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.

Molecular complexity of mammary glands development: a review of lactogenic differentiation in epithelial cells.

The mammary gland is a dynamic organ with various physiological processes like cellular proliferation, differentiation, and apoptosis during the pregnancy-lactation-involution cycle. It is essential to understand the molecular changes during the lactogenic differentiation of mammary epithelial cells (MECs, the milk-synthesizing cells). The MECs are organized as luminal milk-secreting cells and basal myoepithelial cells (responsible for milk ejection by contraction) that form the alveoli. The branching morphogenesis and lactogenic differentiation of the MECs prepare the gland for lactation. This process is governed by many molecular mediators including hormones, growth factors, cytokines, miRNAs, regulatory proteins, etc. Interestingly, various signalling pathways guide lactation and understanding these molecular transitions from pregnancy to lactation will help researchers design further research. Manipulation of genes responsible for milk synthesis and secretion will promote augmentation of milk yield in dairy animals. Identifying protein signatures of lactation will help develop strategies for persistent lactation and shortening the dry period in farm animals. The present review article discusses in details the physiological and molecular changes occurring during lactogenic differentiation of MECs and the associated hormones, regulatory proteins, miRNAs, and signalling pathways. An in-depth knowledge of the molecular events will aid in developing engineered cellular models for studies related to mammary gland diseases of humans and animals.

CRISPR-Cas9 based knockout of S100A8 in mammary epithelial cells enhances cell proliferation and triggers oncogenic transformation via the PI3K-Akt pathway: Insights from a deep proteomic analysis.

S100A8 is a calcium-binding protein with multiple functions, including being a chemoattractant for phagocytes and playing a key role in the inflammatory response. Its expression has been shown to influence epithelial-mesenchymal transition (EMT) and metastasis in colorectal cancer. However, the role of S100A8 in cell proliferation and differentiation remains unknown. In this study, we used the CRISPR-Cas9 system to knock out S100A8 in healthy mammary epithelial cells and investigated the resulting changes in proteome profiling and signaling pathways. Our results showed that S100A8 knockout led to an increase in cell proliferation and migration, reduced cell-cell adhesion, and increased apoptosis compared to wildtype cells. Proteomics data indicated that S100A8 significantly affects cell cycle progression, cell proliferation, and cell survival through the PI3K-Akt pathway. Furthermore, our findings suggest that S100A8 function is associated with Pten expression, a negative regulator of the PI3K-Akt pathway. These results indicate that S100A8 dysregulation in healthy cells can lead to altered cellular physiology and higher proliferation, similar to cancerous growth. Therefore, maintaining S100A8 expression is critical for preserving healthy cell physiology. This study provides novel insights into the role of S100A8 in cell proliferation and differentiation and its potential relevance to cancer biology. SIGNIFICANCE: The study suggests that maintaining S100A8 expression is critical for preserving healthy cell physiology, and dysregulation of S100A8 in healthy cells can lead to altered cellular physiology and higher proliferation, similar to cancerous growth. Therefore, targeting the PI3K-Akt pathway or regulating Pten expression, a negative regulator of the PI3K-Akt pathway, may be potential strategies for cancer treatment by controlling S100A8 dysregulation. Additionally, S100A8 and S100A9 have been shown to promote metastasis of breast carcinoma by forming a metastatic milieu. However, the differential expression of S100A8 in tumors and its dual effects of antitumor and protumor make the relationship between S100A8 and tumors complicated. Currently, most research focuses on the function of S100A8 as a secretory protein in the microenvironment of tumors, and its function inside healthy cells without forming dimers remains unclear. Furthermore, the study provides insight into the role of S100A8 in cell proliferation and differentiation, which may have implications for other diseases beyond cancer. The functional role of S100A8 in normal mammary epithelial cells remains completely uncertain. Therefore, the objective of this study is to investigate the function of S100A8 on proliferation in mammary epithelial cells after its deletion and to elucidate the underlying proteins involved in downstream signaling. Our findings indicate that the deletion of S100A8 leads to excessive proliferation in normal mammary epithelial cells, reduces apoptosis, and affects cell-cell adhesion molecules required for cellular communication, resulting in a cancer-like phenotype.

