Probiotic microorganisms favorably alter the intestinal microflora balance, promote intestinal integrity and mobility, inhibit the growth of harmful bacteria and increase resistance to infection. ...Probiotics are increasingly used in nutraceuticals, functional foods or in microbial interference treatment. However, the effectiveness of probiotic organism is considered to be population-specific due to variation in gut microflora, food habits and specific host-microbial interactions. Most of the probiotic strains available in the market are of western or European origin, and a strong need for exploring new indigenous probiotic organisms is felt.
An indigenous isolate Lp9 identified as Lactobacillus plantarum by molecular-typing methods was studied extensively for its functional and probiotic attributes, viz., acid and bile salt tolerance, cell surface hydrophobicity, autoaggregation and Caco-2 cell-binding as well as antibacterial and antioxidative activities. Lp9 isolate could survive 2 h incubation at pH 1.5-2.0 and toxicity of 1.5-2.0% oxgall bile. Lp9 could deconjugate major bile salts like glycocholate and deoxytaurocholate, indicating its potential to cause hypocholesterolemia. The isolate exhibited cell-surface hydrophobicity of approximately 37% and autoaggregation of approximately 31%. Presence of putative probiotic marker genes like mucus-binding protein (mub), fibronectin-binding protein (fbp) and bile salt hydrolase (bsh) were confirmed by PCR. Presence of these genes suggested the possibility of specific interaction and colonization potential of Lp9 isolate in the gut, which was also suggested by a good adhesion ratio of 7.4+/-1.3% with Caco-2 cell line. The isolate demonstrated higher free radical scavenging activity than standard probiotics L. johnsonii LA1 and L. acidophilus LA7. Lp9 also exhibited antibacterial activity against E. coli, L. monocytogenes, S. typhi, S. aureus and B. cereus.
The indigenous Lactobacillus plantarum Lp9 exhibited high resistance against low pH and bile and possessed antibacterial, antioxidative and cholesterol lowering properties with a potential for exploitation in the development of indigenous functional food or nutraceuticals.
The rapid developments in the Internet of Medical Things (IoMT) help the smart healthcare systems to deliver more sophisticated real-time services. At the same time, IoMT also raises many privacy and ...security issues. Also, the heterogeneous nature of these devices makes it challenging to develop a common security standard solution. Furthermore, the existing cloud-centric IoMT healthcare systems depend on cloud computing for electrical health records (EHR) and medical services, which is not suggestible for a decentralized IoMT healthcare systems. In this article, we have proposed a blockchain-based novel architecture that provides a decentralized EHR and smart-contract-based service automation without compromising with the system security and privacy. In this architecture, we have introduced the hybrid computing paradigm with the blockchain-based distributed data storage system to overcome blockchain-based cloud-centric IoMT healthcare system drawbacks, such as high latency, high storage cost, and single point of failure. A decentralized selective ring-based access control mechanism is introduced along with device authentication and patient records anonymity algorithms to improve the proposed system's security capabilities. We have evaluated the latency and cost effectiveness of data sharing on the proposed system using Blockchain. Also, we conducted a logical system analysis, which reveals that our architecture-based security and privacy mechanisms are capable of fulfilling the requirements of decentralized IoMT smart healthcare systems. Experimental analysis proves that our fortified-chain-based H-CPS needs insignificant storage and has a response time in the order of milliseconds as compared to traditional centralized H-CPS while providing decentralized automated access control, security, and privacy.
