Identifying miRNAs in the modulation of gene regulation associated with ammonia toxicity in catfish, Clarias magur (Linnaeus, 1758).

BACKGROUND: The small non-coding microRNAs play a vital role in post-transcriptional gene regulation associated with different physiological events such as metabolism, stress, etc. The freshwater catfish, Clarias magur, can grow within hyper ammonia containing stagnant water bodies and/or muddy substratum. We intended to identify organ-specific miRNAs associated with ammonia stress management. METHODS AND RESULTS: The miRNA-libraries were generated from QC passed total RNA extracted from liver, muscle, and kidney of ammonia-treated (exposed to 25 mM NH4Cl for 14 days) and untreated catfish. The libraries were validated using High sensitivity D1000 Screen tape. The trimmed quality-filtered reads for control and treated samples of kidney were 19,406,210; 14,904,423; for liver 15,467,727; 18,582,072; and for muscle 25,081,345; 19,782,182 respectively. Total 120 known and 150 novel differentially expressed miRNAs were identified, out of which miR-200, miR-217, miR-122, miR-133, miR-145, miR-221, miR-19, miR-138, miR-34, and miR-184 were predicted to be involved in the metabolism of nitrogen. The key miRNAs targeted several genes associated with urea synthesis like Glutaminase 2, Argininosuccinate lyase, Glutamate dehydrogenase 1, Alanine aminotransferase 2-like, Aspartate aminotransferase, cytoplasmic-like, Glutamate ionotropic receptor NMDA type subunit 2A, etc. CONCLUSIONS: This is the first report of miRNAs, which serve as a vital resource for regulating nitrogen metabolism in freshwater catfish, C. magur. The data will be resourceful for further evaluating the regulatory role of miRNAs in fishes, which grow and reproduce very well in hazardous ammonia-contaminated water bodies.

Differential expression of aquaporin genes and the influence of environmental hypertonicity on their expression in juveniles of air-breathing stinging catfish (Heteropneustes fossilis).

Aquaporins (AQPs) are a superfamily of transmembrane channel proteins that are responsible for the transport of water and some other molecules to and from the cell, mainly for osmoregulation under anisotonicity. We investigated here the expression patterns of different AQP isoforms and also during exposure to hypertonicity (300 mOsmol/L) for 48 h in juvenile stages of air-breathing stinging catfish (Heteropneustes fossilis). A total of 8 mRNA transcripts for different isoforms of AQPs and their translated proteins could be detected in the anterior and posterior regions of S1, S2, and S3 stages of juveniles of stinging catfish at variable levels. In general, more expression of mRNAs for different aqp genes was seen in the S2 and S3 juveniles than in the S1 juveniles. Most interestingly, exposure to hypertonicity of S2 juveniles for a period of 48 h led to increased expression of most of the aqp genes both at transcriptional and translational levels, except for aqp3 in the anterior and posterior regions and aqp1 in the anterior region, showing maximum expression at later stages of hypertonic exposure. Thus, it is evident that AQPs play crucial roles in maintaining the water and ionic balances under anisotonic conditions even at the early developmental stages of stinging catfish as a biochemical adaptational strategy to survive and grow in anisotonic environment.

Genome-wide identification of novel long non-coding RNAs and their possible roles in hypoxic zebrafish brain.

Long non-coding RNAs (lncRNAs) are the master regulators of numerous biological processes. Hypoxia causes oxidative stress with severe and detrimental effects on brain function and acts as a critical initiating factor in the pathogenesis of Alzheimer's disease (AD). From the RNA-Seq in the forebrain (Fb), midbrain (Mb), and hindbrain (Hb) regions of hypoxic and normoxic zebrafish, we identified novel lncRNAs, whose potential cis targets showed involvement in neuronal development and differentiation pathways. Under hypoxia, several lncRNAs and mRNAs were differentially expressed. Co-expression studies indicated that the Fb and Hb regions' potential lncRNA target genes were involved in the AD pathogenesis. In contrast, those in Mb (cry1b, per1a, cipca) was responsible for regulating circadian rhythm. We identified specific lncRNAs present in the syntenic regions between zebrafish and humans, possibly functionally conserved. We thus identified several conserved lncRNAs as the probable regulators of AD genes (adrb3b, cav1, stat3, bace2, apoeb, psen1, s100b).

