Curcumin induces thermotolerance by reducing oxidative stress, apoptosis, and inflammation in buffalo mammary epithelial cells under heat shock conditions.

The epithelial cell is the main basic unit of the udder in which milk synthesis takes place. Curcumin is well known for its antioxidant, anti-apoptotic, and anti- inflammatory properties. The present study was performed to test whether in vitro curcumin supplementation can alleviate the unfavorable impact of hyperthermia on buffalo mammary epithelial cells (BuMECs). The spontaneously immortalized BuMECs were divided into 7 groups (n = 9); 1) unstressed BuMECs (negative control, 37 °C); 2) BuMECs exposed to hyperthermia without curcumin treatment (positive control); 3-7) BuMECs cultured with different concentrations of curcumin (5, 10, 20, 40 and 60 µM), respectively, followed by hyperthermic exposure (42ºC) for 1 h and then returned to 37ºC. Changes in viability (MTT assay), proliferation (BrdU colorimetric immunoassay) and concentrations of antioxidant enzymes, CAT, and SOD (ELISA) of BuMECs were recorded. The gene expression study was performed using qRT-PCR. Lower concentrations of curcumin (5, 10 µM) maintained viability, enhanced proliferation, and content of antioxidant enzymes of heat stressed BuMECs. Curcumin induced thermotolerance and antioxidant status by upregulating the expression of antioxidants genes, anti-apoptotic genes and heat shock proteins in heat stressed BuMECs compared to the positive control group. Besides, curcumin reduced apoptosis and inflammation in BuMECs exposed to hyperthermia by downregulating the expression of genes and transcriptional factors associated with apoptosis and inflammatory immune response. The results reveal the potential roles of curcumin in eliminating the negative impact of hyperthermia on BuMECs by regulating the pathways of apoptosis, inflammation, and oxidative stress.

Comparative Proteome Profiling of Saliva Between Estrus and Non-Estrus Stages by Employing Label-Free Quantitation (LFQ) and Tandem Mass Tag (TMT)-LC-MS/MS Analysis: An Approach for Estrus Biomarker Identification in Bubalus bubalis.

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.

TMT based deep proteome analysis of buffalo mammary epithelial cells and identification of novel protein signatures during lactogenic differentiation.

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.

Crystal structure of breast regression protein 39 (BRP39), a signaling glycoprotein expressed during mammary gland apoptosis, at 2.6 Å resolution.

Breast regression protein 39 (BRP39) is a 39 kDa protein that is a member of chitolectin class of glycosyl hydrolase family 18 (GH18). High expression levels of BRP39 have been detected in breast carcinoma. It helps in proliferation of cells during the progression of this disease and may act as a signaling factor. BRP39 may act as a potential candidate for rational structure-based drug design against breast carcinoma. In this study, we report the crystal structure of mouse recombinant BRP39 expressed in E. coli. The structure was solved by molecular replacement and refined to 2.6 Å resolution. The overall structure of BRP39 consisted of two globular domains: a large (ß/α)8 triosephosphate isomerase (TIM) barrel domain and a small (α + ß) domain. Three non-proline cis-peptides were detected in the sugar-binding cleft of BRP39, including Ser57-Phe58, Leu141-Tyr142, and Trp353-Ala354. The latter residues were conserved in other GH18 family members. It was notable that the conformation of critical Trp100 residue within the sugar-binding cleft was oriented away from the barrel. The side-chain conformation was found to be similar to that observed in chitinases, however, it was oriented into the barrel in other chitinase-like proteins (CLPs). The conformation of this critical residue may have significant implications in sugar binding. Further, two amino acid substitutions were observed in the sugar-binding groove of BRP39. The conserved Asn100 and Arg263 in Hcgp39 and other CLPs proteins (SPX-40 structures) were substituted by Lys101 and Lys264 in BRP39 which may have a significant impact on the sugar-binding properties.

Transcriptional Repression of MFG-E8 Causes Disturbance in the Homeostasis of Cell Cycle Through DOCK/ZP4/STAT Signaling in Buffalo Mammary Epithelial Cells.

