The occurrences of multiple drug-resistant strains have been relentlessly increasing in recent years. The aquaculture industry has encountered major disease outbreaks and crucially affected by this situation. The usage of non-specific chemicals and antibiotics expedites the stimulation of resistant strains. Triggering the natural defense mechanism would provide an effective and safest way of protecting the host system. Hence, we have investigated the innate immune function of serine/threonine-protein kinase (STPK) in Macrobrachium rosenbergii (Mr). The in-silico protein analysis resulted in the identification of cationic antimicrobial peptide, MrSL-19, with interesting properties from STPK of M. rosenbergii. Antimicrobial assay, FACS and SEM analysis demonstrated that the peptide potentially inhibits Staphylococcus aureus by interacting with its membrane. The toxic study on MrSL-19 demonstrated that the peptide is not toxic against HEK293 cells as well as human erythrocytes. This investigation showed the significant innate immune property of an efficient cationic antimicrobial peptide, MrSL-19 of STPK from M. rosenbergii.
Considering the importance of heat shock proteins (HSPs) in the innate immune system of prawn, a comparative molecular approach was proposed to study the crustacean large HSPs 60, 70 and 90. Three different large HSPs were identified from freshwater prawn Macrobrachium rosenbergii (Mr) cDNA library during screening. The structural and functional characteristic features of HSPs were studied using various bioinformatics tools. Also, their gene expression and mRNA regulation upon various pathogenic infections was studied by relative quantification using 2(-ΔΔCT) method. MrHSP60 contains a long chaperonin 60 domain at 46-547 which carries a chaperonin 60 signature motif between 427 and 438, whereas MrHSP70 contains a long HSP70 domain at 21-624 and MrHSP90 carries a HSP90 domain at 188-719. The two dimensional analysis showed that MrHSP60 contains more amino acids (52%) in helices, whereas MrHSP70 (40.6%) and MrHSP90 (51.8%) carried more residues in coils. Gene expression results showed significant (P
In this study, we reported a molecular characterization of three CC chemokines namely, CsCC-Chem14, CsCC-Chem20 and CsCC-Chem25 which are were identified from the established cDNA library of striped murrel Channa striatus. Multiple sequence alignment of all the three chemokines revealed the presence of gene specific domains and motifs including small cytokine domain, IL8 like domain, receptor binding site and glycosaminoglycan (GAG) binding sites. Three dimensional structures of the chemokines under study showed an important facet on their anti-microbial property. Tissue specific mRNA expression showed that the CsCC-Chem14 is highly expressed in spleen, CsCC-Chem20 in liver and CsCC-Chem25 in trunk kidney. On challenge C. striatus with oomycete fungus Aphanomyces invadans, both CsCC-Chem20 and CsCC-Chem25 showed significant (P < 0.05) up-regulation compared to CsCC-Chem14. The increase in the expression levels of CsCC-Chem20 and CsCC-Chem25 due to infection showed that they are antimicrobial proteins. But considering the CsCC-Chem14 expression, it is found to be a constitutive chemokine and is involved in homeostatic function in spleen of C. striatus. C. striatus challenged with bacteria Aeromonas hydrophila also exhibited different up-regulation pattern in all the three chemokines at various time points. However, extensive studies are required to determine the functional activities of CsCC-Chem14, CsCC-Chem20 and CsCC-Chem25 in vitro and in vivo to gain more knowledge at the molecular and proteomic levels.
