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ThesisItem Open Access DECIPHERING THE ROLE OF miRNAs INDUCED BY MILK DERIVED PEPTIDE IN REGULATION OF OSTEOBLAST DIFFERENTIATION AND OSTEOPOROSIS(ICAR-NDRI, KARNAL, 2021) TARUNEET KAUR; SUMAN KAPILABone constantly remodels its formation and resorption, where osteoblasts and osteoclasts exhibit a crucial role and homeostasis for bone differentiation is regulated under normal circumstances. This intricate balance is chiefly sustained by biochemical signalling cascades, and their misregulation or disruption may lead to pathological conditions. Osteoporosis is a wellestablished bone disorder that is shown to be connected with reduced bone mineral density and bone trabecula connectivity loss. MicroRNAs tend to be an important check for gene expression at the post-transcriptional level among many bone signalling pathway regulators, while at the same time addressing genes that regulate bone functioning, with suitable responses to pathogenesis and perhaps bone diseases. Therefore, microRNAs can be exploited as putative therapeutic targets to relieve bone-related diseases, including osteoporosis, in medical conditions. In our laboratory, osteoanabolic action of milk bioactive peptide (PepC) was validated on an in vitro or in vivo platform. PepC possesses a high potential for enhancing osteoblast proliferation and differentiation. Considering the eminence of miRNAs in the bone functioning, we assumed that one of the mechanisms used by PepC is to modify miRNA’s expression in rat osteoblasts. Since miRNAs perform the necessary effect by regulating the respective mRNA or protein targets, it is crucial to accurately identify the high-affinity gene binders in selected miRNAs in vitro and in vivo. The present study was planned, to decipher the function of miRNAs (induced by PepC) in osteoblast differentiation by decrypting their targets via, computational homology or target prediction tools, accompanied by experimental validations with osteoblast cultures and characterization in an osteoporotic rat model. The observations were initially focused on several parameters (high homology scores, fold change, p values, novelty) for screening a repository of 191 profiled miRNA in response to PepC. Five miRNAs were eventually shortlisted (miR300, miR592, miR381, miR369, miR10a). For, rno-miR300 (miR300), target identification was executed based on validated miRNA targets from its homologous miRNA; mmu-miR381 (miR381). Smad3 was recognized as best interactors for miR300 among 291 validated targets of miR381 in silico through RNAhybrid and Sfold tool. miR300 downregulation in response to PepC was confirmed by in vitro validations. Besides, miR300 excess was shown to decrease osteogenic gene and protein expression in rats osteoblasts for 4 days at 50 nM, whereas AntimiR300 (miR300 inhibitor) showed the opposite effect. Smad3 was a validated miR300 target, activating β-catenin and potentiating Runx2. The influence of miR300 on the signalling interaction of Smad3/β-catenin/Runx2 in the induction of osteoblast differentiation was evaluated by immunoblotting and in vivo miRNA antagonism. MicroCT studies showed that the AntimiR300 transfected rat model has improved trabecular microarchitecture in comparison to control. Further, for miR592 its overexpression reduces gene and protein levels of BMP7, phospho-Smad1/5/8 and Runx2 in rat calvarial osteoblasts. Besides, in vivo silencing by AntimiR592, in neonatal pups and adult rats, abolished the expression of BMP7/Smad1/5/8/Runx2 axis by suppressing the osteoblast differentiation. MicroCT displayed deteriorated bone microarchitecture in miR592 transfected ovariectomised rats compared to sham. Thus, miR300 and miR592 regulate the osteogenic differentiation negatively by targeting Smad3 and BMP7 respectively. Finding suggests the regulatory role for miR300 and miR592 in bone differentiation and shows their enormity as a therapeutic target, in management of bonerelated disorders, particularly postmenopausal osteoporosis.ThesisItem Open Access DECIPHERING THE ROLE OF PROBIOTIC LACTOBACILLI ON DNA AND HISTONE MODIFICATIONS IN INTESTINAL EPITHELIAL CELL MODEL(ICAR-NDRI, KARNAL, 2020) ANKITA KUMARI; RAJEEV KAPILAThe gut is the largest immunological organ of the body and its microbiota has an important role in the maturation and maintenance of the immune system. Probiotics are well documented for their role in the maintenance of gut homeostasis which is imperative for a healthy immune system. Probiotic-mediated immunomodulation may occur through the mediation of cytokine secretion which