Molecular characterization and ornithine-urea cycle genes expression in air-breathing magur catfish (Clarias magur) during exposure to high external ammonia.

The air-breathing magur catfish (Clarias magur) is a potential ureogenic teleost because of its functional ornithine-urea cycle (OUC), unlike typical freshwater teleosts. The ability to convert ammonia waste to urea was a significant step towards land-based life forms from aquatic predecessors. Here we investigated the molecular characterization of some OUC genes and the molecular basis of stimulation of ureogenesis via the OUC in magur catfish. The deduced amino acid sequences from the complete cDNA coding sequences of ornithine transcarbamyolase, argininosuccinate synthase, and argininosuccinate lyase indicated that phylogenetically magur catfish is very close to other ureogenic catfishes. Ammonia exposure led to a significant induction of major OUC genes and the gene products in hepatic and in certain non-hepatic tissues of magur catfish. Hence, it is reasonable to assume that the induction of ureogenesis in magur catfish under hyper-ammonia stress is mediated through the activation of OUC genes as an adaptational strategy.

Molecular characterization of superoxide dismutase and catalase genes, and the induction of antioxidant genes under the zinc oxide nanoparticle-induced oxidative stress in air-breathing magur catfish (Clarias magur).

The deduced amino acid sequences from the complete cDNA coding sequences of three antioxidant enzyme genes (sod1, sod2, and cat) demonstrated that phylogenetically the magur catfish (Clarias magur) is very much close to other bony fishes with complete conservation of active site residues among piscine, amphibian, and mammalian species. The three-dimensional structures of three antioxidant enzyme proteins are very much similar to mammalian counterparts, thereby suggesting the functional similarities of these enzymes. Exposure to ZnO NPs resulted in an oxidative stress as evidenced by an initial sharp rise of intracellular concentrations of hydrogen peroxide (H2O2) and malondialdehyde (MDA) but decreased gradually at later stages. The level of glutathione (GSH) also increased gradually in all the tissues examined after an initial decrease. Biochemical and gene expression analyses indicated that the magur catfish has the ability to defend the ZnO NP-induced oxidative stress by inducing the SOD/CAT enzyme system and also the GSH-related enzymes that are mediated through the activation of various antioxidant-related genes both at the transcriptional and translational levels in various tissues. Furthermore, it appeared that the stimulation of NO, as a consequence of induction nos2 gene, under NP-induced oxidative stress serves as a modulator to induce the SOD/CAT system in various tissues of magur catfish as an antioxidant strategy. Thus, it can be contemplated that the magur catfish possesses a very efficient antioxidant defensive mechanisms to defend against the oxidative stress and also from related cellular damages during exposure to ZnO NPs into their natural environment.

Induction of nitric oxide synthesis: a strategy to defend against high environmental ammonia-induced oxidative stress in primary hepatocytes of air-breathing catfish, Clarias magur.

Air-breathing magur catfish (Clarias magur) regularly face the problem of exposure to high environmental ammonia (HEA) as one of the major pollutants in their natural habitats that causes considerable toxic effects at the cellular level, including that of oxidative stress. The major objective of the present study was to demonstrate the antioxidant activity of endogenously produced nitric oxide (NO) to defend against ammonia-induced oxidative stress in primary hepatocytes of magur catfish during exposure to HEA. Exposure to NH4Cl (5 mmol l-1) led to a significant increase in intracellular ammonia concentration with a sharp rise of hydrogen peroxide (H2O2) and malondialdehyde (MDA) concentrations within 3 h in primary hepatocytes, which decreased gradually at later stages of treatment. This phenomenon was accompanied by a significant increase in superoxide dismutase (SOD) and catalase (CAT) activity as a consequence of induction of corresponding genes. HEA exposure also led to the stimulation of NO production due to induction of inducible nitric oxide synthase (iNOS) activity, as a consequence of up-regulation of the nos2 gene. Most interestingly, when NO production by hepatocytes under ammonia stress was blocked by adding certain inhibitors [aminoguanidine and 3-(4-methylphenylsulfonyl)-2-propenenitrile] to the culture medium, there was a further rise of H2O2 and MDA concentrations in hepatocytes. These were accompanied by the lowering of SOD and CAT activity with less expression of corresponding genes. Thus, it can be contemplated that magur catfish use the strategy of stimulation of NO production, which ultimately induces the SOD-CAT enzyme system to defend against ammonia-induced oxidative stress.