This study reports the comprehensive comparative information of two different detoxification enzymes such as glutathione S-transferases (GSTs) delta and kappa from freshwater giant prawn Macrobrachium rosenbergii (designated as MrGSTD and MrGSTK) by investigating their in-silico characters and mRNA modulation against various biotic and abiotic oxidative stressors. The physico-chemical properties of these cDNA and their polypeptide structure were analyzed using various bioinformatics program. The analysis indicated the variation in size of the polypeptides, presence or absence of domains and motifs and structure. Homology and phylogenetic analysis revealed that MrGSTD shared maximum identity (83%) with crustaceans GST delta, whereas MrGSTK fell in arthropods GST kappa. It is interesting to note that MrGSTD and MrGSTK shared only 21% identity; it indicated their structural difference. Structural analysis indicated that MrGSTD to be canonical dimer like shape and MrGSTK appeared to be butterfly dimer like shape, in spite of four β-sheets being conserved in both GSTs. Tissue specific gene expression analysis showed that both MrGSTD and MrGSTK are highly expressed in immune organs such as haemocyte and hepatopancreas, respectively. To understand the role of mRNA modulation of MrGSTD and MrGSTK, the prawns were inducted with oxidative stressors such as bacteria (Vibrio harveyi), virus [white spot syndrome virus (WSSV)] and heavy metal, cadmium (Cd). The analysis revealed an interesting fact that both MrGSTD and MrGSTK showed higher (P
The antioxidant role of sulfite reductase (SiR) derived from Arthrospira platensis (Ap) was identified through a short peptide, TL15. The study showed that the expression of ApSiR was highly expressed on day ten due to sulfur deprived stress in Ap culture. TL15 peptide exhibited strong antioxidant activity when evaluated using antioxidant assays in a concentration ranging from 7.8 and 125 μM. Further, the cytotoxicity of TL15 peptide was investigated, even at the higher concentration (250 μM), TL15 did not exhibit any toxicity, when tested in vitro using human leucocytes. Moreover, a potential reduction in reactive oxygen species (ROS) production was observed due to the treatment of TL15 peptide (>15.6 μM) to H2O2 exposed leucocytes. For the in vivo assessment of TL15 toxicity and antioxidant ability, experiments were performed in zebrafish (Danio rerio) larvae to analyse the developmental toxicity of TL15 peptide. Results showed that, exposure to TL15 peptide in tested concentrations ranging from 10, 20, 40, and 80 μM, did not affect the development and physiological parameters of the zebrafish embryo/larvae such as morphology, survival, hatching and heart rate. Fluorescent assay was performed using DCFH-DA (2,7-dichlorodihydrofluorescein diacetate) to examine the production of intracellular reactive oxygen species (ROS) in zebrafish treated with TL15 peptide during the embryo-larval stages. Fluorescent images showed that pre-treatment with TL15 peptide to attenuate the H2O2 induced ROS levels in the zebrafish larvae in a dose-dependent manner. Further to uncover the underlying biochemical and antioxidant mechanism, the enzyme activity of superoxide dismutase (SOD), catalase (CAT) and lipid peroxidation (LPO) levels were studied in zebrafish larvae. TL15 pre-treated groups showed enhanced antioxidant enzyme activity, while the hydrogen peroxide (H2O2) exposed larvae showed significantly diminished activity. Overall results from the study revealed that, TL15 act as a potential antioxidant molecule with dose-specific antioxidant property. Thus, TL15 peptide could be an effective and promising source for biopharmaceutical applications.
In aquaculture, accumulation of antibiotics resulted in development of resistance among bacterial pathogens. Consequently, it became mandatory to find alternative to synthetic antibiotics. Antimicrobial peptides (AMPs) which are described as evolutionary ancient weapons have been considered as promising alternates in recent years. In this study, a novel antimicrobial peptide had been derived from goose type lysozyme (LyzG) which was identified from the cDNA library of freshwater fish Channa striatus (Cs). The identified lysozyme cDNA contains 585 nucleotides which encodes a protein of 194 amino acids. CsLyzG was closely related to Siniperca chuatsi with 92.8% homology. The depicted protein sequence contained a GEWL domain with conserved GLMQ motif, 7 active residues and 2 catalytic residues. Gene expression analysis revealed that CsLyzG was distributed in major immune organs with highest expression in head kidney. Results of temporal expression analysis after bacterial (Aeromonas hydrophila) and fungal (Aphanomyces invadans) challenges indicated a stimulant-dependent expression pattern of CsLyzG. Two antimicrobial peptides IK12 and TS10 were identified from CsLyzG and synthesized. Antibiogram showed that IK12 was active against Salmonella enterica, a major multi-drug resistant (MDR) bacterial pathogen which produces beta lactamase. The IK12 induced loss of cell viability in the bacterial pathogen. Flow cytometry assay revealed that IK12 disrupt the membrane of S. enterica which is confirmed by scanning electron microscope (SEM) analysis that reveals blebs around the bacterial cell membrane. Conclusively, CsLyzG is a potential innate immune component and the identified antimicrobial peptide has great caliber to be used as an ecofriendly antibacterial substance in aquaculture.
