Optimum dietary sources and levels of selenium improve growth, antioxidant status, and disease resistance: re-evaluation in a farmed fish species, Nile tilapia (Oreochromis niloticus).

The objective of this study was to investigate the effects of sources and levels of selenium (Se) on juvenile Nile tilapia (Oreochromis niloticus). A completely randomized design involving a 2 × 3 factorial arrangement of treatments was used in this study. Organic Se (L-selenomethionine; SeMet) and inorganic Se (sodium selenite; Na2SeO3) were each added to the basal diet at 1, 3 and 5 mg Se/kg. The basal diet, without Se supplementation, was used as a control. There was a total of 7 experimental diets, and each was fed in triplicate to groups of fish with an initial average body weight of 13.5 g for 8 weeks. The results showed that growth performance was significantly affected by dietary sources and levels of Se (P < 0.05). Fish fed diets supplemented with SeMet of 1.0 mg Se/kg resulted in higher growth performance compared to basal diet (P < 0.05), but Na2SeO3 supplementation did not affect growth. The feed conversion ratio was significantly decreased as dietary sources of SeMet (P < 0.05). Interestingly, fish fed diets supplemented with both forms of Se had lower cholesterol levels than those fed the basal diet (P < 0.05). Moreover, dietary sources and levels of Se significantly increased (P < 0.05) the antioxidant enzyme activities such as lysozyme, catalase, myeloperoxidase, superoxide dismutase and glutathione peroxidase. Dietary sources and levels of Se significantly could enhance the Nile tilapia resistance against Streptococcusagalactiae infection (P < 0.05). Overall, it can be concluded that the inclusion level of 1.0 mg Se/kg of organic Se in the diet is suggested to be the optimal level for the growth performance and immune response of Nile tilapia. Therefore, dietary supplementation with Se is useful for improving growth, antioxidant status, immune response, and disease resistance.

Impacts of Aegle marmelos fruit extract as a medicinal herb on growth performance, antioxidant and immune responses, digestive enzymes, and disease resistance against Streptococcus agalactiae in Nile tilapia (Oreochromis niloticus).

An experiment was conducted to investigate the effects of Aegle marmelos fruit (AMF) extract on the growth performance, biochemical parameters, immune response, antioxidative capacity, and digestive enzyme activity of Nile tilapia (Oreochromis niloticus). Fish were fed a diet supplemented with AMF at concentrations of 0 (AMF0; control), 5 (AMF5), 10 (AMF10), 15 (AMF15), or 20 (AMF20) g/kg for 8 weeks. The results show that the final body weight, weight gain, specific growth rate, average daily gain, and feed conversion ratio were significantly higher in fish fed AMF15 and AMF20 compared to those fed the control diet (P < 0.05). Moreover, significant increases in antioxidant enzyme activities and non-specific immune responses were observed in groups fed AMF15 and AMF20. Interestingly, the level of cholesterol decreased with increasing AMF concentrations in the diet. As dietary AMF levels increased, digestive enzyme activities significantly improved. After the feeding trial, fish were injected intraperitoneally with Streptococcus agalactiae, and the 14-day cumulative mortality was calculated. A high survival rate after challenge with S. agalactiae was observed in all groups that received AMF-supplemented feed. Therefore, the present study suggests that supplementing the diet of Nile tilapia with AMF at a concentration of 20 g/kg could encourage their growth, improve their immunity and antioxidant status, and provide strong protection against S. agalactiae.

The nitrated fatty acid, 10-nitrooleate inhibits the neutrophil chemotaxis via peroxisome proliferator-activated receptor gamma in CLP-induced sepsis in mice.