Antioxidant activity of endogenously produced nitric oxide against the zinc oxide nanoparticle-induced oxidative stress in primary hepatocytes of air-breathing catfish, Clarias magur.

The facultative air-breathing magur catfish (Clarias magur) regularly encounter various environmental challenges including the exposure to nanomaterials discarded as industrial wastes in water bodies. The present investigation aimed at determining the possible ZnO NP-induced oxidative stress and also the antioxidant strategy of nitric oxide (NO), generated endogenously, in primary hepatocytes of magur catfish. Exposure of primary hepatocytes to different concentrations of ZnO NPs (5 and 10 µg/mL) led to a sharp rise of intracellular concentrations of hydrogen peroxide (H2O2) and malondialdehyde (MDA) within 6 h, which decreased gradually at later stages. This phenomenon was accompanied by an initial decrease of superoxide dismutase (SOD) and catalase (CAT) activities, the expression of their corresponding genes and the enzyme protein levels, with a subsequent significant increase of all these parameters at later stages. Most interestingly, exposure to ZnO NPs also stimulated the NO production by the primary hepatocytes as a consequence of induction of inducible nitric oxide synthase (iNOS) activity, higher expression of nos2 gene and iNOS protein. Furthermore, when the NO production by the hepatocytes was inhibited by either aminoguanidine (inhibitor for iNOS) or BAY (inhibitor for NFκB) in the presence of ZnO NPs, the intracellular concentrations of H2O2 and MDA was significantly elevated. This elevation was accompanied by a subsequent decrease of sod and cat genes expression, thereby suggesting that the inhibition of NO production leads to oxidative stress. Thus, it is believed that the magur catfish uses the strategy of stimulation of endogenous NO production by inducing the nos2 gene and simultaneous NO-mediated induction of sod and cat genes to defend against the NP-induced oxidative stress. It is the first report of such NO-mediated antioxidant strategy in any teleost fish to defend against the NP-induced oxidative stress and corresponding cellular damages.

Highly Selective Detection of Hypochlorous Acid by a Bis-heteroleptic Ru(II) Complex of Pyridyl-1,2,3-triazole Ligand via C(sp2)-H Hydroxylation.

A Ru(II) complex (Ru-1) of a substituted pyridyl-1,2,3-triazole ligand (BtPT) for highly selective "light-up" detection of hypochlorous acid is presented. An unusual anti-Markovnikov HOCl addition to the C═C bond of 1,2,3-triazole and a highly specific C(sp2)-H hydroxylation over epoxidation made Ru-1 a highly selective luminescent HOCl probe. The abnormal regio- and stereoselective HOCl addition and subsequent hydroxylation mechanism in detail is supported by the combination of ESI-MS, 1H/13C NMR spectroscopy, and 1H NMR titration. The hydroxylation at the C5 center in 1,2,3-triazole increases the electron density and makes BtPT a better σ-donor as well as π-donor, which in turn increases the 3MC-3MLCT energy gap and inhibits the nonradiative decay from the excited state of Ru-1 and is the key reason for luminescence light-up. Most importantly, the exogenous and endogenous HOCl imaging in the living HEK293T cells is also demonstrated. The probe showed low cytotoxicity and efficiently permeated the cell membrane. The cell-imaging experiments revealed rapid staining of the extranuclear region of HEK293T cells which clearly indicates the presence of cytoplasmic HOCl. The endogenous HOCl generation and imaging, stimulated by lipopolysaccharides (LPS) and paraquat in the HEK293T cells, is also demonstrated.

