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Beta-casomorphins (BCMs) are the bio-active peptides having opioid properties which are formed by the proteolytic digestion of ß-caseins in milk. BCM-7 forms when A1 milk is digested in the small intestine due to a histidine at the 67th position in ß-casein, unlike A2 milk, which has proline at this position and produces BCM-9. BCM-7 has further degraded into BCM-5 by the dipeptidyl peptidase-IV (DPP-IV) enzyme in the intestine. The opioid-like activity of BCM-7 is responsible for eliciting signaling pathways which enable a wide range of physiological effects. The aim of our study was to find out the differential role of BCMs (BCM-7, BCM-9 and BCM-5) on pancreatic ß-cell proliferation, insulin secretion, and opioid peptide binding receptors from ß-cells (RIN-5F cell line) in normal (5.5 mM) and high glucose (27.5 mM) concentrations. Our results showed that BCM-7/9/5 did not affect ß-cell viability, proliferation, and insulin secretion at normal glucose level. However, at higher glucose concentration, BCMs significantly protected ß-cells from glucotoxicity but did not affect the insulin secretion. Interestingly, in the presence of Mu-opioid peptide receptor antagonist CTOP, BCMs did not protect ß-cells from glucotoxicity. The results suggest that BCMs protect ß-cells from glucotoxicity via non-opioid mediated pathways because BCMs did not modulate the gene expression of the mu, kappa and delta opioid peptide receptors.
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Acrylamide, a Maillard reaction product, formed in fried food poses a serious concern to food safety due to its neurotoxic and carcinogenic nature. A "Green Approach" using L-Asparaginase enzyme from GRAS-status bacteria synergized with hydrocolloid protective coating could be effective in inhibiting acrylamide formation. To fill this void, the present study reports a new variant of type-II L-asparaginase (AsnLb) from Levilactobacillus brevis NKN55, a food-grade bacterium isolated using a unique metabolite profiling approach. The recombinant AsnLb enzyme was characterized to study acrylamide inhibition ability and showed excellent specificity towards L-asparagine (157.2 U/mg) with Km, Vmax of 0.833 mM, 4.12 mM/min respectively. Pretreatment of potato slices with AsnLb (60 IU/mL) followed by zein-pectin nanocomplex led to >70% reduction of acrylamide formation suggesting synergistic effect of this dual component system. The developed strategy can be employed as a sustainable treatment method by food industries for alleviating acrylamide formation and associated health hazard in fried foods.
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Acrilamida , Asparaginase , Coloides , Pectinas , Zeína , Asparaginase/química , Asparaginase/metabolismo , Acrilamida/química , Pectinas/química , Zeína/química , Coloides/química , Solanum tuberosum/química , CulináriaRESUMO
Nucleic acid aptamers have been used in the past for the development of diagnostic methods against a number of targets such as bacteria, pesticides, cancer cells etc. In the present study, six rounds of Cell-SELEX were performed on a ssDNA aptamer library against X-enriched sperm cells from Sahiwal breed cattle. Sequencing was used to examine the aptamer sequences that shown affinity for sperm carrying the X chromosome in order to find any possible X-sperm-specific sequences. Out of 35 identified sequences, 14 were selected based on bioinformatics analysis like G-Score and Mfold structures. Further validation of their specificity was done via fluorescence microscopy. The interaction of biotinylated-aptamer with sperm was also determined by visualizing the binding of streptavidin coated magnetic beads on the head region of the sperm under bright field microscopy. Finally, a real-time experiment was designed for the validation of X-sperm enrichment by synthesized aptamer sequences. Among the studied sequences, aptamer 29a exhibited a higher affinity for X sperm compared to Y sperm in a mixed population of sperm cells. By using aptamer sequence 29a, we obtained an enrichment of 70% for X chromosome bearing sperm cells.
