Exploring aptamers for targeted enrichment of X sperm in bovine: unraveling selective potential.

Nucleic acid aptamers have been used in the past for the development of diagnostic methods against a number of targets such as bacteria, pesticides, cancer cells etc. In the present study, six rounds of Cell-SELEX were performed on a ssDNA aptamer library against X-enriched sperm cells from Sahiwal breed cattle. Sequencing was used to examine the aptamer sequences that shown affinity for sperm carrying the X chromosome in order to find any possible X-sperm-specific sequences. Out of 35 identified sequences, 14 were selected based on bioinformatics analysis like G-Score and Mfold structures. Further validation of their specificity was done via fluorescence microscopy. The interaction of biotinylated-aptamer with sperm was also determined by visualizing the binding of streptavidin coated magnetic beads on the head region of the sperm under bright field microscopy. Finally, a real-time experiment was designed for the validation of X-sperm enrichment by synthesized aptamer sequences. Among the studied sequences, aptamer 29a exhibited a higher affinity for X sperm compared to Y sperm in a mixed population of sperm cells. By using aptamer sequence 29a, we obtained an enrichment of 70% for X chromosome bearing sperm cells.

Peptidome Profiling of Bubalus bubalis Urine and Assessment of Its Antimicrobial Activity against Mastitis-Causing Pathogens.

Urinary proteins have been studied quite exhaustively in the past, however, the small sized peptides have remained neglected for a long time in dairy cattle. These peptides are the products of systemic protein turnover, which are excreted out of the body and hence can serve as an important biomarker for various pathophysiologies. These peptides in other species of bovine have been reported to possess several bioactive properties. To investigate the urinary peptides in buffalo and simultaneously their bioactivities, we generated a peptidome profile from the urine of Murrah Buffaloes (n = 10). Urine samples were processed using <10 kDa MWCO filter and filtrate obtained was used for peptide extraction using Solid Phase Extraction (SPE). The nLC-MS/MS of the aqueous phase from ten animals resulted in the identification of 8165 peptides originating from 6041 parent proteins. We further analyzed these peptide sequences to identify bioactive peptides and classify them into anti-cancerous, anti-hypertensive, anti-microbial, and anti-inflammatory groups with a special emphasis on antimicrobial properties. With this in mind, we simultaneously conducted experiments to evaluate the antimicrobial properties of urinary aqueous extract on three pathogenic bacterial strains: S. aureus, E. coli, and S. agalactiae. The urinary peptides observed in the study are the result of the activity of possibly 76 proteases. The GO of these proteases showed the significant enrichment of the antibacterial peptide production. The total urinary peptide showed antimicrobial activity against the aforementioned pathogenic bacterial strains with no significant inhibitory effects against a buffalo mammary epithelial cell line. Just like our previous study in cows, the present study suggests the prime role of the antimicrobial peptides in the maintenance of the sterility of the urinary tract in buffalo by virtue of their amino acid composition.

Simple method for isolation and culture of primary buffalo (Bubalus bubalis) endometrial epithelial cells (pBuEECs) and its characterization using high throughput proteomics approach.

Early embryonic mortality resulting from insufficient interaction between the embryo and the uterus leads to the failure of pregnancy in livestock animals. Thus, it is imperative to comprehend the multifaceted process of implantation at molecular levels, which requires synchronized feto-maternal interaction. The in-vitro models serve as valuable tools to investigate the specific stages of implantation. The present study was undertaken to develop a simple method to isolate and culture the primary buffalo endometrial epithelial cells (pBuEECs), followed by proteome profiling of the proliferating cells. Collagenase I was used to separate uterine epithelial cells (UECs) from the ipsilateral uterine horn, and then the cells were separated using a cell strainer. After being seeded on culture plates, UECs developed colonies with characteristic epithelial shape and expressed important markers such as cytokeratin 18 (KRT18), progesterone receptor (PGR), ß-estrogen receptor (ESR1), and leukemia inhibitory factor (LIF), which were confirmed by PCR. The purity of epithelial cells was assessed using cytokeratin 18 immunostaining, which indicated approximately 99% purity in cultured cells. The proteome profiling of pBuEECs via high-throughput tandem mass spectrometry (MS), identified a total of 3383 proteins. Bioinformatics analysis revealed enrichment in various biological processes, including cellular processes, metabolic processes, biological regulation, localization, signaling, and developmental processes. Moreover, the KEGG pathway analysis highlighted associations with the ribosome, proteosome, oxidative phosphorylation, spliceosome, and cytoskeleton regulation pathways. In conclusion, these well characterized cells offer valuable in-vitro model to enhance the understanding of implantation and uterine pathophysiology in livestock animals, particularly buffaloes.

