Biomedical subjects

Mary K Young

Publications and source records attributed to Mary K Young.

6 recordsLinked to original sources

PubMed2025

Fecal microbiota transplantation promotes type 2 mucosal immune responses with colonic epithelium proliferation in patients with recurrent Clostridioides difficile.

BACKGROUNDFecal microbiota transplantation (FMT) is the most effective therapy for recurrent Clostridioides difficile infection (rCDI), yet its mechanism of action remains poorly understood.METHODSWe report the results of a clinical trial of patients undergoing FMT therapy for rCDI (n = 16), which analyzed colon biopsies, plasma, PBMCs, and stool at the time of FMT and 2-month follow-up. Plasma and colon biopsy samples were also collected from healthy controls for comparison with patients with rCDI. Microbiome composition, colonic gene expression, and immune changes were evaluated through high-throughput sequencing and immunoprofiling via flow cytometry.RESULTSNo patients experienced recurrence at follow-up. FMT significantly altered the intestinal microbiome but had no significant impact on the systemic immune system. In contrast, FMT promoted broad changes in colonic transcriptional profiles compared with both pre-FMT and healthy control biopsies, inhibiting genes associated with proinflammatory signaling and upregulating type 2 immunity and proliferative pathways (Myc and mTORC1). FMT increased expression of IL-33 and the type 2 immune EGFR family ligand amphiregulin, potentially explaining upregulation of Myc and mTORC1 pathways. Spatial transcriptomics demonstrated that these changes were localized to the colonic epithelium. Comparison of transcriptional profiles with available single-cell gene sets determined that post-FMT biopsies were enriched in signatures associated with proliferative cell types while repressing signatures of differentiated colonocytes.CONCLUSIONWe conclude that FMT promotes proliferation of the colonic epithelium in patients with rCDI, which may drive regeneration and protect against subsequent CDI.TRIAL REGISTRATIONClinicaltrials.gov NCT02797288.FUNDINGThis work was funded by grants from the NIH.

Adult↗

PubMed2003

Gingipain RgpB is excreted as a proenzyme in the vimA-defective mutant Porphyromonas gingivalis FLL92.

We have previously shown that the unique vimA (virulence-modulating) gene could modulate proteolytic activity in Porphyromomas gingivalis. Although a reduction in cysteine protease activity was observed in the vimA-defective mutant, P. gingivalis FLL92, compared to that of the wild-type strain, no changes were seen in the expression of the gingipain genes. This result might suggest posttranscriptional regulation of protease expression. To determine whether there was a defect in the translation, transport, or maturation of the gingipains, P. gingivalis FLL92 was further characterized. In contrast to the wild-type strain, a 90% reduction was seen in both Rgp and Kgp protease activities in strain FLL92 during the exponential growth phase. These activities, however, increased to approximately 60% of that of the wild-type strain during stationary phase. Throughout all the growth phases, Rgp and Kgp activities were mostly soluble, in contrast to those of the wild-type strain. Western blot analyses identified unique Rgp- and Kgp-immunoreactive bands in extracellular protein fractions from FLL92 grown to late exponential phase. Also, the RgpB proenzyme was identified in this fraction by mass spectrometry. In addition, in vitro protease activity could be induced by a urea denaturation-renaturation cycle in this fraction. These results indicate that protease activity in P. gingivalis may be growth phase regulated, possibly by multiple mechanisms. Furthermore, the gingipain RgpB is excreted in an inactive form in the vimA mutant. In addition, these results provide the first evidence of posttranslational regulation of protease activity in P. gingivalis and may suggest an important role for the vimA gene in protease activation in this organism.

Adhesins, Bacterial↗

PubMed2003

A multiprotein nuclear complex connects Fanconi anemia and Bloom syndrome.

Bloom syndrome (BS) is a genetic disorder associated with dwarfism, immunodeficiency, reduced fertility, and an elevated risk of cancer. To investigate the mechanism of this disease, we isolated from human HeLa extracts three complexes containing the helicase defective in BS, BLM. Interestingly, one of the complexes, termed BRAFT, also contains five of the Fanconi anemia (FA) complementation group proteins (FA proteins). FA resembles BS in genomic instability and cancer predisposition, but most of its gene products have no known biochemical activity, and the molecular pathogenesis of the disease is poorly understood. BRAFT displays a DNA-unwinding activity, which requires the presence of BLM because complexes isolated from BLM-deficient cells lack such an activity. The complex also contains topoisomerase IIIalpha and replication protein A, proteins that are known to interact with BLM and could facilitate unwinding of DNA. We show that BLM complexes isolated from an FA cell line have a lower molecular mass. Our study provides the first biochemical characterization of a multiprotein FA complex and suggests a connection between the BLM and FA pathways of genomic maintenance. The findings that FA proteins are part of a DNA-unwinding complex imply that FA proteins may participate in DNA repair.