Hyper-ammonia stress causes induction of inducible nitric oxide synthase gene and more production of nitric oxide in air-breathing magur catfish, Clarias magur (Hamilton).

Nitric oxide (NO) is an important signalling molecule that plays diverse physiological functions in several vertebrates including that of adaptation to various stressful stimuli. The air-breathing magur catfish (Clarias magur) is known to tolerate a very high external ammonia (HEA) stress in its natural habitats. We report here the possible induction of inducible nitric oxide (inos) gene and more generation of NO in magur catfish exposed to HEA. Exposure to HEA (25 mM NH4Cl) for 14 days led to the higher accumulation of NO in different tissues of magur catfish and also more efflux of NO from the perfused liver of NH4Cl-treated fish as a consequence of high build of toxic ammonia in body tissues. More synthesis and accumulation of NO in body tissues was associated with the induction of iNOS activity, which otherwise was not detectable in control fish. The stimulation of iNOS activity in HEA exposed fish was mainly due to induction of inos gene as evidenced by more expression of inos mRNA and also more abundance of iNOS protein in different tissues of magur catfish. Immunocytochemical analysis indicated the zonal specific expression of iNOS protein in different tissues of magur catfish. The augmentation of iNOS in the fish under HEA could be an adaptive strategy of the fish to defend against the ammonia stress through the generation of NO. Therefore, the present finding identifies the potential role of iNOS to enhance the adaptive capacity and survivability of catfish under various adverse environmental and pathological conditions that it faces in its natural habitats.

Ammonia stress under high environmental ammonia induces Hsp70 and Hsp90 in the mud eel, Monopterus cuchia.

The obligatory air-breathing mud eel (Monopterus cuchia) is frequently being challenged with high environmental ammonia (HEA) exposure in its natural habitats. The present study investigated the possible induction of heat shock protein 70 and 90 (hsp70, hsc70, hsp90α and hsp90ß) genes and more expression of Hsp70 and Hsp90 proteins under ammonia stress in different tissues of the mud eel after exposure to HEA (50 mM NH4Cl) for 14 days. HEA resulted in significant accumulation of toxic ammonia in different body tissues and plasma, which was accompanied with the stimulation of oxidative stress in the mud eel as evidenced by more accumulation of malondialdehyde (MDA) and hydrogen peroxide (H2O2) during exposure to HEA. Further, hyper-ammonia stress led to significant increase in the levels of mRNA transcripts for inducible hsp70 and hsp90α genes and also their translated proteins in different tissues probably as a consequence of induction of hsp70 and hsp90α genes in the mud eel. However, hyper-ammonia stress was neither associated with any significant alterations in the levels of mRNA transcripts for constitutive hsc70 and hsp90ß genes nor their translated proteins in any of the tissues studied. More abundance of Hsp70 and Hsp90α proteins might be one of the strategies adopted by the mud eel to defend itself from the ammonia-induced cellular damages under ammonia stress. Further, this is the first report of ammonia-induced induction of hsp70 and hsp90α genes under hyper-ammonia stress in any freshwater air-breathing teleost.

Influence of environmental hypertonicity on the induction of ureogenesis and amino acid metabolism in air-breathing walking catfish (Clarias batrachus, Bloch).

Effect of environmental hypertonicity, due to exposure to 300 mM mannitol solution for 7 days, on the induction of ureogenesis and also on amino acid metabolism was studied in the air-breathing walking catfish, C. batrachus, which is already known to have the capacity to face the problem of osmolarity stress in addition to other environmental stresses in its natural habitats. Exposure to hypertonic mannitol solution led to reduction of ammonia excretion rate by about 2-fold with a concomitant increase of urea-N excretion rate by about 2-fold. This was accompanied by significant increase in the levels of both ammonia and urea in different tissues and also in plasma. Further, the environmental hypertonicity also led to significant accumulation of different non-essential free amino acids (FAAs) and to some extent the essential FAAs, thereby causing a total increase of non-essential FAA pool by 2-3-fold and essential FAA pool by 1.5-2.0-fold in most of the tissues studied including the plasma. The activities of three ornithine-urea cycle (OUC) enzymes such as carbamoyl phosphate synthetase, argininosuccinate synthetase and argininosuccinate lyase in liver and kidney tissues, and four key amino acid metabolism-related enzymes such as glutamine synthetase, glutamate dehydrogenase (reductive amination), alanine aminotransaminase and aspartate aminotransaminase were also significantly up-regulated in different tissues of the fish while exposing to hypertonic environment. Thus, more accumulation and excretion of urea-N observed during hypertonic exposure were probably associated with the induction of ureogenesis through the induced OUC, and the increase of amino acid pool was probably mainly associated with the up-regulation of amino acid synthesizing machineries in this catfish in hypertonic environment. These might have helped the walking catfish in defending the osmotic stress and to acclimatize better under hypertonic environment, which is very much uncommon among freshwater teleosts.

