Comparative evaluation of milk proteins and oil-seed-cake-derived proteins extracted by chemical and biological methods for obesity management.

BACKGROUND: In light of the exponential rise in global population, there is a critical requirement to reduce food waste on a global scale. According to studies, agricultural wastes such as oil-seed cakes offer great nutritional value. Acid precipitation (A) and alkaline extraction methods (traditional methods) were used to extract protein from oil-seed cakes; however, both procedures are linked to decreased protein quality and quantity, which prompted the development of a novel strategy known as the biological/microbial/probiotic (B) method. Therefore, the present study aimed to highlight the optimal way of protein extraction from oil-seed cakes and the effect of extraction methods on protein efficacy against obesity. The outcomes were also compared with milk proteins. RESULTS: In vitro study provided evidence that proteins from both sources (plant and milk) suppressed adipogenesis and stimulated adipolysis in 3T3L-1 cells. For the in vivo study, mice were fed with different protein extracts: soya protein preparation (SPP), ground protein preparation (GPP), whey protein (WP) and casein protein (CP) containing 40% of their calories as fat. Body weight decreased significantly in all the rats except CP-fed rats. Body mass index, atherogenic index, plasma triglyceride and very-low-density lipoprotein cholesterol level decreased significantly in all the groups in comparison to the model group (high-fat-diet group), but the decrease was more pronounced in plant proteins than milk proteins. In hepatocytes, the expression of fasting-induced adipose factor, carnitine palmitoyltransferase I and peroxisome proliferator-activated receptor α genes was increased significantly in SPP-fed groups. Adiponectin gene expression was upregulated significantly in visceral fat tissue in groups fed SPP-B, GPP-A and CP, whereas leptin gene was downregulated significantly in all groups except SPP-A. CONCLUSION: This study demonstrates that SPP-B showed the most effective anti-obesity property, followed by WP. Additionally, we found that the biological precipitation approach produced better outcomes for plant proteins isolated from oil-seed cakes than the acid precipitation method. © 2023 Society of Chemical Industry.

Indoleamine 2, 3-dioxygenase 1 mediated alterations in the functionality of immune cells, deciphers the pregnancy outcomes in crossbred dairy cows.

Pregnancy establishment in bovines requires maternal immune cell modulation. Present study investigated possible role of immunosuppressive indolamine-2, 3-dioxygenase 1 (IDO1) enzyme in the alteration of neutrophil (NEUT) and peripheral blood mononuclear cells (PBMCs) functionality of crossbred cows. Blood was collected from non-pregnant (NP) and pregnant (P) cows, followed by isolation of NEUT and PBMCs. Plasma pro-inflammatory (IFNγ and TNFα) and anti-inflammatory cytokines (IL-4 and IL-10) were estimated by ELISA and analysis of IDO1 gene in NEUT and PBMCs by RT-qPCR. Neutrophil functionality was assessed by chemotaxis, measuring activity of myeloperoxidase and ß-D glucuronidase enzyme and evaluating nitric oxide production. Changes in PBMCs functionality was determined by transcriptional expression of pro-inflammatory (IFNγ, TNFα) and anti-inflammatory cytokine (IL-4, IL-10, TGFß1) genes. Significantly elevated (P < 0.05) anti-inflammatory cytokines, increased IDO1 expression, reduced NEUT velocity, MPO activity and NO production observed only in P cows. Significantly higher (P < 0.05) expression of anti-inflammatory cytokines and TNFα genes were observed in PBMCs. Study highlights possible role of IDO1 in modulating the immune cell and cytokine activity during early pregnancy and may be targeted as early pregnancy biomarkers.

Probiotic lactobacilli mediate their immunoregulatory functions in intestinal cells via modulation of H3 histone acetylation.

