Toxicological analysis of synthetic dye orange red on expression of NFκB-mediated inflammatory markers in Wistar rats.

Orange red is a food and cosmetic coloring agent made by the amalgamation of two azo dyes carmoisine and sunset yellow. The current study demonstrates the effect of different concentrations of orange red on antioxidant status, inflammatory biomarkers (TNFα, IFNγ, IL1ß, IL6, COX-2, iNOS, and NFκB/p65), biochemical enzymes, and liver histology. In totality, 25 male Wistar rats were procured and arbitrarily alienated into 5 different groups each with 5 animals. Group I was taken as the control. Groups II-V were designated as treatment groups. Groups II and III were administered with (5 and 25 mg/kg b.wt.) and groups IV and V with (150 and 300 mg/kg b.wt.) of orange red via oral gavage for 30 days. It was observed that both low and high concentrations of orange red (25, 150, and 300 mg/kg) remarkably augmented the levels of serum inflammatory cytokines (TNFα, IFNγ, IL1ß, and IL6) and the protein and gene expression of COX-2, iNOS, and NFκB/p65. A significant decrease in glutathione reductase, glutathione peroxidase, glutathione-S-transferase, superoxidase dismutase, and catalase activity was observed with increasing concentration of orange red. Furthermore, an increase in the level of several vital biochemical parameters and damage severity to hepatic tissue was also found dose dependent.

Potential Efficacy of Chitosan-Poly (Lactide-Co-Glycolide)-Encapsulated Trivalent Immersion Vaccine in Olive Flounder (Paralichthys olivaceus) Against Viral Hemorrhagic Septicemia Virus, Streptococcus parauberis Serotype I, and Miamiensis avidus (Scuticociliate).

Olive flounder (Paralichthys olivaceus) is the most valuable aquaculture species in Korea, corresponding to ~60% of its total production. However, infectious diseases often break out among farmed flounders, causing high mortality and substantial economic losses. Although some deleterious pathogens, such as Vibrio spp. and Streptococcus iniae, have been eradicated or contained over the years through vaccination and proper health management, the current disease status of Korean flounder shows that the viral hemorrhagic septicemia virus (VHSV), Streptococcus parauberis, and Miamiensis avidus are causing serious disease problem in recent years. Furthermore, these three pathogens have differing optimal temperature and can attack young fingerlings and mature fish throughout the year-round culture cycle. In this context, we developed a chitosan-poly(lactide-co-glycolide) (PLGA)-encapsulated trivalent vaccine containing formalin-killed VHSV, S. parauberis serotype-I, and M. avidus and administered it to olive flounder fingerlings by immersion route using a prime-boost strategy. At 35 days post-initial vaccination, three separate challenge experiments were conducted via intraperitoneal injection with the three targeted pathogens at their respective optimal temperature. The relative percentages of survival were 66.63%, 53.3%, and 66.75% in the group immunized against VHSV, S. parauberis serotype-I, and M. avidus, respectively, compared to the non-vaccinated challenge (NVC) control group. The immunized fish also demonstrated significantly (p < 0.05) higher specific antibody titers in serum and higher transcript levels of Ig genes in the mucosal and systemic tissues than those of NVC control fish. Furthermore, the study showed significant (p < 0.05) upregulation of various immune genes in the vaccinated fish, suggesting induction of strong protective immune response, ultimately leading to improved survival against the three pathogens. Thus, the formulated mucosal vaccine can be an effective prophylactic measure against VHS, streptococcosis, and scuticociliatosis diseases in olive flounder.

Comparative study on antigen persistence and immunoprotective efficacy of intramuscular and intraperitoneal injections of squalene - aluminium hydroxide (Sq + Al) adjuvanted viral hemorrhagic septicaemia virus vaccine in olive flounder (Paralichthys olivaceus).