The mammary gland is a unique apocrine gland made up of a branching network of ducts that end in alveoli. It is an ideal system to study the molecular mechanisms associated with cell proliferation, differentiation, and oncogenesis. MFG-E8, also known as Lactadherin, is a vital glycoprotein related to the milk fat globule membrane and initially identified to get secreted in bovine milk. Our previous report suggests that a high level of MFG-E8 is indicative of high milk yield in dairy animals. Here, we showed that MFG-E8 controls the cell growth and morphology of epithelial cells through a network of regulatory transcription factors. To understand the comprehensive action, we downregulated its expression in MECs by MFG-E8 specific shRNA. We generated a knockdown proteome profile of differentially expressed proteins through a quantitative iTRAQ experiment on a high-resolution mass spectrometer (Q-TOF). The downregulation of MFG-E8 resulted in reduced phagocytosis and cell migration ability, whereas it also leads to more lifespan to knockdown vis-a-vis healthy cells, which is confirmed through BrdU, MTT, and Caspase 3/7. The bioinformatics analysis revealed that MFG-E8 knockdown perturbs a large number of intracellular signaling, eventually leading to cessation in cell growth. Based on the directed network analysis, we found that MFG-E8 is activated by CX3CL1, TP63, and CSF2 and leads to the activation of SOCS3 and CCL2 for the regulation of cell proliferation. We further proved that the depletion of MFG-E8 resulted in activated cytoskeletal remodeling by MFG-E8 knockdown, which results in the activation of three independent pathways ZP4/JAK-STAT5, DOCK1/STAT3, and PIP3/AKT/mTOR. Overall, this study suggests that MFG-E8 expression in mammary epithelial cells is an indication of intracellular deterioration in cell health. To date, to the best of our knowledge, this is the first study that explores the downstream targets of MFG-E8 involved in the regulation of mammary epithelial cell health.

Proteome dynamics and transcriptome profiling in sorghum [Sorghum bicolor (L.) Moench] under salt stress.

Sorghum is a C4 cereal grain crop which is well adapted to harsh environment. It is a potential model for gaining better understanding of the molecular mechanism due to its wider adaptability to abiotic stresses. In this study, protein extraction was standardized using different methods to study the electrophoretic pattern of sorghum leaves under different salinity levels. The extraction of soluble protein with lysis buffer, followed by its clean-up was found to be the most effective method. The different profiles of salt-responsive proteins were analyzed in G-46 and CSV 44F sorghum genotypes based on their tolerance behavior towards salinity. The kafirin level also changed depending upon the concentration and exposure time to salts suggesting the stored proteins as energy source under stress conditions. The relative expression of salt-responsive genes was studied using Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) which might be used as a molecular screening tool for identification of salt-tolerant genotypes in affected areas. The validated responses were examined in terms of metabolic changes and the expression of stress-induced proteins-viz. heat shock proteins (hsp) via immunoblotting assay. The results showed that the two sorghum genotypes adopted distinct approaches in response to salinity, with G-46 performing better in terms of leaf function. Also, we have standardized different protein extraction methods followed by their clean-up for electrophoretic profiling.

In-depth proteome analysis of more than 12,500 proteins in buffalo mammary epithelial cell line identifies protein signatures for active proliferation and lactation.

The mature mammary gland is made up of a network of ducts that terminates in alveoli. The innermost layer of alveoli is surrounded by the differentiated mammary epithelial cells (MECs), which are responsible for milk synthesis and secretion during lactation. However, the MECs are in a state of active proliferation during pregnancy, when they give rise to network like structures in the mammary gland. Buffalo (Bubalus bubalis) constitute a major source of milk for human consumption, and the MECs are the major precursor cells which are mainly responsible for their lactation potential. The proteome of MECs defines their functional state and suggests their role in various cellular activities such as proliferation and lactation. To date, the proteome profile of MECs from buffalo origin is not available. In the present study, we have profiled in-depth proteome of in vitro cultured buffalo MECs (BuMECs) during active proliferation using high throughput tandem mass spectrometry (MS). MS analysis identified a total of 8330, 5970, 5289, 4818 proteins in four sub-cellular fractions (SCFs) that included cytosolic (SCF-I), membranous and membranous organelle's (SCF-II), nuclear (SCF-III), and cytoskeletal (SCF-IV). However, 792 proteins were identified in the conditioned media, which represented the secretome. Altogether, combined analysis of all the five fractions (SCFs- I to IV, and secretome) revealed a total of 12,609 non-redundant proteins. The KEGG analysis suggested that these proteins were associated with 325 molecular pathways. Some of the highly enriched molecular pathways observed were metabolic, MAPK, PI3-AKT, insulin, estrogen, and cGMP-PKG signalling pathway. The newly identified proteins in this study are reported to be involved in NOTCH signalling, transport and secretion processes.