can also affect the proliferation and differentiation of immune cells. Epigenetic machinery plays a significant role in the modulation of both innate and adaptive immune systems. Various environmental factors including nutrients, microbes as probiotics or pathogens may have profound effects on epigenetic signatures of the intestinal epithelium to mediate their either health-promoting effects or increase susceptibility to diseases. Based upon this information, the present study was aimed to investigate DNA and histone modifications in intestinal epithelial Caco-2 cells by two indigenous probiotic strains Lactobacillus fermentum (MTCC 5898) and Lactobacillus rhamnosus (MTCC 5897) in comparison to inflammatory agent E. coli (ATCC 14849). Under this study, initially Caco-2 cells were separately treated with L. fermentum (1x109 CFU/ml), L. rhamnosus (1x109 CFU/ml) and E. coli (1x109 CFU/ml) for different durations (3, 6, 12, 18 and 24 h) and the temporal changes in gene expression of DNA (DNMT1 and TET2) and histone modifiers (p300, HDAC1, KMT2A, KDM5B, EzH2 and JMJD3) were assessed. Based upon the above results the exclusion of E.coli was performed with L. fermentum on the intestinal cells to study gene expression of DNA and histone modifiers by RT-qPCR. Later, the effect of probiotic induced DNA and histone modifications on the expression of immune related genes (IL6, IL8, hBD-2, NF-κB) was assessed by using epigenetic inhibitors. Finally, the modulation in levels of H3 acetylation by probiotic bacteria and E. coli on the promoter region of immune genes was confirmed by ChIP assay. Treatment with probiotic L. fermentum resulted in a significant (P<0.05) increase in expression of DNA (DNMT1 and TET2) and histone modifiers (p300, HDAC1, KMT2A, KDM5B, EzH2 and JMJD3) in contrast to E. coli which suppressed their mRNA expression at 12h. Moreover, the exclusion of E. coli with L. fermentum maintained the expression of TET2 and restored the expression of DNMT1, p300, and EzH2 close to control. However, L. fermentum was not effective in restoring the gene expression of HDAC1, KMT2A, KDM5B and JMJD3 during the above assay. In the case of L. rhamnosus incubation with intestinal cells did not bring any above changes in transcriptional activities. Later, in order to further validate the impact of epigenetic modifiers on the transcriptional expression of immune genes, intestinal cells were incubated with specific epigenetic inhibitors (RG108, C646, MS-275 and GSK-J4) for 18 h followed by probiotic lactobacilli (L. fermentum and L. rhamnosus) and E. coli respectively for 6 h. RG108 (DNMT1 inhibitor) did not affect transcription of intestinal immune genes either individually or in the presence of probiotic lactobacilli but, it inhibited the mRNA expression that occurred due to E. coli exposure. On the other hand, C646 (p300 inhibitor) inhibited the expression of hBD-2 and NF-κB which remained unaffected even after exposure to either of the lactobacilli or E. coli respectively. However, the expression of cytokine (IL6 and IL8) genes showed contrasting results which were further enhanced (P<0.01) synergistically after L. rhamnosus treatment. Amongst the two probiotic strains, L. fermentum significantly (P<0.05) reduced the MS-275 (HDAC1&3 inhibitor) mediated increased expression of immune related genes (IL6, IL8, hBD-2, NF-κB) in contrast to E. coli which synergistically enhanced their transcriptional activities. In the case of GSK-J4 (JMJD3 inhibitor), both probiotic strains recovered (P<0.05) the inhibitor mediated suppressed (P<0.01) mRNA expression of IL6 and NF-κB, whereas the expression of other immune genes remained suppressed. GSK-J4 also inhibited the stimulation of tested immune genes mediated by E. coli. Further, ChIP analysis also confirmed modulations in histone H3 acetylation of intestinal cells at IL6, IL8, and hBD-2 gene promoters which correspond to transcriptional activity brought by probiotic Lactobacilli and E. coli respectively. In conclusion, probiotics modulated the histone modifications in intestinal cells in a strain-specific manner as L. fermentum regulated immune genes by reducing H3 acetylation however, L. rhamnosus exhibited its immunomodulatory role by increasing the levels of H3 acetylation.ThesisItem Open Access ROLE OF PROBIOTIC LACTOBACILLI ON IMMUNE SIGNALING IN INTESTINAL EPITHELIAL CELLS(ICAR-NDRI, KARNAL, 2020) TARUNA GUPTA; RAJEEV KAPILAIntestinal epithelium plays essential part as a regulator of immune homeostasis. In presense of pathogen, intestinal epithelial cells secretes variety of factors which lead to innate or adaptive