AIMS: Probiotics are known to maintain intestinal homeostasis through the regulation of the immune response of the host. Hence, the role of histone modifications as epigenetic agents on immune modulations by potential probiotic bacteria has been investigated. METHODS AND RESULTS: Human colonic epithelial cells (Caco-2) pre-treated with class I histone deacetylase (HDAC) specific inhibitor, MS-275, were incubated either with potential probiotic bacteria (Limosilactobacillus fermentum MTCC 5898 and Lacticaseibacillus rhamnosus MTCC 5897) or Escherichia coli (ATCC 14948) as an inflammatory agent. Initially, transcriptional expression of potential immune-related genes (IL-6, IL-8, and hBD-2) was analyzed using RT-qPCR, and later H3 histone acetylation (H3Ac) at the promoter region of these genes was confirmed with a chromatin immunoprecipitation (ChIP) assay respectively. Potential probiotic L. fermentum (MTCC 5898) significantly suppressed (P < 0.05) the inhibitor-mediated elevated expression of immune-related genes while another strain L. rhamnosus (MTCC 5897), did not influence these gene expression results. In contrast, as an inflammatory agent, E. coli (ATCC 14948) synergistically augmented the expression of immune-related genes. Later, ChIP analysis confirmed the occurrence of H3 acetylation at these genes' promoter regions, which was directly related to the transcriptional activity of host epithelial cells stimulated by L. fermentum and E. coli, respectively. But in the case of L. rhamnosus, MTCC 5897, acetylation did not follow the transcription pattern and potentiated H3Ac on the promoter regions of these genes. CONCLUSIONS: Potential probiotics used in the study were found to regulate the immune response of host cells through histone acetylation in a strain-specific manner. SIGNIFICANCE AND IMPACT OF STUDY: Occurrence of probiotic-mediated regulation of immune genes by H3 acetylation in a strain-specific manner.

Lactobacillus fermentum (MTCC-5898) based fermented whey renders prophylactic action against colitis by strengthening the gut barrier function and maintaining immune homeostasis.

Nutritional intervention using probiotic fermented dairy product has emerged as a promising prophylactic strategy to curb inflammatory bowel diseases. Under present investigation, the potential of fermented whey prepared with probiotic Lactobacillus fermentum (LF:MTCC-5898) was investigated on dextran sodium sulfate (DSS) induced impaired intestinal barrier function in mice. Probiotic fermented whey (PFW) consumption improved the symptoms of colitis-associated with intestinal inflammation by significantly (p < 0.01) diminishing the percent loss in body weight, disease activity index and spleen index with improved colon length besides hematological and histopathological score. Likewise, pre-treatment with PFW improved the barrier integrity (p < 0.01) in contrast to leaky condition induced by DSS administration which increased the FITC-dextran permeability across gut epithelium. PFW consumption also provided the gut immune protection through significantly increased (p < 0.05) TLR-2 expression and stimulated T-regulatory response by producing TGF-ß (p < 0.01) and, potently suppressed (p < 0.01) inflammatory response (TNF-α, IL-4 and C-reactive protein). Further, PFW intake significantly enhanced (p < 0.05) immunoglobulin (sIgA) secretion and concomitantly restored the Occludin, ZO-1 (p < 0.01) and Claudin-1(p < 0.05) transcriptional expression as compared to colitis mice. Additionally, immune-fluorescence further established the presence of intact actin cytoskeleton and tight junction proteins (claudin-1, occludin and ZO-1) after PFW consumption. Thus, PFW rectified the impaired and leaky barrier junctions not only through modulation of transcriptional expression of tight junction genes but also with reduced secretion of inflammatory mediators and helped in ameliorating the colitis. Hence, probiotic fermented whey prepared with L.fermentum (MTCC-5898) could be used as potential prophylactic functional food in the prevention of gut ailments.

A novel gut inflammatory rat model by laparotomic injection of peptidoglycan from Staphylococcus aureus.