The inhibition of polymorphonuclear neutrophils' (PMNs) migration to the source of injury is among the most prominent aspects of immunosuppression following sepsis, although the precise mechanisms involved remain unclear and multifaceted. Increasing evidence connects this immunosuppression to nitric oxide (NO), as NO production is a classic feature of inflammation probably through neutrophil activation and migration. Nitrated fatty acids (NFA) such as 10-nitrooleate (OA-NO2), nitrolinoleic acid etc. produced endogenously by the non-enzymatic reaction of NO with unsaturated fatty acids, are found to be potent activators of the transcription factor, peroxisome proliferator-activated receptor gamma (PPARγ). Upregulation of PPARγ during immunosuppression and the subsequent inhibition of neutrophil migration in sepsis have been reported. However, the interplay of OA-NO2, NO and PPARγ in polymicrobial-induced immunosuppression has not been established. Hence to understand this, we have studied the role of OA-NO2 in blood PMNs migration, the effects of iNOS inhibitor on PMNs migration and PPARγ activity in cecal ligation and puncture (CLP)-induced sepsis in mice. We found increased expression of PPARγ and its DNA-binding activity in the lungs and blood PMNs from CLP mice. CLP or OA-NO2 treatment inhibited PMNs' migration in response to fMLP stimulation. Pharmacological inhibition of iNOS resulted in decreased PPARγ DNA-binding activity with a concomitant increase in the migration of PMNs to the site of infection. OA-NO2 treatment also inhibited the production of inflammatory cytokines (TNFα and IL-1ß) secretion from PMNs stimulated with lipopolysaccharide. We have also established that, OA-NO2 mediated inhibition of PMNs migration in vivo and ex vivo are regulated through PPARγ-dependent pathway. This study further highlights the fact that the activation of PPARγ by the NFA has a pivotal role in PMNs' migration and immunosuppression.

Polysaccharide fraction from the Indian mistletoe, Dendrophthoe falcata (L.f.) Ettingsh enhances innate immunity and disease resistance in Oreochromis niloticus (Linn.).

The polysaccharide fraction (PF) isolated from the hemiparasitic mistletoe, Dendrophthoe falcata (L.f.) Ettingsh (DF) leaves was tested for its immunostimulatory properties in Oreochromis niloticus (Linn.). Different groups of experimental fish were fed for 1, 2 or 3 weeks with three different doses [low (0.01%), mid (0.1%), or high (1%)] of D. falcata polysaccharide fraction (DFPF) - supplemented diet. After every feeding regimen, the fish were assessed for non-specific immunological parameters, immune related gene expression and disease protection. The DFPF treated groups showed significant (P < 0.05) enhancement of non-specific immune parameters. Significant (P < 0.05) upregulation of lysozyme and TNF-α gene expression was observed in DFPF treated groups. In pathogen challenge studies using Aeromonas hydrophila, the DFPF treated groups displayed significant (P < 0.05) decrease in percentage mortality and the consequent increase in relative percent survival (RPS). Supplementation of 1% DFPF in the feed for a week conferred the maximum protection against the virulent pathogen challenge, recording a RPS of 100. These results suggest that DFPF has the potential to be used as an immunostimulating feed additive in finfish aquaculture.

Characterisation of rainbow trout peripheral blood leucocytes prepared by hypotonic lysis of erythrocytes, and analysis of their phagocytic activity, proliferation and response to PAMPs and proinflammatory cytokines.

Rapid and high quality preparation of peripheral blood leucocytes (PBL) is important in fish immunology research and in particular for fish vaccine development, where multiple immune parameters can be monitored on the same fish over time. Fish PBL are currently prepared by density separation using Percoll or Hispaque-1.077, which is time consuming, costly and prone to erythrocyte contamination. We present here a modified PBL preparation method that includes a 20 s hypotonic lysis of erythrocytes and a subsequent separation of PBL from cell debris by a cell strainer. This method is simple, rapid and cost effective. The PBL obtained are similar in cellular composition to those prepared by density separation but have less erythrocyte contamination as demonstrated by FACS analysis and the expression of cell marker genes. Marker gene analysis also suggested that PBL prepared by hypotonic lysis are superior to those obtained by the gradient method in that some high-density cells (certain B cell types and neutrophils) might be lost using the latter. The PBL prepared in this way can proliferate in response to the T cell mitogen PHA, and both lymphoid and myeloid cells can phagocytose fluorescent beads and bacteria, with the latter enhanced by treatment with pro-inflammatory cytokines (IL-1ß and IL-6). Furthermore, the PBL can respond to stimulation with PAMPs (LPS, poly I:C) and cytokines (IL-1ß and IFNγ) in terms of upregulation of proinflammatory cytokine gene expression. Such data demonstrate the utility of this approach (hypotonic lysis of erythrocytes) for PBL isolation and will enable more studies of their role in disease protection in future immunological and vaccine development research in fish.

Modulation of the innate immune responses in the striped snakehead murrel, Channa striata upon experimental infection with live and heat killed Aeromonas hydrophila.