Adenosine Triphosphatases↗

PubMed2002

Qscore: an algorithm for evaluating SEQUEST database search results.

A scoring procedure is described for measuring the quality of the results for protein identifications obtained from spectral matching of MS/MS data using the Sequest database search program. The scoring system is essentially probabilistic and operates by estimating the probability that a protein identification has come about by chance. The probability is based on the number of identified peptides from the protein, the total number of identified peptides, and the fraction of distinct tryptic peptides from the database that are present in the identified protein. The score is not strictly a probability, as it also incorporates information about the quality of the individual peptide matches. The result of using Qscore on a large test set of data was similar to that achieved using approaches that validate individual spectral matches, with only a narrow overlap in scores between identified proteins and false positive matches. In direct comparison with a published method of evaluating Sequest results, Qscore was able to identify an equivalent number of proteins without any identifiable false positive assignments. Qscore greatly reduces the number of Sequest protein identifications that have to be validated manually.

Algorithms↗

PubMed2002

Composition and functional characterization of the yeast spliceosomal penta-snRNP.

Pre-mRNA introns are spliced in a macromolecular machine, the spliceosome. For each round of splicing, the spliceosome assembles de novo in a series of ATP-dependent steps involving numerous changes in RNA-RNA and RNA-protein interactions. As currently understood, spliceosome assembly proceeds by addition of discrete U1, U2, and U4/U6*U5 snRNPs to a pre-mRNA substrate to form functional splicing complexes. We characterized a 45S yeast penta-snRNP which contains all five spliceosomal snRNAs and over 60 pre-mRNA splicing factors. The particle is functional in extracts and, when supplied with soluble factors, is capable of splicing pre-mRNA. We propose that the spliceosomal snRNPs associate prior to binding of a pre-mRNA substrate rather than with pre-mRNA via stepwise addition of discrete snRNPs.

Protein Biosynthesis↗

PubMed2002

Proteins released by Helicobacter pylori in vitro.

Secretion of proteins by Helicobacter pylori may contribute to gastric inflammation and epithelial damage. An in vitro analysis was designed to identify proteins released by mechanisms other than nonspecific lysis. The radioactivity of proteins in the supernatant was compared with that of the intact organism by two-dimensional gel phosphorimaging following a 4-h pulse-chase. The ratio of the amount of UreB, a known cytoplasmic protein, in the supernatant to that in the pellet was found to be 0.25, and this was taken as an index of lysis during the experiments (n = 6). Ratios greater than that of UreB were used to distinguish proteins that were selectively released into the medium. Thus, proteins enriched more than 10-fold in the supernatant compared to UreB were identified by mass spectrometry. Sixteen such proteins were present in the supernatant: VacA; a conserved secreted protein (HP1286); putative peptidyl cis-trans isomerase (HP0175); six proteins encoded by HP0305, HP0231, HP0973, HP0721, HP0129, and HP0902; thioredoxin (HP1458); single-stranded-DNA-binding 12RNP2 precursor (HP0827); histone-like DNA-binding protein HU (HP0835); ribosomal protein L11 (HP1202); a putative outer membrane protein (HP1564); and outer membrane proteins Omp21 (HP0913) and Omp20 (HP0912). All except HP0902, thioredoxin, HP0827, HP0835, and HP1202 had a signal peptide. When nalidixic acid, a DNA synthesis inhibitor, was added to inhibit cell division but not protein synthesis, to decrease possible contamination due to outer membrane shedding, two outer membrane proteins (Omp21 and Omp20) disappeared from the supernatant, and the amount of VacA also decreased. Thus, 13 proteins were still enriched greater than 10-fold in the medium after nalidixic acid treatment, suggesting these were released specifically, possibly by secretion. These proteins may be implicated in H. pylori-induced effects on the gastric epithelium.

Bacterial Proteins↗