Host-microbe interaction and pathogen exclusion mediated by an aggregation-prone surface layer protein of Lactobacillus helveticus.

Probiotic surface layer proteins (Slps) have multiple functions and bacterial adhesion to host cells is one of them. The precise role of Slps in cellular adhesion is not well understood due to its low native protein yield and self-aggregative nature. Here, we report the recombinant expression and purification of biologically active Slp of Lactobacillus helveticus NCDC 288 (SlpH) in high yield. SlpH is a highly basic protein (pI = 9.4), having a molecular weight of 45 kDa. Circular Dichroism showed a prevalence of beta-strands in SlpH structure and resistance to low pH. SlpH showed binding to human intestinal tissue, enteric Caco-2 cell line, and porcine gastric mucin, but not with fibronectin, collagen type IV and laminin. SlpH inhibited the binding of the enterotoxigenic E. coli by 70 % and 76 % and that of Salmonella Typhimurium SL1344 by 71 % and 75 % to enteric Caco-2 cell line in the exclusion and competition assays, respectively. The pathogen exclusion and competition activity and tolerance to harsh gastrointestinal conditions show the potential for developing SlpH as a prophylactic or therapeutic agent against enteric pathogens.

Identification of bioactive components behind the antimicrobial activity of cow urine by peptide and metabolite profiling.

OBJECTIVE: Cow urine possesses several bioactive properties but the responsible components behind these bioactivities are still far from identified. In our study, we tried to identify the possible components behind the antimicrobial activity of cow urine by exploring the peptidome and metabolome. METHODS: We extracted peptides from the urine of Sahiwal cows belonging to three different physiological states viz heifer, lactation, and pregnant, each group consisting of 10 different animals. The peptides were extracted using the solid phase extraction technique followed by further extraction using ethyl acetate. The antimicrobial activity of the aqueous extract was evaluated against different pathogenic strains like Staphylococcus aureus, Escherichia coli, and Streptococcus agalactiae. The safety of urinary aqueous extract was evaluated by hemolysis and cytotoxicity assay on the BuMEC cell line. The urinary peptides were further fractionated using high-performance liquid chromatography (HPLC) to identify the fraction(s) containing the antimicrobial activity. The HPLC fractions and ethyl acetate extract were analyzed using nLC-MS/MS for the identification of the peptides and metabolites. RESULTS: A total of three fractions were identified with antimicrobial activity, and nLC-MS/MS analysis of fractions resulted in the identification of 511 sequences. While 46 compounds were identified in the metabolite profiling of organic extract. The urinary aqueous extract showed significant activity against E. coli as compared to S. aureus and S. agalactiae and was relatively safe against mammalian cells. CONCLUSION: The antimicrobial activity of cow urine is a consequence of the feeding habit. The metabolites of plant origin with several bioactivities are eliminated through urine and are responsible for their antimicrobial nature. Secondly, the plethora of peptides generated from the activity of endogenous proteases on protein shed from different parts of tissues also find their way to urine. Some of these sequences possess antimicrobial activity due to their amino acid composition.

Production of biologically active recombinant buffalo leukemia inhibitory factor (BuLIF) in Escherichia Coli.

