Postbiotics Implication in the Microbiota-Host Intestinal Epithelial Cells Mutualism.

To sustain host health and provide the microbial community with a nutrient-rich environment, the host and gut microbiota must interact with one another. These interactions between commensal bacterial and intestinal epithelial cells (IECs) serve as the first line of defense against gut microbiota in preserving intestinal homeostasis. In this microenvironment, the post-biotics and similar molecules such as p40 exert several beneficial effects through regulation of IECs. Importantly, post-biotics were discovered to be transactivators of the EGF receptor (EGFR) in IECs, inducing protective cellular responses and alleviating colitis. The transient exposure to post-biotics such as p40 during the neonatal period reprograms IECs by upregulation of a methyltransferase, Setd1ß, leading to a sustained increase in TGF- ß release for the expansion of regulatory T cells (Tregs) in the intestinal lamina propria and durable protection against colitis in adulthood. This crosstalk between the IECs and post-biotic secreted factors was not reviewed previously. Therefore, this review describes the role of probiotic-derived factors in the sustainability of intestinal health and improving gut homeostasis via certain signaling pathways. In the era of precision medicine and targeted therapies, more basic, preclinical, and clinical evidence is needed to clarify the efficacy of probiotics released as functional factors in maintaining intestinal health and preventing and treating disease.

Identification of a functional peptide of a probiotic bacterium-derived protein for the sustained effect on preventing colitis.

Several probiotic-derived factors have been identified as effectors of probiotics for exerting beneficial effects on the host. However, there is a paucity of studies to elucidate mechanisms of their functions. p40, a secretory protein, is originally isolated from a probiotic bacterium, Lactobacillus rhamnosus GG. Thus, this study aimed to apply structure-functional analysis to define the functional peptide of p40 that modulates the epigenetic program in intestinal epithelial cells for sustained prevention of colitis. In silico analysis revealed that p40 is composed of a signal peptide (1-28 residues) followed by a coiled-coil domain with uncharacterized function on the N-terminus, a linker region, and a ß-sheet domain with high homology to CHAP on the C-terminus. Based on the p40 three-dimensional structure model, two recombinant p40 peptides were generated, p40N120 (28-120 residues) and p40N180 (28-180 residues) that contain first two and first three coiled coils, respectively. Compared to full-length p40 (p40F) and p40N180, p40N120 showed similar or higher effects on up-regulating expression of Setd1b (encoding a methyltransferase), promoting mono- and trimethylation of histone 3 on lysine 4 (H3K4me1/3), and enhancing Tgfb gene expression and protein production that leads to SMAD2 phosphorylation in human colonoids and a mouse colonic epithelial cell line. Furthermore, supplementation with p40F and p40N120 in early life increased H3K4me1, Tgfb expression and differentiation of regulatory T cells (Tregs) in the colon, and mitigated disruption of epithelial barrier and inflammation induced by DSS in adult mice. This study reveals the structural feature of p40 and identifies a functional peptide of p40 that could maintain intestinal homeostasis.

Multi-Faceted Bioactivity Assessment of an Exopolysaccharide from Limosilactobacillus fermentum NCDC400: Antioxidant, Antibacterial, and Immunomodulatory Proficiencies.

Exopolysaccharides (EPS) are acknowledged for their diverse functional and technological properties. This study presents the characterization of EPS400, an acidic exopolysaccharide sourced from the native probiotic Limosilactobacillus fermentum NCDC400. Notably, this strain has demonstrated previous capabilities in enhancing dairy food texture and displaying in vivo hypocholesterolemic activity. Our investigation aimed to unveil EPS400's potential biological roles, encompassing antioxidant, antibacterial, and immunomodulatory activities. The results underscore EPS400's prowess in scavenging radicals, including the 2,2-diphenyl-1-picrylhydrazyl radical, 2,2'-azino-di-(3-ethylbenzthiazoline sulfonic acid) radical, superoxide radical, hydroxyl radical, and chelating activity targeting the ferrous ion. Furthermore, EPS400 displayed substantial antibacterial effectiveness against prevalent food spoilage bacteria such as Pseudomonas aeruginosa NCDC105 and Micrococcus luteus. Remarkably, EPS400 exhibited the ability to modulate cytokine production, downregulating pro-inflammatory cytokines TNF-α, IL-1ß, IL-6, and nitric oxide, while concurrently promoting the release of anti-inflammatory cytokine IL-10 within lipopolysaccharide-activated murine primary macrophages. Additionally, EPS400 significantly (p ≤ 0.05) enhanced the phagocytic potential of macrophages. Collectively, our findings spotlight EPS400 as a promising contender endowed with significant antioxidant, antibacterial, and immunomodulatory attributes. These characteristics propose EPS400 as a potential pharmaceutical or bioactive component, with potential applications in the realm of functional food development.

