Abiotic factors and heavy metals defining eco-physiological niche in fish.

Ecosystem dynamics undergoing alterations in structure and function highlights the need to look into the relations between ecological parameters and organismal fitness and tolerance. Ecophysiological studies are used to understand how organisms adapt to and cope up with environmental stress. Current study uses a process-based approach to model physiochemical parameters regarding seven different fish species. Species respond to climatic variations via acclimation or adaptation through physiological plasticity. Four sites are differentiated into two types based on the water quality parameters and metal contamination. Seven fish species are clustered into two groups, each group depicting separate pattern of response in similar habitat. In this manner, biomarkers from three different physiological axes- stress, reproduction, and neurology were taken to determine the organism's ecological niche. Cortisol, Testosterone, Estradiol, and AChE are the signature molecules estimated for the said physiological axes. The ordination technique, nonmetric multidimensional scaling, has been utilized to visualize the differentiated physiological response to changing environmental conditions. Then, Bayesian Model Averaging (BMA) was used to identify the factors that play a key role in refining the stress physiology and determining the niche. Current study confirms different species belonging to similar habitats respond to various environmental and physiological factors in a different manner as various biomarkers respond in a species-specific pattern that induces the choice of habitat preference controlling its ecophysiological niche. In the present study, it is quite apparent that adaptive mechanism of fish to environmental stress is achieved through modification of physiological mechanisms through a panel of biochemical markers. These markers organize a cascade of physiological event at various levels including reproduction.

Bioaccumulation pattern of heavy metals in fish tissues and associated health hazards in human population.

The study vigilantly considered the load of Pb, Cd, Cr, Cu, and Zn in a variety of tissues (muscle, gills, and liver) of 5 fish species (Mystus gulio, Notopterus notopterus, Notopterus chitala, Mugil cephalus, and Glossogobius giuris) collected from six sites in the lower Gangetic area. The study showed the lowest concentration of metals in the muscles. The accumulated patterns of heavy metals differed in different regions and concentrations fluctuated between the liver and gills. The target hazard quotient (THQ) value has been measured in contaminated fish. The THQ values for all the metals in respective fishes are below 1 that indicate that indirect intake of metals by consuming these selected fishes will not result in potential health hazard in human. The estimated daily intake (EDI) results were also calculated. EDI levels of all elements are lower than the permissible limit indicating a lower chance for health risk to occur. However, doses below the recommended levels do not indicate that they are completely safe for consumption or those above are not to be used. Thus, it can be demonstrated that occurrence of Cd, Cr, Pb, Cu, and Zn in the preferred tissues of the selected fish species in the present study may not pretense severe human health risk after consumption at its existing concentration.

RNA Interactome Identification via RNA-BioID in Mouse Embryonic Fibroblasts.

Cytoplasmic localization of mRNAs is common to all organisms and serves the spatial expression of genes. Cis-acting RNA signals (mostly found in the mRNA's 3'-UTR), called zipcodes recruit trans-acting RNA-binding proteins that facilitate the localization of the mRNA. UV-cross-linking or affinity purification has been applied to identify such proteins but suffer from the need for stable RNA-protein binding or direct contact of protein and RNA. To identify stably or transiently interacting proteins that directly or indirectly associated with the localization elements and the body of the mRNA, we developed an in vivo proximity labeling method we call RNA-BioID. In RNA-BioID, we tether a fusion of the BirA* biotin ligase and the MS2 coat protein (MCP) at the 3'-UTR of MS2-tagged ß-actin mRNA in vivo. Exposing BirA* expressing cells to biotin in the media and induces biotinylation of ß-actin mRNA-associated proteins that can be isolated with streptavidin beads. This technique allowed us to identify by mass spectrometry analysis the ß-actin mRNA 3'-UTR-interacting proteome in fibroblasts. The protocol can be useful to identify the interacting proteome of any mRNA in mammalian cells.

Physiological response of fish under variable acidic conditions: a molecular approach through the assessment of an eco-physiological marker in the brain.