We investigated the role of protein arginine methylation (PAM) in estrogen receptor (ER)-positive breast cancer cells through pharmacological intervention. Tamoxifen (TAM) or adenosine dialdehyde (ADOX), independently, triggered cell cycle arrest and down-regulated PAM, as reduced protein arginine methyltransferase1 (PRMT1) mRNA and asymmetric dimethylarginine (ADMA) levels. Synergistic effect of these compounds elicited potent anti-cancer effect. However, reduction in ADMA was not proportionate with the compound-induced down-regulation of PRMT1 mRNA. We hypothesized that the disproportionate effect is due to the influence of the compounds on other methyltransferases, which catalyze the arginine dimethylation reaction and the diversity in the degree of drug-protein interaction among these methyltransferases. In silico analyses revealed that independently, ADOX or TAM, binds with phosphatidylethanolamine-methyltransferase (PEMT) or betaine homocysteine-methyl transferase (BHMT); and that the binding affinity of ADOX with PEMT or BHMT is prominent than TAM. These observations suggest that in breast cancer, synergistic effect of ADOX + TAM elicits impressive protective function by regulating PAM; and plausibly, restoration of normal enzyme activities of methyltransferases catalyzing arginine dimethylation could have clinical benefits.
Understanding the mechanism by which the exogenous biomolecule modulates the GLUT-4 signalling cascade along with the information on glucose metabolism is essential for finding solutions to increasing cases of diabetes and metabolic disease. This study aimed at investigating the effect of hamamelitannin on glycogen synthesis in an insulin resistance model using L6 myotubes. Glucose uptake was determined using 2-deoxy-D-[1-3H] glucose and glycogen synthesis were also estimated in L6 myotubes. The expression levels of key genes and proteins involved in the insulin-signaling pathway were determined using real-time PCR and western blot techniques. The cells treated with various concentrations of hamamelitannin (20 µM to 100 µM) for 24 h showed that, the exposure of hamamelitannin was not cytotoxic to L6 myotubes. Further the 2-deoxy-D-[1-3H] glucose uptake assay was carried out in the presence of wortmannin and Genistein inhibitor for studying the GLUT-4 dependent cell surface recruitment. Hamamelitannin exhibited anti-diabetic activity by displaying a significant increase in glucose uptake (125.1%) and glycogen storage (8.7 mM) in a dose-dependent manner. The optimum concentration evincing maximum activity was found to be 100 µm. In addition, the expression of key genes and proteins involved in the insulin signaling pathway was studied to be upregulated by hamamelitannin treatment. Western blot analysis confirmed the translocation of GLUT-4 protein from an intracellular pool to the plasma membrane. Therefore, it can be conceived that hamamelitannin exhibited an insulinomimetic effect by enhancing the glucose uptake and its further conversion into glycogen by regulating glucose metabolism.
This study demonstrates both the antioxidant and anticancer potential of the novel short molecule YT12 derived from peroxiredoxin (Prx) of spirulina, Arthrospira platensis (Ap). ApPrx showed significant reduction in reactive oxygen species (ROS) against hydrogen peroxide (H2 O2 ) stress. The complementary DNA sequence of ApPrx contained 706 nucleotides and its coding region possessed 546 nucleotides between position 115 and 660. Real-time quantitative reverse transcription polymerase chain reaction analysis confirmed the messenger RNA expression of ApPrx due to H2 O2 exposure in spirulina cells at regular intervals, in which the highest expression was noticed on Day 20. Cytotoxicity assay was performed using human peripheral blood mononuclear cells, and revealed that at 10 μM, the YT12 did not exhibit any notable toxicity. Furthermore, ROS scavenging activity of YT12 was performed using DCF-DA assay, in which YT12 scavenged a significant amount of ROS at 25 μM in H2 O2 -treated blood leukocytes. The intracellular ROS in human colon adenocarcinoma cells (HT-29) was regulated by oxidative stress, where the YT12 scavenges ROS in HT-29 cells at 12.5 μM. Findings show that YT12 peptide has anticancer activity, when treated against HT-29 cells. Through the MTT assay, YT12 showed vital cytotoxicity against HT-29 cells. These finding suggested that YT12 is a potent antioxidant molecule which defends ROS against oxidative stress and plays a role in redox balance.
