Chitosan-Based Highly Sensitive Viable Humidity Sensor for Human Health Monitoring.

We report a sustainable resistive-type humidity sensor based on chitosan (CS) film deposited on an interdigitated Ti/Au electrode coated SiO2 substrate using a simple drop cast approach for human health monitoring. The sensor revealed remarkably high sensitivity (5.8 MΩ/%RH), fast response/recovery time (21 s/25 s), low hysteresis (∼9.3%), excellent reversibility, wide detecting range (11-95% RH), and high selectivity toward water vapor. The calculated associated uncertainty at different %RH indicates the excellent repeatability and stable performance of the sensor. The developed sensor is tested for different human breath patterns, and it is found that the sensor can clearly distinguish between the variations in rate and depth of respiration patterns during normal, fast, deep, and nasal breathing and can monitor for apnea-like situations. The sensor is also utilized to perform noncontact skin humidity sensing. Overall, the developed CS film humidity sensor provides a viable approach for the detection of respiratory disorders and human health issues, detected by skin moisture, in a noninvasive manner.

ddRAD sequencing based genotyping of six indigenous dairy cattle breeds of India to infer existing genetic diversity and population structure.

The present investigation aimed to identify genome wide SNPs and to carry out diversity and population structure study using ddRAD-seq based genotyping of 58 individuals of six indigenous milch cattle breeds (Bos indicus) such as Sahiwal, Gir, Rathi, Tharparkar, Red Sindhi and Kankrej of India. A high percentage of reads (94.53%) were mapped to the Bos taurus (ARS-UCD1.2) reference genome assembly. Following filtration criteria, a total of 84,027 high quality SNPs were identified across the genome of 6 cattle breeds with the highest number of SNPs observed in Gir (34,743), followed by Red Sindhi (13,092), Kankrej (12,812), Sahiwal (8956), Tharparkar (7356) and Rathi (7068). Most of these SNPs were distributed in the intronic regions (53.87%) followed by intergenic regions (34.94%) while only 1.23% were located in the exonic regions. Together with analysis of nucleotide diversity (π = 0.373), Tajima's D (D value ranging from - 0.295 to 0.214), observed heterozygosity (HO ranging from 0.464 to 0.551), inbreeding coefficient (FIS ranging from - 0.253 to 0.0513) suggested for the presence of sufficient within breed diversity in the 6 major milch breeds of India. The phylogenetic based structuring, principal component and admixture analysis revealed genetic distinctness as well as purity of almost all of the 6 cattle breeds. Overall, our strategy has successfully identified thousands of high-quality genome wide SNPs that will further enrich the Bos indicus representation basic information about genetic diversity and structure of 6 major Indian milch cattle breeds which should have implications for better management and conservation of valuable indicine cattle diversity.

Expression profile of different classes of proteases in milk derived somatic cells across different lactation stages of indigenous cows (Bos indicus) and riverine buffaloes (Bubalus bubalis).

Proteases play a significant role in milk and its products by affecting flavor, texture and longevity. The expression of endogenous proteases varies across different stages of lactation. The study was conducted to understand the transcriptional pattern of different classes of protease-pathways associated genes (CTSB, CTSD, CTSH, CTSL, CTSK, CTSS, CTSZ, PLAU, PLAT) and potential protease inhibitors (SERPIN E2 and SERPIN F2) in 40 milk somatic cells (MSC) samples isolated during early, peak, mid and late lactation stages of Sahiwal cows and Murrah buffaloes - the two most important dairy breeds of India. In Sahiwal cows, except CTSK and PLAU, the expression of other proteases class was not affected significantly (p > 0.05) across lactation stages. However, in Murrah buffaloes, the expression of different proteases increased as the lactation progressed. Most of the proteases showed lower expression during early and peak lactation stages while their expression tends to increase during mid to late lactation stages. The overall trend was somewhat similar in both the dairy species albeit the level of expression was higher in buffalo MSC as compared to cow MSC. The study has provided valuable information on expression kinetics of different proteases in milk somatic cells of two major dairy breeds of India.

Demographic pattern of A1/A2 beta casein variants indicates conservation of A2 type haplotype across native cattle breeds (Bos indicus) of India.

Genetic variations of the beta casein gene hold importance because of their probable association with human health. Comparative sequence analysis of ß-casein gene across Indian native, crossbred and exotic breeds in India revealed 15 SNPs and 4 INDELs corresponding to 14 haplotypes. The frequency of A2 type haplotype was maximum (0.941) across all Indian native breeds. Among the 15 variants reported for taurine breeds, only three (A1, A2 and B) were observed in analysed populations. Allelic profiling of A1/A2 ß-casein variants in ~ 4000 animals belonging to three cattle types and breeding bulls also revealed the predominance of A2 allele (0.95) in Indian cattle. The high proportion of A2 allele/haplotype indicates that Indian native cattle are the best suited to meet the demands for A2 milk globally. However, a higher percentage of heterozygous genotype (A1A2) in breeding bulls warrants the need to screen sire lines so as to drift the herd towards A2. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03232-0.

Milk-derived mammary epithelial cells as non-invasive source to define stage-specific abundance of milk protein and fat synthesis transcripts in native Sahiwal cows and Murrah buffaloes.

