Inhibition of SRC-mediated integrin signaling in bone marrow niche enhances hematopoietic stem cell function.

Interaction with microenvironmental factors is crucial for the regulation of hematopoietic stem cell (HSC) function. Stroma derived factor (SDF)-1α supports HSCs in the quiescent state and is central to the homing of transplanted HSCs. Here, we show that integrin signaling regulates Sdf-1α expression transcriptionally. Systemic deletion of Periostin, an Integrin-αv ligand, showed increased expression of Sdf-1α in bone marrow (BM) niche. Pharmacological inhibition or CRISPR-Cas9-mediated deletion of SRC, resulted in a similar increase in the chemokine expression in vitro. Importantly, systemic SRC-inhibition led to increase in SDF-1α levels in BM plasma. This resulted in a robust increase (14.05 ± 1.22% to 29.11 ± 0.69%) in the homing efficiency of transplanted HSCs. In addition, we observed enhancement in the recovery of blood cell counts following radiation injury, indicating an enhanced hematopoietic function. These results establish a role of SRC-mediated integrin signaling in the transcriptional regulation of Sdf-1α. This mechanism could be harnessed further to improve the hematopoietic function.

Nrf2 dependent antiaging effect of milk-derived bioactive peptide in old fibroblasts.

Prolonged passaging of primary fibroblast cells totally shapes the natural biological phenomena and leads to the appearance of features related to senescence. As a result, it is a good natural tool to delineate the molecular mechanism of cellular aging. The present investigation revealed the antiaging effect of milk-derived novel bioactive peptide (VLPVPQK). The peptide played an important role in downregulating apoptosis-related markers in late passages of cultured fibroblast cells. The peptide treatment to aged fibroblasts caused enhancement in cell migration, DNA integrity, and decrease in the lipid peroxidation, reactive oxygen species, nitric oxide production as well as pro-inflammatory cytokines, TNF-α and IL-6. Moreover, the peptide decreased the expression of apoptotic caspases, Bax, and senescence-associated ß-galactosidase (SA-ß-gal) proteins. The peptide pretreatment also enhanced the extracellular collagen protein and antiapoptotic, Bcl-xL. In addition, the peptide treatment reversed the senescence-related activity in fibroblasts by stimulating Nrf2 mediated antioxidative defense system and inhibiting the action of NFkB/p38MAPK signaling, similar to the commercially available inhibitor (SB203580) of p38MAPK. Thus, the peptide exhibits the antiaging effect in dermal fibroblast cells.

Interferon-tau stimulated gene expression: A proxy to predict embryonic mortality in dairy cows.

The embryonic mortality in cows is a growing concern for an ever-expanding dairy industry. The current study was an attempt to shorten the open period of dairy cows having suffered embryonic loss by diagnosing them at an earlier stage. The blood samples were collected from the Karan Fries (KF) cows on days 0 (day of AI/estrus), 4, 8, 12, 14, 16, 18, 21, 24, 28, 35 and 42 post insemination. The experimental animals were then categorized into pregnant (P), conception failure/early embryonic mortality (EEM) and late embryonic mortality cows (LEM), based on progesterone assay, ultrasonography and per-rectal palpation. There were 6 animals in each group. The plasma progesterone was higher in pregnant than EEM and LEM cows. Plasma Interferon-tau concentration was significantly (p < 0.05) lower in LEM than pregnant cows where it could be detected from day 14-21 but was non-detectable in EEM cows. The mRNA expression of ISG15, OAS1, MX1 and MX2 in blood neutrophils was significantly (p < 0.05) higher from day 8-42 as against day 0 in pregnant cows. The highest expression was observed around day 18-21 in pregnant cows. The ISG15, OAS1, MX1 and MX2 mRNA expression was significantly (p < 0.05) higher from day 4-42 as compared to day 0 in LEM cows, whereas in EEM cows the expression stayed close to that of day 0 (1.00 ±â€¯0.00). The mRNA expression of ISG15, OAS1, MX1 and MX2 started to decline from day 24 onwards. The degree of expression of Interferon-tau stimulated genes was higher in pregnant and LEM cows than EEM cows. The study reveals that the Interferon tau stimulated gene expression in neutrophils can act as peripheral biomarkers for detecting the embryonic mortality in dairy cows.