It is well-known that the innate immune mechanisms in fish serve as the first line of defence against wide variety of pathogens. In most of the situations, innate responses get induced and enhanced after the pathogen invasion. It would be interesting to look into the inducibility of various innate immune mechanisms and the level of enhancement after infection with the pathogen. Hence, in the present investigation, modulation of the innate immune responses in the striped snakehead murrel, Channa striata on experimental challenge with either live virulent or heat killed Aeromonas hydrophila at a dose of 1x107 CFU (suspended in 0.2 mL PBS) were measured. Most of the non-specific (both humoral and cellular) immune responses tested were substantially induced or enhanced in both the experimental groups in comparison with the unchallenged control group. Significant increase in the lysozyme, total peroxidase, antiprotease and respiratory burst activities were observed after the pathogen challenge. Thus, most of the innate non-specific immune responses are inducible though they are constitutive of fish immune system exhibiting a basal level of activity even in the absence of pathogen challenge.

Curcumin Nanotechnologies and Its Anticancer Activity.

Cancer is one of the leading causes of death worldwide. Curcumin is a well-established anticancer agent in vitro but its efficacy is yet to be proven in clinical trials. Poor bioavailability of curcumin is the principal reason behind the lack of efficiency of curcumin in clinical trials. Many studies prove that the bioavailability of curcumin can be improved by administering it through nanoparticle drug carriers. This review focuses on the efforts made in the field of nanotechnology to improve the bioavailability of curcumin. Nanotechnologies of curcumin come in various shapes and sizes. The simplest curcumin nanoparticle that increased the bioavailability of curcumin is the curcumin-metal complex. On the other hand, we have intricate thermoresponsive nanoparticles that can release curcumin upon stimulation (analogous to a remote control). Future research required for developing potent curcumin nanoparticles is also discussed.

Cytotoxic effects of Aeromonas hydrophila culture supernatant on peripheral blood leukocytes of Nile tilapia (Oreochromis niloticus): Possible presence of a secreted cytotoxic lectin.

Number of exotoxins like haemolysin, leukocidin, aerolysin etc. were reported from Aeromonas hydrophila. In this study, we report the haemolytic and cytotoxic effect of A. hydrophila culture supernatant (CS) that is specifically inhibited by lactose and also by serum and mucus of Nile tilapia (Oreochromis niloticus). Hence, we assume the presence of a secreted lectin in the CS. CS is toxic to peripheral blood leukocytes (PBL) of O. niloticus as revealed by MTT assay and by flow cytometry. DNA laddering assay indicates that CS causes necrosis to PBL. As a result of necrosis, CS treated PBL showed increased production of reactive oxygen species as indicated by nitroblue tetrazolium and 2',7' -dichlorofluorescin diacetate assays. CS treated PBL showed reduced mRNA expression of TNF-α and IFN-γ genes. When CS was subjected to polyacrylamide gel electrophoresis, it showed a single band corresponding to the molecular weight of 45 kDa. However, upon concentrating the CS by ultrafiltration, many bands were visualized. Further studies at molecular level are required to unravel this macromolecular-leukocyte interaction which would ultimately benefit the aquaculture industry.

Polysaccharides from marine macroalga, Padina gymnospora improve the nonspecific and specific immune responses of Cyprinus carpio and protect it from different pathogens.

Immunostimulation by plant-derived compounds presents a fascinating alternative to vaccines and antibiotics in aquaculture. Fish farmers are longing for immunostimulants that activate both specific and nonspecific immune responses of fish and protect fishes from all possible infections. In this study, we observed that polysaccharide fraction from marine macroalga, Padina gymnospora stimulated the immune response of common carp Cyprinus carpio (Filed for patent, Indian patent no. 201641027311 dated:10-Aug-2016). Our results indicate that fish fed with polysaccharides as feed supplement improved all the immune parameters tested which include serum lysozyme, myeloperoxidase activities and antibody response. Further, polysaccharide fraction protected the fish from its common bacterial pathogens namely Aeromonas hydrophila and Edwardsiella tarda with relative percent survival (RPS) values of 80 and 60 respectively. Gene expression studies, indicate that the immunostimulation by P. gymnospora might be at least in part due to the upregulation of the cytokine interleukin-1ß (IL-1ß) and antimicrobial peptide lysozyme-C.

Methanol extract of Nyctanthes arbortristis seeds enhances non-specific immune responses and protects Oreochromis mossambicus (Peters) against Aeromonas hydrophila infection.