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.

Probiotic and prebiotic supplementation ameliorates chronic restraint stress-induced male reproductive dysfunction.

Restraint stress (RS) can induce male reproductive deficits by activating the hypothalamic-pituitary-adrenal (HPA) axis and causing oxidative stress. Previous studies have shown that probiotics can alleviate neurological and metabolic disorders induced by stress. However, the effects of probiotics on RS-induced reproductive deficits have not been fully elucidated. This study aimed to investigate whether Lactobacillus rhamnosus NCDC-610 (Probiotic-1) and Lactobacillus fermentum NCDC-400 (Probiotic-2) with prebiotic (fructooligosaccharides (FOS)) could prevent RS-induced reproductive deficits. C57BL6/J mice were subjected to RS for four hours daily before oral administration of probiotics (4 × 109 CFU per mice) either separately or concurrently with FOS. The results showed that oral administration of Probiotic-1 and Probiotic-2 protected against RS-induced sperm deficits, including sperm count, motility, morphology, and histopathology of testes, and improved intestinal health. Furthermore, Probiotic-1 and Probiotic-2 prevented RS-induced changes in testosterone levels by up-regulating the expressions of steroidogenic acute regulatory protein (StAR), cytochrome P450 side-chain cleavage enzyme (P450scc), and 17ß-hydroxysteroid dehydrogenase (17ßHSD) in the testes. Additionally, Probiotic-1 and Probiotic-2 increased the activities of catalase and superoxide dismutase and reduced the fold change of interleukin-6 (IL-6), interleukin-10 (IL-10), and tumor necrosis factor-alpha (TNF-α), indicating a protective effect against RS-induced oxidative stress. Oral administration of Probiotic-1 and Probiotic-2, either separately or concurrently with FOS (probiotic dose of 4 × 109 CFU per mice and prebiotic 5% w/v), prevented RS-induced activation of the HPA axis and improved male fertility. These findings suggest that L. rhamnosus NCDC-610 and L. fermentum NCDC-400 are safe and effective probiotics for mitigating stress-induced male reproductive deficits.

Mature white adipocyte plasticity during mammary gland remodelling and cancer.

Mammary gland growth and differentiation predominantly rely on stromal-epithelial cellular communication. Specifically, mammary adipocytes play a crucial role in ductal morphogenesis, as well as in the proliferation and differentiation of mammary epithelial cells. The process of lactation entails a reduction in the levels of white adipose tissue associated with the MG, allowing for the expansion of milk-producing epithelial cells. Subsequently, during involution and the regression of the milk-producing unit, adipocyte layers resurface, occupying the vacated space. This dynamic phenomenon underscores the remarkable plasticity and expansion of adipose tissue. Traditionally considered terminally differentiated, adipocytes have recently been found to exhibit plasticity in certain contexts. Unraveling the significance of this cell type within the MG could pave the way for novel approaches to reduce the risk of breast cancer and enhance lactation performance. Moreover, a comprehensive understanding of adipocyte trans- and de-differentiation processes holds promise for the development of innovative therapeutic interventions targeting cancer, fibrosis, obesity, type 2 diabetes, and other related diseases. Additionally, adipocytes may find utility in the realm of regenerative medicine. This review article provides a comprehensive examination of recent advancements in our understanding of MG remodelling, with a specific focus on the tissue-specific functions of adipocytes and their role in the development of cancer. By synthesizing current knowledge in this field, it aims to consolidate our understanding of adipocyte biology within the context of mammary gland biology, thereby fostering further research and discovery in this vital area.

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.

Physicochemical and rheological characterizations of a novel exopolysaccharide EPSKar1 and its iron complex EPSKar1-Fe: Towards potential iron-fortification applications.

