Sasmita Mohanty

Immunomodulatory effect of arsenic on cytokine and HSP gene expression in Labeo rohita fingerlings.

Immune system is fundamental for survival of an organism against invading pathogens and other harmful agents. Cytokines, the signaling proteins that are produced transiently after cell activation and exert pleiotropic effects on cells of the immune system, are important mediators of cell mediated immune response. When expressed in a dysregulated fashion cytokines can underlie either immunodeficient or immunopathologic states. Heat shock proteins (stress proteins, HSPs) are also key proteins, which play important role in immunomodulation, apoptosis and influence the immune responses. Arsenic is a major toxic environmental contaminant and a human carcinogen. Prolonged drinking of arsenic-contaminated water leads to chronic arsenic toxicity (arsenicosis). Arsenic is also immunotoxic and renders the host immunocompromised. Arsenic exposure has been reported to result in growth retardation, gross pathology including skin and eye lesions, ulcerations, cataract development etc. in different fish species. The present study was undertaken to investigate the effect of arsenic exposure on the expression of immune genes IFN-γ, IL-4, IL-10, IL-12, complement C3a and HSP genes HSP47, HSP60, HSP70, HSC71, HSP78, and HSP90 in Labeo rohita, an important aquacultured species, as such information is not available on this major carp. Cytokine and HSP gene expression analyses were carried out in kidney and liver tissues, respectively, in arsenic-exposed fishes by RT-PCR and HSPs were analyzed by immunoblotting. It was observed that arsenic has a generalized immune-suppressive effect leading to down regulation of both Th1 and Th2 cytokines; besides, it led to up regulation of the HSP genes indicating arsenic-induced cellular stress. Thus arsenic exposure makes L. rohita immunocompromised and could increase its susceptibility to pathogen attacks.

Investigating hsp Gene Expression in Liver of Channa striatus under Heat Stress for Understanding the Upper Thermal Acclimation

Changes in hsp gene expression profiles in murrel Channa striatus experimentally exposed to temperature stress (36°C) for 4, 15, and 30 days were investigated; fish collected from aquaculture ponds and maintained in laboratory at the pond temperature (25 ± 1°C) served as control. Channa collected from a hot spring runoff (36°C) was included in the study to examine the hsp profiles beyond 30 days of exposure. Gene expression analyses of a battery of hsps in liver tissues were carried out by quantitative RT-PCR and protein expressions were analyzed by immunoblotting. hsps could be grouped into three clusters based on similarity in response to heat stress: hsp70, hsp78, and hsp60, whose transcript level continued to increase with duration of exposure; hsp90 and hsp110 that increased to a much higher level and then decreased; hsp27 and hsp47 that did not significantly vary as compared to control. The results suggest that Hsp70, Hsp78, and Hsp60 are involved in thermal acclimation and long term survival at high temperature. Fish living in the hot spring runoff appears to continuously express hsps that can be approximated by long term induction of hsps in farmed fish if temperature of their environment is raised to 36°C.

Proteomic Analysis of Sarcoplasmic Peptides of Two Related Fish Species for Food Authentication

Detection of species-specific sarcoplasmic peptides can be used as proteomic markers for fish food authentication and identification of species of origin in processed products. In the present study, proteomics technology was employed for differential characterization of sarcoplasmic peptides of two closely related fish species, Sperata seenghala and Sperata aor. Species-specific peptides were searched in white muscle extracts of the two species for identification of unique peptides that might aid in differentiation of the species, under two-dimensional gel electrophoresis platform. A total of 19 proteins were identified by combined matrix-assisted laser desorption ionization time-of-flight mass spectrometry and liquid chromatography-tandem mass spectrometry, of which nine and two proteins were found to be unique to S. seenghala and S. aor, respectively. One of the proteins, triosephosphate isomerase (TPI) was found to have three isoforms, out of which two were specific to S. aor, and one was specific to S. seenghala. All the three isoforms of TPI were present in the mixed samples of raw protein extracts of S. seenghala and S. aor, an observation that can be exploited to differentiate between the species and detection of deceptive practices of fraudulent substitution of commercially valuable fish species with inferior ones and differential characterization between closely related fish species.