The current study demonstrates oxidative damage and associated neurotoxicity following pH stress in two freshwater carp Labeo rohita and Cirrhinus cirrhosus. Carp (n = 6, 3 replicates) were exposed to four different pH (5.5, 6, 7.5, and 8) against control (pH 6.8 ± 0.05) for 7 days. After completion of treatment, levels of enzymatic (superoxide dismutase [SOD], catalase [CAT], glutathione reductase [GRd]) and non-enzymatic antioxidants (malondialdehyde [MDA], glutathione [GSH]), brain neurological parameters (Na+-K+ATPase, acetylcholinesterase [AcHE], monoamine oxidase [MAO], and nitric oxide [NO]), xanthine oxidase (XO), heat shock proteins (HSP70 and HSP90), and transcription factor NFkB were measured in carp brain. Variation in the pH caused a significant alteration in the glutathione system (glutathione and glutathione reductase), SOD-CAT system, and stress marker malondialdehyde (MDA). Xanthine oxidase was also induced significantly after pH exposure. Brain neurological parameters (MAO, NO, AChE, and Na+-K+ATPase) were significantly reduced at each pH-treated carp group though inhibition was highest at lower acidic pH (5.5). Cirrhinus cirrhosus was more affected than that of Labeo rohita. Molecular chaperon HSP70 expression was induced in all pH-treated groups though such induction was more in acid-stressed fish. HSP90 was found to increase only in acid-stressed carp brain. Expression of NFkB was elevated significantly at each treatment group except for pH 7.5. Finally, both acidic and alkaline pH in the aquatic system was found to disturb oxidative balance in carp brain which ultimately affects the neurological activity in carp. However, acidic environment in the aquatic system was more detrimental than the alkaline system regarding oxidative damage and subsequent neurotoxicity in carp brain.

ß-Actin mRNA interactome mapping by proximity biotinylation.

The molecular function and fate of mRNAs are controlled by RNA-binding proteins (RBPs). Identification of the interacting proteome of a specific mRNA in vivo remains very challenging, however. Based on the widely used technique of RNA tagging with MS2 aptamers for RNA visualization, we developed a RNA proximity biotinylation (RNA-BioID) technique by tethering biotin ligase (BirA*) via MS2 coat protein at the 3' UTR of endogenous MS2-tagged ß-actin mRNA in mouse embryonic fibroblasts. We demonstrate the dynamics of the ß-actin mRNA interactome by characterizing its changes on serum-induced localization of the mRNA. Apart from the previously known interactors, we identified more than 60 additional ß-actin-associated RBPs by RNA-BioID. Among these, the KH domain-containing protein FUBP3/MARTA2 has been shown to be required for ß-actin mRNA localization. We found that FUBP3 binds to the 3' UTR of ß-actin mRNA and is essential for ß-actin mRNA localization, but does not interact with the characterized ß-actin zipcode element. RNA-BioID provides a tool for identifying new mRNA interactors and studying the dynamic view of the interacting proteome of endogenous mRNAs in space and time.

Physiological and behavioural responses to acid and osmotic stress and effects of Mucuna extract in Guppies.

Variation in pH (acidification) and salinity conditions have severe impact at different levels of biological organization in fish. Present study focused to assess the effects of acidification and salinity changes on physiological stress responses at three different levels of function: i) hormonal and oxidative response, ii) osmoregulation and iii) reproduction, in order to identify relevant biomarkers. Second objective of the study was to evaluate the efficacy of plant (Mucuna pruriens) extract for alleviating pH and salinity related stress. Guppies (Poecilia reticulata) were exposed to different pH (6.0, 5.5, 5.0) and salinity (1.5, 3.0, 4.5 ppt) for 7, 14 and 21 days. Following exposure to stress for respective duration, fish were fed diet containing methanol extract of Mucuna seeds (dose 0.80 gm/kg feed) for 7, 14 and 21 days to measure their possible recovery response. Stress hormone (cortisol), hepatic oxidative stress parameters [superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GRd), glutathione peroxidise (GPx), glutathione S-transferase (GST), malondialdehyde (MDA), glutathione (GSH)], gill osmoregulatory response (Na+-K+ATPase activity), sex steroid profiles and mating behaviours (gonopodial thrust and gestation period) were estimated. Cortisol and MDA levels increased with dose and duration of acid and salinity stress, and cortisol levels were higher in males than in females. Effect on Na+-K+ATPase activity was more intense by salinity stress rather than pH induced stress. Both acid and salinity stress reduced sex steroid levels, and mating response was highly affected by both stresses in a dose- and duration-dependent manner. Mucuna treatment reduced stress-induced alteration of cortisol, MDA, Na+-K+ATPase activity and reproductive parameters. Dietary administration of Mucuna seed extract decreased the intensity of environmental stressors at all three functional levels. Mucuna treatment was more effective against salinity stress than acid stress. Thus, cortisol, oxidative stress marker MDA and Na+-K+ATPase could be effective indicators for acid and salinity stress in wild and domestic fish populations. Dietary administration of Mucuna extract may limit the detrimental effects of acidification and salinity variations that are the inevitable outcomes expected under global climate change conditions.