BACKGROUND: Leukemia inhibitory factor (LIF) is a multifunctional cytokine which plays multiple roles in different biological processes such as implantation, bone remodeling, and hematopoiesis. The buESCs are difficult to culture due to lack of proper understanding of the culture conditions. LIF is one of the important factors which maintain the pluripotency in embryonic stem cells and commercial LIF from murine and human origin is used in the establishment of buffalo embryonic stem cells (buESCs). The LIF from a foreign origin is not able to maintain pluripotency and proliferation in buESCs for a long term which is contributed by difference in the binding sites on LIF; therefore, culture medium supplemented with buffalo-specific LIF may enhance the efficiency of buESCs by improving the environment of culture conditions. The high cost of LIF is another major drawback which restricts buESCs research, thus limits the scope of buffalo stem cell use. Various methods have been developed to produce human and murine LIF in prokaryotic system. However, Buffalo leukemia inhibitory factor (BuLIF) has not been yet produced in prokaryotic system. Here, we describe a simple strategy for the expression and purification of biologically active BuLIF in Escherichia coli (E. coli). RESULTS: The BuLIF cDNA from buffalo (Bubalus bubalis) was cloned into pET22b(+) and expressed in E. coli Lemo-21(DE3). The expression of BuLIF was directed into periplasmic space of E. coli which resulted in the formation of soluble recombinant protein. One step immobilized metal affinity chromatography (IMAC chromatography) was performed for purification of BuLIF with ≥ 95% of homogeneity. The recombinant protein was confirmed by western blot and identified by mass spectroscopy. The biological activity of recombinant BuLIF was determined on murine myeloid leukemic cells (M1 cells) by MTT proliferation assay. The addition of BuLIF increased the reduction of MTT by stimulated M1 cells in a dose-dependent manner. The BuLIF induced the formation of macrophage like structures from M1 cells where they engulfed fluorescent latex beads. The recombinant BuLIF successfully maintained pluripotency in buffalo embryonic stem cells (buESCs) and were positive for stem cells markers such as Oct-4, Sox-2, Nanog, and alkaline phosphatase activity. CONCLUSIONS: The present study demonstrated a simple method for the production of bioactive BuLIF in E. coli through single step purification. BuLIF effectively maintained buffalo embryonic stem cells pluripotency. Thus, this purified BuLIF can be used in stem cell study, biomedical, and agricultural research.

Recombinant expression and molecular characterization of buffalo sperm lysozyme-like protein 1.

Several sperm lysozyme-like genes evolved from lysozyme by successive duplications and mutations; however their functional role in the reproduction of farm animals is not well understood. To understand the function and molecular properties of buffalo sperm lysozyme-like protein 1 (buSLLP1), it was expressed in E. coli; however, it partitioned to inclusion bodies. Lowering of temperature and inducer concentration did not help in the recovery of the expressed protein in the biologically active form. Therefore, buSLLP1 was cloned and expressed in Pichiapink system based on auxotrophic Pichia pastoris in a labscale fermenter. The expressed protein was obtained in flow-through by using a 30 kDa ultrafiltration membrane followed by MonoQ anion exchange chromatography, resulting in a homogenous preparation of 40 mg recombinant buSLLP1 per liter of initial spent culture-supernatant. Circular dichroism spectroscopy showed that recombinant buSLLP1 possessed a native-like secondary structure. The recombinant buSLLP1 also showed thermal denaturation profile typical of folded globular proteins; however, the thermal stability was lower than the hen egg white lysozyme. Binding of buSLLP1 to chitin and zona pellucida of buffalo oocytes showed that the recombinant buSLLP1 possessed a competent binding pocket, therefore, the produced protein could be used to study its functional role in the reproduction of farm animals.

Peptide profiling in cow urine reveals molecular signature of physiology-driven pathways and in-silico predicted bioactive properties.

