatory cytokines in bacterial endotoxin lipopolysaccharide (LPS)-stimulated mouse macrophages. The presence of TH2-3 in LPS-stimulated cells reduced the amount of tumor necrosis factor (TNF)-α secretion. From a microarray, real-time polymerase chain reaction (PCR), and cytokine array studies, we showed down-regulation of the proinflammatory cytokines TNF-α, interleukin (IL)-1α, IL-1β, IL-6, and the prostaglandin synthesis gene, cyclooxygenase (COX)-2, by TH2-3. Studies with the COX-2-specific inhibitor, melaxicam, and with COX-2-overexpressing cells demonstrated the positive regulation of TNF-α and negative regulation of cAMP degradation-specific phosphodiesterase (PDE) 4D by COX-2. In LPS-stimulated cells, TH2-3 acts like melaxicam and down-regulates COX-2 and up-regulates PDE4D. The reduction in intracellular cAMP by TH2-3 or melaxicam in LPS-stimulated cells supports the negative regulation of PDE4D by COX-2 and TH2-3. This demonstrates that the inhibition of COX-2 is among the mechanisms through which TH2-3 controls TNF-α release. At 1 h after treatment, the presence of TH2-3 in LPS-stimulated cells had suppressed the induction of pERK1/2 and prevented the LPS-stimulated nuclear accumulation of NF-κB family proteins of p65, NF-κB2, and c-Rel. In conclusion, TH2-3 inhibits TNF-α and other proinflammatory cytokines through COX-2-, PDE4D-, and pERK1/2-dependent mechanisms.

We tested the activity of epinecidin-1, a novel antimicrobial peptide structurally related to pleurocidin, in commercial cleaning solutions stored at 4 and 25 degrees C for 7 and 14 days. The peptide's activities against Enterococcus faecalis, Escherichia coli, Klebsiella oxytoca, Pseudomonas aeruginosa, Staphylococcus aureus, Propionibacterium acnes, and Candida albicans were measured in a minimum inhibitory concentration (MIC) determination, minimal bactericidal concentration (MBC) determination, disk diffusion test, and a count of the bacterial numbers. Exposure to epinecidn-1 in a cleaning solution following MIC value comparisons in the disk diffusion test and counts of bacterial numbers after 16, 24, 48, and 72 h suggested that bacterial numbers were much lower than those treated with only commercial cleaning solutions for all bacteria. The efficacy of the antimicrobial activities of inhibiting bacterial numbers by epinecidin-1 in cleaning solutions at a low pH and a low temperature was not affected. Given its simple structure and antimicrobial activity, epinecidin-1 may be a useful component of microbicides designed to prevent pathogen infections and/or remediate abnormal vaginal or skin flora.

Antimicrobial peptides (AMP ), also known as host defense peptides or alarmins, are among the first lines of defense against infection in many organisms. Marine organisms have proved to be a rich source of AMPs, and several uniquely structured marine AMPs have been isolated using biochemical, in silico and genetic approaches. Hemolymph is the main source of AMPs in marine invertebrates, although other tissues may also contain these peptides. Based on their three-dimensional structure, AMPs may be classified into peptides with α-helix structures, peptides with β-sheets and cysteine residues, peptides enriched for modified and rare amino acids, and peptides with ring structure amino acids. This chapter describes AMPs of each structure, in terms of their antibacterial, antiviral, antifungal, insecticidal, nematicidal, and immunomodulatory functions. It is hoped that the diversity of marine AMPs may facilitate the identification of effective substitutes to existing antibiotics, thereby helping to minimize the development of antibiotic-resistant pathogen strains in response to the continuous application of a single antibiotic therapy.

Nervous necrosis virus (NNV) infects a wide range of larval and juvenile fish species, thereby causing enormous economic losses in the aquaculture industry. Possible solutions to this problem include the use of antimicrobial peptides (AMPs), which directly inhibit bacterial growth, and also modulate host signaling mechanisms. The AMPs epinecidin (Epi)-1 and Tilapia hepcidin (TH) 1-5 have been demonstrated to be effective against Nervous necrosis virus infection in medaka (Oryzias latipes). However, the underlying molecular mechanisms are yet to be explored. Here, microarray analyses were performed to examine how NNV infection and/or epinecidin-1 or TH1-5 treatment affects gene expression in medaka; such analyses enabled the prediction of host signaling pathways affected by virus infection and/or regulated by epinecidin-1 and TH1-5. Transcriptome analysis revealed altered expression of genes involved in B cell activation, T cell activation, adipocytokine signaling, and mast cell activation. We subsequently used real-time PCR to analyze expression of key genes involved in these signaling mechanisms. Medaka infected with NNV exhibited up-regulation of PVALB, CEBPA, IFIM, IFN, IL-6ST, NF-kB2, SOC3, SP1, and TGFB1, and such increases were prevented by pre-treatment with epinecidin-1 or TH1-5. Immunohistochemistry using the anti-NNV antibody to stain brain and eye sections revealed that epinecidin-1 treatment during or after infection clears viral load, while TH1-5 treatment only reduces viral numbers if applied during infection. These observations demonstrate that epinecidin-1 and TH1-5 modulate NNV-induced host signaling mechanisms, thereby preventing viral multiplication in host organisms.