immune response in the gut. Unregulated and uncontrolled immune response lead to tissue damage and disruption of epithelial barrier function which further cause various gastrointestinal dysfunctions. Probiotic, also known as immunobiotic, as a part of natural microbiota display better potential to treat these conditions. Based upon this knowledge, the present investigation was carried out to delineate the basic mechanism associated with immunomodulatory effect of two indigenous probiotic strains of Lactobacillus (LR: L. rhamnosus MTCC 5897 and LF: L. fermentum MTCC 5898) during exclusion, competition and displacement of E.coli (1х 109 cfu/ml) and LPS (1μg/ml) as inflammatory agents respectively using Caco-2 as intestinal cell model. In order to assess immune signalling in intestinal epithelial cells modulation in expression of genes related to pro-inflammatory cytokines, pathogen recognisation receptors (PRR’s) and other genes associated with NF-κβ pathway were evaluated by qRT-PCR. Likewise, release of cytokines from intestinal cells was also determined by ELISA. Later, downstream signalling in intestinal cells resulting in immunomodulation was assessed through nuclear translocation of NF-kb (p-65 subunit) by immunofluorescence and western blotting respectively. Initially, optimal expression of immunomodulatory genes (IL-8,IL-6, TLR-2 and NF-kβ) in intestinal cells were observed at 3 and 6 hr exposure of respective probiotic lactobacilli (LF and LR) during temporal assessment. Similarly, ideal expression of immune genes in intestinal cells were found during 3 hr of incubation with inflammatory agents (E.coli / LPS). Pre-exposure of intestinal cells with either of probiotic lactobacilli followed by incubation with any of the inflammatory agents under exclusion assay reduced the expression (p<0.05) of inflammatory cytokines IL-8, TNF-α and IL-23 respectively. Similarly, modulation in expression of TLR-2 and TLR-4 genes in intestinal cells also occurred during this assay. In competition assay during simultaneous incubation of either the probiotic lactobacilli with any of the inflammatory agents, the mRNA levels of inflammatory cytokines TNF-α and TLR-4 diminished significantly (p<0.05). However, post treatment to inflamed cells with probiotic lactobacilli under displacement assay did not appear very effective in maintainace of immune homeostasis in intestinal epithelial cells based upon mRNA expression of immune related genes. Exposure of intestinal cells to either of probiotic lactobacilli strains also reduced the secretion of TNF-α irrespective of the mode of challenge with inflammatory agents. However, L.fermentum decreased (p<0.05) the release of IFN-ϒ also during exclusion and competition assays in a strain specific manner. On the other hand, release of anti-inflammatory cytokines (IL-10 and TGF-β) increased significantly (p<0.05) by L.fermentum during exclusion, competition and displacement assays respectively conducted in presence of E.coli. In case of NF-kβ as master regulator of immune modulation, mRNA expression declined significantly (p<0.05) during competition as compared to other treatments. While SIGIRR, negative regulator of inflammatory pathway, showed increased mRNA expression in presense of either of lactobacilli strains during various modes of challenge with both inflammatory agents respectively. Further western blotting showed that inflammatory agents (E.coli / LPS) significantly translocated p-65 sub-unit of NF-kβ from cytoplasm to nucleus. Contrarily, L.fermentum incubated individually with intestinal cells or during exclusion, competition and displacement assays respectively attenuated translocation of p-65 sub-unit into nucleus. Likewise, treatment with probiotic L.rhamnosus under exclusion assay with E.coli and LPS also appeared to resist nuclear translocation of NF-kβ sub-unit. These results were also confirmed by immunofluorescent staining. Hence, it is concluded that E.coli/ LPS induced inflammatory response in intestinal epithelial cells were potentially attenuated in a strain specific manner where L.fermentum was found effective during exclusion and competition assays and L. rhamnosus was more potent during exclusion assay to maintain gut homeostasis.ThesisItem Open Access NANOSAFETY OF NANOFERTILIZER AND NANOCARRIER IN RATS MEDIATED THROUGH BREAST MILK(ICAR-NDRI, KARNAL, 2019) AHMAD HUSSAIN; GAUTAM KAULNanotechnology is a promising tool in every aspect of modern life including agriculture. Nanoproducts like nanofertilizers (nutrients in nanoform) and nanocarriers (nanoporous materials for controlled release) are