Effect of buffalo casein-derived novel bioactive peptides on osteoblast differentiation.

PURPOSE: Epidemiological and intervention studies show that milk consumption in childhood and during adolescence is related to higher bone mineral density. Milk and milk products prevent the bone loss in pre- and postmenopausal women. Apart from calcium, there are other biologically active compounds in milk such as bioactive peptides which may play a role in promoting bone health. Casein is the major protein in milk which has also been reported to have numerous biological active peptides within it. The hypothesis of the present study was to identify the key peptides behind osteoanabolic nature of the milk protein, which further can be used to prepare functional foods to alleviate bone diseases like osteoporosis. Hence, this study was carried out to investigate osteogenic nature of four novel bioactive peptides [PEP1 (EDVPSER), PEP2 (NAVPITPTL), PEP3 (VLPVPQK) and PEP4 (HPHPHLSF)] derived from buffalo casein by in vitro osteoblast differentiation model. METHODS: Calvaria cells were isolated from 3-day-old rat pups, cultured under in vitro conditions till confluence and further used for experiments. Calvarial osteoblast cells were cultured in the presence or absence of peptides including positive controls up to 21 days. Effect of peptides was checked at regular intervals by quantifying osteoblast differentiation marker genes (ALP, OCN and COL-1) expression, alkaline phosphatase activity, osteocalcin level in culture supernatants, mineral deposition by alizarin red staining and caspase-3 and 9 assays. RESULTS: The osteoblast differentiation marker genes (ALP, OCN and COL-1) expression was significantly [(p < 0.01) (p < 0.001)] up-regulated in the presence of these peptides. The peptides also significantly induced alkaline phosphatase activity, osteocalcin level and mineral deposition in comparison with the control. It was also observed that all the four peptides did not show any cytotoxic effect during 21-day treatment period. CONCLUSION: All peptides enhanced osteoblast differentiation along with the positive controls. These results hold an immense scope to use peptides as preventive measure for reducing incidence of osteoporosis. These peptides can also be used as drugs and can be utilized as functional ingredients in functional foods preparation for osteoporosis therapy, but in vivo studies are required for further confirmation.

Antioxidative peptide from milk exhibits antiosteopenic effects through inhibition of oxidative damage and bone-resorbing cytokines in ovariectomized rats.

OBJECTIVES: Oxidative stress has been implicated as a crucial pathogenic factor in the development of postmenopausal osteoporosis. Milk-derived antioxidative peptides are gaining much attention toward the development of prodrugs for alleviating several human diseases, including osteoporosis. The aim of the present study was to determine whether antioxidant peptides are good candidates for alleviating postmenopausal osteoporosis. METHODS: In the present study, an ovariectomized (OVX) osteoporotic rat model was used to investigate the protective effects of buffalo milk casein-derived novel peptide VLPVPQK (PEP) against OVX-induced bone loss and the related mechanisms. RESULTS: Results of the present study indicated that daily administration of antioxidative peptide PEP at 50 and 100 µg/kg for 8 wk prevents body weight gain, uterine weight loss, and atrophy of endometrial lumen. Moreover, PEP increased femur dry weight, ash weight, bone ash calcium, and serum calcium and phosphorus level. Interestingly, PEP increased bone mineral density and improved trabecular microarchitecture in both femur and tibia of OVX rats. Additionally, PEP increased bone strength, reduced serum bone turnover markers, inhibited bone resorbing cytokines and decreased malondialdehyde level in OVX rat. Furthermore, PEP-elevated serum transforming growth factor-ß, increased, reduced glutathione levels, superoxide dismutase, and catalase activity altered by OVX. CONCLUSION: We demonstrated that PEP exhibits antiosteopenic effects via enhancement of antioxidant activity and reduction of bone-resorbing cytokines expression.

Buffalo casein derived peptide can alleviates H2O2 induced cellular damage and necrosis in fibroblast cells.

