Biology subjects

Sharma, N. K.

Publications and source records attributed to Sharma, N. K..

3 recordsLinked to original sources

bioRxiv2019

Detection of urinary metabolites of metabolic pathway disorders by using VTGE and LC-HRMS techniques

BackgroundIn recent, various human health disorders including cancer, diabetes, neurodegenerative and metabolic diseases are noticed among human populations. Currently, genetic and proteomic approaches are highly reported to detect metabolic disorders that also include inborn error of metabolisms. These existing detection methods are faced with cost issue and time consuming factors. Therefore, metabolites as biomarkers are one of potential avenues to detect metabolic disorders. Further, exploitation of urine as potential source of metabolite biomarkers, there are limitation in this area of research due to abundance of non-metabolite components such as proteins and nucleic acids. Hence, methods and processes are required to precisely fractionate metabolites from urine of inborn error of metabolism patients and then identified by analytical tools such as LC-HRMS and GC-MS.\n\nMethodsSterile filtered urine samples (750 {micro}l) mixed with (250 {micro}l) loading buffer were electrophoresed on VTGE that uses acrylamide gel (acrylamide:bisacrylamide, 30:1) as matrix of 15%. Further, vertical tube gel electrophoresis (VTGE) technique combined with LC-HR-MS to identify metabolites that are known as the biomarkers of metabolic disorders was carried out.\n\nResults and DiscussionThe authors provide evidence on the use of novel VTGE coupled with LC-HRMS to detect metabolites among metabolic disorders. Data suggest the applicability of VTGE coupled with LC-HRMS technique to detect metabolites such as 2-methyluridine, 2-Methylglutaric acid, 2-Methyl citric acid, 2-Hydroxyglutaric acid in case of metabolic disorders.\n\nConclusionThis preliminary work is suggested to be extended to large clinical samples to validate application of this method to detect metabolic disorders including inborn error of metabolisms.

biochemistry↗

bioRxiv2019

Distinct DNA metabolism and anti-proliferative effects of goat urine metabolites: An explanation for xeno-tumor heterogeneity

BackgroundThe tumor microenvironment, including microbiome populations in the local niche of several types of solid tumors like mammary and colorectal cancer are distinct. The occurrence of one type of cancer over another varies from animals to human individuals. Further, clinical data suggest that specific cancer types such as mammary and colorectal cancer are rare in ruminant like goat.\n\nMethodsFresh urine samples were collected from healthy ruminates including cow, goat, buffalo, ox, horse, jenny and human and subjected to fractionation using drying, vortexing, centrifugation and sterile filtration in DMSO solvent. Collected urine DMSO fraction (UDF) samples from all sources were subjected DNA metabolizing assay with plasmid DNA pBR322 and genomic DNA of MCF-7 cells. Further, based on the discernible DNA metabolizing effects, goat UDF was tested for anti-proliferative effects upon HCT-116 and MCF-7 cells using Trypan blue due exclusion assay.\n\nResultsThis paper reports that goat UDF possesses very clear DNA metabolizing effects (up to 95%) upon plasmid and genomic DNA compared to other ruminants and human UDF samples. Interestingly autoclaving of goat UDF and other sample results in the significant loss of DNA metabolizing effects. In this way, data potentially indicate that the goat UDF sample contains metabolite or similar organic compounds. Further, in vitro treatment of the goat UDF sample shows discernible anti-proliferative effects upon HCT-116 (up to 75%) and MCF-7 (up to 40%).\n\nConclusionThis study signifies the clear differences in DNA metabolizing effects of goat UDF and well correlated with anti-proliferative effects upon HCT-116 and MCF-7 cells. This study is of first report to show the comparison of urine metabolites and an indirect link to support the possible reasons behind xeno-tumor heterogeneity as rare occurrences of colorectal and mammary cancer in goat over other ruminants and human.

cancer biology↗

bioRxiv2019

A novel method to detect intracellular metabolite alterations in MCF-7 cells by doxorubicin induced cell death

Metabolic reprogramming within cancer cells is suggested as a potential barrier to chemotherapy. Additionally, metabolic tumor heterogeneity is one of factor behind discernible hallmarks such as drug resistance, relapse of tumor and the formation of secondary tumors. In this paper, cell based assays including PI/annexin V staining and immunoblot assay were performed to show the apoptotic cell death in MCF-7 cells treated with DOX. Further, MCF-7 cells were lysed in hypotonic buffer and whole cell lysate was purified by a novel and specifically designed metabolite (100 to 1000 Da) fractionation system as vertical tube gel electrophoresis (VTGE). Further, purified intracellular metabolites were subjected to identification by LC-HRMS technique. The authors show the presence of cleaved PARP 1 in MCF-7 cells treated with DOX. Concomitantly, data show the absence of active caspase 3 in MCF-7 cells. Novel findings are to identify key intracellular metabolites assisted by VTGE system that include lipid (CDP-DG, phytosphingosine, dodecanamide), non-lipid (N-acetyl-D-glucosamine, N1-acetylspermidine and gamma-L-glutamyl-L-cysteine) and tripeptide metabolites in MCF-7 cells treated by DOX. Interestingly, the authors report a first evidence of doxorubicinone, an aglycone form of DOX in MCF-7 cells that is potentially linked to the mechanism of cell death in MCF-7 cells. This paper reports on novel methods and processes that involve VTGE system based purification of hypotonically lysed novel intracellular metabolites of MCF-7 cells treated by DOX. Here, these identified intracellular metabolites corroborate to caspase 3 independent and mitochondria induced apoptotic cell death in MCF-7 cells.\n\nSIGNIFICANCE STATEMENTMetabolic reprogramming in cancer cells is implicated in various tumor hallmarks. Interestingly, thousands of research have addressed the molecular basis of drug treatment and resistance in chemotherapy. But, there is a significant gap in the precise methodologies and approaches in addressing intracellular metabolite alterations. This paper reports on a novel approach that helped reveal new findings on intracellular metabolite changes in case of doxorubicin (DOX) induced cell death in MCF-7 cells. This paper highlights the additional insights on debatable findings available in literature in the contexts of DOX induced cell death mechanisms. In this paper, novel and specifically designed vertical tube gel electrophoresis (VTGE) system is claimed to purify intracellular metabolites and this method is compatible with other biological system.

cancer biology↗