Inflammatory bowel disease includes ulcerative colitis and Crohn's disease, and is globally increasing. An appropriate model system is required to dissect the disease pathogenesis and drugs screening for adequate treatment. In the present study, we established a novel model of gut inflammation by injecting peptidoglycan from Staphylococcus aureus using laparotomic procedure. For this, three different doses of peptidoglycan, i.e., 2.5, 5 and 10 mg/kg body weight were used. The treatment effect was evaluated by studying the macrophage phagocytic function, spleen lymphocytes' proliferation and qRT-PCR for the assessment of peritoneal cells' gene expression. In addition, histological analysis of gut sections, gastric acidity, immunoglobulins and cytokines were assessed. There was significant increase in phagocytic activity in 10 mg/kg body weight PGN group. A dose dependent increase in spleen lymphocytes' proliferation and a significant increase in total acid secretion in 5 and 10 mg/kg body weight PGN treated rats were observed. In addition, a significant increment in TLR-2 and CD-14 mRNA expression in peritoneal cells, TNF-α, IL-6 and IFN-γ level and maximum distortion of gut architecture was observed in 10 mg/kg body weight PGN group. Hence, peptidoglycan from S. aureus can be used for establishing the screening model to study the action and mechanism of anti-inflammatory food products and drugs.

Macrophage-activating factor of bovine colostrum promotes phagocytic activity of murine macrophages and bovine phagocytes.

Periparturient dairy cows and their newborn calves are highly prone to health complications. Enhancing the innate immune system of these animals is essential to mitigate the transition period stress and promote their health. Macrophage activating factor (MAF) possess immunomodulatory properties and is believed to enhance immune response. In the present study, the impact of different concentrations (10, 50, 100 ng) of MAF on the phagocytic activity (PA) of murine and bovine phagocytoses was explored. MAF synthesized from IgA of cow colostrum was studied for its effect on the phagocytic index (PI) of cow colostrum macrophages (Mφ) and blood neutrophils (sick and healthy calves) under in vitro conditions. Besides, the impact of MAF on the PI of peritoneal Mφ of healthy and immunocompromised mice was studied. PI of healthy Mφ (mice peritoneal and cow colostrum) and healthy neutrophils (blood calf) increased significantly (P < 0.05) after MAF supplementation. MAF also significantly (P < 0.05) increased the PI of neutrophils and Mφ obtained from sick calf and immunocompromised mice, respectively. Results indicate that colostrum MAF can be used as a potential immune modulator to promote immunity and fight infections in dairy animals.

Strain-specific effects of probiotic Lactobacilli on mRNA expression of epigenetic modifiers in intestinal epithelial cells.

The epigenome of an organism is as important as the genome for the normal development and functioning of an individual. The human epigenome can be affected by various environmental factors including nutrients, microbiota and probiotics through epigenetic modifiers and mediates various health-promoting effects. The present study was aimed to explore the temporal changes in DNA and histone modifiers (DNMT1, TET2, p300, HDAC1, KMT2A, KDM5B, EzH2 and JMJD3) in intestinal epithelial cells (Caco-2) by probiotic lactobacilli (Limosilactobacillus fermentum MTCC 5898 and Lacticaseibacillus rhamnosus MTCC 5897) in comparison to opportunistic commensal pathogen Escherichia coli (ATCC 14849). Cells were treated separately with probiotic strains and E. coli for different durations and temporal changes in gene expression among DNA and histone modifiers were measured. Time-dependent studies showed that L. fermentum enhanced the transcription of epigenetic modifiers at 12 h of treatment (P < 0.05) contrary to E. coli which reduced the expression of these genes during the same duration of treatment. On the other hand, probiotic L. rhamnosus was not able to induce any significant changes in gene expression of these modifiers. Furthermore, during the exclusion of E. coli by L. fermentum, the probiotic was found to resist the changes made by E. coli in the transcription of some of the epigenetic modifiers. Thus, it is concluded that the probiotics modulated the mRNA expression of DNA and histone modifiers contrarily to E. coli in a strain-specific manner.