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Aptâmeros de Nucleotídeos , Técnica de Seleção de Aptâmeros , Espermatozoides , Cromossomo X , Masculino , Animais , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Espermatozoides/química , Bovinos , Cromossomo X/genética , Técnica de Seleção de Aptâmeros/métodosRESUMO
Urinary proteins have been studied quite exhaustively in the past, however, the small sized peptides have remained neglected for a long time in dairy cattle. These peptides are the products of systemic protein turnover, which are excreted out of the body and hence can serve as an important biomarker for various pathophysiologies. These peptides in other species of bovine have been reported to possess several bioactive properties. To investigate the urinary peptides in buffalo and simultaneously their bioactivities, we generated a peptidome profile from the urine of Murrah Buffaloes (n = 10). Urine samples were processed using <10 kDa MWCO filter and filtrate obtained was used for peptide extraction using Solid Phase Extraction (SPE). The nLC-MS/MS of the aqueous phase from ten animals resulted in the identification of 8165 peptides originating from 6041 parent proteins. We further analyzed these peptide sequences to identify bioactive peptides and classify them into anti-cancerous, anti-hypertensive, anti-microbial, and anti-inflammatory groups with a special emphasis on antimicrobial properties. With this in mind, we simultaneously conducted experiments to evaluate the antimicrobial properties of urinary aqueous extract on three pathogenic bacterial strains: S. aureus, E. coli, and S. agalactiae. The urinary peptides observed in the study are the result of the activity of possibly 76 proteases. The GO of these proteases showed the significant enrichment of the antibacterial peptide production. The total urinary peptide showed antimicrobial activity against the aforementioned pathogenic bacterial strains with no significant inhibitory effects against a buffalo mammary epithelial cell line. Just like our previous study in cows, the present study suggests the prime role of the antimicrobial peptides in the maintenance of the sterility of the urinary tract in buffalo by virtue of their amino acid composition.
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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.
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Búfalos , Queratina-18 , Gravidez , Feminino , Animais , Búfalos/fisiologia , Queratina-18/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Endométrio/metabolismo , Implantação do Embrião/fisiologia , Células Epiteliais/metabolismoRESUMO
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.
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Células Epiteliais , Leite , Animais , Humanos , Feminino , Gravidez , Diferenciação Celular , Apoptose , Proliferação de CélulasRESUMO
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.
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Calgranulina A , Carcinogênese , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Humanos , Calgranulina A/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Sistemas CRISPR-Cas , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol 3-Quinases/farmacologia , Proteômica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Técnicas de Inativação de GenesRESUMO
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.
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Lactobacillus helveticus , Probióticos , Animais , Humanos , Suínos , Proteínas de Membrana , Lactobacillus helveticus/genética , Escherichia coli , Células CACO-2 , Interações entre Hospedeiro e Microrganismos , Aderência Bacteriana , Probióticos/metabolismoRESUMO
In recent years, beta-casomorphin peptides (BCM7/BCM9) derived from the digestion of cow milk have drawn a lot of attention world over because of their proposed impact on human health. In order to evaluate the transcriptional modulation of target genes through RT-qPCR in response to these peptides, availability of appropriate reference or internal control genes (ICGs) will be the key. The present study was planned to identify a panel of stable ICGs in the liver tissue of C57BL/6 mice injected with BCM7/BCM9 cow milk peptides for 3 weeks. A total of ten candidate genes were evaluated as potential ICGs by assessing their expression stability using software suites; geNorm, NormFinder and BestKeeper. The suitability of the identified ICGs was validated by assessing the relative expression levels of target genes, HP and Cu/Zn SOD. Based on geNorm, PPIA and SDHA gene pair was identified to be most stably expressed in liver tissue during the animal trials. Similarly, NormFinder analysis also identified PPIA as the most stable gene. BestKeeper analysis showed crossing point SD value for all the genes in the acceptable range that is closer to 1. Overall, the study identified a panel of stable ICGs for reliable normalization of target genes expression data in mice liver tissues during BCM7/9 peptides trial.