The profitability of the olive flounder (Paralichthys olivaceus) aquaculture industry in Korea depends on high production and maintenance of flesh quality, as consumers prefer to eat raw flounders from aquaria and relish the raw muscles as 'sashimi'. For sustaining high production, easy-to-deliver and efficient vaccination strategies against serious pathogens, such as viral hemorrhagic septicemia virus (VHSV), is very important as it cause considerable losses to the industry. Whereas, a safe and non-invasive vaccine formulation that is free from unacceptable side-effects and does not devalue the fish is needed to maintain flesh quality. We previously developed a squalene-aluminium hydroxide (Sq + Al) adjuvanted VHSV vaccine that conferred moderate to high protection in flounder, without causing any side effects when administered through the intraperitoneal (IP) injection route. However, farmers often demand intramuscular (IM) injection vaccines as they are relatively easy to administer in small fishes. Therefore, we administered the developed vaccine via IP and IM routes and investigated the safety and persistency of the vaccine at the injection site. In addition, we conducted a comparative analysis of vaccine efficacy and serum antibody response. The clinical and histological observation of the IM and IP groups showed that our vaccine remained persistence at the injection sites for 10-17 weeks post vaccination (wpv), without causing any adverse effects to the fish. The relative percentage of survival were 100% and 71.4% for the IP group and 88.9% and 92.3% for the IM group at 3 and 17 wpv, respectively. Thus, considering the persistency period (24 wpv) and both short and long-term efficacy of our vaccine, the present study offers an option to flounder farmers in selecting either IM or IP delivery strategy according to their cultured fish size and harvesting schedule - IM vaccination for small-sized fish and IP vaccination for table-sized fish.

Active Cousinia thomsonii Extracts Modulate Expression of Crucial Proinflammatory Mediators/Cytokines and NFκB Cascade in Lipopolysaccharide-Induced Albino Wistar Rat Model.

INTRODUCTION: Chronic inflammation is implicated in a multitude of diseases, including arthritis, neurodegeneration, autoimmune myositis, type 2 diabetes, rheumatic disorders, spondylitis, and cancer. Therefore, strategies to explore potent anti-inflammatory regimens are pivotal from a human-health perspective. Medicinal plants represent a vast unexplored treasure trove of therapeutically active constituents with diverse pharmacological activities, including anti-inflammatory properties. Herein, we evaluated Cousinia thomsonii, an edible medicinal herb, for its anti-inflammatory/immunomodulatory properties. METHODS: Soxhlet extraction was used to obtain different solvent extracts (hexane, ethyl acetate, ethanol, methanol, and aqueous extract) in increasing order of polarity. In vitro anti-inflammatory assays were performed to investigate the effects of extracts on protein denaturation, proteinase activity, nitric oxide surge, and erythrocyte-membrane stabilization. The most effective extracts, ie, ethyl acetate (CTEA) and ethanol (CTE) extracts (150-200 g) were selected for further in vivo analysis using albino Wistar rats. Wistar rats received varying concentrations of CTEA and CTE (25, 50, and 100 mg/kg) for 3 weeks, followed by a single subplantar injection of lipopolysaccharide. Dexamethasone served as positive control. Blood was obtained from the retro-orbital plexus and serum separated for estimation of proinflammatory cytokines (IL6, IL1ß, IFNγ and TNFα). Western blotting was performed to study expression patterns of crucial proteins implicated in the NFκB pathway, ie, NFκB p65, NFκB1 p50, and NFκB2 p52. Histopathological examination was done and gas chromatography-mass spectrometry (GC-MS) carried out to reveal the identity of compounds responsible for ameliorating effects of C. thomsonii. RESULTS: Among five tested extracts, CTEA and CTE showed marked inhibition of protein denaturation, proteinase activity, nitric oxide surge and erythrocyte-membrane hemolysis at 600 µg/mL (P<0.001). Both these extracts showed no toxic effects up to a dose of 2,500 mg/kg. Extracts exhibited concentration-dependent reductions in expression of IL6, IL1ß, IFNγ, TNFα, NFκB-p65, NFκB1, and NFκB2 (P<0.05). Healing effects of extracts were evident from histopathological investigation. GC-MS analysis revealed the presence of important anti-inflammatory compounds, notably stigmast-5-en-3-ol, oleate, dotriacontane, ascorbic acid, n-hexadecanoic acid, and α-tocopherol, in C. thomsonii. CONCLUSION: C. thomsonii possesses significant anti-inflammatory/immunomodulatory potential by virtue of modifying levels of proinflammatory cytokines/markers and NFκB proteins.

Identification, functional characterization and expression profiling of cytochrome p450 1A (CYP1A) gene in Labeo rohita against emamectin benzoate.