Biofunctional Attributes of Surface Layer Protein and Cell-Bound Exopolysaccharide from Probiotic Limosilactobacillus fermentum (MTCC 5898).

The probiotic extracellular matrix components (ECM) have been considered as an important factor in eliciting the beneficial roles of the bacteria. The study involved the growth phase-dependent extraction of the surface layer protein (SLP) and cell-bound exopolysaccharide (EPS-b) from novel Limosilactobacillus fermentum (MTCC 5898). Both SLP and EPS-b were optimally extracted at the late logarithmic phase of the bacteria upon 8 h of incubation. Furthermore, the adhesive, immunomodulatory, and anti-oxidative potential of the extracted components were evaluated using in vitro models. The major role of SLP was evidenced on bacterial adhesion to mucin and was related to its hydrophobic character. Under in vitro conditions, no effect of SLP and EPS-b was observed on the proliferation of murine splenocytes; however, both the components stimulated the phagocytosis of murine peritoneal macrophages at varying concentrations. Furthermore, all the components exhibited strong radical scavenging, chelating, and reducing potential with increasing concentration. Therefore, the ECM components of L. fermentum exhibited a variable biofunctional effect, providing crucial information to enable its further use as functional foods and overcome the challenges posed by probiotics.

Health-promoting role of dietary bioactive compounds through epigenetic modulations: a novel prophylactic and therapeutic approach.

The epigenome is an overall epigenetic state of an organism, which is as important as that of the genome for normal development and functioning of an individual. Epigenetics involves heritable but reversible changes in gene expression through alterations in DNA methylation, histone modifications and regulation of non-coding RNAs in cells, without any change in the DNA sequence. Epigenetic changes are owned by various environmental factors including pollution, microbiota and diet, which have profound effects on epigenetic modifiers. The bioactive compounds present in the diet mainly include curcumin, resveratrol, catechins, quercetin, genistein, sulforaphane, epigallocatechin-3-gallate, alkaloids, vitamins, and peptides. Bioactive compounds released during fermentation by the action of microbes also have a significant effect on the host epigenome. Besides, recent studies have explored the new insights in vitamin's functions through epigenetic regulation. These bioactive compounds exert synergistic, preventive and therapeutic effects when combined as well as when used with chemotherapeutic agents. Therefore, these compounds have potential of therapeutic agents that could be used as "Epidrug" to treat many inflammatory diseases and various cancers where chemotherapy results have many side effects. In this review, the effect of diet derived bioactive compounds through epigenetic modulations on in vitro and in vivo models is discussed.

Potential probiotic Lacticaseibacillus rhamnosus MTCC-5897 attenuates Escherichia coli induced inflammatory response in intestinal cells.

Probiotics are microbes having tremendous potential to prevent gastrointestinal disorders. In current investigation, immunomodulatory action of probiotic Lacticaseibacillus rhamnosus MTCC-5897 was studied during exclusion, competition and displacement of Escherichia coli on intestinal epithelial (Caco-2) cells. The incubation of intestinal cells with Escherichia coli, enhanced downstream signalling and activated nuclear factor kappa B (NF-κB). This significantly increased (p < 0.01) the pro-inflammatory cytokines (IL-8, TNF-α, IFN-ϒ) expression. While, incubation of epithelial cells with Lacticaseibacillus rhamnosus during exclusion and competition with Escherichia coli, counteracted these enhanced expressions. The immunomodulatory feature of Lacticaseibacillus rhamnosus was also highlighted with increased (p < 0.05) transcription of toll-like receptor-2 (TLR-2) and single Ig IL-1-related receptor (SIGIRR) along with diminished expression of TLR-4. Likewise, attenuation (p < 0.05) of E. coli-mediated enhanced nuclear translocation of NF-κB p-65 subunit by Lacticaseibacillus rhamnosus during exclusion was confirmed with western blotting. Thus, present finding establishes the prophylactic potential of Lacticaseibacillus rhamnosus against exclusion of Escherichia coli in intestinal cells.