becoming centre of attraction due to their role in precision agriculture. There is dire need of safety assessment before their application due to environmental exposure and possible health hazard to life forms including humans. Keeping all this in view, the present investigation was designed to assess the safety of ZnO nanofertilizer (ZNF) and mesoporous silica nanocarrier (MSN) taking both in vitro and in vivo model into account. Under in vitro study, cytotoxicity of ZNF and MSN on immune as well as male germ cell was evaluated by MTT, WST8 and neutral red assay. The cell viability in both cases decreased significantly by time (1hr, 4hr) and concentration (1, 5, 10, 25, 50 and 100 μg/ml) dependent treatment of ZNF compared to control. The anti-oxidative enzymes (SOD: Superoxide dismutase, GPX: Glutathione peroxidase) increased significantly (P < 0.01) after 1 hour incubation with 50 μg/ml of ZNF. Lethal Concentration (100 μg/ml) obtained from percentage hemolysis (%hemolysis) assay of RBCs confirms that ZNF is relative toxic than MSN. In neutrophils and male germ cells treated upto 100 μg/ml MSN no significant changes was observed in %viability, %hemolysis as well as in the activities of oxidative stress enzymes as compared to control. Additionally, ZNF at higher concentration leads to significant loss (p<0.05) in viability and membrane integrity of male germ cell as depicted by Eosin-Nigrosin assay and hypo-osmotic swelling test, respectively. In case of in vivo study, lactating nulliparous rats were orally administered 5, 25 and 50mg/kg b.w. ZNF as well as MSN for consecutively 19 days from the first day of parturition. ZNF was distributed significantly to breast milk with increasing dose as quantified by inductively coupled plasma mass spectrometry (ICPMS). Further, ZNF were absorbed by the small intestine and distributed to liver of breast-fed pups. Histopathology study of intestine and liver of offsprings depicted the gross pathological changes caused by milk mediated transfer of ZNF. Most of the ZNF and MSN were excreted through feces of dams. IgA secretion in the intestinal fluid of offsprings increased significantly on increasing dose of ZNF to dams. Liver injury marker enzymes (AST, ALT) increased significantly in dams and offspring postnatally. The level of cytokines (TNF-α, IL-1β) in serum increased significantly in the offsprings of 25mg/kg and 50mg/kg bw group. The total cytochrome P450 reductase and total antioxidant capacity decreased significantly in the offsprings of 50mg/kg ZNF given dams group. Further, nitric oxide production and phagocytosis activity of macrophages from offsprings of 50mg/kg ZNF dams group decreased significantly (p<0.05). Real-time polymerase chain reaction indicated milk mediated expression of anti-oxidative enzymes (SOD, GPX), apoptosis (BAX) and cytokines (TNFα, IL1β) genes in offspring neutrophils after oral administration of 50/mg kg b.w ZNF to dams. These results provide information that MSN is comparatively safer than ZNF upto 100 μg/ml concentrations in vitro and 50mg/kg body weight in vivo intubation to dams after parturition.ThesisItem Open Access UNDERSTANDING THE INTERNALIZATION OF FOLLICULAR FLUID EXTRACELLULAR miRNAs IN BUFFALO GRANULOSA CELLS(ICAR-NDRI, KARNAL, 2020) SUMAN KUMARI; DHEER SINGHFollicular uid granulosa cells (GC) cross-talk is important for the regulation of folliculogenesis. The cross-talk is primarily mediated by hormones and signaling molecules (e.g., cytokines and chemokines) and they act in autocrine, paracrine and endocrine manner. Recently, extracellular microRNAs (miRNAs) have been studied widely across di erent cells and species, primarily because of their high stability in biological uids. The high stability of miRNAs is likely because of their association with exosomes and Ago-2 proteins (non-exosomal fraction), which protects them from degradation. In the present study, we aimed to identify the exosomal and non-exosomal miRNAs in bu alo follicular uid and elucidated the mechanism of their internalization in granulosa cells. Exosomes were rst characterized and then total RNA was isolated from both exosomal and non-exosomal fraction of follicular uid, followed by next generation sequencing (NGS). NGS analysis identi ed 45 and 141 miRNAs in exosmal and non-exosomal fractions of follicular uid, respectively. Among these miRNAs, bta-1246 and bta-148 were found to be the most abundant miRNAs in exosomal and non-exosomal fractions, respectively. Abundant and less abundant miRNAs were validated using real-time PCR. Results showed a consistent expression of miRNAs in both fractions