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.

Proteomic Approaches to Unravel the Molecular Dynamics of Early Pregnancy in Farm Animals: An In-Depth Review.

Infertility is a major problem in farm animals, which has a negative economic effect on farm industries. Infertility can be defined as the inability of animals to achieve a successful pregnancy. Early pregnancy is crucial to establish a successful pregnancy, and it is reported that 70-80% and 20-30% of total embryonic loss occur in cattle and pigs, respectively, during the first month of pregnancy. The advanced high-throughput proteomics techniques provide valuable tools for in-depth understanding of the implantation process in farm animals. In the present review, our goal was to compile, assess, and integrate the latest proteomic research on farm animals, specifically focused on female reproduction, which involves endometrial tissues, uterine fluids, oviductal fluids, and microRNAs. The series of studies has provided in-depth insights into the events of the implantation process by unfolding the molecular landscape of the uterine tract. The discussed data are related to pregnant vs. non-pregnant animals, pregnancy vs. oestrous cycle, different days of the early pregnancy phase, and animals with uterine infections affecting reproduction health. Some of the studies have utilized non-invasive methods and in vitro models to decipher the molecular events of embryo-maternal interaction. The proteomics data are valuable sources for discovering biomarkers for infertility in ruminants and new regulatory pathways governing embryo-uterine interaction, endometrium receptivity, and embryonic development. Here, we envisage that the identified protein signatures can serve as potential therapeutic targets and biomarkers to develop new therapeutics against pregnancy diseases.

Critical Review on Physiological and Molecular Features during Bovine Mammary Gland Development: Recent Advances.

The mammary gland is a unique organ with the ability to undergo repeated cyclic changes throughout the life of mammals. Among domesticated livestock species, ruminants (cattle and buffalo) constitute a distinct class of livestock species that are known milk producers. Cattle and buffalo contribute to 51 and 13% of the total milk supply in the world, respectively. They also play an essential role in the development of the economy for farming communities by providing milk, meat, and draft power. The development of the ruminant mammary gland is highly dynamic and multiphase in nature. There are six developmental stages: embryonic, prepubertal, pubertal, pregnancy, lactation, and involution. There has been substantial advancement in our understanding of the development of the mammary gland in both mouse and human models. Until now, there has not been a thorough investigation into the molecular processes that underlie the various stages of cow udder development. The current review sheds light on the morphological and molecular changes that occur during various developmental phases in diverse species, with a particular focus on the cow udder. It aims to explain the physiological differences between cattle and non-ruminant mammalian species such as humans, mice, and monkeys. Understanding the developmental biology of the mammary gland in molecular detail, as well as species-specific variations, will facilitate the researchers working in this area in further studies on cellular proliferation, differentiation, apoptosis, organogenesis, and carcinogenesis. Additionally, in-depth knowledge of the mammary gland will promote its use as a model organ for research work and promote enhanced milk yield in livestock animals without affecting their health and welfare.

Deep Insight of the Pathophysiology of Gestational Diabetes Mellitus.

Diabetes mellitus is a severe metabolic disorder, which consistently requires medical care and self-management to restrict complications, such as obesity, kidney damage and cardiovascular diseases. The subtype gestational diabetes mellitus (GDM) occurs during pregnancy, which severely affects both the mother and the growing foetus. Obesity, uncontrolled weight gain and advanced gestational age are the prominent risk factors for GDM, which lead to high rate of perinatal mortality and morbidity. In-depth understanding of the molecular mechanism involved in GDM will help researchers to design drugs for the optimal management of the condition without affecting the mother and foetus. This review article is focused on the molecular mechanism involved in the pathophysiology of GDM and the probable biomarkers, which can be helpful for the early diagnosis of the condition. The early diagnosis of the metabolic disorder, most preferably in first trimester of pregnancy, will lead to its effective long-term management, reducing foetal developmental complications and mortality along with safety measures for the mother.