Transcriptome analysis of gills reveals novel insights into the molecular response of stinging catfish (Heteropneustes fossilis) to environmental hypertonicity.

The stinging catfish Heteropneustes fossilis is a champion survivor under hypertonic stress and is suggested to be a profitable candidate for culture in slightly saline water in coastal regions. Fish gills are an essential site of osmoregulation and other physiological processes. To investigate the stress responses and mechanisms of salinity tolerance in stinging catfish, we sampled gills tissues from control and hypertonicity (100 mM NaCl solution) treated adult catfish and assessed for transcriptomic profiling by high throughput sequencing. The raw data generated was filtered and assembled for de novo transcriptome assembly. The final contig assembly produced a total of 1,71,478 unigene transcripts with an average transcript length of 898 bp and a GC content of 45%. A total of 22,231 transcripts matched with Chordata with BLAST search and were functionally annotated, out of which 21,814 were best-hit transcripts aligned with the UniProt database. Comparative transcriptomic analysis revealed that a total of 1951 genes were differentially expressed in the gills of NaCl-treated fish compared to the control. Functional and enrichment analysis of the Differentially expressed genes demonstrated that several GO pathway terms were significantly over-represented, such as 'catalytic activity', 'hydrolase activity' in molecular function category, 'membrane', 'integral component of membrane' in cellular component category and 'metabolic process', 'regulation of transcription' in biological process category. The functional analysis study of DEGs demonstrated that tolerance to hypertonic stress by stinging catfish is associated with a few pathways related to stress response, immune response, biosynthesis, metabolism, molecular transport, cytoskeleton remodeling, apoptosis, cell signaling, transcriptional regulation, etc. The present study provides a novel insight into the molecular responses of the air-breathing stinging catfish against salinity stress, which could elucidate the underlying mechanisms of adaptation of this stenohaline species under various environmental constraints.

Deciphering the role of aquaporin 1 in the adaptation of the stinging catfish Heteropneustes fossilis to environmental hypertonicity by molecular dynamics simulation studies.

A thorough investigation of the water permeability of H. fossilis aquaporin 1 (hfAQP1) in a hypertonic environment can provide a useful insight into the understanding of the underlying molecular mechanism of its high tolerance to salinity. Here, we constructed a 3 D homology model of hfAQP1 by taking Bos taurus AQP1, AQP0, and human AQP2 as templates using I-TASSER. The model obtained has similar structural organizations with mammalian AQP1s in all aspects. We investigated the water permeability of the modeled hfAQP1 in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membrane under neutral and 100 mM hypersalinity by subjecting each system to a 100 ns molecular dynamics simulation. Our results show that hypersalinity hinders water permeation across the membrane through the hfAQP1 channel. A change in the intermolecular distance between key residues of the ar/R selectivity filter along with charge redistribution resulted in the accommodation of only 2-6 water molecules inside the channel at once under hypersaline conditions. We investigated the mRNA expression pattern of hfaqp1 in osmoregulatory organs of H. fossilis in response to 100 mM hypertonicity by using qPCR analysis. The transcript was downregulated in kidney and GI tract, but upregulated in the Gills. Thus, the catfish survive in a hypertonic environment by reducing the transport of water in its cellular systems and downregulating the expression of the hfaqp1 gene. The results observed in our study can shed more light on the functionality of AQP1 in catfishes under salinity stress and aid in future researches on solving more gating mechanisms involved in its regulation.Communicated by Ramaswamy H. Sarma.

