Effect of thermal stress on HSP70 expression in dermal fibroblast of zebu (Tharparkar) and crossbred (Karan-Fries) cattle

The present studies were conducted to investigate the difference response of dermal fibroblasts to heat stress in Tharparkar and Karan-Fries cattle. Skin is the most important environmental interface providing a protective envelope to animals. In skin, dermal fibroblasts are the most regular cell constituent of dermis that is crucial for temperature homeostasis. The study aimed to examine the reactive oxygen species (ROS) formation, cytotoxicity (%) and heat shock protein 70 (HSP70) genes expression in dermal fibroblast of Tharparkar and Karan-Fries cattle and to assess whether resistance of dermal fibroblast to heat stress is breed specific. Dermal fibroblasts from ear pinna of Tharparkar and Karan-Fries cattle were exposed at 25 °C, 37 °C, 40 °C and 44 °C for 3 h to measure the ROS, cytotoxicity (%) and HSP 70 (HSPA1A, HSPA2 and HSPA8) genes’ expression. The results showed that ROS formation at low temperature (25 °C) decreased in both breeds as compared to control (37 °C) and the differences were significant (P<0.0001). Heat stress at 40 °C did not increase ROS formation significantly in Tharparkar but increased significantly (P<0.001) in Karan-Fries cattle. The overall cytotoxicity (%) was also found to be significantly different (P<0.001) between Tharparkar and Karan-Fries cattle, and on exposure to different temperatures (P<0.001). The cytotoxicity (%) in dermal fibroblast cells of Karan-fries cows was more than Tharparkar. The expression studies indicated that all HSP70 genes (HSPA8, HSPA1A and HSPA2) were up-regulated at different temperatures in both breeds. In Tharparkar, the relative mRNA expression of HSPA8 gene was higher but HSPA1A and HSPA2 genes were low as compared to Karan-Fries cattle. At 40 and 44 °C, the relative expressions of inducible HSP 70 genes (HSPA1A and HSPA2) were higher in Karan-Fries than Tharparkar. In summary, dermal fibroblast resistance to heat shock differed between breeds. Dermal fibroblasts of Tharparkar were observed to be more heat tolerant than crossbred Karan-Fries cattle. The study concludes that zebu cattle (Tharparkar) dermal fibroblasts are more adapted to tropical climatic condition than crossbreed cattle (Karan-Fries). Differences exist in dermal fibroblasts of heat adapted and non-adapted cattle.     

Expression of HSP70 genes in skin of zebu (Tharparkar) and crossbred (Karan Fries) cattle during different seasons under tropical climatic conditions

Skin is most important environmental interface providing a protective envelope to animals. It's always under the influence of both internal and external stressors. Heat shock proteins (HSP) are highly conserved stress proteins which play crucial roles in environmental stress tolerance and thermal adaptation. Present study was planned to observe the relative mRNA expression of inducible (HSP70.1 and HSP70.2) and constitutive (HSP70.8) HSP in skin of zebu (Tharparkar) and crossbred (Karan Fries) cattle during different seasons. Skin biopsies were collected from rump region of each animal, aseptically during winter, spring and summer season. Quantitative real time polymerase chain reaction was performed to examine the gene expression of constitutive (HSP70.8) and inducible (HSP70.1 and HSP70.2) HSP in skin of both the breeds during different seasons. Present study observed higher expression of both constitutive and inducible HSP genes in both the breeds during summer and winter than spring season, but magnitude of increase was higher during summer than winter. During summer season, expression pattern of HSPs in skin showed breed differences, where constitutive HSP expression was higher in Tharparkar than Karan Fries and that of inducible HSP was higher in Karan Fries than Tharparkar. Hence, present study suggested that HSP may be conveniently used as biomarkers for assessing protective response of skin against heat stress in zebu and crossbred cattle. Variation in expression between breeds is associated with their heat tolerance and thermal adaptability. In summary, skin of zebu cattle (Tharparkar) is more resistant to summer stress than crossbred (Karan Fries), providing greater protection against heat stress during summer season. Superior skin protective mechanism of zebu (Tharparkar) than crossbred (Karan-Fries) cattle against heat stress may contribute to superior adaptability of zebu cattle to tropical climatic conditions than crossbreed.     