Climate Change: Impacts on Fisheries and Aquaculture

Climate change has been recognized as the foremost environmental problem of the twentyfirst century and has been a subject of considerable debate and controversy. It is predicted to lead to adverse, irreversible impacts on the earth and the ecosystem as a whole. Although it is difficult to connect specific weather events to climate change, increases in global temperature has been predicted to cause broader changes, including glacial retreat, arctic shrinkage and worldwide sea level rise. Climate change has been implicated in mass mortalities of several aquatic species including plants, fish, corals and mammals. The present chapter has been divided in to two parts; the first part discusses the causes and general concerns of global climate change and the second part deals, specifically, on the impacts of climate change on fisheries and aquaculture, possible mitigation options and development of suitable monitoring tools.

Evaluation of housekeeping genes as references for quantitative real-time PCR analysis of gene expression in the murrel Channa striatus under high-temperature stress

Quantitative real-time polymerase chain reaction is the most advanced method of quantifying gene expression studies; however, the significance of the obtained results strongly depends on the normalization of the data to compensate for differences between the samples. In the present study, expression analysis of six different constitutively expressed genes viz. 18S ribosomal RNA, glyceraldehyde-3-phosphate dehydrogenase (gapdh), beta actin (βactin), ribosomal binding protein L13, tubulin and TATA-box-binding protein (tbp) were carried out to test their efficacy as reference genes in three different tissues, namely liver, gill and muscle of murrel Channa striatus exposed to high temperature for variable time periods. The stability and suitability of the genes were determined by using bioinformatic tools: GeNorm, NormFinder and BestKeeper. Based on the results, tub/βactin could be used as the reference genes for liver and gill tissues and βactin/gapdh could be the reference genes for muscle tissues in Channa striatus under both short- and long-term thermal stress.

Proteomic changes in the liver of Channa striatus in response to high temperature stress

The present study was undertaken to investigate the proteomic changes in the liver of murrel Channa striatus exposed to high temperature stress. Fishes were exposed to 36°C for 4 days and liver proteome changes were analyzed using gel‐ based proteomics, i.e. 2DE, MALDI‐TOF/TOF‐MS, and validation by transcript analysis. The study showed, besides others, increased abundance of two sets of proteins, the antioxidative enzymes superoxide dismutase (SOD), ferritin, cellular retinol binding protein (CRBP), glutathione‐S‐transferase (GST), and the chaperones HSP60 and protein disulfide isomerase; this was validated by transcript analysis. The proteome data are available via ProteomeXchange with identifier PXD002608. Further, gene expression analysis was also carried out in the fishes exposed to thermal stress for longer durations (30 days experimental exposure in laboratory and for 30 days beyond, taking Channa collected from a hot spring runoff at 36–38°C); sod, gst, crbp, and hsp60 were found to continue to remain upregulated at eight‐, 2.5‐, 2.4‐, and 2.45‐fold, respectively, in the hot spring runoff fish. Pathway analysis showed that the upregulations of the antioxidant enzymes as well as molecular chaperones are induced by the transcription factor Nrf2 (nuclear factor erythroid 2‐related factor 2). Thus, while short‐term heat stress tolerance involves the antioxidative enzymes SOD, ferritin, CRBP, GST, and chaperones HSP60 and protein disulfide isomerase, adaptation under chronic heat stress is associated with SOD, CRBP, GST, and HSP60.

Effect of storage temperature as a preanalytical variable on the lens crystallins protein quality for proteomic studies

Purpose: To study the effect of storage temperature on lens crystallins quality for proteomic analysis, using αA‐crystallins as internal marker.