Functional expression, localization, and biochemical characterization of thioredoxin glutathione reductase from air-breathing magur catfish, Clarias magur.

The glutathione (GSH) and thioredoxin (Trx) systems regulate cellular redox homeostasis and maintain antioxidant defense in most eukaryotes. We earlier reported the absence of gene coding for the glutathione reductase (GR) enzyme of the GSH system in the facultative air-breathing catfish, Clarias magur. Here, we identified three thioredoxin reductase (TrxR) genes, one of which was later confirmed as a thioredoxin glutathione reductase (TGR). We then characterized the novel recombinant TGR enzyme of C. magur (CmTGR). The tissue-specific expression of the txnrd genes and the tissue-specific activity of the TrxR enzyme were analyzed. The recombinant CmTGR is a dimer of ~133 kDa. The protein showed TrxR activity with 5,5'-diothiobis (2-nitrobenzoic acid) reduction assay with a Km of 304.40 µM and GR activity with a Km of 58.91 µM. Phylogenetic analysis showed that the CmTGR was related to the TrxRs of fishes and distantly related to the TGRs of platyhelminth parasites. The structural analysis revealed the conserved glutaredoxin active site and FAD- and NADPH-binding sites. To our knowledge, this is the first report of the presence of a TGR in any fish. This unusual presence of TGR in C. magur is crucial as it helps maintain redox homeostasis under environmental stressors-induced oxidative stress.

Expression of inducible nitric oxide synthase and nitric oxide production in the mud-dwelled air-breathing singhi catfish, Heteropneustes fossilis under condition of water shortage.

Nitric oxide (NO) is known to be an important regulator molecule for regulating the multiple signaling pathways and also to play diverse physiological functions in mammals including that of adaptation to various stresses. The present study reports on the production of nitric oxide (NO) and the expression of inducible nitric oxide synthase (iNOS) enzyme that produces NO from l-arginine in the freshwater air-breathing catfish (Heteropneustes fossilis) while dwelling inside the mud peat under semidry conditions. Desiccation stress, due to mud-dwelling for 2 weeks, led to significant increase of NO concentration in different tissues and in plasma of singhi catfish, and also the increase of NO efflux from the perfused liver with an accompanying increase of toxic ammonia level in different tissues. Mud-dwelling also resulted to induction of iNOS activity, expression of iNOS protein in different tissues after 7 days with further increase after 14 days, which otherwise was not detectable in control fish. Further, mud-dwelling also resulted to a significant expression of iNOS mRNA after 7 days with a more increase of mRNA level after 14 days, suggesting that the desiccation stress caused transcriptional regulation of iNOS gene. Immunocytochemical analysis indicated the zonal specific expression of iNOS protein in different tissues. Desiccation stress also led to activation and nuclear translocation of nuclear factor кB (NFкB) in hepatic cells. These results suggest that the activation of iNOS gene under desiccation-induced stresses such as high ammonia load was probably mediated through the activation of one of the major transcription factors, the NFкB. This is the first report of desiccation-induced induction of iNOS gene, iNOS protein expression leading to more generation of NO while living inside the mud peat under condition of water shortage in any air-breathing teleosts.

Functional ureogenesis and changes of amino acid metabolism in amphihaline shad hilsa (Tenualosa ilisha, Hamilton-Buchanan) while inhabiting in estuary and freshwater habitats.