Oxidative stress is one of a critical pathogenic factor in the progression of aging and chronic diseases such as cancer, myocardial inflammation and diabetes. In the present scenario, peptides with short half life and more biological specificities are gaining much attention as prodrugs. Thus, the present investigation carried out to screen potential antioxidative peptide, VLPVPQK to cope with the cellular oxidative damage. Our results showed that treatment of rat fibroblast cells with 0.2mM H2O2 for 6h significantly declined different oxidative stress biomarkers such as SOD, CAT, GSH, and promoted LDH activity. In addition, ROS and TNF-α levels were also increased upon H2O2 exposure for 6h and thereby, it induced cell death. Amazingly, pretreatment of the peptide (VLPVPQK) significantly elevated cell survivability, by reversing all H2O2 induced alterations in fibroblast cells. Therefore, our results indicated that, the peptide (VLPVPQK) acted as a potential cytoprotective agent, who restored redox balance and cell homeostasis in cultured fibroblast cells, even after H2O2 exposure, suggesting that the peptide can be valuable as an effective remedy in treatment of oxidative stress related diseases and skin inflammation related disorders.

Aloe vera (Aloe barbadensis Miller) supplemented probiotic lassi prevents Shigella infiltration from epithelial barrier into systemic blood flow in mice model.

The aim of present work was to investigate preventive role of orally administered Aloe vera supplemented probiotic lassi (APL) on Shigella dysenteriae infection in mice. At the end of experimental period (2, 5 and 7 days of challenging), different organs such as spleen, liver, small intestine, large intestine, and peritoneal fluid were collected and assessed for Shigella colonization. Secretary IgA was estimated in intestinal fluid. Blood was collected in heparinized tubes for various haematological studies. Oral administration of APL showed a significant (p < 0.05) reduction in the Shigella counts (log cfu/mL) in all organs as compared to other treatment groups at different intervals after post feeding. Similarly, secretary IgA antibody levels (µg/mL) in intestinal fluid were significantly (p < 0.05) increased in case of APL fed mice. Further, feeding of APL also demonstrated a positive effect on different haematological parameters viz. Hb (gm %), RBC and WBC count. The results indicated the immunoprotective effects of APL against Shigella dysenteriae induced infection in mice.

Akt drives buffalo casein-derived novel peptide-mediated osteoblast differentiation.

Milk is a potential nutraceutical with wide range of bioactive compounds that are antioxidative, antimicrobial, antithrombotic, immunomodulatory, opioid and antihypertensive. Various intervention studies with milk reflect its stupendous role in elevating bone mineral density. Milk and milk products have shown a preventive effect in bone loss during pre- and postmenopausal women. Since, milk is proved to have a vital role in bone health promotion, there is a need to identify bioactive compounds within it. Recently we have reported four novel peptides from milk casein for their osteoblast proliferation activity. Their role in differentiation and the signaling cascade evoked by them have not been studied. Thus, the present study has been designed to investigate the differentiation potential and signaling cascade of one of the novel peptides, that is, NAVPITPTL by analyzing osteoblast differentiation markers such as alkaline phosphatase activity, osteocalcin and mineral deposition. All the experimentations suggested a significant role of this peptide in osteoblast differentiation. The inhibitor studies, immunocytochemistry and immunoblotting have proven that the peptide-induced differentiation through pAkt signaling cascade as pAkt was observed in nucleus. Moreover, the peptide was found to be bioaccessible up to 1%.

Incidence of mastitis and activity of milk neutrophils in Tharparkar cows reared under semi-arid conditions.

Rearing of indigenous Tharparkar (TP) cows (native of arid Thar deserts) under high humid conditions (>75 % humidity) has increased the incidence of mammary infections in them. A study was undertaken to see the number, activity, and expression of milk neutrophils isolated from healthy and mastitic cows. There was a significant (P < 0.05) influx in milk somatic cell counts (SCC) and neutrophils in sub-clinical and clinical mastitis cows. No change was observed in the phagocytic activity (PA) of milk neutrophils between healthy and sub-clinical mastitis (SCM) cows, but these activities decreased significantly (P < 0.05) in clinical cases. Chemotactic activity showed a significant difference between all the groups. Lactose varied significantly (P < 0.05) between healthy, sub-clinical, and clinical mastitis (CM) cows. Expression of chemokine receptor (CXCR1) was more in mastitis cows and also higher as compared to CXCR2. No change was observed in cluster of differentiation molecule (CD62L) among all the three groups of TP cows. Expression of interleukin (IL-8) and CD11b was low in healthy cows, increased significantly (P < 0.05) in both sub-clinical and mastitis cows. This study indicates that low producing TP cows are also prone to mammary infections when reared under semi-arid conditions.

