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Shrestha, H. K.

Publications and source records attributed to Shrestha, H. K..

2 recordsLinked to original sources

Meta-omics-aided isolation of an elusive anaerobic arsenic-methylating soil bacterium

Soil microbiomes harbor unparalleled functional and phylogenetic diversity and are sources of novel metabolisms. However, extracting isolates with a targeted function from complex microbiomes is not straightforward, particularly if the associated phenotype does not lend itself to high-throughput screening. Here, we tackle the methylation of arsenic (As) in anoxic soils. By analogy to mercury, As methylation was proposed to be catalyzed by sulfate-reducing bacteria. However, to date, there are no anaerobic isolates capable of As methylation, whether sulfate-reducing or otherwise. The isolation of such a microorganism has been thwarted by the fact that the anaerobic bacteria harboring a functional arsenite S-adenosylmethionine methyltransferase (ArsM) tested to date did not methylate As in pure culture. Additionally, fortuitous As methylation can result from the release of non-specific methyltransferases upon lysis. Thus, we combined metagenomics, metatranscriptomics, and metaproteomics to identify the microorganisms actively methylating As in anoxic soil-derived microbial cultures. Based on the metagenome-assembled genomes of microorganisms expressing ArsM, we isolated Paraclostridium sp. strain EML, which was confirmed to actively methylate As anaerobically. This work is an example of the application of meta-omics to the isolation of elusive microorganisms.

microbiology↗

Development and Validation of an LC-MS/MS Based Quantitative Assay for Marmoset Insulin

IntroductionInsulin is a peptide hormone that is secreted by the {beta} cells of the pancreas and is essential to the metabolism of carbohydrates, fats, and proteins in the body. The marmoset insulin peptide is not homologous with human insulin and therefore commonly available assays do not work for this species. Due to the increasing popularity of marmoset research, a simple, specific assay for the quantitation of marmoset insulin is needed. This study aimed to develop and validate a bottom-up proteomic workflow with trypsin digestion and analysis using LC coupled with triple quadrupole mass spectrometry (LC-MS/MS). MethodsMarmoset serum proteins were subjected to denaturation, reduction, and enzymatic cleavage to extract a unique, 7 amino acid peptide for quantitation of marmoset insulin.Resolution of the peptide was achieved by LC-MS/MS using electrospray ionization operating in positive mode. Calibration was achieved by aliquot dilution of fully synthetic marmoset insulin tryptic peptide into macaque serum. A stable-isotope labeled (13C, 15N) synthetic marmoset insulin tryptic peptide was used as internal standard. ResultsThe assay was fully validated according to bioanalytical method validation guidelines for linearity, precision, and dilution linearity using purified marmoset insulin. The limit of detection was 4.28 pmol/L and the limit of quantitation was 8.62 pmol/L. Biological validation was achieved by comparison of samples previously run by radioimmunoassay and measurement of the marmoset insulin response to glucose via an oral glucose tolerance test (OGTT). The physiological range of marmoset insulin was shown to be 84.5 to 1222 pmol/L. ConclusionsThis paper presents a simple, reproducible method to measure marmoset insulin using LC-MS/MS.

biochemistry↗