Physicochemical Characteristics of Novel Cell-Bound Exopolysaccharide from Probiotic Limosilactobacillus fermentum (MTCC 5898) and Its Relation to Antioxidative Activity.

This study investigated the physicochemical characteristics and antioxidative role of novel acidic cell-bound exopolysaccharide (EPS-b) from probiotic Limosilactobacillus fermentum (MTCC 5898) and gained an insight into the structure-function relationship. The physicochemical analysis of EPS-b isolated by ultrasonication method revealed a heteropolysaccharide molecule with an average MW of 96.97 kDa composed of glucose and galactose subunits present in random-coiled conformation. Fourier transform infrared (FTIR) and nuclear magnetic resonance (NMR) analyses further supported the observation and indicated the presence of α-(1 → 6) linkages. The analyses implicated the significant influence of structural features on the antioxidative activity of EPS-b by showing remarkable ABTS scavenging, reducing, and metal chelating potential with increasing concentration. Besides, the EPS-b by its scavenging potential also maintained the oxidative balance in the Caco-2 cells under oxidative stress and preserved the cellular antioxidative defense system (CAT, GPx, SOD, HO1, and GCLC) at the basal level.

Protective effects of potential probiotic Lactobacillus rhamnosus (MTCC-5897) fermented whey on reinforcement of intestinal epithelial barrier function in a colitis-induced murine model.

Fermented foods provide essential nutritional components and bioactive molecules that have beneficial effects on several gastrointestinal disorders. In the present investigation, the potential protective effects of whey fermented with probiotic Lactobacillus rhamnosus MTCC-5897 on gastrointestinal health in a murine ulcerative colitis model induced by dextran sulfate sodium (DSS) were evaluated. Pre-consumption of whey fermented with probiotic L. rhamnosus (PFW) before colitis induction significantly reduced (p < 0.01) the disease activity index and improved (p < 0.05) the hematological parameters and histological scores. The considerably diminished levels (p < 0.01) of pro-inflammatory markers (IL-4, TNF-α, CRP and MPO activity) and the enhanced (p < 0.05) levels of the anti-inflammatory cytokine TGF-ß with IgA in the intestine upon feeding PFW appeared to prevent inflammation on colitis induction. Transcriptional modulations in pathogen recognition receptors (TLR-2/4) and tight junctional genes (ZO-1, occludin, claudin-1) along with localized distribution of junctional (claudin-1, occludin and ZO-1) and cytoskeleton (actin) proteins improved immune homeostasis and intestinal barrier integrity. Besides, significantly reduced (p < 0.05) levels of the FITC-dextran marker in serum upon consumption of PFW directly confirmed the healthy status of the host gut.

miR300 intervenes Smad3/ß-catenin/RunX2 crosstalk for therapy with an alternate function as indicative biomarker in osteoporosis.

The study reports a theranostic nature of rno-miR-300 (miR300) in the osteoblast functioning, by influencing the signaling pathway(s), associated with osteoblast differentiation. Excessive expression of miR300 suppresses osteoblast functions. Smad3 served as a validated target for miR300, on homology-based computational analysis and experimental testimony, which activates ß-catenin, and subsequently potentiates Runx2. The impact of miR300 on the Smad3/ß-catenin/Runx2 signaling interactions in the induction of osteoblast differentiation was scrutinized by immunoblotting and in vivo miRNA antagonism. Overexpression of miR300 in the rat calvarial osteoblasts decreases the protein levels of Smad3, ß-catenin and Runx2. Besides, in vivo silencing of miR300 in the neonatal pups and adult rats by AntimiR300 abolishes the suppressing action of miR300 on the osteoblast differentiation and expressions of Smad3/ß-catenin/Runx2 axis. MicroCT studies showed improved trabecular microarchitecture in the AntimiR300 transfected ovariectomised rat model compared to sham and negative control. Furthermore, expression levels of miR300 were evaluated in serum samples from an independent set of 30 osteoporotic patients followed by a Receiver Operating Characteristic Curve (ROC) based analysis for the diagnostic efficiency of miR300. Interestingly, the results exhibited high levels of miR300 (p < 0.0001) in the serum samples from osteoporotic patients relative to non-osteoporotic subjects (AUC = 0.9689). Thus, miR300 negatively regulates the differentiation of osteoblasts by targeting crosstalk among Smad3, ß-catenin and Runx2, unveiling an enormous ability to serve as a therapeutic target for bone-related disorder management strategies. Besides, miR300 may potentially function for the diagnosis of osteoporosis as a non-invasive biomarker.