The possible occurrence of a functional ornithine-urea cycle (OUC) and changes of activity of key amino acid metabolism-related enzymes were studied in the amphihaline shad hilsa (Tenualosa ilisha) that were collected from estuarine water of Kakdwip and from freshwater river basin of Bhrahmaputra during the breeding season. Very high concentration of urea was detected in different tissues and plasma of shad hilsa collected from estuarine water compared to the one collected from freshwater river basin. This observation clearly suggests that the shad hilsa has the potential of synthesizing and retaining urea inside the body for the purpose of osmoregulation while living in hypertonic saline environment of estuary. This was accompanied by the presence of high activity of all the five OUC enzymes in hepatic and in certain non-hepatic tissues such as the kidney and muscle of shad hilsa in support of its potential ureogenic capacity while inhabiting in estuarine water. The activities of different key amino acid metabolism-related enzymes such as glutamine synthetase, glutamate dehydrogenase, alanine aminotransaminase and aspartate aminotransaminase were also found to be significantly higher in shad hilsa of estuarine water compared to the one collected from freshwater habitat. Thus the adjustment to amino acid metabolism in shad hilsa in different environmental salinities appears to play significant roles for osmotic balance and also for proper energy supply in addition to the presence of a functional OUC while migrating between marine and freshwater habitats throughout their life cycle.

Influence of increased environmental water salinity on gluconeogenesis in the air-breathing walking catfish, Clarias batrachus.

The present study was aimed at determining the effect of hypertonicity due to increased environmental water salinity on gluconeogenesis in air-breathing walking catfish (Clarias batrachus). In situ exposure to hypertonic saline solution (150 mM NaCl) led to a significant stimulation of glucose efflux due to gluconeogenesis from the liver after 7 days with further elevation after 14 days in the presence of each of the three potential gluconeogenic substrates (lactate, pyruvate, and glutamate). This was accompanied by significant increase of activities of three key gluconeogenic enzymes, namely phosphoenolpyruvate carboxykinase (PEPCK), fructose 1,6-biphosphatase (FBPase), and glucose 6-phosphatase (G6Pase) in liver and kidney by about twofold to threefold. Environmental hypertonicity also led to a significant elevation in the levels of PEPCK, FBPase, and G6Pase enzyme proteins in both the tissues by about 2- to 2.75-fold, accompanied by a significant elevation in the level of PEPCK mRNA by about 2- to 2.5-fold after 7 days, and further enhancement to about 3.5- to 4-fold after 14 days. Thus, the upregulation of PEPCK, FBPase. and G6Pase activities appears to be a result of transcriptional regulation of these genes. The induction of gluconeogenesis under environmental hypertonicity, which this catfish faces regularly in its natural habitat, possibly occurs as a consequence of changes in hydration status/cell volume of different cell types. This would certainly assist in maintaining glucose homeostasis, and also for a proper energy supply to support metabolic demands for ion transport and other altered metabolic processes under various environmental hypertonic stress-related insults.

Increase in PPARγ inhibitory phosphorylation by Fetuin-A through the activation of Ras-MEK-ERK pathway causes insulin resistance.

Obesity induced insulin resistance is primarily regulated by the inhibitory phosphorylation of peroxisome proliferator-activated receptor γ at serine 273 (PPARγS273) which has been shown to be regulated by MEK and ERK. An upstream regulatory molecule of this pathway could be a therapeutic option. Here we analyzed the involvement of Fetuin-A (FetA), a key hepato-adipokine implicated in insulin resistance, as an upstream regulator molecule for the regulation of PPARγ inhibitory phosphorylation. Mice fed with standard diet (SD), high fat diet (HFD) and HFD with FetA knockdown (HFD-FetAKD) were used to examine the role of FetA on PPARγS273 phosphorylation in adipocytes. The mechanism of regulation and its effect on skeletal muscle were studied using primary adipocytes, 3T3-L1 (preadipocyte) and C2C12 (myotube) cell lines. Increased FetA in HFD mice strongly correlated with augmentation of PPARγS273 phosphorylation in inflamed adipocytes while knockdown of FetA suppressed it. This effect of FetA was mediated through the activation of Ras which in turn activated MEK and ERK. On addressing how FetA could stimulate activation of Ras, we found that FetA triggered TNFα in inflamed adipocytes which induced Ras activation. The ensuing sharp fall in adiponectin level attenuated AMPK activation in skeletal muscle cells affecting mitochondrial ATP production. Our data reveal the essential role of FetA induced activation of Ras in regulating PPARγ inhibitory phosphorylation through Ras-MEK-ERK pathway which downregulates adiponectin disrupting skeletal muscle mitochondrial bioenergetics. Thus, FetA mediated PPARγ inactivation has adverse consequences upon adipocyte-myocyte crosstalk leading to disruption of energy homeostasis and loss of insulin sensitivity.