Peptidomics allows the identification of peptides that are derived from proteins. Urinary peptidomics has revolutionized the field of diagnostics as the samples represent complete systemic changes happening in the body. Moreover, it can be collected in a non-invasive manner. We profiled the peptides in urine collected from different physiological states (heifer, pregnancy, and lactation) of Sahiwal cows. Endogenous peptides were extracted from 30 individual cows belonging to three groups, each group comprising of ten animals (biological replicates n = 10). Nano Liquid chromatography Mass spectrometry (nLC-MS/MS) experiments revealed 5239, 4774, and 5466 peptides in the heifer, pregnant and lactating animals respectively. Urinary peptides of <10 kDa size were considered for the study. Peptides were extracted by 10 kDa MWCO filter. Sequences were identified by scanning the MS spectra ranging from 200 to 2200 m/z. The peptides exhibited diversity in sequences across different physiological states and in-silico experiments were conducted to classify the bioactive peptides into anti-microbial, anti-inflammatory, anti-hypertensive, and anti-cancerous groups. We have validated the antimicrobial effect of urinary peptides on Staphylococcus aureus and Escherichia coli under an in-vitro experimental set up. The origin of these peptides was traced back to certain proteases viz. MMPs, KLKs, CASPs, ADAMs etc. which were found responsible for the physiology-specific peptide signature of urine. Proteins involved in extracellular matrix structural constituent (GO:0005201) were found significant during pregnancy and lactation in which tissue remodeling is extensive. Collagen trimers were prominent molecules under cellular component category during lactation. Homophilic cell adhesion was found to be an important biological process involved in embryo attachment during pregnancy. The in-silico study also highlighted the enrichment of progenitor proteins on specific chromosomes and their relative expression in context to specific physiology. The urinary peptides, precursor proteins, and proteases identified in the study offers a base line information in healthy cows which can be utilized in biomarker discovery research for several pathophysiological studies.

Antimicrobial Peptides in Farm Animals: An Updated Review on Its Diversity, Function, Modes of Action and Therapeutic Prospects.

Antimicrobial peptides (AMPs) are the arsenals of the innate host defense system, exhibiting evolutionarily conserved characteristics that are present in practically all forms of life. Recent years have witnessed the emergence of antibiotic-resistant bacteria compounded with a slow discovery rate for new antibiotics that have necessitated scientific efforts to search for alternatives to antibiotics. Research on the identification of AMPs has generated very encouraging evidence that they curb infectious pathologies and are also useful as novel biologics to function as immunotherapeutic agents. Being innate, they exhibit the least cytotoxicity to the host and exerts a wide spectrum of biological activity including low resistance among microbes and increased wound healing actions. Notably, in veterinary science, the constant practice of massive doses of antibiotics with inappropriate withdrawal programs led to a high risk of livestock-associated antimicrobial resistance. Therefore, the world faces tremendous pressure for designing and devising strategies to mitigate the use of antibiotics in animals and keep it safe for posterity. In this review, we illustrate the diversity of farm animal-specific AMPs, and their biochemical foundations, mode of action, and prospective application in clinics. Subsequently, we present the data for their systematic classification under the major and minor groups, antipathogenic action, and allied bioactivities in the host. Finally, we address the limitations of their clinical implementation and envision areas for further advancement.

Tandem Mass Tag (TMT)-based quantitative proteomics reveals potential targets associated with onset of Sub-clinical Mastitis in cows.

Bovine milk is vital for infant nutrition and is a major component of the human diet. Bovine mastitis is a common inflammatory disease of mammary gland in cattle. It alters the immune profile of the animal and lowers the quality and yield of milk causing huge economic losses to dairy industry. The incidence of sub-clinical mastitis (SCM) is higher (25-65% worldwide) than clinical mastitis (CM) (>5%), and frequently progresses to clinical stage due to lack of sensitive and specific detection method. We used quantitative proteomics to identify changes in milk during sub-clinical mastitis, which may be potential biomarkers for developing rapid, non-invasive, sensitive detection methods. We performed comparative proteome analysis of the bovine milk, collected from the Indian hybrid cow Karan Fries. The differential proteome in the milk of Indian crossbred cows during sub-acute and clinical intramammary gland infection has not been investigated to date. Using high-resolution mass spectrometry-based quantitative proteomics of the bovine whey proteins, we identified a total of 1459 and 1358 proteins in biological replicates, out of which 220 and 157 proteins were differentially expressed between normal and infected samples. A total of 82 proteins were up-regulated and 27 proteins were down-regulated, having fold changes of ≥2 and ≤0.8 respectively. Among these proteins, overexpression of CHI3L1, LBP, GSN, GCLC, C4 and PIGR proteins was positively correlated with the events that elicit host defence system, triggering production of cytokines and inflammatory molecules. The appearance of these potential biomarkers in milk may be used to segregate affected cattle from the normal herd and may support mitigation measures for prevention of SCM and CM.