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Perfilação da Expressão Gênica , Fígado , Animais , Feminino , Bovinos , Camundongos , Humanos , Camundongos Endogâmicos C57BL , Expressão Gênica , Reação em Cadeia da Polimerase em Tempo Real , Padrões de ReferênciaRESUMO
Lumpy skin disease (LSD) was reported for the first time in India in 2019 and since then, it has become endemic. Since a homologous (LSD-virus based) vaccine was not available in the country, goatpox virus (GPV)-based heterologous vaccine was authorized for mass immunization to induce protection against LSD in cattle. This study describes the evaluation of safety, immunogenicity and efficacy of a new live-attenuated LSD vaccine developed by using an Indian field strain, isolated in 2019 from cattle. The virus was attenuated by continuous passage (P = 50) in Vero cells. The vaccine (50th LSDV passage in Vero cells, named as Lumpi-ProVacInd) did not induce any local or systemic reaction upon its experimental inoculation in calves (n = 10). At day 30 post-vaccination (pv), the vaccinated animals were shown to develop antibody- and cell-mediated immune responses and exhibited complete protection upon virulent LSDV challenge. A minimum Neethling response (0.018% animals; 5 out of 26,940 animals) of the vaccine was observed in the field trials conducted in 26,940 animals. There was no significant reduction in the milk yield in lactating animals (n = 10108), besides there was no abortion or any other reproductive disorder in the pregnant animals (n = 2889). Sero-conversion was observed in 85.18% animals in the field by day 30 pv.
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Doença Nodular Cutânea , Vírus da Doença Nodular Cutânea , Vacinas Virais , Animais , Bovinos , Feminino , Chlorocebus aethiops , Doença Nodular Cutânea/prevenção & controle , Doença Nodular Cutânea/epidemiologia , Vírus da Doença Nodular Cutânea/genética , Vacinas Atenuadas/efeitos adversos , Células Vero , Vacinas Virais/administração & dosagemRESUMO
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.
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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.
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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.
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Búfalos , Glândulas Mamárias Humanas , Gravidez , Feminino , Bovinos , Animais , Camundongos , Humanos , Glândulas Mamárias Animais , Lactação , LeiteRESUMO
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.
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Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Animais , Aptâmeros de Nucleotídeos/genética , Técnicas Biossensoriais/métodos , Técnicas Biossensoriais/veterinária , Endorfinas , Humanos , Ligantes , Técnica de Seleção de Aptâmeros/métodos , Técnica de Seleção de Aptâmeros/veterináriaRESUMO
Pseudomonas aeruginosa (PA) is an opportunistic pathogen that causes high mortality in cystic fibrosis patients. Treatment failures often occur due to the emergence of antibiotic resistance. Inhibition of virulence factors production without suppressing the growth of the pathogens is a potential alternative strategy to control the antibiotic resistance. In order to accomplish, three different interaction studies were performed using Bacillus subtilis BR4, PA and their extracellular contents. Firstly, co-cultivation was performed with different cell density of BR4 or PA. In co-culture setup (F), high cell density of BR4 significantly inhibits the biofilm formation of PA in a growth-independent manner (p < 0.01). To substantiate the biofilm inhibition, LC-MS/MS was performed and metabolic profile of monocultures and cocultures were compared. Multivariate analysis corroborated that metabolic profile of coculture setup (F) is drastically different from other coculture and monoculture setups. To check the effect of extracellular content of PA on BR4, supernatant of PA was extracted with ethyl acetate and different concentration of that extract (PA-EXT) was supplemented with BR4 culture. Exogenous supplementation PA-EXT (40 µg/mL) led to increased biofilm inhibitory activity (p < 0.01) in BR4. Further, to check the effect of extracellular content of BR4, PA was grown in the supernatant of BR4. PA survives in the spent media of BR4 without biofilm formation. Though 50% spent media of BR4 was replaced with fresh media, PA could not produce biofilm. In support of this, LC-MS/MS analysis has revealed that abundance of quorum sensing (QS) signals was reduced in the spent media grown PA than control. Furthermore, BR4 protects zebrafish larvae (Danio rerio) against PA infection and increases their survival rate (p < 0.05). We found that PA-induced oxidative stress and apoptosis were also significantly reduced in the BR4-pretreated larval group than control group. These results clearly indicate that BR4 exerts growth-independent QS inhibition in PA, suggesting that it could be used as a probiotic for future therapeutic interventions.