Proteomics fingerprints of systemic mechanisms of adaptation to bile in Lactobacillus fermentum.

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. BIOLOGICAL SIGNIFICANCE: 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.

Evaluation of Milk Colostrum Derived Lactoferrin of Sahiwal (Bos indicus) and Karan Fries (Cross-Bred) Cows for Its Anti-Cancerous Potential.

Lactoferrin (Lf) is an iron-binding glycoprotein protein known to have immune-modulatory role and recently, its anticancerous effect against different cancer cell types was emphasized. In the present investigation, a comparative evaluation of anticancer potential of colostrum-derived lactoferrin from Indian native zebu cow (Sahiwal, SAC), crossbred (Karan Fries, KFC) and commercially available (C-Lf) lactoferrin from exotic cow using cellular models was made. A protocol was standardized successfully to purify Lf protein from colostrum of both breeds using HPLC and purity was confirmed by LC-MS. A standardized dose of 750 µg/mL Lf was used to treat two cell types MDA-MB-231 and MCF-7 with Lf from three different sources; SAC-Lf, KFC-Lf and C-Lf for 48 h and 72 h. Different cellular parameters including cytotoxicity, viability, apoptosis and cell proliferation were determined. Comparatively, Lf from commercial source (C-Lf) had maximum effect in both cell types followed by SAC-Lf and KFC-Lf. Further, transcriptional changes in genes associated with apoptosis (Bax and Bcl-2), tumor progression (p53, p21, CD44 and NF-κß) and survival (survivin) were evaluated in Lf treatment. The overall results strongly emphasized to the fact that Lf purified from cow colostrum has the capacity to inhibit the in vitro growth of cancerous cell lines albeit to a varied extent.

Structural and functional characterization of buffalo oviduct-specific glycoprotein (OVGP1) expressed during estrous cycle.

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 (ß/α)8 TIM barrel sub-domain and a small (α+ß) 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+ß 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.

Functional analysis of recombinant buffalo lactoferrin and monoferric lobes and their cytotoxic effect on buffalo mammary epithelial cells.

Lactoferrin (Lf) has been involved in diverse type of cellular activities and its biochemical properties are species specific. Lf is a bilobal molecule in which each lobe binds with one Fe2+/Fe3+ ion. A lot of physiological effects of Lf are regulated by its iron binding and release properties; however these properties are species-specific. To understand the iron-binding, thermal stability and cytotoxic effect of buffalo Lf (buLf) and contribution of individual N- and C-terminal lobes therein, buLf and the truncated monoferric lobes were expressed in Kluyveromyces lactis or Pichia pastoris yeast expression systems. The iron-uptake/release behavior and thermal stability of recombinant buLf was observed similar to the Lf purified from buffalo milk. Supplementation of recombinant buLf to the buffalo mammary epithelial cells (BuMEC) culture decreased their proliferation and the cell viability in a dose dependent manner. The cell growth decreased by 37% at 1.0 mg/ml Lf. C-lobe decreased the viability of BuMEC by 15% at 1 mg/ml. The C-lobe showed greater cytotoxic effect against BuMEC in comparison to N-lobe. buLf caused a reduced expression of the casein in BuMEC. At 1.0 mg/ml of buLf, CSN2 transcript level was reduced by 74% and 78% in the normal and hormone free media, respectively. The expression of IL-1ß gene in BuMEC increased by 4-5 fold in the presence of 1.0 mg/ml of Lf. The effect was similar to that observed in the involutory mammary gland, suggesting the role of elevated level of Lf in remodeling of buffalo mammary tissue during involution.