Iron is a micronutrient essential for human health and physiology. Iron-deficiency anemia, the most common form of anemia, may occur from an iron homeostasis imbalance. Iron fortification is a promising and most sustainable and affordable solution to tackle the global prevalence of this anemia. Herein, we investigate physicochemical, rheological and stability characteristics of a novel exopolysaccharide 'EPSKar1' (derived from Lacticaseibacillus rhamnosus strain Kar1) and its iron complex 'EPSKar1-Fe (II)'. Our findings demonstrate that EPSKar1 is a high molecular-weight (7.8 × 105 Da) branched-chain heteropolysaccharide composed of galactose, N-acetylglucosamine, and mannose in a molar ratio of 8:4:1, respectively, and exhibits strong emulsifying and water-holding capacities. We find that EPSKar1 forms strong complexes with Fe, wherein the interactions between EPSKar1-Fe (II) complexes are mediated by sulfate, carboxyl, and hydroxyl groups. The rheological analyses reveal that the EPSKar1 and EPSKar1-Fe (II) complexes exhibited shear thickening and thinning properties in skim milk and water, respectively; however, the suspension of EPSKar1 in skim milk is viscoelastic with predominantly elastic response (G'>G" and tan Î´ < 1). In comparison, EPSKar1-Fe (II) complex exhibits remarkable stability under various processing conditions, highlighting its usefulness for the development of fortified dairy products. Together, these findings underpin considerable prospects of EPSKar1-Fe (II) complex as a novel iron-fortifier possessing multifarious rheological benefits for food applications.

Interactions between the gut microbiota-derived functional factors and intestinal epithelial cells - implication in the microbiota-host mutualism.

Mutual interactions between the gut microbiota and the host play essential roles in maintaining human health and providing a nutrient-rich environment for the gut microbial community. Intestinal epithelial cells (IECs) provide the frontline responses to the gut microbiota for maintaining intestinal homeostasis. Emerging evidence points to commensal bacterium-derived components as functional factors for the action of commensal bacteria, including protecting intestinal integrity and mitigating susceptibility of intestinal inflammation. Furthermore, IECs have been found to communicate with the gut commensal bacteria to shape the composition and function of the microbial community. This review will discuss the current understanding of the beneficial effects of functional factors secreted by commensal bacteria on IECs, with focus on soluble proteins, metabolites, and surface layer components, and highlight the impact of IECs on the commensal microbial profile. This knowledge provides a proof-of-concept model for understanding of mechanisms underlying the microbiota-host mutualism.

Multifaceted Pharmacological Potentials of Curcumin, Genistein, and Tanshinone IIA through Proteomic Approaches: An In-Depth Review.

Phytochemicals possess various intriguing pharmacological properties against diverse pathological conditions. Extensive studies are on-going to understand the structural/functional properties of phytochemicals as well as the molecular mechanisms of their therapeutic function against various disease conditions. Phytochemicals such as curcumin (Cur), genistein (Gen), and tanshinone-IIA (Tan IIA) have multifaceted therapeutic potentials and various efforts are in progress to understand the molecular dynamics of their function with different tools and technologies. Cur is an active lipophilic polyphenol with pleiotropic function, and it has been shown to possess various intriguing properties including antioxidant, anti-inflammatory, anti-microbial, anticancer, and anti-genotoxic properties besides others beneficial properties. Similarly, Gen (an isoflavone) exhibits a wide range of vital functions including antioxidant, anti-inflammatory, pro-apoptotic, anti-proliferative, anti-angiogenic activities etc. In addition, Tan IIA, a lipophilic compound, possesses antioxidant, anti-angiogenic, anti-inflammatory, anticancer activities, and so on. Over the last few decades, the field of proteomics has garnered great momentum mainly attributed to the recent advancement in mass spectrometry (MS) techniques. It is envisaged that the proteomics technology has considerably contributed to the biomedical research endeavors lately. Interestingly, they have also been explored as a reliable approach to understand the molecular intricacies related to phytochemical-based therapeutic interventions. The present review provides an overview of the proteomics studies performed to unravel the underlying molecular intricacies of various phytochemicals such as Cur, Gen, and Tan IIA. This in-depth study will help the researchers in better understanding of the pharmacological potential of the phytochemicals at the proteomics level. Certainly, this review will be highly instrumental in catalyzing the translational shift from phytochemical-based biomedical research to clinical practice in the near future.

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.

Promising role of defensins peptides as therapeutics to combat against viral infection.

Antimicrobial peptides (AMPs) are ubiquitously present small peptides, which play a critical function in the innate immune system. The defensin class of AMPs represented an evolutionarily ancient family containing cationic cysteine residue and frequently expressed in epithelial or neutrophils cells. It plays myriad functions in host innate immune responses against various infection. Defensin has a broad spectrum of antimicrobial activities, including anti-bacteria, anti-viruses (AVPs), anti-fungi, anti-cancers, and also overcoming bacterial drug resistance. In this review, we compiled the progress on defensin, particularly incorporating the mechanism of action, their application as an antiviral agent, prospects in different areas, and limitations to be solved as an antiviral peptide. Defensins were explored, in particular, their capacity to stimulate innate and adaptive immunity by trigging as anti-coronavirus (COVID-19) peptides. The present review summarised its immunomodulatory and immunoenhancing properties and predominantly focused on its promising therapeutic adjuvant choices for combat against viral infection.