The present study aims to identify the heat responsive genes and biological pathways in heat stressed buffalo mammary epithelial cells (MECs). The primary mammary epithelial cells of riverine buffalo ...were exposed to thermal stress at 42°C for one hour. The cells were subsequently allowed to recover at 37°C and harvested at different time intervals (30 min to 48 h) along with control samples (un-stressed). In order to assess the impact of heat stress in buffalo MECs, several in-vitro cellular parameters (lactate dehydrogenase activity, cell proliferation assay, cellular viability, cell death and apoptosis) and transcriptional studies were conducted. The heat stress resulted in overall decrease in cell viability and cell proliferation of MECs while induction of cellular apoptosis and necrosis. The transcriptomic profile of heat stressed MECs was generated using Agilent 44 K bovine oligonucleotide array and at cutoff criteria of ≥3-or ≤3 fold change, a total of 153 genes were observed to be upregulated while 8 genes were down regulated across all time points post heat stress. The genes that were specifically up-regulated or down-regulated were identified as heat responsive genes. The upregulated genes in heat stressed MECs belonged to heat shock family viz., HSPA6, HSPB8, DNAJB2, HSPA1A. Along with HSPs, genes like BOLA, MRPL55, PFKFB3, PSMC2, ENDODD1, ARID5A, and SENP3 were also upregulated. Microarray data revealed that the heat responsive genes belonged to different functional classes viz., chaperons; immune responsive; cell proliferation and metabolism related. Gene ontology analysis revealed enrichment of several biological processes like; cellular process, metabolic process, response to stimulus, biological regulation, immune system processes and signaling. The transcriptome analysis data was further validated by RT-qPCR studies. Several HSP (HSP40, HSP60, HSP70, HSP90, and HSPB1), apoptotic (Bax and Bcl2), immune (IL6, TNFα and NF-kβ) and oxidative stress (GPX1 and DUSP1) related genes showed differential expression profile at different time points post heat stress. The transcriptional data strongly indicated the induction of survival/apoptotic mechanism in heat stressed buffalo MECs. The overrepresented pathways across all time points were; electron transport chain, cytochrome P450, apoptosis, MAPK, FAS and stress induction of HSP regulation, delta Notch signaling, apoptosis modulation by HSP70, EGFR1 signaling, cytokines and inflammatory response, oxidative stress, TNF-alpha and NF- kB signaling pathway. The study thus identified several genes from different functional classes and biological pathways that could be termed as heat responsive in buffalo MEC. The responsiveness of buffalo MECs to heat stress in the present study clearly suggested its suitability as a model to understand the modulation of buffalo mammary gland expression signature in response to environmental heat load.
An early and accurate diagnosis of reproductive dysfunctions or aberrations is crucial to better reproductive management in livestock. High reproductive efficiency is a prerequisite for high ...life-time production in dairy animals. Early pregnancy diagnosis is key to shorten the calving interval through early identification of open animals and their timely treatment and rebreeding so as to maintain a postpartum barren interval close to 60 days. A buffalo, the most important dairy animal in the Indian subcontinent, is known for problems related to high calving interval, late puberty, and high incidence of anestrus. Lack of reliable cow-side early pregnancy diagnosis methods further aggravates the situation. Several methods of pregnancy diagnosis are being practiced in bovine species, yet none qualifies as the ideal pregnancy diagnosis method due to the inherent limitations of sensitivity, accuracy, specificity, speed, and ease of performing the test. The advancement of molecular techniques like proteomics and their applications in animal research has given a new hope to look for pregnancy biomarker molecules in these animals. This review attempts to examine common pregnancy diagnosis methods available for dairy animals, while assessing the usefulness of the modern technologies in detecting novel pregnancy markers and designing future strategies for research in this area.
Mammary gland is made up of a branching network of ducts that end with alveoli which surrounds the lumen. These alveolar mammary epithelial cells (MEC) reflect the milk producing ability of farm ...animals. In this study, we have used 2D-DIGE and mass spectrometry to identify the protein changes in MEC during immediate early, peak and late stages of lactation and also compared differentially expressed proteins in MEC isolated from milk of high and low milk producing cows. We have identified 41 differentially expressed proteins during lactation stages and 22 proteins in high and low milk yielding cows. Bioinformatics analysis showed that a majority of the differentially expressed proteins are associated in metabolic process, catalytic and binding activity. The differentially expressed proteins were mapped to the available biological pathways and networks involved in lactation. The proteins up-regulated during late stage of lactation are associated with NF-κB stress induced signaling pathways and whereas Akt, PI3K and p38/MAPK signaling pathways are associated with high milk production mediated through insulin hormone signaling.