New insights into the catalytic inactivity of mammary gland protein-40, a chitinase-like protein expressed during mammary gland involution.

MGP-40 is a mammary gland-specific glycoprotein which is expressed during involution and is an important marker for mammary gland apoptosis. It is an inactive chitinase-like protein belonging to Glycosyl Hydrolase family 18. The present study reports sequence characterization, tissue-specific expression analysis, production of recombinant MGP-40 and its mutant (A117D and L119E) in both E. coli and COS1 cells for their chitin-binding and chitinase activity analysis. The cDNA of buffalo MGP-40 was cloned and sequenced which corresponded to 1803 bp with an open reading frame of 1152 bp (361 aa), signal sequence of 63 bp (21 aa), 5' and 3' UTR of 144 bp and 507 bp, respectively. The 3' UTR analysis revealed potential sites for high level expression and stability during involution. The half-life of buffalo MGP-40 was found to be 11.7 h. MGP-40 was highly expressed in mammary gland followed by small intestine, spleen and mammary epithelial cells. The purified recombinant MGP-40 and its mutant expressed in E.coli were observed to bind chitin efficiently, however, no chitinase activity was observed. Further, chitinase activity was also not observed by expressing mutant recombinant MGP-40 in COS1 cells ruling out the possible role of post-translational modifications. Structure-based in-silico mutagenesis by FoldX algorithm showed a drastic decrease in overall fold stability which might be a possible reason for inability to recover its activity. Therefore, chitinase activity could not be restored in MGP-40 even after reverting back two critical residues in active site which may be due to detrimental effect of mutations on structural stability.

Identification and characterization of vaccine candidates against Hyalomma anatolicum-Vector of Crimean-Congo haemorrhagic fever virus.

Crimean-Congo haemorrhagic fever (CCHF) is a tick borne viral disease reported from different parts of the world. The distribution of the CCHF cases are linked with the distribution of the principal vector, Hyalomma anatolicum in the ecosystem. Presently, vector control is mainly dependent on repeated application of acaricides, results in partial efficacy and generated acaricide resistant tick strains. Amongst the different components of integrated management programme, immunization of hosts is considered as one of the sustainable component. To restrict CCHF virus spreading, use of anti-Hyalomma vaccines appears as a viable solution. Accordingly, present study was under taken to characterize and evaluate vaccine potential of two conserved molecules, ferritin2 (FER2) and tropomyosin (TPM). Silencing of the genes conferred a cumulative reduction (rejection + unable to engorge) of 61.3% in FER2 and 70.2% in TPM respectively. Furthermore, 44.2% and 72.7% reduction in engorgement weight, 63.6% and 94.9% reduction in egg masses in FER2 and TPM silenced ticks in comparison to LUC-control group was recorded. The recombinant protein, rHaFER2 was characterized as 35 kDa protein with pI of 5.84 and possesses iron binding domains. While rHaTPM is a 51kDa protein with pI of 4.94 having calcium binding domains. Immunization of cross-bred calves by rHaFER2 conferred 51.7% and 51.2% protection against larvae and adults of H. anatolicum challenge infestations. While rHaTPM conferred 63.7% and 66.4% protection against larvae and adults infestations, respectively. The results were comparable with the data generated by RNAi and it clearly showed the possibility for the development of anti-hyalomma vaccine to manage CCHF virus and Theileria annulata infection in human and animals.

Primary structures of different isoforms of buffalo pregnancy-associated glycoproteins (BuPAGs) during early pregnancy and elucidation of the 3-dimensional structure of the most abundant isoform BuPAG 7.