Identification of potential biomarkers of hepatotoxicity by plasma proteome analysis of arsenic-exposed carp Labeo rohita

Arsenic (As) is a toxic environmental contaminant and potential human carcinogen. Chronic intake of arsenic-contaminated water and food leads to arsenicosis, a major public health problem in many parts of the world. Early detection of arsenic toxicity would greatly benefit patients; however, the detection of arsenicosis needs to be done early before onset of severe symptoms in which case the tools used for detection have to be both sensitive and reliable. In this context, the present study investigated plasma proteome changes in arsenic-exposed Labeo rohita, with the aim of identifying biomarkers for arsenicosis. Changes in the plasma proteome were investigated using gel-based proteomics technology. Using quantitative image analysis of the 2D proteome profiles, 14 unique spots were identified by MALDI-TOF/TOF MS and/or LC-MS/MS which included Apolipoprotein-A1 (Apo-A1) (6 spots), α-2 macroglobulin-like protein (A2ML) (2 spots), transferrin (TF) (3 spots) and warm-temperature acclimation related 65kDa protein (Wap65). The proteome data are available via ProteomeXchange with identifier PXD003404. Highly abundant protein spots identified in plasma from arsenic-exposed fish i.e. Apo-A1 (>10-fold), A2ML (7-fold) and Wap65 (>2-fold) indicate liver damage. It is proposed that a combination of these proteins could serve as useful biomarkers of hepatotoxicity and chronic liver disease due to arsenic exposure.

The membrane tethered transcription factor EcbZIP17 from finger millet promotes plant growth and enhances tolerance to abiotic stresses

The occurrence of various stresses, as the outcome of global climate change, results in the yield losses of crop plants. Prospecting of genes in stress tolerant plant species may help to protect and improve their agronomic performance. Finger millet (Eleusine coracana L.) is a valuable source of superior genes and alleles for stress tolerance. In this study, we isolated a novel endoplasmic reticulum (ER) membrane tethered bZIP transcription factor from finger millet, EcbZIP17. Transgenic tobacco plants overexpressing this gene showed better vegetative growth and seed yield compared with wild type (WT) plants under optimal growth conditions and confirmed upregulation of brassinosteroid signalling genes. Under various abiotic stresses, such as 250 mM NaCl, 10% PEG6000, 400 mM mannitol, water withdrawal, and heat stress, the transgenic plants showed higher germination rate, biomass, primary and secondary root formation, and recovery rate, compared with WT plants. The transgenic plants exposed to an ER stress inducer resulted in greater leaf diameter and plant height as well as higher expression of the ER stress-responsive genes BiP, PDIL, and CRT1. Overall, our results indicated that EcbZIP17 improves plant growth at optimal conditions through brassinosteroid signalling and provide tolerance to various environmental stresses via ER signalling pathways.

Expression patterns and mutation analysis of p53 in fish Rita rita from polluted riverine environment

The present study was undertaken to investigate the alterations in gene expression patterns and for mutation analysis of p53 in the riverine catfish Rita rita collected from polluted riverine habitat. The partial p53 gene sequence of Rita rita generated showed a high degree of similarities with the DNA binding domains of fishes, mice and human. Transcriptomic analysis, carried out by quantitative real-time Polymerase Chain Reaction (RT-qPCR), showed significant down-regulation of p53 in fishes collected from most of the polluted stretches. Similar trend in protein abundance was observed by western blot analysis. Down-regulation of p53 was more pronounced in gill than liver. Expression patterns of p53 suggest that exposure to a multitude of contaminants in the natural riverine ecosystem could suppress the expression of p53. Genomic DNA showed a low stained smear pattern upon electrophoresis, with no evidence of DNA fragmentation. For mutation analysis PCR-SSCP followed by sequence analysis was carried out, which identified eight mutations; two at codon level and six missense mutations in the DNA binding domain IV and V. Secondary structure prediction showed that these mutations could lead to impairment of protein structure. Thus, the present study indicated that aquatic pollution has impacted these lower vertebrates which are reflected by the down-regulation of tumor suppressor protein (p53) in majority of the stretches studied.