Immunostimulation using medicinal plant extracts is a promising approach for prevention and control of diseases with reference to sustainable fish farming. Oreochromis mossambicus, dubbed as aquatic chicken is a cultured fish worldwide and a laboratory model organism. Aeromonas hydrophila is one of the major bacterial pathogens in fish farming that causes huge loss to aquaculture industries. In this study, we investigated the efficacy of methanol extract of Nyctanthes arbortristis seeds on disease resistance of O. mossambicus against live virulent A. hydrophila. We also investigated its effect on the non-specific immune parameters such as serum lysozyme, myeloperoxidase, antiprotease and specific immune parameters in terms of specific serum antibody titres assayed by bacterial agglutination test. Our studies indicate that intra-peritoneal administration of 20mg/kg methanol extract increases the Relative Percent Survival (RPS) of O. mossambicus challenged with LD80 of A. hydrophila. Further, both non-specific and specific immune parameters were enhanced by the methanol extract. Further experiments at molecular levels in the laboratory and also efficacy testing at field level are essential before applying this plant product in aquaculture industry.

Molecular docking and simulation of Curcumin with Geranylgeranyl Transferase1 (GGTase1) and Farnesyl Transferase (FTase).

Protein prenylation is a posttranslational modification that is indispensable for translocation of membrane GTPases like Ras, Rho, Ras etc. Proteins of Ras family undergo farnesylation by FTase while Rho family goes through geranylgeranylation by GGTase1. There is only an infinitesimal difference in signal recognition between FTase and GGTase1. FTase inhibitors mostly end up selecting the cells with mutated Ras proteins that have acquired affinity towards GGTase1 in cancer microcosms. Therefore, it is of interest to identify GGTase1 and FTase dual inhibitors using the docking tool AutoDock Vina. Docking data show that curcumin (from turmeric) has higher binding affinity to GGTase1 than that of established peptidomimetic GGTase1 inhibitors (GGTI) such as GGTI-297, GGTI-298, CHEMBL525185. Curcumin also interacts with FTase with binding energy comparable to co-crystalized compound 2-[3-(3-ethyl-1-methyl-2-oxo-azepan-3-yl)-phenoxy]-4-[1-amino-1-(1-methyl-1h-imidizol-5-yl)-ethyl]-benzonitrile (BNE). The docked complex was further simulated for 10 ns using molecular dynamics simulation for stability. Thus, the molecular basis for curcumin binding to GGTase1 and FTase is reported.

Effect of UV-B radiation on the antibody response of fish - implication on high altitude fish culture.

Literally, all living forms are either directly or indirectly dependent upon sun for energy. Radiation from sun is differentiated into several components of a spectrum based on the wavelength. Ultraviolet (UV) radiation may be one of the infamous radiations emitted by the sun. Ozone depletion is another critical factor by which UV induced ill-effects are intensified. Though there are numerous studies on effects of UV radiation on terrestrial organisms, its effect on freshwater and aquaculture ecosystems has been largely neglected. Here, we report that enhanced UV irradiation may suppress the primary and secondary antibody responses to a soluble protein antigen in fish. Fishes exposed for longer periods (80min) were particularly very sensitive to infection, as shown by our sensitivity index.

The role of nitrated fatty acids and peroxisome proliferator-activated receptor gamma in modulating inflammation.

Nitrated fatty acids (NFAs), thought to be produced by nonenzymatic reactions of endogenous nitric oxide (NO) with naturally present unsaturated fatty acids, have recently been identified as one of the largest single pools of biologically active NO derivatives in human plasma. As the biological role of NFAs is unknown, initial in vitro studies have shown them to be potent suppressors of inflammatory responses. The aim of the study was to collect all the literature on NFAs and its interactions with peroxisome proliferator-activated receptor gamma (PPAR-γ) and review in detail the anti-inflammatory properties of PPAR-γ interceded by NFAs. A literature survey was performed using PubMed and ScienceDirect to gather complete information on NFAs and their interactions with PPAR-γ. An exhaustive literature survey revealed that NFAs found in human plasma and urine comprises a class of cell signaling mediators that can activate PPAR-γ within its physiological concentration. NFAs exhibit anti-inflammatory and anti-fibrotic effects through PPAR-γ activation in various in vitro models tested. Besides its role in inflammation other properties of NFAs such as inhibition of enzymes, inducer of gene expression, etc., were discussed. NFAs are good electrophiles with pleiotropic biological activities. Hence NFAs can be treated as potent drug candidates.

Molecular docking of Glyceroneogenesis pathway intermediates with Peroxisome Proliferator- Activated Receptor-Alpha (PPAR-α).