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.

Non-SELEX method for aptamer selection against ß-casomorphin-7 peptide.

The non-systematic evolution of ligands by the exponential enrichment (non-SELEX) method was used in the present study for the selection of ß-casomorphin-7 (BCM-7)-specific aptamers. These aptamers were tested to evaluate their ability to detect BCM-7 peptide in the human urine sample. The method did not employ aptamer amplification and counterselection as used in conventional SELEX but included a negative round of selection. The selection was performed in a single day, and after 5 rounds, a total of 16 numbers of aptamer were identified through Sanger sequencing. Newly selected aptamers named sequence ID no. 3 have performed better than other aptamers in detecting the BCM-7 peptide. Sequence ID no. 3 was also compared with previously selected aptamers through the SELEX method and its performance was found to be better than old aptamers. The sensing experiment was tried on different platforms from magnetic beads to the membrane. In each strategy, satisfactory results were obtained with aptamers that recognized BCM-7 spiked in a human urine sample at a very low amount. The non-SELEX method is an easy and time-saving process for aptamer selection. Selection of viable aptamers from a large pool of sequences for sensing experiments is a tedious job; however, an attempt has been made to select aptamers on the basis of In Silico (http://www.unafold.org/, https://bioinformatics.ramapo.edu/QGRS/index.php) information, observing DNA band intensity on agarose gel and colorimetric results obtained on magnetic beads and membrane. These aptamers have the potential in biosensor making for detecting BCM-7 peptide in urine samples of autistic patients.

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.

Role of Natural Killer Cells during Pregnancy and Related Complications.

A high number of leucocytes reside in the human endometrium and are distributed differentially during the menstrual cycle and pregnancy. During early pregnancy, decidual natural killer (dNK) cells are the most common type of natural killer (NK) cells in the uterus. The increase in the number of uterine NK (uNK) cells during the mid-secretory phase of the menstrual cycle, followed by further increase of dNK cells in early pregnancy, has heightened interest in their involvement during pregnancy. Extensive research has revealed various roles of dNK cells during pregnancy including the formation of new blood vessels, migration of trophoblasts, and immunological tolerance. The present review article is focused on the significance of NK cells during pregnancy and their role in pregnancy-related diseases. The article will provide an in-depth review of cellular and molecular interactions during pregnancy and related disorders, with NK cells playing a pivotal role. Moreover, this study will help researchers to understand the physiology of normal pregnancy and related complications with respect to NK cells, so that future research work can be designed to alleviate the complications.

Aptamers based sensing of pregnancy associated glycoproteins (PAG) of bovine for early pregnancy detection.

Tosyl activated magnetic beads were used for aptamer selection against PAG- 7 and 18 proteins of bovine origin. PAG proteins were immobilized on beads with further addition of biotin tagged aptamer library. The recognition of aptamers with PAG was identified by ST-HRP based approach which was colorimetric in nature. The selected aptamers were sequenced and at the same time several new aptamers were identified. Later M-fold structure and G-quadruplex score of aptamers were analyzed for their selection. Those aptamers having high G value and complex structure were chosen. In dot blot assay, aptamers recognized PAG protein in an animal after 42 days of artificial insemination which later given birth to a healthy calf. Further the cross reactivity with serum of 0th day animal (post AI) or with non pregnant animal serum was minimal. Aptamers have also shown interaction with PAG protein of buffalo origin. These selected aptamers have commercial application especially in development of biosensors for early detection of pregnancy in bovine.

Role of Fibulins in Embryonic Stage Development and Their Involvement in Various Diseases.