The cytochrome p450 1A (CYP1A) plays vital role in detoxification of xenobiotic compounds in living organisms. In the present study, full-length CYP1A gene was sequenced from liver of Labeo rohita and mRNA expression analysis were carried out at 0, 2, 4, 8, 12, 24, 48, 72, 96 and 120 h (h) time points after emamectin benzoate treatment. The full-length cDNA sequence of CYP1A was 1741 bp which consist of open reading frame (ORF) of 1618 bp, 5'-untranslated region (UTR) 48 bp and 75 bp 3'-UTR respectively. ORF encodes 526 amino acids with a molecular mass a 59.05 kDa and an isoelectric point of 8.74. The subcellular localization confirmed presence of the CYP1A protein was higher in plasma membrane (45.8%), followed by the mitochondrial region (13.9%) and nuclear region (9.2%). The CYP1A protein interaction was found to intermingle more with other CYP family proteins. Analysis of tissue distribution revealed that CYP1A gene was predominantly expressed in the liver compared to other tissues kidney, gills, muscle and intestine. Furthermore, present study reveals that CYP1A mRNA level in emamectin benzoate treated group @ 20 mgkg-1 body was significantly (p < 0.05) higher compared with the control. The CYP1A mRNA expression levels were found upregulating with time and highest expression levels at 24 h. Histological examination found that emamectin benzoate treated liver revealed vacuolisation, hepatocyte infiltrations, cytoplasmic degeneration of hepatocytes compared to control. Overall, present results lay a strong basis for CYP1A is important biomarker for drug detoxification in aquatic animals.

Molecular and computational analysis of Ghrelin, growth hormone Secretagogues receptor and mRNA expression of Growth-related genes after exogenous administered ghrelin peptide in Labeo rohita.

Ghrelin is a peptide hormone secreted primarily by the stomach and is involved in controlling growth by governing different functions in vertebrates including feed intake and metabolism in vertebrates. This work was aimed to identify sequences of ghrelin gene and growth hormone secretagogue receptor (GHSR) in Labeo rohita. The full-length cDNA sequence of ghrelin is 453 bp including 5'-untranslated region (UTR) of 65 bp, 3'-UTR of 76 bp with a poly-A frame. An open reading frame (ORF) is 312 bp, which encodes a peptide of 103 amino acid residues. A secondary structure of GHSR protein consists of alpha helix 66.0%, 16% disordered and 43% transmembrane helix. Molecular docking and interaction between synthetic ghrelin peptides and GHSR in the contact map revealed 19 amino acid residues closer than 4.5 Šdistance. The mRNA expression level of ghrelin, leptin, GHSR, growth hormone releasing hormone (GHRH) and insulin growth factor-I (IGF-1) revealed significant changes (p < 0.05), in the different treatments. The outcome of the present work contributes to understanding the role of ghrelin and its mechanism of action in regulating the expression of growth-related genes and feed intake in fish.

Expression of Growth and Hunger Related Genes and Physio-Biochemical Responses in Labeo rohita (Hamilton, 1822) Fed with Lysine and Betaine.

BACKGROUND/AIMS: The growth promoting effect of lysine and betaine as well as the expression of candidate genes reflecting their efficacy, such as ghrelin, leptin, Growth Hormone Secretagogue Receptor (GHS-R), Insulin like Growth Factor (IGF- 1) and Growth Hormone Releasing Hormone (GHRH) was examined in Labeo rohita fingerlings. METHODS: One hundred eighty healthy juveniles from a homologous population were randomly distributed to 15 rectangular tanks of 150 litres capacity. The experiment was carried out for 60 days with five treatment groups consisting T1 (0.25% Betaine), T2 (0.5% Betaine), T3 (0.75% Lysine) and T4 (1.5% Lysine) and control group. The experiment was carried out for 60 days with five treatment groups consisting T1 (0.25% Betaine), T2 (0.5% Betaine), T3 (0.75% Lysine) and T4 (1.5% Lysine) and control group. At the end of trial, the growth parameters such as weight gain, SGR, PER were estimated from the weight of the triplicate groups. The digestive, metabolic and antioxidant enzymes were analysed using spectrophotometric methods. The intestine, brain and liver were sampled from the treatments and expression of different genes ghrelin, leptin, GHSR, IGF-1 and GHRH was also performed by realtime PCR. RESULTS: A significant (P<0.05) increase in weight gain, SGR, PER and lowest FCR was found in T4 group which was significantly (p < 0.05) different from other experimental groups. The highest mRNA expression levels of expression were found in T4 group which was similar to that of ghrelin gene mRNA of T2 group. The significantly (p<0.05) highest GHSR, GHRH and IGF-1 gene expression levels were found in T4 treatment group compared to other groups. CONCLUSION: The present study reveals that the lysine and betaine stimulate growth and expression of ghrelin GHRH, GHS-R and IGF-1 genes. The increase of IGF-I mRNA expression with lysine and betaine supplementation revealed that these compounds act as growth modulators. However, lysine was found to be a more potent modulator of growth compared to betaine.

Expression and activity of key lipases during the larval development of walking catfish (Clarias magur).

