Biology subjects

Dudley, A.

Publications and source records attributed to Dudley, A..

5 recordsLinked to original sources

bioRxiv2025

Isolation Methods of Tumor Endothelial Cells Impact AngiomiR Profiles

MicroRNAs (miRNAs) play an important role in endothelial cell growth and differentiation. Tumor angiogenesis-specific miRNAs (angiomiRs) are a subset of miRNAs that are dyregulated in tumor endothelial cells. Because of the importance of angiogenesis in cancer progression, regulation of angiomiRs may have significant therapeutic implications. However, discovery of angiomiRs has often been limited by biased model systems that may not be valid. Here, we evaluated whether the variable expression levels of angiomiRs in endothelial cells were impacted by the isolation methods used to profile them. Using an autochthonous, genetically engineered mouse model of lung adenocarcinoma, we used Nanostring to profile miRNA expression levels of normal lung endothelial cells (NECs) to tumor endothelial cells (TECs) using two endothelial cell (EC) isolation methods: 1) staining and sorting ECs directly from tumors ("in vivo"), and 2) magnetic bead isolation and sub-cloning ECs ("in vitro"). We then compared candidate angiomiRs with the profiles from two orthotopic, immunocompetent lung cancer models. When TECs were directly enriched from tumors ("in vivo" method), three candidate angiomiRs (miR-30b, miR-1981, and miR-707) were significantly lower in TECs than NECs. In contrast, when ECs were isolated and cultured ("in vitro" method), three different candidate angiomiRs (miR-200a, miR-124 and miR-186) were significantly lower in TECs than NECs. Using two independent model systems for validation, we found miR-30b to be significantly reduced in TECs using freshly sorted ECs. Conversely, the in vitro discovered angiomiR candidates did not validate in these model systems, suggesting that TECs grown in vitro may not maintain relevant angiomiR profiles or serve as an adequate method for molecular profiling. Our findings demonstrate that angiomiR expression patterns are impacted by isolation methods. Instead of relying on ECs cultured in vitro, we suggest careful validation studies of cells freshly collected from tumors before determining whether a miRNA is a bona fide angiomiR.

molecular biology↗

bioRxiv2023

Predicting the functional effect of compound heterozygous genotypes from large scale variant effect maps

BackgroundPathogenic variants in PHGDH, PSAT1, and PSPH cause a set of rare, autosomal recessive diseases known as serine biosynthesis defects. Serine biosynthesis defects present in a broad phenotypic spectrum that includes, at the severe end, Neu-Laxova syndrome, a lethal multiple congenital anomaly disease, intermediately in the form of infantile serine biosynthesis defects with severe neurological manifestations and growth deficiency, and at the mild end, as childhood disease with intellectual disability. However, because L-serine supplementation, especially if started early, can ameliorate and in some cases even prevent symptoms, knowledge of pathogenic variants is highly actionable. MethodsRecently, our laboratory established a yeast-based assay for human PSAT1 function. We have now applied it at scale to assay the functional impact of 1,914 SNV-accessible amino acid substitutions. In addition to assaying the functional impact of individual variants in yeast haploid cells, we can assay pairwise combinations of PSAT1 alleles that recapitulate human genotypes, including compound heterozygotes, in yeast diploids. ResultsResults of our assays of individual variants (in haploid yeast cells) agree well with clinical interpretations and protein structure-function relationships, supporting the use of our data as functional evidence under the ACMG interpretation guidelines. Results from our diploid assay successfully distinguish patient genotypes from those of healthy carriers and agree well with disease severity. Finally, we present a linear model that uses individual allele measurements (in haploid yeast cells) to accurately predict the biallelic function (in diploid yeast cells) of ~1.8 million allele combinations corresponding to potential human genotypes. ConclusionsTaken together, our work provides an example of how large-scale functional assays in model systems can be powerfully applied to the study of a rare disease.

genetics↗

bioRxiv2019

Selection of Candida albicans Trisomy during Oropharyngeal Infection Results in a Commensal-Like Phenotype

