Validation of putative reference genes for gene expression studies in heat stressed and α-MSH treated melanocyte cells of Bos indicus using real-time quantitative PCR.

Normalization of cellular mRNA data using internal reference gene (IRG) is an essential step in expression analysis studies. MIQE guidelines ensure that the choice and appropriateness of IRG should be validated for particular tissues or cell types and specific experimental designs. The objective of the present study was to assess 15 IRGs from different functional classes that could serve as best IRGs for Bos indicus (Tharparkar cattle) melanocyte cells under heat stress and hormonal treatment. We implemented the use of geNorm, NormFinder and BestKeeper algorithm to measure the stability of the gene transcript. A total of 15 IRGs (ACTB, BZM, EEF1, GAPDH, GTP, HMBS, HPRT, RPL22, RPL4, RPS15, RPS18, RPS23, RPS9, UBC and UXT) from different functional classes were evaluated. Pair wise comparisons using geNorm revealed that HPRT and RPS23 were the most stable combination of IRGs with M-value of 0.29 followed by UXT (0.30) and RPL4 (0.31). The NormFinder analysis also identified the same set of stably expressed genes (UXT, RPL4, RPS23 and HPRT); however, the rank order was little different. The UXT gene showed lowest crossing point SD and CV values of 0.30 and 1.17, respectively indicating its maximum expression stability through BestKeeper analysis. The present study indicated that, ACTB and HMB were not reliable IRGs for melanocytes cells on account of their lower expression stability. Current study further revealed that UXT, HPRT and RPS23 are the best IRGs for normalization of qPCR data in Bos indicus melanocyte cells under heat stress and hormonal treatment.

Splice variants and seasonal expression of buffalo HSF genes.

In eukaryotes, the heat shock factors (HSFs) are recognized as the master regulator of the heat shock response. In this respect, the genes encoding the heat shock factors seem to be important for adaptation to thermal stress in organisms. Despite this, only few mammalian HSFs has been characterized. In this study, four major heat shock factor genes viz. HSF-1, 2, 4, and 5 were studied. The main objective of the present study was to characterize the cDNA encoding using conserved gene specific primers and to investigate the expression status of these buffalo HSF genes. Our RT-PCR analysis uncovered two distinct variants of buffalo HSF-1 and HSF-2 gene transcripts. In addition, we identified a variant of the HSF5 transcript in buffalo lacking a DNA-binding domain. In silico analysis of deduced amino acid sequences for buffalo HSF genes showed domain architecture similar to other mammalian species. Changes in the gene expression profile were noted by quantitative real-time PCR (qRT-PCR) analysis. We detected the transcript of buffalo HSF genes in different tissues. We also evaluated the seasonal changes in the expression of HSF genes. Interestingly, the transcript level of HSF-1 gene was found upregulated in months of high and low ambient temperatures. In contrast, the expression of the HSF-4 and 5 genes was found to be downregulated in months of high ambient temperature. This suggests that the intricate balance of different HSFs is adjusted to minimize the effect of seasonal changes in environmental conditions. These findings advance our understanding of the complex, context-dependent regulation of HSF gene expression under normal and stressful conditions.

Evaluation of physiological and biochemical responses in different seasons in Surti buffaloes.

AIM: This study was conducted to evaluate the impact of hot dry, hot humid and comfortable season on physiological, hematological, biochemical, and oxidative stress parameters in Surti buffaloes. MATERIALS AND METHODS: Ten lactating Surti buffaloes of similar physiological status were selected. Based on the temperature-humidity index (THI), their natural exposure to the environment was categorized as hot dry (THI1), hot humid (THI2) and moderate winter/comfort season (THI3). Blood/serum samples were collected and analyzed for physiological, hematological, biochemical, and oxidative stress parameters. The results were analyzed using standard statistical methods. RESULTS: With increase in THI, significant rise in physiological parameters such as respiration rate (RR), hematological parameters such as red blood cell (RBC), hematocrit, hemoglobin (Hb) and mean cell Hb concentration (MCHC), biochemical parameters such as alanine aminotransferase (ALT), Na, K, creatinine, blood urea nitrogen, Mn, Cu and Zn, hormones such as cortisol and oxidative stress parameters such as glutathione peroxidase (GPx), superoxide dismutase (SOD), lipid peroxide (LPO) and total antioxidant status (TAS) and significant decline in glucose, cholesterol and triiodothyronine (T3) was observed. CONCLUSION: It was concluded that THI is a sensitive indicator of heat stress and is impacted by ambient temperature more than the relative humidity in buffaloes. Higher THI is associated with significantly increased RR, total RBC count, Hb, hematocrit, MCHC, ALT, urea, sodium, creatinine, triiodothyronine, SOD, GPx, LPO and TAS and with significant decrease in glucose, cholesterol and triiodothyronine (T3).

Seasonal variation in expression pattern of genes under HSP70 : Seasonal variation in expression pattern of genes under HSP70 family in heat- and cold-adapted goats (Capra hircus).

Heat shock protein 70 (HSP70) is one of the most abundant and best characterized heat shock protein family that consists of highly conserved stress proteins, expressed in response to stress, and plays crucial roles in environmental stress tolerance and adaptation. The present study was conducted to identify major types of genes under the HSP70 family and to quantify their expression pattern in heat- and cold-adapted Indian goats (Capra hircus) with respect to different seasons. Five HSP70 gene homologues to HSPA8, HSPA6, HSPA1A, HSPA1L, and HSPA2 were identified by gene-specific primers. The cDNA sequences showed high similarity to other mammals, and proteins have an estimated molecular weight of around 70 kDa. The expression of HSP70 genes was observed during summer and winter. During summer, the higher expression of HSPA8, HSPA6, and HSPA1A was observed, whereas the expression levels of HSPA1L and HSPA2 were found to be lower. It was also observed that the expression of HSPA1A and HSPA8 was higher during winter in both heat- and cold-adapted goats but downregulates in case of other HSPs. Therefore, both heat and cold stress induced the overexpression of HSP70 genes. An interesting finding that emerged from the study is the higher expression of HSP70 genes in cold-adapted goats during summer and in heat-adapted goats during winter. Altogether, the results indicate that the expression pattern of HSP70 genes is species- and breed-specific, most likely due to variations in thermal tolerance and adaptation to different climatic conditions.

New records of Cantharellus species from the northwestern Himalayas of India.

This study investigated several collections of the genus Cantharellus (Cantharellaceae) from the northwestern Himalayas, India, on the basis of morphology and molecular data. Phylogenetic relationships and species limits were investigated by using nuclear ribosomal large subunit sequences (LSU). We recognized 13 species: Cantharellus appalachiensis Petersen, C. cibarius Fries, C. lateritius (Berk) Singer, C. miniatescens Heinem, C. minor Peck, C. pseudoformosus and seven species, C. applanatus sp. nov., C. elongatipes sp. nov., C. fibrillosus sp. nov., C. himalayensis sp. nov., C. indicus sp. nov., C. natarajanii sp. nov., and C. umbonatus sp. nov., as new to science. All these species are described and their taxonomy and ecology are discussed. In addition, a key is provided to all the recognized species. The phylogenetic analysis recovered 10 major supported clades of Cantharellus species.