Development of antimicrobial drugs against multidrug-resistant (MDR) bacteria is a great focus in recent years. TG12, a short peptide molecule used in this study was screened from tachykinin (Tac) protein of an established teleost Channa striatus (Cs) transcriptome. Tachykinin cDNA has 345 coding sequence, that denotes a protein contained 115 amino acids; in which a short peptide (TG12) was identified at 83-94. Tachykinin mRNA upregulated in C. striatus treated with Aeromonas hydrophila and Escherichia coli lipopolysaccharide (LPS). The mRNA up-regulation was studied using real-time PCR. The up-regulation tachykinin mRNA pattern confirmed the immune involvement of tachykinin in C. striatus during infection. Further, the identified peptide, TG12 was synthesized and its toxicity was demonstrated in hemolytic and cytotoxic assays using human erythrocytes and human dermal fibroblast cells, respectively. The toxicity study exhibited that the toxicity of TG12 was similar to negative control, phosphate buffer saline (PBS). Moreover, the antibiogram of TG12 was active against Klebsiella pneumonia ATCC 27736, a major MDR bacterial pathogen. Further, the antimicrobial activity of TG12 against pathogenic bacteria was screened using minimum inhibitory concentration (MIC) and anti-biofilm assays, altogether TG12 showed potential activity against K. pneumonia. Fluorescence assisted cell sorter flow cytometer analysis (FACS) and field emission scanning electron microscopy (FESEM) was carried on TG12 with K. pneumonia; the results showed that TG12 significantly reduced K. pneumonia viability as well as TG12 disrupt its membrane. In conclusion, TG12 of CsTac is potentially involved in the antibacterial immune mechanisms, which has a prospectus efficiency in pharma industry against MDR strains, especially K. pneumonia.
Snakehead murrel, Channa striatus is an economically important aquatic species in Asia and are widely cultured and captured because of its nutritious and medicinal values. Their growth is predominantly affected by epizootic ulcerative syndrome (EUS) which is primarily caused by an oomycete fungus, Aphanomyces invadans. However, the molecular mechanism of immune response in murrel against this infection is still not clear. In this study, transcriptome technique was used to understand the molecular changes involved in C. striatus during A. invadans infection. RNA from the control (CF) and infected fish (IF) groups were sequenced using Illumina Hi-seq sequencing technology. For control group, 28,952,608 clean reads were generated and de novo assembly was performed to produce 60,753 contigs. For fungus infected group, 25,470,920 clean reads were obtained and assembled to produce 58,654 contigs. Differential gene expression analysis revealed that a total of 146 genes were up-regulated and 486 genes were down regulated. Most of the differentially expressed genes were involved in innate immune mechanism such as pathogen recognition, signalling and antimicrobial mechanisms. Interestingly, few adaptive immune genes, especially immunoglobulins were also significantly up regulated during fungal infection. Also, the results were validated by qRT-PCR analysis. These results indicated the involvement of various immune genes involved in both innate and adaptive immune mechanism during fungal infection in C. striatus which provide new insights into murrel immune mechanisms against A. invadans.
GR15 is a short molecule or peptide composed of aliphatic amino acids and possesses to have antioxidant properties. The GR15, 1GGGAFSGKDPTKVDR15 was identified from the protein S-adenosylmethionine synthase (SAMe) expressed during the sulfur departed state of Arthrospira platensis (spirulina or cyanobacteria). The in-silico assessment and the structural features of GR15 showed its antioxidant potency. Real-time PCR analysis found the up-regulation of ApSAMe expression on day 15 against oxidative stress due to 10 mM H2O2 treatment in A. platensis (Ap). The antioxidant activity of GR15 was accessed by the cell-free antioxidant assays such as ABTS, SARS, HRAS and NO; the results showed dose-dependent antioxidant activity. The toxicity assay was performed in both in vitro and in vivo models, in which peptide does not exhibit any toxicity in MDCK cell and zebrafish embryos. The intercellular ROS reduction potential of GR15 peptide was also investigated in both in vitro and in vivo models including LDH assay, antioxidant enzymes (SOD and CAT), and fluorescent staining assay (DCFDA, Hochest and Acridine orange sting) was performed; the results showed that the GR15 peptide was effectively reduced the ROS level. Further, RT-PCR demonstrated that GR15 enhanced the antioxidant property and also up-regulated the antioxidant gene, thus reduced the ROS level in both in vitro and in vivo models. Based on the results obtained from this study, we propose that GR15 has the potential antioxidant ability; hence further research can be directed towards the therapeutic product or drug development against disease caused by oxidative stress.