Methicillin-resistant Staphylococcus aureus (MRSA) causes infections through open skin injuries, and its resistance makes treatment difficult. The antimicrobial peptide Epinecidin-1 (Epi-1) has been reported to possess antibacterial, antifungal, antiviral, and antitumor functions. This study investigated the antimicrobial activity of Epi-1 against skin trauma-mediated MRSA infection in mice. One square centimeter of outer skin was excised from the ventral region of mice, and a lethal dose of MRSA was applied in the presence or absence of methicillin, vancomycin, or Epi-1. While untreated mice and mice treated with methicillin died within four days, mice treated with Epi-1 survived infection. Epi-1 decreased MRSA bacterial counts in the wounded region, enhanced wound closure, and increased angiogenesis at the injury site. Treatment with Epi-1 decreased serum levels of the proinflammatory cytokines TNF-α, IL-6, and MCP-1, and regulated the recruitment of monocytes and clearance of lymphocytes around the wounded region during healing. In conclusion, Epi-1 may be effective at treating clinical MRSA, and may enhance wound recovery when combined with collagen.

The seventh author's name was spelled incorrectly. The correct name is: Shyam S. Mohapatra.

Introduction Pulmonary Arterial Hypertension (PAH) is a progressively devastating disease characterized by excessive proliferation of the Pulmonary Arterial Smooth Muscle Cells (PASMCs). Studies suggest that PAH and cancers share an apoptosis-resistant state featuring excessive cell proliferation. The proliferation of cancer cells is mediated by increased expression of Enhancer of Zeste Homolog 2 (EZH2), a mammalian histone methyltransferase that contributes to the epigenetic silencing of target genes. However, the role of EZH2 in PAH has not been studied. In this study, it is hypothesized that EZH2 could play a role in the proliferation of PASMCs. Methods In the present study, the expression patterns of EZH2 were investigated in normal and hypertensive mouse PASMCs. The effects of EZH2 overexpression on the proliferation of human PASMCs were tested. PASMCs were transfected with EZH2 or GFP using nucleofector system. After transfection, the cells were incubated for 48 hours at 37°C. Proliferation and cell cycle analysis were performed using flow cytometry. Apoptosis of PASMCs was determined using annexin V staining and cell migration was tested by wound healing assay. Results EZH2 protein expression in mouse PASMCs were correlated with an increase in right ventricular systolic pressure and Right Ventricular Hypertrophy (RVH). The overexpression of EZH2 in human PASMCs enhances proliferation, migration, and decrease in the rate of apoptosis when compared to GFP-transfected cells. In the G2/M phase of the EZH2 transfected cells, there was a 3.5 fold increase in proliferation, while there was a significant decrease in the rate of apoptosis of PASMCs, when compared to control. Conclusion These findings suggest that EZH2 plays a role in the migration and proliferation of PASMCs, which is a major hallmark in PAH. It also suggests that EZH2 could play a role in the development of PAH and can serve as a potential target for new therapies for PAH.

The antimicrobial and immunomodulatory functions of the antimicrobial peptide, tilapia hepcidin (TH)2-3, against a bacterial endotoxin under in vitro conditions was previously reported. In this study, we investigated the antibacterial and immunomodulatory functions of TH2-3 in mice infected with the pathogen, Vibrio vulnificus. A TH2-3 injection in V. vulnificus-infected mice produced an increased survival rate compared to mice injected with V. vulnificus only. In addition, a TH2-3 injection increased the bacteriostatic property against V. vulnificus in mice. Gene expressions examined using a microarray demonstrated that TH2-3 modulated several V. vulnificus-responsive genes in the host. A neutralizing antibody assay of mice serum against inactivated V. vulnificus antigen-coated plates demonstrated the induction of an immune response by TH2-3 against the pathogen. Taken together, TH2-3 enhanced the survival rate of mice against the bacterial pathogen V. vulnificus through both antimicrobial and immunomodulatory functions. These properties make the TH2-3 peptide a good candidate for development as a new antimicrobial drug and suggest that TH2-3 can underpin the design of adjuvants for further development of vaccines.