Influence of cell volume changes on protein synthesis in isolated hepatocytes of air-breathing walking catfish (Clarias batrachus).

The present study aimed at determining the effect of cell volume changes on protein synthesis, measured as the incorporation of [(3)H]leucine into acid-precipitable protein, in isolated hepatocytes of air-breathing walking catfish (Clarias batrachus). The rate of protein synthesis, which was recorded to be 10.02 +/- 0.10 (n = 25) nmoles mg(-1) cell protein h(-1) in isotonic incubation conditions, increased/decreased significantly by 18 and 48%, respectively, following hypo- (-80 mOsmol l(-1))/hypertonic (+80 mOsmol l(-1)) incubation conditions (adjusted with NaCl), with an accompanying increase/decrease of hepatic cell volume by 12 and 20%, respectively. Similar cell volume-sensitive changes of protein synthesis were also observed when the anisotonicity of incubation medium was adjusted with mannitol. Increase of hepatic cell volume by 9%, due to addition of glutamine plus glycine (5 mM each) to the isotonic control incubation medium, led to a significant increase of protein synthesis by 14%. Decrease of hepatic cell volume by 15 and 18%, due to addition of dibutyl-cAMP and adenosine in isotonic control incubation medium, led to a significant decrease of protein synthesis by 30 and 34%, respectively. Thus, it appears that the increase/decrease of hepatic cell volume, caused either by changing the extracellular osmolarity or by the presence of amino acids or certain other metabolites, leads to increase/decrease of protein synthesis, respectively, and shows a direct correction (r = 0.99) between the hepatic cell volume and protein synthesis in walking catfish. These cell volume-sensitive changes of protein synthesis probably help this walking catfish in fine tuning the different metabolic pathways for better adaptation during cell volume changes and also to avoid the adverse affects of osmotic stress. This is the first report of cell volume-sensitive changes of protein synthesis in hepatic cells of any teleosts.

Orthopantomography and Cone-Beam Computed Tomography for the Relation of Inferior Alveolar Nerve to the Impacted Mandibular Third Molars.

CONTEXT: Relation of inferior alveolar nerve to the impacted mandibular third molars (IMTMs). AIMS: The aim of this study was to assess the reliability of seven specific radiographic signs of mandibular third molar root that are observed on orthopantomography (OPG) and to predict the proximity and the absence of corticalization between the mandibular canal and IMTM root on cone-beam computed tomography (CBCT) images. SETTINGS AND DESIGN: The present study was conducted in the Department of Oral and Maxillofacial Surgery, Raja Rajeswari Dental College and Hospital. SUBJECTS AND METHODS: Data set of 30 panoramic images was retrieved between the year of 2015 and 2016 indicated for extraction of lower third molars. The sample consisted of 30 individuals, who underwent preoperative radiographic evaluation before the extraction of impacted mandibular third molars (IMTM). Patients aged above 18 years with any of the seven specific signs observed on the panoramic radiograph which includes darkening, deflection, narrowing of roots, bifid root apex, diversion, narrowing of canal and interruption in the white line of the canal were included in the study. If any of the above mentioned seven specific sign were present, the patient was subjected to CBCT. On the CBCT images, the canal was traced in three planes. The acquired images were assessed for the presence or absence of corticalization. STATISTICAL ANALYSIS USED: Descriptive and inferential statistical analyses were used. Proportions were compared using the Chi-square test and Student's t-test. RESULTS: Among the 4 subjects, diagnosed with an absence of corticalization, patients with isolated darkening of root P = 0.001 and patients with isolated interruption in white line P = 0.69. Patients with darkening of root in association with interruption in white lines on OPG showed the absence of corticalization on CBCT findings P = 0.001, respectively. CONCLUSIONS: This study showed the poor reliability of radiographic signs seen on OPG on predicting the proximity of third mandibular root with mandibular canal related to CBCT finding. Four were diagnosed with the absence of corticalization in CBCT findings.