Mechanistic insights into the host-microbe interaction and pathogen exclusion mediated by the Mucus-binding protein of Lactobacillus plantarum.

Surface adhesins of pathogens and probiotics strains are implicated in mediating the binding of microbes to host. Mucus-binding protein (Mub) is unique to gut inhabiting lactic acid bacteria; however, the precise role of Mub proteins or its structural domains in host-microbial interaction is not well understood. Last two domains (Mubs5s6) of the six mucus-binding domains arranged in tandem at the C-terminus of the Lp_1643 protein of Lactobacillus plantarum was expressed in E. coli. Mubs5s6 showed binding with the rat intestinal mucus, pig gastric mucins and human intestinal tissues. Preincubation of Mubs5s6 with the Caco-2 and HT-29 cell lines inhibited the binding of pathogenic enterotoxigenic E. coli cells to the enterocytes by 68% and 81%, respectively. Pull-down assay suggested Mubs5s6 binding to the host mucosa components like cytokeratins, Hsp90 and Laminin. Mubs5s6 was predicted to possess calcium and glucose binding sites. Binding of Mubs5s6 with these ligands was also experimentally observed. These ligands are known to be associated with pathogenesis suggesting Mub might negotiate pathogens in multiple ways. To study the feasibility of Mubs5s6 delivery in the gut, it was encapsulated in chitosan-sodium tripolyphosphate microspheres with an efficiency of 65% and release up to 85% in near neutral pH zone over a period of 20 hours. Our results show that Mub plays an important role in the host-microbial cross-talk and possesses the potential for pathogen exclusion to a greater extent than mediated by L. plantarum cells. The functional and technological characteristics of Mubs5s6 make it suitable for breaking the host-pathogen interaction.

Recombinant purified buffalo leukemia inhibitory factor plays an inhibitory role in cell growth.

Leukemia Inhibitory Factor (LIF) is a polyfunctional cytokine, involved in numerous regulatory effects in vivo and in vitro, varying from cell proliferation to differentiation, and has therapeutic potential for treating various diseases. In the current study, a COS-1 cell line overexpressing recombinant Buffalo LIF (rBuLIF) was established. The rBuLIF was purified to homogeneity from the total cell lysate of COS-1 cells using a two-step affinity chromatography. The purified LIF was confirmed by western blot and mass spectrometer (MS/MS). Particularly, high-resolution MS has identified the rBuLIF with 73% of sequence coverage with highest confidence parameters and with the search engine score of 4580. We determined the molecular weight of rBuLIF protein to be 58.99 kDa and 48.9 kDa with and without glycosylation, respectively. Moreover, the purified rBuLIF was verified to be functionally active by measuring the growth inhibition of M1 myeloid leukemia cells, revealing a maximum inhibition at 72 hours and half-maximal effective concentration (EC50) of 0.0555 ng/ml, corresponding to a specific activity of >1.6×10(7) units/mg. Next, we evaluated the effect of rBuLIF on buffalo mammary epithelial cell lines for its role in involution and also identified the IC50 value for BuMEC migrating cells to be 77.8 ng/ml. Additionally, the treatment of MECs (BuMEC and EpH4) displayed significant (P < 0.05) reduction in growth progression, as confirmed by qRT-PCR analysis, suggesting its strong involvement in the involution of the mammary gland in vivo. Thus, we conclude that the glycosylated rBuLIF, purified from COS-1 cells was found to be functionally active as its natural counterpart.

Expression of fibronectin-binding protein of L. acidophilus NCFM and in vitro refolding to adhesion capable native-like protein from inclusion bodies.