Fish are a major component of the aquatic fauna. Like other organisms, fish secrete different kinds of antimicrobial peptides (AMPs), which are positively charged short amino-acid-chain molecules involved in host defense mechanisms. Environmental hazards and the greenhouse effect have led to increased evolution of drug- and vaccine-resistant pathogenic strains, and it is necessary to find new drugs with structural uniqueness to fight them. Aquatic sources contain thousands of fish species, and each secretes AMPs with structural differences which can be used by the pharmaceutical industry in its search for novel drugs to treat drug-resistant pathogens. Not only limited to antimicrobial functions, AMPs possess other desirable characteristics which may be exploited in the near future. In this review, we list fish AMPs available from published reports, and discuss application-oriented functions of these AMPs. Notably, the possibilities of using fish AMPs as antimicrobial agents, vaccine adjuvants, inactivated vaccines, and antitumor agents are discussed in this review.

The antimicrobial and immunomodulatory functions of the antimicrobial peptide, tilapia hepcidin (TH)2-3, were previously studied. Herein, we report the differential modulation of protein kinase C (PKC)-associated proteins by TH2-3, and the PKC activator, phorbol 12-myristate 13-acetate (PMA), in RAW264.7 macrophages. Treatment with TH2-3 at 40 or 80μg/ml did not affect the cell morphology, but TH2-3 at 120μg/ml produced morphological changes similar to those after treatment with PMA in RAW264.7 cells. The coexistence of the PKC inhibitor, Ro-31-8220, prevented morphological changes induced by either PMA or 120μg/ml TH2-3 in RAW264.7 cells. Since PMA is known to induce expression of the proinflammatory cytokine, tumor necrosis factor (TNF)-α, activation of the TNF-α promoter in response to TH2-3 and PMA treatments in lipopolysaccharide (LPS)-stimulated cells was compared. In LPS-stimulated RAW264.7 cells, TNF-α promoter activity was significantly suppressed by TH2-3, but not by PMA. In addition, PMA activated prostaglandin synthase-associated cyclooxygenase (COX)-2 proteins on the cell surface, while the presence of TH2-3 inhibited its expression. Western blotting demonstrated that the expressions of PKC-μ, phosphorylated (p)-PKCμ at serine (S)-744, and p-PKCδ were activated by PMA, but were suppressed by TH2-3. In addition, p-PKC at S-916 was activated by TH2-3 and inhibited by PMA. In conclusion, the differential regulation of PKC isoforms by PMA and TH2-3 may influence morphological changes and regulation of TNF-α in RAW264.7 cells.

The inhibitory function of tilapia hepcidin (TH)1-5, an antimicrobial peptide, was not examined in previous studies. In this study, we synthesized the TH1-5 peptide and tested TH1-5's antitumor activity against several tumor cell lines. We show that TH1-5 inhibited the proliferation of tumor cells and reduced colony formation in a soft agar assay. Scanning electron microscopy and transmission electron microscopy showed that TH1-5 altered the membrane structure similar to the function of a lytic peptide. Acridine orange/ethidium bromide staining, a wound-healing assay, and a flow cytometric analysis showed that TH1-5 induced necrosis with high-concentration treatment and induced apoptosis with low-concentration treatment. Inflammation is known to be closely associated with the development of cancer. TH1-5 showing anti-inflammatory effects in a previous publication induced us to evaluate the anti-inflammatory effects in cancer cell lines through the expressions of immune-related genes after being treated with the TH1-5 peptide. However, real-time qualitative RT-PCR indicated that TH1-5 treatment induced downregulation of the expressions of interleukin (IL)-6, IL-8, IL-12, IL-15, interferon-γ, CTSG, caspase-7, and Bcl-2, and upregulation of IL-2 and CAPN5 in HeLa cells, and upregulation of IL-8 and CTSG in HT1080 cells. These results suggest that TH1-5 possibly induces an inflammatory response in HeLa cells, but not in HT1080 cells. Overall, these results indicate that TH1-5 possesses the potential to be a novel peptide for cancer therapy.

Antimicrobial peptides, small cysteine-rich molecules, play vital roles in host defense mechanisms against pathogen infection. Recently, tilapia hepcidin (TH)1–5, was characterized, and its antimicrobial functions against several pathogens were reported. Herein, we investigated the antiviral functions of TH1-5 against infectious pancreatic necrosis virus (IPNV) in Chinook salmon embryo (CHSE)-214 cells. The presence of TH1-5 enhanced the survival of CHSE-214 cells infected with IPNV. Additionally, the number of plaques formed by the cytopathic effect of IPNV in CHSE-214 cells decreased when IPNV was preincubated with TH1-5. This observation demonstrates the antiviral function of TH1-5. Real-time PCR studies showed the modulation of interleukin, annexin, and other viral-responsive gene expressions by TH1-5. When TH1-5 and IPNV were used to co-treat CHSE-214 cells, then cells were re-challenged with IPNV at 24 h, the cells did not survive the IPNV infection. This shows that in the absence of TH1-5, viral re-challenge killed CHSE-214 cells. In conclusion TH1-5 protected CHSE-214 cells against IPNV by direct antimicrobial and immunomodulatory functions.