When the fungus Candida albicans proliferates in the oropharyngeal cavity during experimental oropharyngeal candidiasis (OPC), it undergoes large-scale genome changes at a much higher frequency than when it grows in vitro. Previously, we identified a specific whole chromosome amplification, trisomy of Chr 6 (Chr6x3), that was highly overrepresented among strains recovered from the tongues of mice with OPC. To determine the functional significance of this trisomy, we assessed the virulence of two Chr6 trisomic strains and a Chr5 trisomic strain in the mouse model of OPC. We also analyzed the expression of virulence-associated traits in vitro. All three trisomic strains exhibited characteristics of a commensal during OPC in mice. They achieved the same oral fungal burden as the diploid progenitor strain but caused significantly less weight loss and elicited a significantly lower inflammatory host response. In vitro, all three trisomic strains had reduced capacity to adhere to and invade oral epithelial cells and increased susceptibility to neutrophil killing. Whole genome sequencing of pre- and post-infection isolates found that the trisomies were usually maintained. Most post-infection isolates also contained de novo point mutations, but these were not conserved. While in vitro growth assays did not reveal phenotypes specific to de novo point mutations, they did reveal novel phenotypes specific to each lineage. These data reveal that during OPC, clones that are trisomic for Chr5 or Chr6 are selected and they facilitate a commensal-like phenotype.

genomics↗

bioRxiv2018

A polyploid admixed origin of beer yeasts derived from European and Asian wine populations

Strains of Saccharomyces cerevisiae used to make beer, bread and wine are genetically and phenotypically distinct from wild populations associated with trees. The origins of these domesticated populations are not always clear; human-associated migration and admixture with wild populations have had a strong impact on S. cerevisiae population structure. We examined the population genetic history of beer strains and find that ale strains and the S. cerevisiae portion of allotetraploid lager strains were derived from admixture between populations closely related to European grape wine strains and Asian rice wine strains. Similar to both lager and baking strains, ale strains are polyploid, providing them with a passive means of remaining isolated from other populations and providing us with a living relic of their ancestral hybridization. To reconstruct their polyploid origin we phased the genomes of two ale strains and found ale haplotypes to both be recombinants between European and Asian alleles and to also contain novel alleles derived from extinct or as yet uncharacterized populations. We conclude that modern beer strains are the product of a historical melting pot of fermentation technology.

evolutionary biology↗

bioRxiv2018

Exposure to the oral host niche yields rapid phenotypic and genotypic diversification in Candida albicans

In vitro studies suggest that stress may generate random standing variation, and that different cellular and ploidy states may evolve more rapidly under stress. Yet this idea has not been tested with pathogenic fungi growing within their host niche in vivo. Here, we analyzed the generation of both genotypic and phenotypic diversity during exposure of Candida albicans to the mouse oral cavity. Ploidy, aneuploidy, loss of heterozygosity (LOH) and recombination were determined using flow cytometry and ddRADseq. Colony phenotypic changes (CPs) in size and filamentous growth were evident without selection, and were enriched among colonies selected for LOH of the GAL1 marker. Aneuploidy and LOH occurred on all chromosomes (Chrs), with aneuploidy more frequent for smaller Chrs and whole Chr LOH more frequent for larger Chrs. Large genome shifts in ploidy to haploidy often maintained one or more heterozygous disomic Chrs, consistent with random Chr missegregation events. Most isolates displayed several different types of genomic changes, suggesting that the oral environment rapidly generates diversity de novo. In sharp contrast, following in vitro propagation isolates were not enriched for multiple LOH events, except in those that underwent haploidization and/or had high levels of Chr loss. The frequency of events was overall 100 times higher for C. albicans populations following in vivo passage compared to in vitro. These hyperdiverse in vivo isolates likely provide C. albicans with the ability to adapt rapidly to the diversity of stress environments it encounters inside the host.\n\nAuthor summaryAdaption is a continuous dynamic process that requires genotypic and phenotypic variation. Here we studied the effects of a single passage in a mouse oropharyngeal model of infection on the appearance of diversity in C. albicans, a common commensal of the human oral cavity and GI tract. We found that variation could be rapidly detected following oral colonization, with the frequency of genome change being considerably higher with pre-selection for recombination and colony phenotypic changes. Importantly, one third of all isolates had multiple genome changes, significantly higher than expected by chance alone. We suggest that some cells in the population are naturally hypervariable and that they are a major source of diversity upon which selection can act in stressful conditions in vivo and in vitro.

genetics↗