The ability of Lactobacilli to adhere to host epithelial surface and intestinal tracts is important for colonization and persistence of bacteria in the host gut. Extracellular matrix components like fibronectin, mucin, collagen and other adhesion molecules serve as substratum for attachment of bacteria. However, the precise structure, function and mechanism of binding of microbial surface adhesion proteins such as Fibronectin-binding protein (FBP) with host molecules remains unclear. This is primarily due to limitations in high expression of these proteins in biologically active form. To study adhesion of its FBP (64 kDa), the fbp gene of L. acidophilus NCFM was cloned and expressed in E. coli. However, the fibronectin-binding protein expressed in soluble form could not be purified by Ni-NTA affinity chromatography possibly because of partially buried Histidine tag in the recombinant fusion protein. Therefore, the protein was expressed as inclusion bodies (IBs) at 37 °C and solubilized in urea followed by purification in denatured form by Ni-NTA affinity chromatography. The purified denatured protein was refolded in vitro to structurally stable and biologically active form. The conformational properties of the refolded protein were studied by circular dichroism, which showed prominence of α+ ß structural element. The refolded FBP also showed significant binding to human intestinal tissue sections. Our optimized refolding protocol from IBs of this recombinant probiotic FBP led into high amounts of biologically active protein. Our results help in increasing understanding of structure-function relation of surface adhesion proteins and host-microbial interactions.

Examination of pathways involved in leukemia inhibitory factor (LIF)-induced cell growth arrest using label-free proteomics approach.

Leukemia inhibitory factor (LIF) is a multifunctional highly glycosylated protein, synthesized and secreted in various body tissues. Besides the abundance in multiple organs, the molecular mechanism underlying the LIF interactions for cell survival and polarity is poorly understood. In the present study, high-resolution LC-MS/MS based LFQ approach identified 2083 proteins with the overall PSM as 16,032. This proteomics data reviles that LIF promotes the AKT-mTOR signaling pathway. It induces cell growth arrest by an intracellular pathways loop to increase the half-life of the cell. Bioinformatics-based enrichment analysis revealed the involvement of LIF interacting partners in cell survival through increasing the cell cycle length. The anti-proliferative effect of LIF was confirmed by BrdU, MTT and Caspase 3/7 assays and further validated by RT-qPCR. Till date to the best of our knowledge, this is the first study that elucidates LIF-mediated cascade of activation of MEK/ERK, Ras, mTOR, Hippo, and RAP1 pathways. This study further expands the repertoire of signaling pathways known to be subject to activation by LIF. These multiple involvements of pathways through autocrine-paracrine mediated cell cycle arrest additionally suggests a novel means for amplification of a growth arrest stimulus from LIF and its homolog's receptors. BIOLOGICAL SIGNIFICANCE: Leukemia inhibitory factor (LIF) is the polyfunctional cytokine and highly pleiotropic member of the interleukin-6 family. It utilizes a receptor that consists of the LIF receptor b and gp130 and displays diverse effects on target cells. Despite well-known signal transduction mechanisms (JAK/STAT, MAPK, and PI3K) LIF also contains paradoxically opposing influences in several cell types which includes cellular stimulation, inhibition, proliferation, differentiation, and survival. LIF1 is also undergoing clinical trials as a driving force for the embryo implantation in the uterus in women who fail to become pregnant. As LIF can act on the broad spectrum of cell types, it is necessary to understand the basic response mechanism. The available non-canonical regulatory pathways and molecular mechanism associated with LIF are poorly explained. Therefore, we have performed the global proteome analysis of LIF-mediated autocrine-paracrine signaling. The obtained data were examined through advanced bioinformatics tools and LIF inducible changes in terms of pathways were elucidated. The result showed the involvement of cluster of proteins maintaining the Ras/Rap1/STAT3/Hippo pathways which modify the protein component machinery of core histone complexes. This report describes the involvement of proteins responsible for cell growth and progression and defines the LIF-mediated novel autocrine-paracrine signaling loop for cell growth arrest.

Expression of recombinant truncated domains of mucus-binding (Mub) protein of Lactobacillus plantarum in soluble and biologically active form.