of follicular uid with the NGS data. Target scan was further used to identify genes that were targeted by top-10 abundant exosomal and non-exosomal miRNAs. We used Bos taurus as the reference genome. All targets with a context score of less than 0:4 of top-10 abundant miRNAs were considered for network and pathway analysis. Functional annotation and pathway analysis revealed that predicted targets of exosomal miRNAs were enriched in genes that were involved in PI3K-Akt signaling pathways, focal adhesion and Relaxin signaling pathways. On the other hand, the non-exosomal fraction target genes participated in MAPK signaling pathways, FoxO signaling pathways, PI3K-Akt signaling pathways and Progesterone mediated Oocyte maturation and these pathways have been reported in the regulation of ovarian functions such as folliculogenesis, ovulation and cell proliferation. To further understand the interaction between di erent genes, we constructed protein-protein interaction network using STRING software and selected hub genes with signi cant interactions for further analysis. With In silico analysis in both fractions 9 signi cant clusters were perdicted to be involved in important cellular processes. To elucidate the mechanism of exosomes internalization, we studied the uptake of exosomes in granulosa cells. We found that exosomes uptake was a time dependent process. We also found that exosome can induce TLR-4 signaling because the knockdown of TLR-4 blocked the induction of pro-in ammatory cytokines (IL6, IL1 and TNF ). This study clearly demonstrated that follicular uid exosomes triggered the innate immune response through the release of in ammatory cytokines. Since the mode of non-exosomal miRNA internalization is little studied, we also studied the role of neuropilin-1 (NRP-1) as a receptor in internalizing follicular uid non-exosomal miRNAs in GCs.As NRP-1 has been shown to be involved in the internalization of non-exosomal/Ago-2 protein associated miRNAs in cancer cells. We rst con rmed the expression of NRP-1 in GCs during follicular development, followed by its role in the internalization of non-exosomal miR-210. This study showed that GCs incubation with (1) non-exosomal fraction and (2) non-exosomal fraction incubated with miR-210 of follicular uid increased the content of miR-210 in GCs as compared to their control. Results of silencing experiments further showed that there was a signi cant decrease in uptake of miR-210 during NRP-1 knockdown. Downstream expression analysis of miR-210 target genes (CYP19A1, PCNA and EFNA3) further con rmed the involvement of NRP-1 in the internalization of miR-210. In summary, the present study demonstrated that exosomal and non-exosomal miRNAs can act as hormone as they bind to the receptor of target cells to perform their functions.ThesisItem Open Access COMPARATIVE EVALUATION OF GUT HEALTH MODULATING ROLE OF MILK FROM INDIGENOUS AND CROSS BRED COWS(ICAR-NDRI, KARNAL, 2020) SAVITA DEVI; SUMAN KAPILAIndigenous cow milk is considered as powerhouse of health. Charaka in “Charaka Samhita” explains the many properties of milk from Indigenous cows particularly its immunomodulatory property. Furthermore, gut is the most important immune organ associated with the inflammation. Presently, most of the researchers are focusing on the beneficial effects of Indian cow milk in relation to A1/A2 milk or intestinal inflammatory disorders related to A1 milk. But, the breed-wise difference in systemic and localized immunomodulatory properties of milk is not yet investigated. So, the present investigation focuses on the comparative evaluation of Indigenous and crossbred cow milk on gut immune status. To meet the objective, two in-vivo trials were conducted in rats that were fed with milk from different breeds of cow under normal and immunoreactive (Inflammatory) state for a period of 100 and 28 days, respectively. The results depicted a significant decrease in phagocytic activity of SW, GIR and HF milk treated rats than control group (without milk). 