The study was conducted to investigate the expression and activity of key lipolytic enzymes during the ontogenetic development of Clarias magur. After partial characterization, the messenger RNA (mRNA) expression analysis of lipoprotein lipase (LPL), pancreatic triacylglycerol lipase (PL), and bile salt-activated lipase (BAL) genes along with the specific lipase activity were performed in larvae from Day 1 after hatching till 34-day posthatch (dph). Heterogeneous patterns of mRNA expression were shown by the important lipolytic enzymes and were detected before first exogenous feeding during the yolk-sac stage. LPL started increasing from 13 dph and peaked at 16 dph followed by a declining trend till 34 dph. However, the PL observed to be peaking at 9, 22, and 30 dph. Similarly, BAL showed an increasing trend from 11 to 22 dph with a significantly high level of mRNA expression at 16 dph. Later, the specific lipase activity was evaluated which appears at Day 1 after hatching with a progressive increase from 7 to 16 dph and a further declining trend afterwards with a peak at 22 dph. The results indicated the development of exocrine pancreas at 16 dph. Furthermore, the transcript levels and the activity of lipases were regulated with the age. Hence, the present study can be helpful in devising different strategies containing optimum lipid levels at a suitable stage of development for improving the survival during larval rearing. Furthermore, the study could be a baseline for elucidating the optimized dietary lipid levels of this catfish during its larval rearing.

Temporal changes in superoxide dismutase, catalase, and heat shock protein 70 gene expression, cortisol and antioxidant enzymes activity of Labeo rohita fingerlings subjected to starvation and refeeding.

A short term starvation and refeeding experiment was conducted to study the temporal changes in SOD, CAT and HSP70 gene expression of Labeo rohita fingerlings. The study was carried out for 15 days with initial 7 days of starvation and then refeeding up to 15th day of the experimental trial. The expressions of SOD and CAT genes of liver and gills were significantly up-regulated after 7 days of starvation, down-regulated after 3 days of refeeding, and returned to the basal values after 8 days of refeeding. The HSP70 gene expression was significantly (p < 0.05) increased after starvation, with highest mRNA expression found on 7th day and reduced to the levels of control on refeeding. The activities of antioxidant enzymes, SOD and CAT were also studied to correlate with the results of gene expression. The changes in activities of SOD and CAT were found significantly (p < 0.05) higher in the starved group compared to the fed group. The dynamics of AST and ALT in serum revealed a progressive increase till the 7th day and decreased upon refeeding, cortisol level also has shown significant increase up to 7th day of starvation and sharp decline on refeeding. The concentration of blood glucose level start declining on 3rd day onwards with lowest level found on 7th day of starvation and was quickly restored to the levels of control on refeeding. The present study reveals that starvation elicits oxidative stress response as revealed by enhanced expression and activities of antioxidant enzymes, HSP 70 and serum biochemical alterations. However, these alterations were restored upon refeeding of L. rohita within 7 days.

saRNAdb: Resource of Small Activating RNAs for Up-regulating the Gene Expression.

RNA activation (RNAa) is the process of enhancing selective gene expression at transcriptional level using double-stranded RNAs, targeting gene promoter. These RNA molecules are usually 21 nucleotides long and termed as small activating RNAs (saRNAs). They are involved in gene regulation, epigenetics, gain-of-function studies and have potential therapeutic applications for various diseases especially cancer. RNAa is opposite to RNA interference in functionality; however, both processes share some protein machinery. There are many RNA interference centered online resources but no one for saRNAs; therefore, we developed "saRNAdb" database (http://bioinfo.imtech.res.in/manojk/sarna/). It contains 2150 manually curated saRNA entries with detailed information about their nucleotide sequences, activities, corresponding target gene, promoter and other experimental data. Besides, saRNA-promoter binding location, predicted saRNA features, tools (off-target, map) and RNAa-related proteins with their interacting partners are provided. saRNAdb is expected to assist in RNA research especially for nucleic acid-based therapeutics development.

ZikaVR: An Integrated Zika Virus Resource for Genomics, Proteomics, Phylogenetic and Therapeutic Analysis.