Mucins amount to 70% of total proteins present in mammalian mucus and serve as important substrata for bacterial adhesion. In probiotic bacteria such as Lactobacillus plantarum, surface adhesion proteins mediate its adhesion to mucus and adhesion is pivotal in bi-directional host-microbe interactions. Mucus binding (Mub) proteins are a group of bacterial surface adhesion proteins that bind to mucin proteins. The structural framework and functional role of these proteins needs immediate attention but is poorly understood because of their large size, low yield and lack of highly purified protein. The lp_1643 gene of L. plantarum encodes a large Mub protein of 240 kDa and has six mucus binding (Mub) domains in tandem. In this study, the fragment of lp_1643 containing the last two domains with their preceding spacers herein referred to as Mubs5s6 was cloned and expressed in E. coli for probing its functional role in the adhesion of L. plantarum. The protein was expressed with a solubility enhancing maltose binding protein (MBP) fusion tag, yet the MBP-Mubs5s6 protein expressed majorly (>90%) as biologically insoluble inclusion bodies. Thus, extensive optimization of culture conditions was carried out to achieve high level soluble expression (∼70%) of Mubs5s6 protein from its initial low level of solubility. The recombinant protein was purified up to homogeneity by affinity chromatography. Recombinant MBP-Mubs5s6 protein showed strong adhesion potential by binding with human intestinal tissue sections. Our results show a step-by-step hierarchical approach to improve the solubility of difficult-to-express extracellular surface proteins while retaining high functional viability.

Evaluation of casein & whey protein hydrolysates as well as milk fermentates from Lactobacillus helveticus for expression of gut hormones.

BACKGROUND & OBJECTIVES: Milk proteins play a beneficial role in the regulation of food intake, postprandial glycaemia and enteroendocrine hormone secretions and thus are receiving considerable attention for the management of metabolic inflammatory disorders such as type 2 diabetes mellitus (T2DM). The objective of this study was to evaluate the efficacy of peptide/s obtained from milk proteins (casein and whey) as well as from the milk fermented with Lactobacillus helveticus as secretagogues for gut hormones and to purify and characterize the active peptides. METHODS: Effect of hydrolysates of casein protein (CP) and whey protein (WP) and L. helveticus fermented milk on the expression of proglucagon, pro-gastric inhibitory peptide (GIP) and cholecystokinin (CCK) genes was monitored by real-time quantitative polymerase chain reaction. The active glucagon-like peptide-1 (GLP-1) secretion was also quantitatively measured using ELISA. RESULTS: Hydrolysates of CP and WP as well as fermentates of L. helveticus induced the proglucagon, pro-GIP and CCK expression and secretion of GLP-1 in STC-1 (pGIP/Neo) cells. However, intact casein exhibited maximum GLP-1 secretion and proglucagon expression. Two active peptides (F5 and F7) derived from CP1 and WP3 hydrolysates having the ability to upregulate the GLP-1 secretion by 1.6 and 1.8 folds were obtained, and the mass was found to be 786 and 824 Da, respectively, as determined by electrospray ionization-mass spectrometry. However, no single active peptide from L. helveticus fermented milk could be obtained. INTERPRETATION & CONCLUSIONS: Casein as well as fermentates obtained from L. helveticus fermented milk showed higher potential for GLP-1 induction. These can be explored as novel therapeutics to T2DM effectively after demonstrating their in vivo efficacy in appropriate animal models.

Functional characterization of Mammary Gland Protein-40, a chitinase-like glycoprotein expressed during mammary gland apoptosis.