Tmprss2 specific miRNAs as promising regulators for SARS-CoV-2 entry checkpoint.

Tmprss2 is an emerging molecular target which guides cellular infections of SARS-CoV-2, has been earmarked for interventions against the viral pathologies. The study aims to computationally screen and identifies potential miRNAs, following in vitro experimental validation of miRNA-mediated suppression of Tmprss2 for early prevention of COVID-19. Pool of 163 miRNAs, scrutinized for Tmprss2 binding with three miRNA prediction algorithms, ensued 11 common miRNAs. Further, computational negative energies for association, corroborated miRNA-Tmprss2 interactions, whereas three miRNAs (hsa-miR-214, hsa-miR-98 and hsa-miR-32) based on probability scores ≥0.8 and accessibility to Tmprss2 target have been selected in the Sfold tool. Transfection of miRNA(s) in the Caco-2 cells, quantitatively estimated differential expression, confirming silencing of Tmprss2 with maximum gene suppression by hsa-miR-32 employing novel promising role in CoV-2 pathogenesis. The exalted binding of miRNAs to Tmprss2 and suppression of later advocates their utility as molecular tools for prevention of SARS-CoV-2 viral transmission and replication in humans.

Implantation associated changes in expression profile of indoleamine-2, 3-dioxygenase 1, Th1-Th2 cytokines and interferon-stimulated genes on neutrophils and peripheral blood mononuclear cells of crossbred cows.

Effective bidirectional communication between the embryo and dam improves the reproductive efficiency of dairy cows. Possible role of immunosuppressive indolamine-2, 3-dioxygenase 1 (IDO1) enzyme in the regulation of maternal systemic cytokine balance/shift during early pregnancy establishment along with various interferon-stimulated genes (ISGs) expression in neutrophils and peripheral blood mononuclear cell (PBMCs) were investigated in crossbred cows. Blood was collected on days 0 i.e. day of Artificial Insemination (AI), 10, 18 and 36 post-AI followed by isolation of neutrophils and PBMCs for gene expression study of IDO1, anti-inflammatory cytokines (IL-4, IL-10 and TGFß1), pro-inflammatory cytokines (IFNγ and TNFα) and ISGs (ISG15, MX1, MX2, OAS1) in pregnant and non-pregnant cows. Cows were grouped as pregnant and non-pregnant after pregnancy confirmation by non-return to heat, ultrasonography, per rectal examination along with progesterone and IFNτ assay. Significantly (P < 0.05) higher relative mRNA expression of IDO1 and anti-inflammatory cytokines on days 10 and 18 post-AI were observed in both neutrophils and PBMCs of pregnant cows. Pregnant cows showed significantly (P < 0.05) higher mRNA transcripts of IFNγ and TNFα genes on days 18 post-AI in both neutrophils and PBMCs. Expression of ISGs was higher (P < 0.05) on day 10th and 18th post AI in both the neutrophils and PBMCs of pregnant cows. The study indicates that systemic immune regulation by IDO1 (through cytokine shift) and ISGs in peripheral immune cells are essential for the establishment of pregnancy and may be targeted in future as biomarkers for pregnancy diagnosis.