Pregnancy-associated glycoproteins (PAGs) are expressed during pregnancy by the trophoectodermal cells of fetus. Presence of PAGs in dam's circulation has been widely used in pregnancy diagnosis. The present study reports the identification and characterization of different PAG isoforms in buffalo during early stages of pregnancy. The PAG mRNAs isolated from fetal cotyledons (Pregnancy stages: 45, 75 and 90 days) were successfully cloned in pJET1.2 vector and transformed in E. coli. A total of 360 random clones were sequenced and correlated with their stages of expression. A total of 12 isoforms namely, BuPAG 1, 2, 4, 6, 7, 8, 9, 13, 15, 16, 18 and one new isoform were identified. BuPAG 7 was found as the most abundant isoform in all three stages followed by BuPAG 18. Further, a large number of variants were found for most of these isoforms. Phylogenetic relationship of identified BuPAGs showed that BuPAG 2 belonged to an ancient group while other members clustered with modern group. Three-dimensional (3D) structure of BuPAG 7 was determined by homology modeling and molecular dynamic (MD) simulations which displayed a typical fold represented by other aspartic proteinase (AP) family members. Molecular docking of Pepstatin inhibitor with BuPAG 7 revealed to interact through various hydrogen bonding and hydrophobic interactions. Various amino acid substitutions were observed in peptide-binding cleft of BuPAG 7. Superimposition of BuPAG 7 with homologous structures revealed the presence of a 35-41 amino acid long insertion (alpha helix connected by two loops) near the N- terminus which seems to be a unique feature of BuPAG 7 in AP family. This is the first report on identification and sequence characterization of PAG isoforms in buffalo with unique finding that these isoforms represent many transcript variants. We also report 3D structure of the most abundant isoform BuPAG 7 for the first time.

Correction: Impact of Heat Stress on Cellular and Transcriptional Adaptation of Mammary Epithelial Cells in Riverine Buffalo (Bubalus Bubalis).

[This corrects the article DOI: 10.1371/journal.pone.0157237.].

Effect of recombinant and native buffalo OVGP1 on sperm functions and in vitro embryo development: a comparative study.

BACKGROUND: An oviduct- specific glycoprotein, OVGP1, is synthesized and secreted by non-ciliated epithelial cells of the mammalian oviduct which provides an essential milieu for reproductive functions. The present study reports the effects of recombinant buffalo OVGP1 that lacks post-translational modifications, and native Buffalo OVGP1 isolated from oviductal tissue, on frozen- thawed sperm functions and in vitro embryo development. RESULTS: The proportion of viable sperms was greater (P < 0.05) in the recombinant OVGP1-treated group compared to the native OVGP1-treated group at 2 h, 3 h, and 4 h of incubation. The proportion of motile sperms at 3 h and 4 h of incubation; and membrane- intact sperms at 4 h was greater (P < 0.05) in the native OVGP1-treated group compared to the control and recombinant OVGP1-treated groups. The proportion of capacitated and acrosome- reacted sperms was greater (P < 0.05) in the native OVGP1-treated group compared to the recombinant OVGP1 group at 4 h. The rates of cleavage of embryos and their development to the blastocyst stage were greater (P < 0.05) in the presence of either native or recombinant OVGP1 in comparison to control at 10 µg/mL concentration as compared to 5 or 20 µg/mL. CONCLUSIONS: The study suggests that both native and recombinant OVGP1 impart a positive effect on various sperm features and in vitro embryo development. However, native OVGP1 was found to have a more pronounced effect in comparison to recombinant non-glycosylated OVGP1 on various sperm functions except viability. Hence, our current findings infer that glycosylation of OVGP1 might be essential in sustaining the sperm functions but not the in vitro embryo development.

Buffalo Leukemia Inhibitory Factor Induces Differentiation and Dome-Like Secondary Structures in COS-1 Cells.

This study aimed to understand the molecular characteristics of buffalo leukemia inhibitory factor (BuLIF) and the generation of a stably transfected COS-1_BuLIF cell line for its functional characterization. Cumulus cells, isolated from oocytes, were separated, and total cDNA was prepared. The BuLIF gene was ligated into the cloning vector pJET1.2/blunt and expression vector pAcGFP-N1 which was transfected into COS-1 cells and confirmed by qRT-PCR and Western blot. BuLIF was immunoprecipitated and evaluated through a MTT assay. qRT-PCR of STAT3 was performed. The multiple sequence alignment of BuLIF showed high similarity with sheep (98.77%) and cattle (96.62%) compared with other species. The BuLIF gene has an open reading frame of 609 nucleotides coding for 202 amino acids. BuLIF was integrated into the genome of COS-1 cells and resulted in the formation of dome-like secondary structures which are indicative of its functional role mediated through STAT3 proteins. In conclusion, this cell line is suitable for understanding LIF-mediated biological functions.