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Probióticos , Pseudomonas aeruginosa , Animais , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Bacillus subtilis/metabolismo , Biofilmes , Cromatografia Líquida , Humanos , Metabolômica , Percepção de Quorum , Espectrometria de Massas em Tandem , Fatores de Virulência/metabolismo , Peixe-ZebraRESUMO
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.
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Cell-to-cell communication is essentially required in bacteria for the production of multiple virulence factors and successful colonization in the host. Targeting the virulence factors production without hampering the growth of the pathogens is a potential strategy to control pathogenesis. To accomplish this, a total of 43 mangrove isolates were screened for quorum quenching (QQ) activity against Pseudomonas aeruginosa (PA), in which eight bacteria have shown antibiofilm activity without hampering the growth of the PA. Prominent QQ activity was observed in Bacillus subtilis BR4. Previously, we found that BR4 produces stigmatellin Y, a structural analogue of PQS signal of PA, which could competitively bind with PqsR receptor and inhibits the quorum sensing (QS) system of PA. Further, stigmatellin Y containing ethyl acetate extract (S-EAE) (100 µg ml-1 ) of BR4 significantly inhibits (p < 0.001) the biofilm formation of PA. Confocal laser scanning microscope (CLSM) and scanning electron microscope (SEM) analysis also fortified the QQ activity of BR4. Furthermore, S-EAE of BR4 (500 µg ml-1 ) has significantly reduced the production of virulence factors, including protease, elastase, pyocyanin and extracellular polysaccharides substances. Furthermore, liquid chromatography-mass spectrometry (LC-MS)/MS analysis affirms that BR4 intercepts the PQS-mediated QS system by reducing the synthesis of as many PQS signals, including precursor molecule (243.162313 Da) of PQS signal. Thus, S-EAE of B. subtilis BR4 could be used as a promising therapeutic agent to combat QS system-mediated pathogenesis of PA. Further therapeutic potentials of stigmatellin Y to be evaluated in clinical studies for the treatment of multidrug resistant PA.
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Pseudomonas aeruginosa , Percepção de Quorum , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Biofilmes , Polienos , Pseudomonas aeruginosa/metabolismo , Fatores de VirulênciaRESUMO
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.
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Decídua/imunologia , Tolerância Imunológica , Células Matadoras Naturais/imunologia , Complicações na Gravidez/imunologia , Trofoblastos/imunologia , Feminino , Humanos , GravidezRESUMO
Several sperm lysozyme-like genes evolved from lysozyme by successive duplications and mutations; however their functional role in the reproduction of farm animals is not well understood. To understand the function and molecular properties of buffalo sperm lysozyme-like protein 1 (buSLLP1), it was expressed in E. coli; however, it partitioned to inclusion bodies. Lowering of temperature and inducer concentration did not help in the recovery of the expressed protein in the biologically active form. Therefore, buSLLP1 was cloned and expressed in Pichiapink system based on auxotrophic Pichia pastoris in a labscale fermenter. The expressed protein was obtained in flow-through by using a 30 kDa ultrafiltration membrane followed by MonoQ anion exchange chromatography, resulting in a homogenous preparation of 40 mg recombinant buSLLP1 per liter of initial spent culture-supernatant. Circular dichroism spectroscopy showed that recombinant buSLLP1 possessed a native-like secondary structure. The recombinant buSLLP1 also showed thermal denaturation profile typical of folded globular proteins; however, the thermal stability was lower than the hen egg white lysozyme. Binding of buSLLP1 to chitin and zona pellucida of buffalo oocytes showed that the recombinant buSLLP1 possessed a competent binding pocket, therefore, the produced protein could be used to study its functional role in the reproduction of farm animals.
Assuntos
Búfalos/genética , Expressão Gênica , Muramidase , Animais , Escherichia coli/genética , Escherichia coli/metabolismo , Muramidase/biossíntese , Muramidase/química , Muramidase/genética , Muramidase/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Saccharomycetales/genética , Saccharomycetales/metabolismoRESUMO
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.