Modified Veria Technique for Cochlear Implantation by Postaural Approach.

There are various surgical techniques for cochlear implantation, classical one being mastoidectomy and posterior tympanotomy which has some disadvantages and complications like extensive bone work, violation of mastoid air cell system and fear of injury to vital structures like facial nerve. To minimize these problems, various modifications in cochlear implantation surgery has been done which includes introduction of Veria technique which also has certain disadvantages like extensive dissection and prolonged surgical time. In this article we are introducing an innovative technique of cochlear implantation where we have modified the pre-existing Veria technique that has been described in detail in the coming sections. Total 9 cases have been done so far with this modified Veria technique. This technique includes postaural approach with minimal soft tissue and bone work, making the cochlear implantation simple, easily doable, with less operating time, with minimal morbidity, faster healing due to smaller incision and avoiding facial nerve injury.

Repertoire of Structure-Activity-Based Novel Modified Peptides Elicits Enhanced Osteogenic Potential.

Biologically active peptides in milk proteins can be used as effective dietary supplements for management of bone-associated issues including osteoporosis. A bioactive peptide derived from milk, viz. VLPVPQK/PepC, has been validated previously from our lab for its osteoanabolic action. In this study, we report 14 novel variants of PepC, designed in silico, based on the structure-activity relationship, aiming to enhance its osteogenic effect that holds tremendous therapeutic utility for bone-related injuries. PepC was computationally modified at seven positions of its original sequence, resulting in 14 modified synthetic peptides for functional predictions and in vitro assessment by comparative analysis of modified peptides by PepC for improved ability in osteogenic functional assays (proliferation potential, antioxidant ability, gene and protein expression, cytotoxic effect, bone mineralization) using calvarial osteoblasts. For most peptides with the highest Peptide7 response relative to PepC (p < 0.05), enhanced osteoanabolic response was observed. Further observations on Peptide7 have therefore been investigated in depth (qPCR, immunoblotting, LCMS/MS, and PCA analysis). Peptide7 displayed a rise in the expression of osteogenes (Osterix, Opg, Bmp2, and Runx2, p < 0.05) and protein (Runx2 and Bmp2, p < 0.05). Besides, LCMS/MS findings suggest Peptide7 escapes intestinal peptidases degradation. Experimental evidence supports an improved osteological reaction to newly modified peptides and hence exploitation in the preparation of functional foods or supplements.

Proteomics and transcriptomics study reveals the utility of ISGs as novel molecules for early pregnancy diagnosis in dairy cows.

An early and precise diagnosis of pregnancy in cows is critical to short the calving interval and to improve their reproductive efficiency. Neutrophils are the first blood cells to sensitize the embryo in the uterus and participate in maternal recognition of pregnancy after getting induced by interferon tau (IFNτ). To study the protein abundance ratio, blood samples were collected on 0th, 10th, 18th and 36th day post-artificial insemination (AI) from crossbred Karan Fries cows. Neutrophils were isolated through density gradient centrifugation and studied for protein abundance by high-performance liquid chromatography coupled with mass spectrometry (LC-MS). Protein abundance ratios for Myxovirus resistance (MX1 and MX2) were found to be higher (P < 0.05) on day 10 and day 18 post-AI, whereas Oligoadenylate synthetase-1 (OAS1) and Interferon stimulated gene-15 ubiquitin-like modifier (ISG15) proteins were more abundant on day 18 post-AI. The relative mRNA expressions of these molecules were also studied by qPCR. The gene expression of ISG15, MX1, MX2 and OAS1 was found to be higher (P < 0.05) on day 10th, 18th and 36th post-AI compared to day 0. The study indicates that ISGs on blood neutrophils are essential for the establishment of pregnancy and may be targeted as potential biomarkers for pregnancy diagnosis in cows.

pH-dependent inhibition of AHL-mediated quorum sensing by cell-free supernatant of lactic acid bacteria in Pseudomonas aeruginosa PAO1.