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The Inflammatory bowel disease model was established in rats using peptidoglycan (PGN). PGN treatment resulted in significant increase in gut permeability, but feeding of milk from SW, GIR, KF and KS brought about significant reduction in gut permeability. Gastric acidity was found to decrease significantly in different milk treated groups in comparison to PGN. Likewise, significant increase in intestinal GLP-2 in GIR and HF compared to PGN was found. TNF-α and IFN-γ was reduced in TP and GIR milk treated rats, but IgA level was increased in TP and GIR group as compared to PGN rats. The anti-inflammatory property of Sahiwal milk was better among the different types of milk studied. In order to further delineate the anti-inflammatory property of Sahiwal milk, the inflammatory conditions were induced in Caco-2 using PGN. A significant (p<0.05) increase in Zo-1 and Claudin-1 gene expression was observed in Caco-2 cells pre-treated with the digested Sahiwal milk followed by PGN treatment in comparison to control. Whereas, pre-treatment of Caco-2 cells with the digested Sahiwal milk caused a significant reduction in relative gene level expression of TLR-2, TLR-4, NF-kB, TNF-α, IL-6 and IL-8 in comparison to PGN treated cells. Thus, the feeding of cow milk from different breeds showed tolerogenic effect on immune status in rats. While milk from Indigenous Sahiwal breed showed the better anti-inflammatory property in comparison to milk of other breeds of cattle.ThesisItem Open Access EXPLORING MILK EXOSOMAL miRNAs RELATED TO INSULIN AND INFLAMMATORY SIGNALING(ICAR-NDRI, KARNAL, 2020) MONIKA VASHISHT; DHEER SINGHMilk is a part of human diet as it serves as excellent nutritional package. miRNA, as bioactive nucleic acid, has been identified and found to biologically active component in the milk. Recently, we have profiled cow and buffalo milk exosomal miRNAs while functional validation is yet to be carried out. Studies showed that bovine (cow and buffalo) and non-bovine (goat and camel) milk exosomal miRNAs contributes in the insulin and inflammation related studies. Therefore, the aim of proposed study is to explore the role of milk exosomal miRNAs and whether these miRNAs play any role in insulin and inflammatory signaling. First, we did qPCR validation of abundant miRNA in camel and goat milk exosomes which has been already profiled and validated in cow and buffalo milk exosomes in our lab. Result showed that miR-21, miR-182 is most significant abundant and common miRNA present in all species whereas miR- 148a, miR-30f and miR-25 are significant common in all except camel. Further, we identified miRNA related to insulin and inflammatory signaling on literature basis which were further confirmed by target scan analysis and validated by qPCR in all four species. On the basis of Ct value, we found that miR-2478 and miR-21 is evolutionary conserved and shows higher expression as compared to other insulin and inflammatory miRNA respectively and selected as candidate miRNA. For insulin signaling, HepG2 cells were selected and firstly effect of whole exosome has been studied on insulin receptor, intracellular levels of miR- 2478 and viability of cells. Results showed increased expression of insulin receptor, intracellular levels of miR-2478 and decreased viability in exosomes treated cells as compared to control. To confirm whether this effect was due to miR-2478, it was ectopically expressed in HepG2 cells via transfection.The effect of miR-2478 mimic on INSR mRNA and protein expression, glycogen content and enrichment of RNA polymerase II at insulin gene promoter in HepG2 cells were analysed. Results of the current study showed that there was increased expression of insulin receptor gene at both mRNA and protein with increased abundance of bta-miR-2478 in HepG2 cells. Similarly, glycogen content was also found to be increased in bta-miR-2478 transfected cells as compared to control. Results of ChIP assay also showed enrichment of RNA polymerase on insulin receptor promoter in miR-2478 transfected cells as compared to control. For inflammatory signaling first role of milk exosomes on proinflammatory cytokines was evaluated using Caco-2 cell line in vitro. Results showed that, as endotoxin, milk exosomes treatment transiently increased proinflammatory cytokine gene expression. Further, experiments also showed that exosome mediate proinflammatory gene expression via TLR4 signalling pathway. However, pretreatment of milk exosomes attenuated the endotoxin-TLR4 mediated proinflammatory cytokine expression. Bioinformatics approach by target scan showed that candidate miRNA-21 encapsulated in bovine milk exosomes has potential to influence inflammatory signaling via targeting PDCD4. bta-miR-21 down regulates the IL-6, TNF-α, IL-8 cytokines while up regulates the IL-10 production in LPS challenged Caco-2 cells. In addition to them, milk exosomes decrease the fatty acid induced proinflammatory cytokine production in insulin resistant adipocytes. In conclusion, milk exosomes functionally deliver miRNA inside the recipient cells and influence insulin and inflammatory signaling in