Accurate determination of estrus is essentially required for efficient reproduction management of farm animals. Buffalo is a shy breeder and does not manifest overt signs of estrus that make estrus ...detection difficult resulting in a poor conception rate. Therefore, identifying estrus biomarkers in easily accessible biofluid such as saliva is of utmost interest. In the current study, we generated saliva proteome profiles during proestrus (PE), estrus (E), metestrus (ME), and diestrus (DE) stages of the buffalo estrous cycle using both label-free quantitation (LFQ) and labeled (TMT) quantitation and mass spectrometry analysis. A total of 520 proteins were identified as DEPs in LFQ; among these, 59 and four proteins were upregulated (FC ≥ 1.5) and downregulated (FC ≤ 0.5) during E vs. PE, ME, and DE comparisons, respectively. Similarly, TMT-LC-MS/MS analysis identified 369 DEPs; among these, 74 and 73 proteins were upregulated and downregulated during E vs. PE, ME, and DE stages, respectively. Functional annotations of GO terms showed enrichment of glycolysis, pyruvate metabolism, endopeptidase inhibitor activity, salivary secretion, innate immune response, calcium ion binding, oocyte meiosis, and estrogen signaling. Over-expression of SERPINB1, HSPA1A, VMO1, SDF4, LCN1, OBP, and ENO3 proteins during estrus was further confirmed by Western blotting. This is the first comprehensive report on differential proteome analysis of buffalo saliva between estrus and non-estrus stages. This study generated an important panel of candidate proteins that may be considered buffalo estrus biomarkers which can be applied in the development of a diagnostic kit for estrus detection in buffalo.
Lactobacillus fermentum is a natural resident of the human GIT and is used as a probiotic. A unique property of L. fermentum is its ability to tolerate, colonize, and survive in the harsh conditions ...of bile, which facilitates transient colonization of the host colon. In the current study, we investigated the key mechanisms of action involved in bacterial survival in the presence of bile, using high-resolution mass spectrometry. A total of 1071 proteins were identified, among which 378 were up-regulated and 368 down-regulated by ≥2-fold (t-test, p < .05). Differentially regulated proteins comprised both intracellular and surface-exposed (i.e., membrane) proteins (p < .01, t-test for total proteome analysis; p < .05, t-test for membrane proteome analysis). These alterations strengthen the cell envelope and also mediate bile efflux by adjusting carbohydrate metabolic pathways and prevention of protein misfolding. These processes are mainly involved in the active removal of bile salts or amelioration of its adverse effects on cells. Further investigation of mRNA transcript expression levels of selected proteins by quantitative reverse transcriptase-PCR verified the proteomic data. Together, our proteomics findings reveal the roles of post-stress recovery proteins and highlight the interacting pathways responsible for bacterial cell tolerance to bile stress.
Our intestinal tract is a nutrient-rich milieu crowded with up to 100 trillion (1014) of microbes. The fact that we are born germ-free describes that these microbes must colonize our intestinal tract from outside. However, their survival is also complicated because of hazardous conditions in the gut due to the presence of bile acid and others, which exerts a deleterious effect on the beneficial microbial load. While there was limited information available describing the comprehensive mechanism of survival? Furthermore, the imbalance of these micro floras leads to numerous disease conditions. It explains the need for enhanced understanding of host-microbe interactions in the colon. The present study majorly focuses on identifying “how microbes respond to environmental stressors” in this context, particularly bile acid response. This work addresses a fascinating cellular mechanism involved in the complex changes of bile induction in the microbial system; in this case, L. fermentum NCDC 605 a well established probiotic organism. In this article, we decipher the characteristic adaptation mechanism adjusted by probiotics in the harsh condition of 1.2% bile. The generated new knowledge will also improve the potential therapeutic efficacy of probiotics strains in clinical trials for patients of inflammatory bowel diseases (IBD) and related disorders.
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•The proteomic analysis has been investigated to analyze bacterial survival in the presence of 1.2% bile.•Total of 1071 proteins were profiled in L. fermentum 605 with 378 were up-regulated and 368 down-regulated ≥2-fold (t-test, p < .05).•These finding, showed the up-regulation of stress proteins DnaK, GrpE, GroEL, and GroES to overcome the effect of ROS upon 1.2% bile treatment.•This study provides detailed mechanisms that enable L. fermentum to persist under bile stress and adapt to harsh intestinal conditions.