Neutrophil gene dynamics and plasma cytokine levels in dairy cattle during peri-implantation period.

Neutrophils being the first line of cellular defense show significant difference in their expression in pregnant (P) and non-pregnant (NP) cows during the peri-implantation period. To study these changes, blood samples were collected from cows coming in heat and brought for artificial insemination (AI) on day 0 (day of AI), 4, 8, 12, 14, 16, 18, 21, 24, 30 and 40 day post-AI. Pregnancy was confirmed by observing non-return of heat, progesterone assay and ultrasonography. Cows were then categorized into pregnant (n=10) and non-pregnant (n=10) groups. Blood neutrophils were isolated and plasma samples collected from both P and NP cows during this period. RNA from neutrophils was isolated and studied for gene expression of adhesion molecules (CD62L, CD11b), chemokine (IL-8), and interferon stimulated genes (ISG15, OAS1, MX1, MX2, IFI16 and IFI44) using qRT-PCR. Adhesion molecules along with IL-8 showed a higher expression in NP cows. Expression of IFI16 was up-regulated as early as day 8, whereas, that of ISG15, OAS1, MX1 and MX2 were up-regulated on days 12-21 post-AI in P cows. Highest expression was shown by OAS1 on day 18 and by ISG15 and MX2 on day 21 post-AI in P cows. Pro-inflammatory cytokines (TNFα and IL-8) were higher, whereas, anti-inflammatory cytokine (IL-10) levels were lower in plasma samples isolated from NP cows. Our study indicates that blood neutrophils are sensitive to implantation signals received from the conceptus and may play an important role in implantation of the developing conceptus.

Fermented milk with probiotic Lactobacillus rhamnosus S1K3 (MTCC5957) protects mice from salmonella by enhancing immune and nonimmune protection mechanisms at intestinal mucosal level.

We investigated the mechanism by which an Indian indigenous probiotic culture, Lactobacillus rhamnosus S1K3, could overcome the pathogenic strain Salmonella enterica with an emphasis on the response at the intestinal mucosal level after long-term (30days) consumption. S1K3 was able to produce antimicrobial compounds against the pathogens. The probiotic adhered strongly to intestinal epithelium and maintained its integrity in presence of Salmonella through stimulation of tight junction and antimicrobial peptide genes in vitro. Mice prefed for 30days with S1K3-fermented milk exhibited low incidence of pathogenic Salmonella at mucosal and systemic levels. The probiotic induced TLRs transcripts at the Peyer's patches, followed by an increase in the Secretory-IgA in intestinal fluid, the IgA-secreting cells in lamina propria of small intestine and the IgA level in serum. Moreover, S1K3 maintained the protein level of IL-12, increased the IL-4 and reduced the TGF-ß level in intestinal fluid/serum at the later stage of infection. All these actions concurred to lower the count of Salmonella in feces, its invasion in spleen, liver and intestine tissues and improved the health status of probiotic-fed group. In view of this performance, S1K3 appears to be a suitable candidate for the development of nutraceutical food.

Transepithelial transport of milk derived bioactive peptide VLPVPQK.

The transepithelial transport of an antioxidative and ACE inhibitory peptide, VLPVPQK (named peptide C) derived from casein hydrolysates was investigated along with extensively studied opioid peptide ß-casomorphin using a human intestinal cell (Caco-2) monolayer. The susceptibility to the brush-border peptidases and route of transepithelial transport were observed to be the primary factors influencing the transport of these peptides. The apical to basal transport mechanism was studied using bradykinin as control as it shows resistance to cellular peptidases and its route of transepithelial transport had been established. VLPVPQK and BCM 5 were hydrolyzed by cellular peptidases while bradykinin was found intact. The transport of VLPVPQK (1.0%) was found to be relatively much higher than BCM 5 (0.03%) and bradykinin (0.1%). Interestingly the effect of some inhibitors on the transport of VLPVPQK suggested involvement of PepT1 like transporters/SOPT2 while BCM 5, its hydrolytic product and bradykinin were suggested to be transported mainly via the intracellular transcytosis pathway.