Antibiotic mediated therapies target the growth-related processes of the pathogen hence imparting a strong selection pressure on the pathogen to develop antibiotic resistance. Recently anti-virulence strategies have gained lots of attention amongst the scientific community, wherein instead of inhibiting the normal growth of pathogens, it interferes with the regulation of virulence factors of the pathogens and impede their pathogenesis. In Pseudomonas aeruginosa, the virulence mechanism accountable for various types of infections in humans depends on N-acyl homoserine lactone (AHL) mediated quorum sensing. So quenching of these molecules, pose as a promising tool against P. aeruginosa pathogenesis. Lactic acid bacteria cell-free supernatant (acidic and neutralized) were evaluated in quorum quenching of P. aeruginosa PAO1 (MTCC 3541) after their initial screening for anti-biofilm potential against this pathogen.Though the reduction in biofilm formation with acidic and neutralized supernatants of lactic acid bacteria revealed strain specific response but acidic fractions showed much stronger (P ≤ 0.05) inhibition of biofilm irrespective of the type of challenge given to P. aeruginosa with lactic acid bacteria. The acidic fraction of supernatants (L. lactis, L. rhamnosus and L. fermentum) not only showed a significant reduction (P ≤ 0.05) in auto-inducer AHL levels but also diminished elastase activity which was among important virulence characters directly controlled by the quorum sensing signaling. Moreover, significant decrease (P ≤ 0.05) in mRNA expression of lasI and rhlI in presence of acidic fractions of lactic acid bacterial supernatants further confirmed the quorum quenching process in P. aeruginosa.

Dietary intake of pearl millet based weaning food supplemented with iron and vitamin A enhances bioavailability of vitamin A in anemic rats.

The study was aimed to assess vitamin A bioavailability and allergenicity of pearl millet (Pennisetum glaucum) based weaning food (PMWF) fortified with iron and retinyl acetate in male Wistar albino rats. Animals (n = 64) were divided into Normal (NG) and Anemic (AG) groups; further sub-divided into 4 sub-groups having 8 animals each receiving synthetic diet, commercial diet, iron fortified PMWF diet and iron (150.00 ± 0.73 ppm) plus retinyl acetate (393.00 ± 3.07 µg/100 g) fortified PMWF diet (Final diet). Results revealed that anemic sub-groups showed apparent digestibility coefficient (ADC) in the range of 69.5 ± 0.40-93.2 ± 0.79%, which was significantly (P < 0.01) higher than normal sub-groups (65.5 ± 0.62-84.6 ± 0.33%). In both groups, rats fed final diet presented significantly (P < 0.01) higher ADC (84.6 ± 0.33-93.2 ± 0.79%) than that of animals received iron fortified diet (69.0 ± 0.59-76.1 ± 1.02%), indicating higher bioavailability of vitamin A in final diet. Moreover, hepatic vitamin A replenished rapidly in anemic groups (1.79-27.8) when compared to normal rats (1.11-19.4 µg/g liver). Immunoglobulins IgG, IgE in blood serum and IgA in intestinal fluid ranged from 574 ± 6.48 to 603 ± 9.76 µg/ml, 287 ± 4.46 to 309 ± 5.70 ng/ml and 204 ± 10.33 to 255 ± 13.22 µg/ml, respectively. However, no significant (P > 0.05) difference was observed between the groups and/or subgroups, suggesting no allergic response of final diet. Stimulation index triggered by lipopolysaccharide (LPS) ranged from 1.22 ± 0.06 to 1.45 ± 0.09 µg ml-1 in normal sub-groups and 1.16 ± 0.02 to 1.33 ± 0.03 µg ml-1 in anemic sub-groups with no significant (P > 0.05) difference among them. Overall, it can be concluded that retinyl acetate could be an effective fortificant to improve the status of vitamin A in anemic models.