Current Zika virus (ZIKV) outbreaks that spread in several areas of Africa, Southeast Asia, and in pacific islands is declared as a global health emergency by World Health Organization (WHO). It causes Zika fever and illness ranging from severe autoimmune to neurological complications in humans. To facilitate research on this virus, we have developed an integrative multi-omics platform; ZikaVR (http://bioinfo.imtech.res.in/manojk/zikavr/), dedicated to the ZIKV genomic, proteomic and therapeutic knowledge. It comprises of whole genome sequences, their respective functional information regarding proteins, genes, and structural content. Additionally, it also delivers sophisticated analysis such as whole-genome alignments, conservation and variation, CpG islands, codon context, usage bias and phylogenetic inferences at whole genome and proteome level with user-friendly visual environment. Further, glycosylation sites and molecular diagnostic primers were also analyzed. Most importantly, we also proposed potential therapeutically imperative constituents namely vaccine epitopes, siRNAs, miRNAs, sgRNAs and repurposing drug candidates.

SMEpred workbench: A web server for predicting efficacy of chemicallymodified siRNAs.

Chemical modifications have been extensively exploited to circumvent shortcomings in therapeutic applications of small interfering RNAs (siRNAs). However, experimental designing and testing of these siRNAs or chemically modified siRNAs (cm-siRNAs) involves enormous resources. Therefore, in-silico intervention in designing cm-siRNAs would be of utmost importance. We developed SMEpred workbench to predict the efficacy of normal siRNAs as well as cm-siRNAs using 3031 heterogeneous cm-siRNA sequences from siRNAmod database. These include 30 frequently used chemical modifications on different positions of either siRNA strand. Support Vector Machine (SVM) was employed to develop predictive models utilizing various sequence features namely mono-, di-nucleotide composition, binary pattern and their hybrids. We achieved highest Pearson Correlation Coefficient (PCC) of 0.80 during 10-fold cross validation and similar PCC value in independent validation. We have provided the algorithm in the 'SMEpred' pipeline to predict the normal siRNAs from the gene or mRNA sequence. For multiple modifications, we have assembled 'MultiModGen' module to design multiple modifications and further process them to evaluate their predicted efficacies. SMEpred webserver will be useful to scientific community engaged in use of RNAi-based technology as well as for therapeutic development. Web server is available for public use at following URL address: http://bioinfo.imtech.res.in/manojk/smepred .

siRNAmod: A database of experimentally validated chemically modified siRNAs.

Small interfering RNA (siRNA) technology has vast potential for functional genomics and development of therapeutics. However, it faces many obstacles predominantly instability of siRNAs due to nuclease digestion and subsequently biologically short half-life. Chemical modifications in siRNAs provide means to overcome these shortcomings and improve their stability and potency. Despite enormous utility bioinformatics resource of these chemically modified siRNAs (cm-siRNAs) is lacking. Therefore, we have developed siRNAmod, a specialized databank for chemically modified siRNAs. Currently, our repository contains a total of 4894 chemically modified-siRNA sequences, comprising 128 unique chemical modifications on different positions with various permutations and combinations. It incorporates important information on siRNA sequence, chemical modification, their number and respective position, structure, simplified molecular input line entry system canonical (SMILES), efficacy of modified siRNA, target gene, cell line, experimental methods, reference etc. It is developed and hosted using Linux Apache MySQL PHP (LAMP) software bundle. Standard user-friendly browse, search facility and analysis tools are also integrated. It would assist in understanding the effect of chemical modifications and further development of stable and efficacious siRNAs for research as well as therapeutics. siRNAmod is freely available at: http://crdd.osdd.net/servers/sirnamod.

Antidiabetic activity of Artemisia amygdalina Decne in streptozotocin induced diabetic rats.

Artemisia species have been extensively used for the management of diabetes in folklore medicine. The current study was designed to investigate the antidiabetic and antihyperlipidemic effects of Artemisia amygdalina. Petroleum ether, ethyl acetate, methanol, and hydroethanolic extracts of Artemisia amygdalina were tested for their antidiabetic potentials in diabetic rats. The effect of extracts was observed by checking the biochemical, physiological, and histopathological parameters in diabetic rats. The hydroethanolic and methanolic extracts each at doses of 250 and 500 mg/kg b. w significantly reduced glucose levels in diabetic rats. The other biochemical parameters like cholesterol, triglycerides, low density lipoproteins (LDL), serum creatinine, serum glutamate pyruvate transaminase (SGPT), serum glutamate oxaloacetate transaminase (SGOT), and alkaline phosphatise (ALP), were found to be reduced by the hydroethanolic and methanolic extracts. The extracts also showed reduction in the feed and water consumption of diabetic rats when compared with the diabetic control. The histopathological results of treated groups showed the regenerative/protective effect on ß -cells of pancreas in diabetic rats. The current study revealed the antidiabetic potential of Artemisia amygdalina being effective in hyperglycemia and that it can effectively protect against other metabolic aberrations caused by diabetes in rats, which seems to validate its therapeutic traditional use.