The extracellular matrix (ECM) plays an important role in the evolution of early metazoans, as it provides structural and biochemical support to the surrounding cells through the cell-cell and cell-matrix interactions. In multi-cellular organisms, ECM plays a pivotal role in the differentiation of tissues and in the development of organs. Fibulins are ECM glycoproteins, found in a variety of tissues associated with basement membranes, elastic fibers, proteoglycan aggregates, and fibronectin microfibrils. The expression profile of fibulins reveals their role in various developmental processes such as elastogenesis, development of organs during the embryonic stage, tissue remodeling, maintenance of the structural integrity of basement membrane, and elastic fibers, as well as other cellular processes. Apart from this, fibulins are also involved in the progression of human diseases such as cancer, cardiac diseases, congenital disorders, and chronic fibrotic disorders. Different isoforms of fibulins show a dual role of tumor-suppressive and tumor-promoting activities, depending on the cell type and cellular microenvironment in the body. Knockout animal models have provided deep insight into their role in development and diseases. The present review covers details of the structural and expression patterns, along with the role of fibulins in embryonic development and disease progression, with more emphasis on their involvement in the modulation of cancer diseases.

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.

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.

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.

EZH2 knockdown in tamoxifen-resistant MCF-7 cells unravels novel targets for regaining sensitivity towards tamoxifen.

BACKGROUND: Acquired resistance to drug involves multilayered genetic and epigenetic regulation. Inhibition of EZH2 has proven to reverse the tamoxifen resistance back to the sensitive state in breast cancer. However, the molecular players involved in EZH2-mediated effects on tamoxifen-resistant MCF-7 cells are unknown. This study was conducted to understand the global change in proteome profile of tamoxifen-resistant MCF-7 breast cancer cells upon EZH2 knockdown. METHODS: Tamoxifen resistance MCF-7 breast cancer cells were established using increasing concentrations of 4-hydroxy tamoxifen. Using label free proteomics approach, we studied the alteration in total proteome in resistant cells as well as cells transfected with siEZH2 in comparison to sensitive and cells transfected with non-targeting siRNA. RESULTS: Here, we report list of proteins that were previously not recognized for their role in tamoxifen resistance and hold a close association with breast cancer patient survival. Proteins Annexin A2, CD44, nucleosome assembly protein 1, and lamin A/C were among the most upregulated protein in tamoxifen-resistant cells that were found to be abrogated upon EZH2 knockdown. The study suggests the involvement for various proteins in acquiring resistance towards tamoxifen and anticipates further research for investigating their therapeutic potentials. CONCLUSION: Overall, we propose that targeting EZH2 or the molecules down the cascade might be helpful in reacquiring sensitivity to tamoxifen in breast cancer.

Combustion of methylcyclohexane at elevated temperatures to investigate burning velocity for surrogate fuel development.

To overcome the complexity associated with the development of detailed kinetic models for real transportation fuels, surrogate fuel models offer an excellent alternative. The present study reports laminar burning velocity (LBV) measurements of methylcyclohexane (MCH) + air mixtures for mixture temperatures up to 610 K using externally heated diverging channel method (EHDC) method at 1 atm pressure. MCH is a commonly used surrogate blend for aviation fuels, gasoline, and diesel, whose kinetic model is simpler to develop. The measurement of laminar burning velocity forms the basis of kinetic model development for such surrogate fuels. The present work reports the measured LBV values for an equivalence ratio range, φ = 0.7-1.4, and their comparison with available experimental data and detailed kinetic model predictions for a mixture temperature range, 353-610 K. Temperature exponent, α is derived using the power-law correlation and good consistency with kinetic model predictions is observed up to 500 K mixture temperatures. At 610 K mixture temperature, an overprediction of ≈12% at φ = 1.05 is observed with JeTSurF 2.0 (2010) model and 27% overprediction with the kinetic model of PoliMi (2014) φ = 1.1. Overall, the reported LBV measurements show slightly better match with the JeTSurF 2.0 (2010) kinetic model than the Wang (2014) kinetic model. Reaction pathway diagrams are drawn to highlight the importance of C2H4 and C2H3 radicals for an increase in the overall reaction rate at 610 K.

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.