Consequences of oxidative damage and mitochondrial dysfunction on the fatty acid profile of muscle of Indian Major Carps considering metal toxicity.

Current study aims to find interrelation between mitochondrial enzyme function and fatty acid profile in fish muscle and role of antioxidant agents to maintain their balance in response to metal accumulation. Fishes (Labeo rohita, Catla catla, Cirrhinus cirrhosus) were collected from two sites (Nalban Bheri and Diamond Harbour, India). Concentrations of metals (lead, cadmium, copper, nickel, zinc), enzymatic and non-enzymatic antioxidant activity (malondialdehyde, superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase, glutathione S-transferase), muscle enzyme activity (acetylcholinesterase, succinate dehydrogenase, lactate dehydrogenase, Ca2+ATPase, AMP-deaminase, lipoamide reductase, cytochrome C oxidase, aldolase) and fatty acid composition in muscle tissues were analyzed. Metal concentrations were significantly higher (P < 0.05) in fish muscles from Nalban compared to those in Diamond Harbour. Increased activity of antioxidant enzymes was noted with diminished mitochondrial enzymes activity and altered fatty acid composition in response to higher metal accumulation. Higher metal concentration in fish muscle of Nalban seems to significantly (P < 0.05) affect poly and monounsaturated fatty acid content, possibly due to oxidative damage and accumulation of hazardous reactive oxygen species (ROS) molecules. Changes in fatty acid contents following metal accumulation were observed to be species specific. Current study is the first correlative study to illuminate the level of oxidative damage and possible consequences on muscle cellular integrity, mitochondrial functionality and flesh quality against bioaccumulation of different metals in carps. Future studies are needed to quantify the relative contributions of enzymatic and low-molecular-mass antioxidants in protecting mitochondrial function and maintenance of proper fatty acid oxidation during acclimation to long term metal exposure.

Vesicular transport of a ribonucleoprotein to mitochondria.

Intracellular trafficking of viruses and proteins commonly occurs via the early endosome in a process involving Rab5. The RNA Import Complex (RIC)-RNA complex is taken up by mammalian cells and targeted to mitochondria. Through RNA interference, it was shown that mito-targeting of the ribonucleoprotein (RNP) was dependent on caveolin 1 (Cav1), dynamin 2, Filamin A and NSF. Although a minor fraction of the RNP was transported to endosomes in a Rab5-dependent manner, mito-targeting was independent of Rab5 or other endosomal proteins, suggesting that endosomal uptake and mito-targeting occur independently. Sequential immunoprecipitation of the cytosolic vesicles showed the sorting of the RNP away from Cav1 in a process that was independent of the endosomal effector EEA1 but sensitive to nocodazole. However, the RNP was in two types of vesicle with or without Cav1, with membrane-bound, asymmetrically orientated RIC and entrapped RNA, but no endosomal components, suggesting vesicular sorting rather than escape of free RNP from endosomes. In vitro, RNP was directly transferred from the Type 2 vesicles to mitochondria. Live-cell imaging captured spherical Cav1(-) RNP vesicles emerging from the fission of large Cav(+) particles. Thus, RNP appears to traffic by a different route than the classical Rab5-dependent pathway of viral transport.