Chitinases play a vital role during the pathogenic invasion and immunosuppression in various organisms including invertebrates and vertebrates. In this study, we have investigated the participation of MrChit-3 (Macrobrachium rosenbergii Chitinase-3) during host-pathogenic interaction in freshwater prawn, M. rosenbergii. Quantitative real-time PCR analysis showed that the expression of MrChit-3 was up-regulated during bacterial, viral and laminarin challenge. Moreover, to understand the antimicrobial role of the GH18 domain, a putative membrane-targeting antimicrobial peptide (MrVG) was identified from the GH18 domain region of the protein and it was chemically synthesized. Physico-chemical features of the GH18 derived antimicrobial peptide (AMP) was assessed by various in silico tools and the antimicrobial property of the peptide was confirmed from in vitro studies. The membrane targeting mechanism of the peptide was determined by flow cytometry (FACS) and scanning electron microscope (SEM) analysis. Interestingly, the peptide was able to inhibit the growth of a chitinolytic fungal pathogen, Aspergillus niger, which was isolated from the shells of M. rosenbergii. The toxicity studies such as hemolysis activity on human blood erythrocytes and cell viability assay with primary kidney cells, HEK293 of MrVG revealed that the peptide was not involved in inducing any toxicity.
Antioxidant peptides are naturally present in food, especially in fishes, and are considered to contain rich source of various bioactive compounds that are structurally heterogeneous. This study aims to identify and characterize the antioxidant property of the WL15 peptide, derived from Cysteine and glycine-rich protein 2 (CSRP2) identified from the transcriptome of a freshwater food fish, Channa striatus. C. striatus is already studied to contain high levels of amino acids and fatty acids, besides traditionally known for its pharmacological benefits in the Southeast Asian region. In our study, in vitro analysis of WL15 peptide exhibited strong free radical scavenging activity in 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), superoxide anion radical and hydrogen peroxide (H2O2) scavenging assay. Further, to evaluate the cytotoxicity and dose-response, the Human dermal fibroblast (HDF) cells were used. Results showed that the treatment of HDF cells with varying concentrations (10, 20, 30, 40 and 50 μM) of WL15 peptide was not cytotoxic. However, the treatment concentrations showed enhanced antioxidant properties by significantly inhibiting the levels of free radicals. For in vivo assessment, we have used zebrafish larvae for evaluating the developmental toxicity and for determining the antioxidant property of the WL15 peptide. Zebrafish embryos were treated with the WL15 peptide from 4 h of post-fertilization (hpf) to 96 hpf covering the embryo-larval developmental period. At the end of the exposure period, the larvae were exposed to H2O2 (1 mM) for inducing generic oxidative stress. The exposure of WL15 peptide during the embryo-larval period showed no developmental toxicity even in higher concentrations of the peptide. Besides, the WL15 peptide considerably decreased the intracellular reactive oxygen species (ROS) levels induced by H2O2 exposure. WL15 peptide also inhibited the H2O2-induced caspase 3-dependent apoptotic response in zebrafish larvae was observed using the whole-mount immunofluorescence staining. Overall results from our study showed that the pre-treatment of WL15 (50 μM) in the H2O2-exposed zebrafish larvae, attenuated the expression of activated caspase 3 expressions, reduced Malondialdehyde (MDA) levels, and enhanced antioxidant enzymes, including superoxide dismutase (SOD) and catalase (CAT). The gene expression of antioxidant enzymes such as glutathione S-transferase (GST), glutathione peroxide (GPx) and γ-glutamyl cysteine synthetase (GCS) was found to be upregulated. In conclusion, it can be conceived that pre-treatment with WL15 could mitigate H2O2-induced oxidative injury by elevating the activity and expression of antioxidant enzymes, thereby decreasing MDA levels and cellular apoptosis by enhancing the antioxidant response, demonstrated by the in vitro and in vivo experiments.