Expression pattern of bta-mir-2898 miRNA and their correlation with heat shock proteins during summer heat stress among native vs crossbred cattle

Earlier studies identified the role of bta-mir-2898 in bovine. Our earlier study identified that, bta-mir-2898 can be over expressed in crossbred cattle during heat stress. Nevertheless the differential expression of bta-mir-2898 among native vs crossbred cattle during summer stress along with it's correlation with different heat shock proteins (HSPs) is not yet studied. In the present context, we studied the differential expression of bta-mir-2898 among Frieswal (Bos indicus x Bos taurus) and Sahiwal (Bos indicus) breeds of cattle during a range of environmental air temperatures and further investigated the correlation of bta-mir-2898 with different HSPs (HSP70, HSP90, HSP60. HSF, HSPB8 and HSP27). It was observed that, at peak air temperature the relative miRNA expression level (p < 0.05) of bta-mir-2898 was 3.4 ± 0.41 and 0.79 ± 0.22 among Frieswal and Sahiwal, respectively. We also observed significant levels (p < 0.05) of mRNA abundance of HSP70, HSP90, HSPB8 and HSP27 among the breeds. In all the cases Sahiwal found to exhibited higher level of HSPs in comparison to Frieswal. Studies revealed that the expression profile of bta-mir-2898 was negatively correlated with the expression of all the HSPs during thermal stress in post anti-mir2898 treated PBMC invitro cultured model originated from both Frieswal and Sahiwal cattle breeds. However, significantly (p < 0.05) higher negative correlations were observed between bta-mir-2898 and HSP70, HSP60 and HSPB8. Present findings highlighted the preliminary role of overexpressed bta-mir-2898 in cattle during thermal stress and its impact on different heat shock proteins.     

Seasonal variation in HSP70 expression and oxidative stress in skin of zebu (Tharparkar) and crossbred (Karan Fries) cattle under tropical climate

The study aimed to evaluate inducible HSP70 (HSP70.1 and HSP70.2) gene expression and oxidative stress status in skin of cattle during different seasons. Ten each of Tharparkar (zebu) and Karan Fries (crossbred) heifers were selected from NDRI herd, Karnal. Animals were maintained under standard managemental practices followed at the farm. Skin biopsies were aseptically collected from each animal during winter, spring, and summer. Real time PCR was performed to examine HSP70 expression. Reactive oxygen species (ROS) and antioxidant enzymes (SOD and CAT) were determined by ELISA. In both the breeds, significantly higher (p < 0.05) levels of HSP70 expression, ROS, caspases, and antioxidant enzymes were observed during summer followed by winter and spring. Breeds showed no significant difference during winter and spring. During summer, HSP70 expression, ROS, and antioxidant enzymes were higher (p < 0.05) in Karan Fries than Tharparkar, whereas caspases levels were higher in Tharparker than Karan Fries. The study concludes that levels of HSP70 expression, ROS, caspases, and antioxidant enzymes in skin of cattle were strongly affected by seasonal change in temperature. Differences exist in skin tissue thermotolerance of Tharparkar and Karan Fries cattle. This might be an efficient and centrally important mechanism for better adaptability of zebu cattle to heat stress.     

Seasonal variation in expression pattern of genes under HSP70

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.     

Impact of season on antioxidants,nutritional metabolic status,cortisol and heat shock proteins in Hariana and Sahiwal cattle

The aim of this study was to evaluate the effects of summer and winter seasons on antioxidant status, body reserve mobilization and biomarkers of stress in Hariana and Sahiwal cows. Twelve lactating cows (six of each Hariana and Sahiwal cows) were included in summer (May to July) and winter season (November to January) study. Microclimatic observations were recorded on daily basis during the experimental period. In both seasons, blood samples were collected at fortnightly intervals for analysis of total antioxidant activity, non-esterified fatty acids (NEFA), β-Hydroxybutyric acid (BHBA), heat shock protein 70 and 90 (HSP70 and HSP90). Antioxidant activity reduced significantly (p < 0.05) in Hariana cattle during summers as compared to winters; whereas, seasonal variation exerts no effect on antioxidant activity in Sahiwal. Blood NEFA concentration was similar among both the breeds over both the seasons but reduced significantly (p < 0.05) during summer season as compared to winters in both the breeds. BHBA concentration was significantly higher (p < 0.05) in Hariana cows than Sahiwal cows during winters, however, no effect on BHBA level was observed during summer season in both the breeds. Significantly, lower plasma cortisol level (p < 0.05) was found during winter season in Sahiwal as well as Hariana cows. Further, Sahiwal exhibited lower plasma cortisol as compared to Hariana in both the seasons. HSP 70 and 90 showed non-significant differences between breeds within both the seasons. However, significantly, lower plasma HSP 70 levels (p < 0.05) were reported during winter season in Sahiwal as well as in Hariana cows. Results of present study revealed that indigenous Sahiwal is more heat tolerant as compared to Hariana breed.     

A minimally invasive method of piscine tissue collection and an analysis of long-term field-storage conditions for samples

Background

Improvement of efficiency and economic returns is an important goal in dairy farming, as in any agricultural enterprise. The primary goal of dairy industry has been to identify an efficient and economical way of increasing milk production and its constituents without increasing the size of the dairy herd. Selection of animals with desirable genotypes and mating them to produce the next generation has been the basis of livestock improvement and this would continue to remain the same in the coming years. The use of polymorphic genes as detectable molecular markers is a promising alternative to the current methods of trait selection once these genes are proven to be associated with traits of interest in animals. The point mutations in exon IV of bovine β-Lactoglobulin gene determine two allelic variants A and B. These variants were distinguished by Polymerase Chain Reaction and Restriction Fragment Length Polymorphism (PCR-RFLP) analysis in two indigenous Bos indicus breeds viz. Sahiwal and Tharparkar cattle. DNA samples (228 in Sahiwal and 86 in Tharparkar) were analyzed for allelic variants of β-Lactoglobulin gene. Polymorphism was detected by digestion of PCR amplified products with Hae III enzyme, and separation on 12% non-denaturing gels and resolved by silver staining.