MGP-40 is a chitinase-like protein which is over expressed during mammary gland involution. However, its physiological function in the mammary gland is poorly understood. In the present investigation, we have reported the functional significance of buffalo specific MGP-40 in the mammary gland by using an in vitro model of the buffalo mammary epithelial cell (BuMEC) line. MGP-40 was highly up regulated in BuMECs in serum starved condition as well as after treatment with prolactin suggesting its role in the stress response. Subsequently, to study the effect of MGP-40 on BuMECs, the cells were transfected with a mammalian expression construct of pCI neo harboring MGP-40 gene. It was observed that over expression of MGP-40 enhanced proliferation of BuMECs and protected the cells from apoptosis under serum free condition. In contrast, MGP-40 attenuated the mitogenic effect of insulin in BuMECs. Besides, over expression of the MGP-40 reduced dome formation, acinar polarization and casein synthesis in BuMECs in the presence of lactogenic hormones, it also induced Stat3 phosphorylation and epithelial to mesenchymal transition (EMT) -like features. Together, our data suggest that MGP-40 is involved in protection of BuMECs under stress conditions, inhibits cellular differentiation and induces EMT-like features. A schematic diagram depicting possible association of MGP-40 in various molecular pathways has been presented.

Generation of parthenogenetic goat blastocysts: effects of different activation methods and culture media.

The present study was carried out to investigate the effects of different activation methods and culture media on the in vitro development of parthenogenetic goat blastocysts. Calcium (Ca2+) ionophore, ethanol or a combination of the two, used as activating reagents, and embryo development medium (EDM), modified Charles Rosenkrans (mCR2a) medium and research vitro cleave (RVCL) medium were used to evaluate the developmental competence of goat blastocysts. Quantitative expression of apoptosis, stress and developmental competence-related genes were analysed in different stages of embryos. In RVCL medium, the cleavage rate of Ca2+ ionophore-treated oocytes (79.61 ± 0.86) was significantly (P < 0.05) higher than in ethanol (74.90 ± 1.51) or in the combination of both Ca2+ ionophore and ethanol. In mCR2a or EDM, hatched blastocyst production rate of Ca2+ ionophore-treated oocytes (8.33 ± 1.44) was significantly higher than in ethanol (6.46 ± 0.11) or in the combined treatment (6.70 ± 0.24). In ethanol, the cleavage, blastocyst and hatched blastocyst production rates in RVCL medium (74.90 ± 1.51, 18.30 ± 1.52 and 8.24 ± 0.15, respectively) were significantly higher than in EDM (67.81 ± 3.21, 14.59 ± 0.27 and 5.59 ± 0.42) or mCR2a medium (65.09 ± 1.57, 15.36 ± 0.52 and 6.46 ± 0.11). The expression of BAX, Oct-4 and GlUT1 transcripts increased gradually from 2-cell stage to blastocyst-stage embryos, whereas the transcript levels of Bcl-2 and MnSOD were significantly lower in blastocysts. In addition, different activation methods and culture media had little effect on the pattern of variation and relative abundance of the above genes in different stages of parthenogenetic activated goat embryos. In conclusion, Ca2+ ionophore as the activating agent, and RVCL as the culture medium are better than other tested options for development of parthenogenetic activated goat blastocysts.

Role of surface layer collagen binding protein from indigenous Lactobacillus plantarum 91 in adhesion and its anti-adhesion potential against gut pathogen.

Human feacal isolates were ascertain as genus Lactobacillus using specific primer LbLMA1/R16-1 and further identified as Lactobacillus plantarum with species specific primers Lpl-3/Lpl-2. 25 L. plantarum strains were further assessed for hydrophobicity following the microbial adhesion to hydrocarbons (MATH) method and colonization potentials based on their adherence to immobilized human collagen type-1. Surface proteins were isolated from selected L. plantarum 91(Lp91) strain. The purified collagen binding protein (Cbp) protein was assessed for its anti-adhesion activity against enteric Escherichia coli 0157:H7 pathogen on immobilized collagen. Four L. plantarum strains displayed high degree of hydrophobicity and significant adhesion to collagen. A 72 kDa protein was purified which reduced 59.71% adhesion of E. coli 0157:H7 on immobilized collagen as compared to control well during adhesion assay. Cbp protein is the major influencing factor in inhibition of E. coli 0157:H7 adhesion with extracellular matrix (ECM) components. Hydrophobicity and adhesion potential are closely linked attributes precipitating in better colonization potential of the lactobacillus strains. Cbp is substantiated as a crucial surface protein contributing in adhesion of lactobacillus strains. The study can very well be the platform for commercialization of indigenous probiotic strain once their functional attributes are clinically explored.