Peroxisome proliferator-activated receptor alpha (PPAR-α) belongs to the nuclear receptor superfamily of proteins. It is one of the principle regulators of metabolism and lipid homeostasis whose malfunction leads to complications including obesity and type 2 diabetes. In the adipose tissue, glyceroneogenesis is a unique pathway through which pyruvate is converted into glycerol-3- phosphate (G3P) in a multistep process. Previous findings demonstrated that glyceroneogenesis regulates triacylglycerol synthesis and adipogenesis. This led us to hypothesize that one of the pathway intermediate is physiologically relevant PPAR-α ligand. In the present study using in silico docking, we proved that glycerate, dihydroxy acetone phosphate, glyceraldehyde-3-phosphate, and G3P are key glyceroneogenesis pathway intermediates which bind to PPAR-α. They bind PPAR-α with comparable binding energy and docking score to that of (2s)-2-ethoxy-3-[4-(2-{4-[(methylsulfonyl)oxy]phenyl}ethoxy)phenyl]propanoic acid(AZ-2), a synthetic high affinity ligand of PPAR-α. These intermediates could be studied further as potential physiologically relevant activators of PPAR-α in vitro and in vivo.

The need for physiologically relevant peroxisome proliferator-activated receptor-gamma (PPAR-γ) ligands.

Peroxisome proliferator-activated receptor-γ (PPAR-γ) is a nuclear transcription factor which is involved in the differentiation of fibroblasts to adipocytes in vitro. PPAR-γ also plays a pivotal role in inflammation and macrophage activation. Furthermore, type 2 diabetes mellitus (T2DM), a condition in which an individual's ability to respond to insulin is lowered, is treated by drugs called thiazolidinediones (TZDs) that are known to activated PPAR-γ, thus augmenting insulin signaling and glucose uptake by adipose tissue. Unfortunately, these otherwise effective drugs are responsible for side effects such as obesity and cardiovascular diseases. The ligand-binding ability of PPAR-γ is different from other nuclear receptors since it can bind to a wide variety of ligands. Although a number of compounds have been shown to activate PPAR-γ, knowledge of its endogenous ligands and their physiological functions is lacking. The known ligands were either ambiguous or found to produce ill effects in vivo. In this review we discuss the structure and functions of PPAR-γ, ligands discovered so far, and focus on the importance of identification of physiologically relevant endogenous ligands.

Challenges of curcumin bioavailability: novel aerosol remedies.

Nanoparticles are promising aids for drug delivery for previously challenging diseases, and many incurable ones. Curcumin (diferuloylmethane) is a pleiotropic molecule having various target molecules in the body. Despite its effects, curcumin-based drugs are not readily available in the market because of their low bioavailability. Although dietary intake and knowledge about the potential of curcumin are high in countries like India, studies indicate that the bioavailability problem still persists. However, administration of curcumin through inhalation has received little consideration. In this review we discuss the potential of curcumin, approaches made to overcome the bioavailability challenges, and novel approaches that could be applied in order to deliver curcumin in a pressurized metered dose inhaler (pMDI).

A Homology Based Model and Virtual Screening of Inhibitors for Human Geranylgeranyl Transferase 1 (GGTase1).

Protein prenylation is a post translational modification that is indispensable for Ras-Rho mediated tumorigenesis. In mammals, three enzymes namely protein farnesyltransferase (FTase), geranylgeranyl transferase1 (GGTase1), and geranylgeranyl transferase2 (GGTase2) were found to be involved in this process. Usually proteins of Ras family will be farnesylated by FTase, Rho family will be geranylgeranylated by GGTase1. GGTase2 is exclusive for geranylgeranylating Rab protein family. FTase inhibitors such as FTI- 277 are potent anti-cancer agents in vitro. In vivo, mutated Ras proteins can either improve their affinity for FTase active site or undergo geranylgeranylation which confers resistance and no activity of FTase inhibitors. This led to the development of GGTase1 inhibitors. A well-defined 3-D structure of human GGTase1 protein is lacking which impairs its in silico and rational designing of inhibitors. A 3-D structure of human GGTase1 was constructed based on primary sequence available and homology modeling to which pubchem molecules library was virtually screened through AutoDock Vina. Our studies show that natural compounds Camptothecin (-8.2 Kcal/mol), Curcumin (-7.3 Kcal/mol) have higher binding affinities to GGTase-1 than that of established peptidomimetic GGTase-1 inhibitors such as GGTI-297 (-7.5 Kcal/mol), GGTI-298 (-7.5 Kcal/mol), CHEMBL525185 (-7.2 Kcal/mol).