Results

The allele B of β-Lactoglobulin occurred at a higher frequency than the allele A in both Sahiwal and Tharparkar breeds. The genotypic frequencies of AA, AB, and BB in Sahiwal and Tharparkar breeds were 0.031, 0.276, 0.693 and 0.023, 0.733, 0.244 respectively. Frequencies of A and B alleles were 0.17 and 0.83, and 0.39 and 0.61 in Sahiwal and Tharparkar breeds respectively. The Chi-square test results (at one degree of freedom at one per cent level) revealed that the Tharparkar population was not in Hardy-Weinberg equilibrium as there was a continuous migration of animals in the herd studied, where as, the results are not significant for the Sahiwal population.

Conclusion

Genotype frequencies of AA were the lowest compared to that of BB genotype in Sahiwal cattle while AB genotypes were more frequent in Tharparkar cattle. The frequency of A allele was found to be lower than that of B allele in both the breeds studied. These results further confirm that Bos indicus cattle are predominantly of β-Lactoglobulin B type than Bos taurus breeds.     

Evolution and diversity studies of innate immune genes in Indian buffalo (<Emphasis Type="Italic">Bubalus bubalis</Emphasis>) breeds using next generation sequencing

Indian buffalo (Bubalus bubalis) breeds are acknowledged for their disease resistance power, hardiness and considerable milk production. River buffaloes are more disease resistance despite of tropical environment of India than cattle breeds of the world. The genetic polymorphisms of innate immune genes play a prominent role in disease susceptibility of livestock. Here in our study, we mainly focused on SNPs in ten selected candidate innate immune genes viz CHGA, CHGB, CHGC, Slc11a1, Slc11a2, DEFB1, BNBD4, BNBD5, LAP and TAP among three most economically important Indian buffalo breeds viz., Jaffrabadi, Mehsani and Murrah using amplicon sequencing on Ion torrent PGM sequencing platform. Polymorphism identification using by GATK tool of Unified Genotyper revealed 274, 245 and 224 high quality SNPs in Jaffrabadi, Mehsani and Murrah buffalo breeds, respectively as well as 168 common SNPs among these three breeds and uniquely detected SNPs were 31, 15 and 37 SNPs in Jaffrabadi, Mehsani and Murrah breeds respectively. Consequences of non-synonymous SNPs were analysed using several computational tools viz., I-Mutant, SIFT, PolyPhen and PROVEAN which identified three deleterious nsSNPs. This wealth of sequence information will be productive for conservation studies, selective breeding and designing future strategies for identifying disease associations.     

Inhibition of Inducible Heat Shock Protein-70 (Hsp72) Enhances Bortezomib-Induced Cell Death in Human Bladder Cancer Cells

The proteasome inhibitor bortezomib (Velcade) is a promising new agent for bladder cancer therapy, but inducible cytoprotective mechanisms may limit its potential efficacy. We used whole genome mRNA expression profiling to study the effects of bortezomib on stress-induced gene expression in a panel of human bladder cancer cell lines. Bortezomib induced strong upregulation of the inducible HSP70 isoforms HSPA1A and HSPA1B isoforms of Hsp72 in 253J B-V and SW780 (HSPA1A^high) cells, but only induced the HSPA1B isoform in UM-UC10 and UM-UC13 (HSPA1A^low) cells. Bortezomib stimulated the binding of heat shock factor-1 (HSF1) to the HSPA1A promoter in 253JB-V but not in UM-UC13 cells. Methylation-specific PCR revealed that the HSPA1A promoter was methylated in the HSPA1A^low cell lines (UM-UC10 and UM-UC13), and exposure to the chromatin demethylating agent 5-aza-2′-deoxycytidine restored HSPA1A expression. Overexpression of Hsp72 promoted bortezomib resistance in the UM-UC10 and UM-UC13 cells, whereas transient knockdown of HSPA1B further sensitized these cells to bortezomib, and exposure to the chemical HSF1 inhibitor KNK-437 promoted bortezomib sensitivity in the 253J B-V cells. Finally, shRNA-mediated stable knockdown of Hsp72 in 253J B–V promoted sensitivity to bortezomib in vitro and in tumor xenografts in vivo. Together, our results provide proof-of-concept for using Hsp72 inhibitors to promote bortezomib sensitivity in bladder cancers and suggest that selective targeting of HSPA1B could produce synthetic lethality in tumors that display HSPA1A promoter methylation.