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Kerala Veterinary and Animal Sciences University, Wayanad
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ThesisItem Open Access ANALYSIS OF DIFFERENTIAL EXPRESSION OF microRNA IN LIPOPOLYSACCHARIDE CHALLENGED LYMPHOCYTES OF VECHUR AND CROSSBRED CATTLE(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2023-03-23) DIVYA P. D; Dr. Shynu M.The present study was carried out to analyse the differences in the expression of miRNAs in response to the in vitro LPS stimulation of PBMC cultures of crossbred and Vechur cattle. The study was performed in adult, apparently healthy, female crossbred and Vechur cattle maintained at University Livestock Farm and Fodder Research and Development Scheme, Mannuthy, and Vechur Conservation Unit, Kerala Veterinary and Animal Sciences University, respectively. The known/ novel miRNAs and the differential expression of miRNAs in response to bacterial endotoxin, LPS in PBMCs of the two genetic groups were identified by Short-read Illumina Next Generation Sequencing. Validation of NGS data of selected differentially expressed miRNAs was carried out by qRT-PCR assay. Prediction of target genes of differentially expressed miRNAs, functional gene enrichment analysis, analysis of cellular pathways involved and protein-protein interaction (PPI) network evaluation of the predicted target genes were also studied using various online bioinformatics tools. Cytokines associated with the immune related pathways of targets of differentially expressed miRNAs were analysed by ELISA. The differences in cytokine expression were also measured after overexpression of selected miRNA with miRNA mimics in the PBMC cultures of Vechur cattle. A total of 0.47 and 0.64 million clean reads, with an average Phred score of 34.92 and 34.75 corresponding 55.3 and 62.1 per cent of the adapter trimmed reads, respectively, for crossbred and Vechur samples were retrieved by NGS. Analysis of miRNAome identified 979 and 853 known miRNAs, and 393 and 139 novel miRNAs in samples from Vechur and crossbred cattle, respectively. Differential expression studies of NGS data revealed significant variation in the expression of miRNAs in LPS challenged PBMCs cultures of Vechur cows with respect to crossbred cattle. The results of real time validation of the expression of selected miRNAs by qRT-PCR assay were also consistent with the results of NGS. Functional gene enrichment analysis, analysis of pathways associated to the targets of differentially expressed miRNAs also revealed significant enrichment of targets of differentially expressed miRNAs in many immune related GO terms, immune associated cellular pathways as well as major cell signalling pathways. The PPI network analysis also showed active involvement of proteins encoded by these target genes in many of the important immune mechanisms. The study could identify differences in the immune related pathways associated to target genes of both up regulated and down regulated miRNAs though some pathways were found to be identical among both. Assessment of cytokines associated to the pathways regulated by the target genes of differentially expressed miRNAs in the supernatants from LPS treated PBMC cultures of also showed significant variations in the level of cytokines viz; TNF α, IL-4 and IFN γ among the crossbred and Vechur samples when compared to the control groups within the breed as well as between the breed. A significantly higher level of TNF α was noticed in LPS treated PBMCs of crossbred cattle whereas, IL-4 level was found to be significantly increased in LPS stimulated PBMCs of Vechur cattle compared to the LPS untreated cells from both groups. However, the present study could not detect any significant difference in IFN γ level among the LPS treated and untreated cells of both crossbred and Vechur cattle. The overexpression studies of miRNAs in Vechur PBMCs by transfecting with selected miRNA mimic could identify significant differences in IL-4 level while the changes were negligible with respect to other cytokines. The findings of the current research work suggest that both Vechur and crossbred cattle are having differences in their potential to tackle the immunological changes in response to an acute inflammation caused by the bacterial endotoxin; LPS. These differences might be contributing to the alleged immunological sturdiness to Vechur cattle compared to crossbred animals although, the specifics needs to be further validated.ThesisItem Open Access EVALUATION OF ANTICANCER PROPERTY OF RECOMBINANT GOAT LACTOFERRIN(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2023-02-08) VASUDHAR BHAT S. V.; Dr. Uma R.The present study was conducted with the objective to express and purifyrecombinant Malabari goat lactoferrin in Escherichia coli (E. coli). The study was carried out in three phases. In the phase I, a portion of lactoferrin (Lf) gene encoding the N lobe of Malabari goat lactoferrin (cLf-N) was cloned into pJET1.2/blunt cloning vector and transformed into E. coli strain JM109 and was sequenced which revealed a 793 bp fragment with 100 per cent similarity to goat Lf in the database. This ampliconwas subcloned into pET28a(+) expression vector and transformed into BL-21 (DE3) pLysS bacterial host. The transformed E. coli BL21 (DE3) pLysS containing the recombinant plasmid expressed maximum recombinant protein (rcLf-N) upon induction with 1mM IPTG at 37°C for five hours. The recombinant protein, rcLf-N, containing polyhistidine tag was purified using Ni-NTA affinity column chromatography and confirmed as a derivative of Lf by Western blotting. In phase II, rcLf-N was analysed for its in vitro anticancer activity in Daltons Lymphoma Ascites (DLA) cell lines. In MTT assay there was a significant (p<0.05) reduction in the per cent inhibition of cell proliferation in a concentration-dependent manner from 800μg/mL to 8.75μg/mL, indicating the cytotoxic effect of rcLf-N in a dose-dependent manner upon DLA cell lines. The half-maximal inhibitory concentration (IC50) value was found to be 263.5 μg/mL using Graph pad prism. Further, upon Acridine orange/ Ethidium bromide (AO/EB) and Hoechst staining of the treated cells, the apoptotic changes produced by rcLf-N and standard drug Cisplatin in DLA cells were highlighted. During phase III trial, the in vivo anticancer activity of rcLf-N was analysed on Swiss albino mice bearing DLA induced solid tumour. Based on preliminary studies the concentrations 50μg and 75μg of rcLf-N per animal were selected for comparison with the standard drug Cisplatin. A significant (p<0.05) reduction in tumour weight, tumour volume and tumour weight to body weight ratio was observed in the rcLf-N and cisplatin treated groups compared to the control group. Maximum reduction was observed in group treated with rcLf-N @75μg intratumourally for three days. Histopathological examination of tumour tissue in all the groups treated with rcLf-N and cisplatin showed the presence of apoptotic changes with decreased spread of neoplastic cells into surrounding tissues and decreased neovasculature.Relative expression of VEGF and Caspase-3 genes was analysed in both in vivo and in vitro studies with GAPDH as the reference gene via quantitative real-time PCR. A dose-dependent downregulation of VEGF and upregulation of Caspase-3 was revealed in the cells/tissues treated with IC50 and double IC50 doses of rcLf-N both in vitro and in vivo. Similar results were observed with IC50 Cisplatin. On comparison between intratumoural and intraperitoneal routes of treatment in vivo, the intratumoural route of treatment was better in downregulating VEGF and activating Caspase-3 on day 7 and 14, although the fold change was non-significant. From the present study, it could beconcluded that the novel recombinant protein produced antineoplastic activity through apoptosis and rcLf-N @75μg exhibited most potent anticancer activity against DLA cells.