vitro. However, further functional validation of candidate milk exosomal miRNA is needed using animal model for therapeutics.ThesisItem Open Access DEVELOPMENT OF EXOSOME-ENCAPSULATED ANTIBIOTIC BASED THERAPEUTICS FOR MASTITIS(ICAR-NDRI, KARNAL, 2022) JITENDRA KUMAR; DHEER SINGHIncreasing antibiotic load in dairy animal following conventional prolonged treatment of mastitis with a higher dosage of antibiotics is a major public health issue throughout the globe. Antibiotic therapy for mastitis is currently ineffective due to antibiotic resistance, intracellular persistence of Staphylococcus aureus and less bioavailability of antibiotics to the targeted site. Therefore, the aim of present study was to develop an effective therapeutics for intracellular delivery of antibiotic into mammary epithelial cells to tackle S.aureus. In this study, the milk exosome (mEs), pre-characterized by zetasizer and high resolution transmission electron microscopy were used for encapsulation of D-(-)-α- aminobenzylpenicillin (AMP) antibiotic. The killing efficacy of exosome-encapsulated ampicillin (mEs-AMPs) was evaluated on pathogenic S.aureus. The results demonstrated that, mEs-AMP had a significantly (p<0.001) higher killing rate than the unencapsulated-AMP at the similar dosage and frequency of treatment. The minimum inhibitory concentration of mEs-AMP was 4.08 times (75.16%) lower than that of only ampicillin. The validation of antimicrobial efficacy of mEs-AMP was tested against subclinical and clinical mastitis Sahiwal cows. The mEs-AMPs exhibited higher killing efficacy (74.35% vs 34.46%) on S. aureus as compared to unencapsulated AMP. Further, encapsulated mEs-AMP significantly reduced somatic cell count (SCC) (4.53 ± 0.69 lakh/mL to 3.39 ± 0.51 lakh/mL; P<0.001) and bacterial load (log CFU; (2.2 ± 0.15 to 1.7 ± 0.37; p<0.05) in the milk of treated animals in comparison with uncapsulated AMP. Therapeutic responses measured by SCC and log CFU count showed that mEs-AMP had significantly reduced S.aureus count in both subclinical and clinical mastitis animals. In conclusion, exosomes encapsulated-AMP was found more effective in reducing SCC, CFU, and MIC of AMP resulting in high therapeutic efficacy of the antibiotic against S.aureus. Therefore, we anticipate that this technology can be further adapted as a therapeutic tool for the effective management of mastitis in dairy cattleThesisItem Open Access EXPLORING THE PREDOMINANT TOXIN IN MARKET MILK OF NORTH INDIA AND ITS CYTOTOXIC EFFECT ON AN INTESTINAL CELL LINE SYSTEM(ICAR-NDRI, KARNAL, 2022) LAL KRISHAN KUMAR; SUNEEL KUMAR ONTERUMilk is easily available in the market in pasteurized form. As many studies reported milk contamination even post-pasteurization, the present study was designed to explore the predominant toxicants in the marketed milk of North India. 92 pasteurized milk samples were collected across the seven states of North India and toxicants were analyzed by two approaches. Initially, 320 pesticides, 63 antibiotic residues, seven mycotoxins, and six heavy metals were analyzed in overall pooled milk sample. Targeted UPLC-based analysis detected chloramphenicol, paraquat, oxacillin, AFM1, BPA, and maneb residues above MRL in state-wise pooled milk samples. Samples from six states had oxacillin, chloramphenicol, ciprofloxacin, and maneb, and samples from 3 states had paraquat and AFM1 levels above MRL. As aflatoxin M1 (AFM11) was found (145 ng/kg) in both the approaches, it was considered as the predominant toxicant and selected for the toxicity study. Checking its digestive fate by an in vitro digestion model system showed that AFM1 (2ug/L) in milk might be completely digested. Additionally, we checked the cytotoxic effects of AFM1 on intestinal cells such as CaCo2 at different dosages ranging from 5-2000 ng/L for different time intervals i.e., 6hrs, 12hrs, and 24hrs of incubation time, and morphology, viability, ROS and 8 transcript-markers were examined. The results suggested that AFM1 has no effect on cell morphology and viability, but the cellular ROS levels were increased after 6hrs of incubation with AFM1. Gene expression study showed an increased expression of cytokeratin, villin, vimentin, junctional adhesion molecule-1 and CYP1A1 genes and a decreased expression of claudin, occludin, and ZO1 at 24hrs of incubation as the concentration of AFM1 increases. Further, trans-well experiments suggested an increase in the permeability of CaCo2 cells by AFM1. The study concludes that AFM1 contaminated milk consumption may cause intestinal barrier dysfunction and makes the individual susceptible to leaky gut.