The molecular physiology of milk production of two important dairy species; Sahiwal cows (Bos indicus) and Murrah buffaloes (Bubalus bubalis) are not fully understood due to constraints in obtaining mammary tissue samples because of sacred and ethical reasons. The present study suggests the use of milk-derived mammary epithelial cells (MECs) as a non-invasive method to understand molecular aspects of lactation biology in dairy animals. A total of 76 MECs were collected from five different lactation periods viz. colostrum (0-2), early (5-20), peak (30-50), mid (90-140) and late lactation (> 215 days) stages from Sahiwal cows and Murrah buffaloes to study the transcription kinetics of milk protein, fat synthesis, and their regulatory genes. Significant changes were observed in milk composition of both dairy species with lactation stages. High mRNA abundance of all milk protein and fat synthesis genes was observed in MECs of Murrah buffaloes as compared to Sahiwal cows. The mRNA abundance of caseins (CSN1S1, CSN1S2, CSN2, and CSN3) and whey protein (LALBA, LF) were higher in early lactation stage. Similarly, the expression of milk fat synthesis genes (SCD, BTN1A1, ACACA, GPAM, FAPB3, FASN) was also high in early lactation stage. The relative abundance of 4 regulatory genes (JAK2, STAT5, SREBF1 and EIF4BP41) remained high during early lactation indicating their regulatory roles in lactogenesis process. Overall, results suggested a significant effect of lactation stages on milk composition and transcription abundance of milk protein and fat synthesis genes. The present study establishes the fact that milk-derived MECs could be utilized as a valuable source to understand mammary gland functioning of native cows and buffaloes.

Selection of suitable reference genes for normalization of quantitative RT-PCR (RT-qPCR) expression data across twelve tissues of riverine buffaloes (Bubalus bubalis).

Selection of reference genes has become an integral step in any real time quantitative PCR (RT-qPCR) based expression studies. The importance of this study stems from the fact that riverine buffaloes are major dairy species of Indian sub-continent and the information generated here will be of great interest to the investigators engaged in functional genomic studies of this important livestock species. In this study, an effort was made to evaluate a panel of 10 candidate reference genes (glyceraldehyde 3-phosphate dehydrogenase (GAPDH), beta- actin (ACTB), ubiquitously expressed transcript (UXT), ribosomal protein S15 (RPS15), ribosomal protein L-4 (RPL4), ribosomal protein S9 (RPS9), ribosomal protein S23 (RPS23), hydroxymethylbilane synthase (HMBS), ß2 Microglobulin (ß2M) and eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) across 12 tissues (mammary gland, kidney, spleen, liver, heart, intestine, ovary, lung, muscle, brain, subcutaneous fat and testis) of riverine buffaloes. In addition to overall analysis, tissue wise evaluation of expression stability of individual RG was also performed. Three different algorithms provided in geNorm, NormFinder and BestKeeper softwares were used to evaluate the stability of 10 potential reference genes from different functional classes. The M-value given by geNorm ranged from 0.9797 (RPS9 and UXT) to 1.7362 (RPS15). From the most stable to the least stable, genes were ranked as: UXT/RPS9> RPL4> RPS23> EEF1A1> ACTB> HMBS> GAPDH> B2M> RPS15. While NormFinder analysis ranked the genes as: UXT> RPS23> RPL4> RPS9> EEF1A1> HMBS> ACTB> ß2M> GAPDH> RPS15. Based on the crossing point SD value and range of fold change expression, BestKeeper analysis ranked the genes as: RPS9> RPS23/UXT> RPL4> GAPDH> EEF1A1> ACTB> HMBS> ß2M> RPS15. Overall the study has identified RPS23, RPS9, RPL4 and UXT genes to be the most stable and appropriate RGs that could be utilized for normalization of transcriptional data in various tissues of buffaloes. This manuscript thus provide useful information on panel of reference genes that could be helpful for researchers conducting functional genomic studies in riverine buffaloes.

Peripheral blood mononuclear cells: a potential cellular system to understand differential heat shock response across native cattle (Bos indicus), exotic cattle (Bos taurus), and riverine buffaloes (Bubalus bubalis) of India.

Circulating leukocytes can be used as an effective model to understand the heat stress response of different cattle types and buffaloes. This investigation aimed to determine the temporal profile of HSPs (HSP40, HSP60, HSP70, and HSP90) expression in circulating peripheral blood mononuclear cells (PBMCs) of Murrah buffaloes, Holstein-Friesian (HF), and Sahiwal cows in response to sublethal heat shock at 42 °C. The viability data indicated HF PBMCs to be the most affected to the heat shock, whereas Sahiwal PBMCs were least affected, indicating its better survivability during the heat stress condition. The qRT-PCR expression data showed significant increase in mRNA expression of the analyzed HSPs genes after heat stimuli to the PBMCs under in vitro condition. In each case, the HSPs were most upregulated at 2 h after the heat stress. Among the HSPs, HSP70 was relatively more expressed followed by HSP60 indicating the action of molecular chaperones to stabilize the native conformation of proteins. However, PBMCs from different cattle types and buffaloes showed difference in the extent of transcriptional response. The level of expression of HSPs throughout the time period of heat stress was highest in buffaloes, followed by HF and Sahiwal cows. The higher abundance of HSP70 mRNA at each time point after heat stress showed prolonged effect of heat stress in HF PBMCs. The data presented here provided initial evidence of transcriptional differences in PBMCs of different cattle types and buffaloes and warrant further research.

Selection of stable reference genes in heat stressed peripheral blood mononuclear cells of tropically adapted Indian cattle and buffaloes.

Eleven reference genes from different functional categories were assessed for their stable expression pattern in heat stressed PBMCs across Indian buffalo and cattle using GeNorm, NormFinder and BestKeeper algorithms. As the first report, we suggest B2M, RPS9 and RPS15a as suitable reference genes for accurate normalization of PBMC transcript data while the ACTB gene is not recommended.