Extending the Depth of Human Plasma Proteome Coverage Using Simple Fractionation Techniques.

Human plasma is one of the most widely used tissues in clinical analysis, and plasma-based biomarkers are used for monitoring patient health status and/or response to medical treatment to avoid unnecessary invasive biopsy. Data-driven plasma proteomics has suffered from a lack of throughput and detection sensitivity, largely due to the complexity of the plasma proteome and in particular the enormous quantitative dynamic range, estimated to be between 9 and 13 orders of magnitude between the lowest and the highest abundance protein. A major challenge is to identify workflows that can achieve depth of plasma proteome coverage while minimizing the complexity of the sample workup and maximizing the sample throughput. In this study, we have performed intensive depletion of high-abundant plasma proteins or enrichment of low-abundant proteins using the Agilent multiple affinity removal liquid chromatography (LC) column-Human 6 (Hu6), the Agilent multiple affinity removal LC column-Human 14 (Hu14), and ProteoMiner followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE) and C18 prefractionation techniques. We compared the performance of each of these fractionation approaches to identify the method that satisfies requirements for analysis of clinical samples and to include good plasma proteome coverage in combination with reasonable sample output. In this study, we report that one-dimensional (1D) gel-based prefractionation allows parallel sample processing and no loss of proteome coverage, compared with serial chromatographic separation, and significantly accelerates analysis time, particularly important for large clinical projects. Furthermore, we show that a variety of methodologies can achieve similarly high plasma proteome coverage, allowing flexibility in method selection based on project-specific needs. These considerations are important in the effort to accelerate plasma proteomics research so as to provide efficient, reliable, and accurate diagnoses, population-based health screening, clinical research studies, and other clinical work.

DeSUMOylase SENP7-Mediated Epithelial Signaling Triggers Intestinal Inflammation via Expansion of Gamma-Delta T Cells.

Inflammatory bowel disease (IBD) is a complex autoimmune disorder recently shown to be associated with SUMOylation, a post-translational modification mechanism. Here, we have identified a link between epithelial deSUMOylases and inflammation in IBD. DeSUMOylase SENP7 was seen to be upregulated specifically in intestinal epithelial cells in both human IBD and a mouse model. In steady state, but not IBD, SENP7 expression was negatively regulated by a direct interaction and ubiquitination by SIAH2. Upregulated SENP7 in inflamed tissue displayed a distinct interactome. These changes led to an expansion of localized proinflammatory γδ T cells. Furthermore, in vivo knockdown of SENP7 or depletion of γδ T cells abrogated dextran sulfate sodium (DSS)-induced gut inflammation. Strong statistical correlations between upregulated SENP7 and high clinical disease indices were observed in IBD patients. Overall, our data reveal that epithelial SENP7 is necessary and sufficient for controlling gut inflammation, thus highlighting its importance as a potential drug target.

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.

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.

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.

Characterization and use of HapT1-derived homologous tumors as a preclinical model to evaluate therapeutic efficacy of drugs against pancreatic tumor desmoplasia.

Desmoplasia in human pancreatic cancer (PC) promotes cancer progression and hinders effective drug delivery. The objectives of this study were to characterize a homologous orthotopic model of PC in Syrian golden hamster and investigate the effect of anti-fibrotic (pirfenidone), antioxidant (N-acetyl cysteine, NAC) and anti-addiction (disulfiram, DSF) drugs on desmoplasia and tumor growth in this model. The HapT1 PC cells when implanted orthotopically into hamsters formed tumors with morphological, cellular and molecular similarities to human PC. Protein profiling of activated hamster pancreatic stellate cells (ha-PSCs) revealed expression of proteins involved in fibrosis, cancer cells growth and metastasis. Pirfenidone, suppressed growth of HapT1 cells and the desmoplastic response in vivo; these effects were enhanced by co-administration of NAC. Disulfiram alone or in combination with copper (Cu) was toxic to HapT1 cells and PSCs in vitro; but co-administration of DSF and Cu accelerated growth of HapT1 cells in vivo. Moreover, DSF had no effect on tumor-associated desmoplasia. Overall, this study identifies HapT1-derived orthotopic tumors as a useful model to study desmoplasia and tumor-directed therapeutics in PC. Pirfenidone in combination with NAC could be a novel combination therapy for PC and warrants investigation in human subjects.