Identification of serum protein markers for early diagnosis of pregnancy in buffalo.

Improper or delayed pregnancy diagnosis has significant impact over animal production, particularly in buffaloes which inherently suffer from several reproductive inefficiencies. Thus the present study has undertaken to identify serum protein markers pertaining to early pregnancy diagnosis in buffaloes. Serum samples were collected from 10 pregnant Murrah Buffalo heifers at weekly intervals from days 0-35 post-artificial insemination and from 12 inseminated non-pregnant cyclic buffalo heifers on days 0, 7, 14 and 21. Two-dimensional gel electrophoresis and densitometric analysis revealed the presence of five protein spots showing average density fold change of ≥4 during early pregnancy. Mass spectrometry analysis identified these up-regulated proteins as anti-testosterone antibody light chain, apolipoprotein A-II precursor, serum amyloid A, cytokeratin type II, component IV isoform 1, which are have established roles in embryogenesis, but over-expression of the fifth identified protein immunoglobulin lambda light chain in pregnancy has been elucidated as a novel finding in the current study. Further, with bioinformatics analysis, potential antigenic B-cell epitopes were predicted for all these five proteins. An antibody cocktail-based approach involving antibodies against all these five up-regulated entire proteins or their epitopes could be developed for early detection of pregnancy in buffaloes. © 2016 Japanese Society of Animal Science.

Structural, Functional and Phylogenetic Analysis of Sperm Lysozyme-Like Proteins.

Sperm lysozyme-like proteins belonging to c-type lysozyme family evolved in multiple forms. Lysozyme-like proteins, viz., LYZL2, LYZL3 or SLLP1, LYZL4, LYZL5 and LYZL6 are expressed in the testis of mammals. Not all members of LYZL family have been uniformly and unambiguously identified in the genome and proteome of mammals. Some studies suggested a role of SLLP1 and LYZL4 in fertilization; however, the function of other LYZL proteins is unknown. We identified all known forms of LYZL proteins in buffalo sperm by LC-MS/MS. Cloning and sequence analysis of the Lyzl cDNA showed 38-50% identity at amino acid level among the buffalo LYZL paralogs, complete conservation of eight cysteines and other signature sequences of c-type lysozyme family. Catalytic residues in SLLP1, LYZL4 and LYZL5 have undergone replacement. The substrate binding residues showed significant variation in LYZL proteins. Residues at sites 62, 101, 114 in LYZL4; 101 in SLLP1; 37, 62, and 101 in LYZL6 were more variable among diverse species. Sites 63 and 108 occupied by tryptophan were least tolerant to variation. Site 37 also showed lower tolerance to substitution in SLLP1, LYZL4 and LYZL5, but more variable in non-testicular lysozymes. Models of LYZL proteins were created by homology modeling and the substrate binding pockets were analyzed in term of binding energies and contacting residues of LYZL proteins with tri-N-acetylglucosamine (NAG)3 in the A-B-C and B-C-D binding mode. Except LYZL6, LYZL proteins did not show significant difference in binding energies in comparison to hen egg white lysozyme in the A-B-C mode. (NAG)3 binding energy in the B-C-D mode was higher by 1.3-2.2 kcal/mol than in A-B-C mode. Structural analysis indicated that (NAG)3 was involved in making more extensive interactions including hydrogen bonding with LYZL proteins in B-C-D mode than in A-B-C mode. Despite large sequence divergence among themselves and with respect to c-type lysozymes, substrate binding residues as well as hydrogen bonding network between (NAG)3 and proteins were mostly conserved. LYZL5 in buffalo and other mammalian species contained additional 10-12 amino acid sequence at c-terminal that matched with ankyrin repeat domain-containing protein 27. Phylogenetic analysis indicated LYZL2 to be most ancient among all the LYZL proteins and that the evolution of LYZL proteins occurred through several gene duplications preceding the speciation of mammals from other vertebrates as distant as reptiles and amphibians.