Urine is a non-invasive source of biological fluid, which reflects the physiological status of the mammals. We have profiled the cow urinary proteome and analyzed its functional significance. The ...urine collected from three healthy cows was concentrated by diafiltration (DF) followed by protein extraction using three methods, namely methanol, acetone, and ammonium sulphate (AS) precipitation and Proteo Spin urine concentration kit (PS). The quality of the protein was assessed by two-dimensional gel electrophoresis (2DE). In-gel digestion method revealed more proteins (1191) in comparison to in-solution digestion method (541). Collectively, 938, 606 and 444 proteins were identified in LC-MS/MS after in-gel and in-solution tryptic digestion of proteins prepared by AS, PS and DF methods, respectively resulting in identification of a total of 1564 proteins. Gene ontology (GO) using Panther7.0 grouped the majority of the proteins into cytoplasmic (location), catalytic activity (function), and metabolism (biological processes), while Cytoscape grouped proteins into complement and coagulation cascades; protease inhibitor activity and wound healing. Functional significance of few selected proteins seems to play important role in their physiology. Comparative analysis with human urine revealed 315 overlapping proteins. This study reports for the first time evidence of more than 1550 proteins in urine of healthy cow donors.
This article is part of a Special Issue entitled: Proteomics in India.
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•Ammonium sulphate, proteo spin and daifiltration methods used for protein extraction•Ammonium sulphate precipitation method yielded maximum proteins.•Identified a total of 1564 proteins in urine of healthy cows for the first time•Comparison with human urine revealed 315 overlapping and 1256 new proteins.•Biological significance and biomarker potential of cow urinary proteins reported
The lactating mammary gland harbours numerous matured alveoli with their lumen surrounded by differentiated mammary epithelial cells (MECs), which are exclusively involved in milk synthesis and ...secretion. Buffalo (Bubalus bubalis) is the second major milk‐producing animal, and its physiology is different from cattle. The complete protein machinery involved in MECs differentiation is still not defined in ruminants, in particular, buffalo. Therefore, we have studied the differential expression of regulated proteins in the in vitro grown buffalo MECs (BuMECs) at different time points (on 3, 6, 12, and 15 days) of their differentiation in the presence of lactogenic hormones. TMT‐based MS analysis identified 4,934 proteins; of them, 681 were differentially expressed proteins (DEPs). The principal component analysis suggested a highly heterogeneous expression of DEPs at the four‐time points of hormone treatment, with most of them (307) attained the highest expression on 12 days. Bioinformatics analysis revealed the association of DEPs with 24 KEGG pathways. We observed few new proteins, namely ABCA13, IVL, VPS37, CZIB, RFX7, Rab5, TTLL12, SMEK1, GDI2, and TMEM131 in BuMECs. The function of one of the highly upregulated proteins, namely involucrin in the differentiation of BuMECs was confirmed based on biochemical inhibition assay. The results further conclude that the proteins with higher abundance can be considered as the potential biomarkers for differentiation, and they may have a significant association with the lactation process in buffalo too. The proteome dataset obtained can be used to understand the species‐specific variations among other lactating animals.
Oviduct-specific glycoprotein (OVGP1) is a high molecular weight chitinase-like protein belonging to GH18 family. It is secreted by non-ciliated epithelial cells of oviduct during estrous cycle ...providing an essential milieu for fertilization and embryo development. The present study reports the characterization of buffalo OVGP1 through structural modeling, carbohydrate-binding properties and evolutionary analysis. Structural model displayed the typical fold of GH18 family members till the boundary of chitinase-like domain further consisting of a large (β/α)8 TIM barrel sub-domain and a small (α+β) sub-domain. Two critical catalytic residues were found substituted in the catalytic centre (Asp to Phe118, Glu to Leu120) compared with the active chitinase. The carbohydrate-binding groove in TIM barrel was lined with various conserved aromatic residues. Molecular docking with different sugars revealed the involvement of various residues in hydrogen-bonding and non-bonded contacts. Most of the substrate-binding residues were conserved except for a few replacements (Ser13, Lys48, Asp49, Pro50, Asp167, Glu199, Gln272 and Phe275) in comparison with other GH18 members. The residues Trp10, Trp79, Asn80, Gln272, Phe275 and Trp334 were involved in recognition of all six ligands. The α+β sub-domain participated in sugar-binding through Thr270, Gln272, Tyr242 and Phe275. The binding assays revealed significant sugar-binding with purified native and recombinant OVGP1. Phylogenetic analysis revealed that OVGP1 was closely related to AMCases followed by other CLPs and evolution of OVGP1 occurred through several gene duplications. This is the first study describing the structural characteristics of OVGP1 that will further help to understand its interaction with gametes to perform crucial reproductive functions.
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