Biology subjects

Freed, S.

Publications and source records attributed to Freed, S..

3 recordsLinked to original sources

bioRxiv2025

Genomic Characterization of a Severe West Nile Virus Transmission Season using a Single Reaction Amplicon Sequencing Approach

West Nile virus (WNV) is an endemic arthropod-borne virus that has routinely caused seasonal outbreaks in the United States since it was first detected in 1999. While phylogenetic studies have shown how WNV has diversified and undergone genotype replacement since introduction, more geographically focused studies are needed to understand intricate transmission dynamics at local and regional scales. In this study, we validate the IDT xGen WNV panel, a novel single reaction amplicon-based Next-Generation Sequencing approach, to generate high-quality WNV genomes and compare it to the "Primal Scheme" assay for WNV, a common amplicon sequencing strategy. We show that the IDT xGen WNV panel generated complete and accurate WNV genomes and was more robust to amplicon drop out compared to the current sequencing approaches. Additionally, we used this approach to generate 100 complete WNV genomes from surveillance pools of mosquitoes collected in Nebraska during the 2023 outbreak. Our discrete phylogeographic analysis revealed substantial genetic diversity in WNV genomes from 2023 with minimal clustering across the state. This study demonstrated the utility of a single reaction amplicon-based sequencing approach to generate quality WNV genomes from routine surveillance samples and characterize WNV transmission dynamics in a high-incidence setting.

genomics↗

bioRxiv2023

The Overexpression of AmpC is Not Required but May Contribute to Imipenem/Relebactam Non-susceptibility

In the US, carbapenem resistance in Pseudomonas aeruginosa is strongly linked to the regulation of chromosomal resistance determinants, AmpC and OprD. The {beta}-lactamase AmpC requires overexpression and genetic modifications to be capable of inhibiting imipenem activity. The outer membrane porin OprD can be downregulated or undergo genetic modifications that strongly correlate with imipenem non-susceptibility. Co-administration of imipenem and the {beta}-lactamase inhibitor, relebactam, can lower imipenem MICs and restore susceptibility. However, how this occurs in P. aeruginosa isolates that do not overproduce AmpC or produce a functional OprD for imipenem entry is not understood. Therefore, we investigated whether imipenem could enter P. aeruginosa in the absence of OprD and whether any of the chromosomal {beta}-lactamases (AmpC, OXA-51, PIB-1) contributed to imipenem and/or imipenem/relebactam non-susceptibility. This investigation evaluated 17 imipenem non-susceptible clinical isolates and 3 laboratory strains of PAO1, two of which were porin deletion mutants for either oprD or opdP. Expression of OXA-50 and PIB-1 RNA was similar to PAO1. However, all 20 isolates exhibited AmpC induction under sub-lethal exposure to imipenem. This occurred in the absence of detectable OprD protein in 18 isolates. Collectively, our data identify that OprD is not the only channel required for imipenem entry and that in many isolates the restored susceptibility to imipenem by imipenem/relebactam was due to the interaction of relebactam on the overexpression of AmpC due to imipenem induction.

microbiology↗

bioRxiv2020

Algorithmic Assessment of Missense Mutation Severity in the Von-Hippel Lindau Protein

Von Hippel-Lindau disease (VHL) is an autosomal dominant rare disease that causes the formation of angiogenic tumors. When functional, pVHL acts as an E3 ubiquitin ligase that negatively regulates hypoxia inducible factor (HIF). Genetic mutations that perturb the structure of pVHL result in dysregulation of HIF, causing a wide array of tumor pathologies including retinal angioma, pheochromocytoma, central nervous system hemangioblastoma, and clear cell renal carcinoma. These VHL-related cancers occur throughout the lifetime of the patient, requiring frequent intervention procedures, such as surgery, to remove the tumors. Although VHL is classified as a rare disease (1 in 39,000 to 1 in 91,000 affected) there is a large heterogeneity in genetic mutations listed for observed pathologies. Understanding how these specific mutations correlate with the myriad of observed pathologies for VHL could provide clinicians insight into the potential severity and onset of disease. Using a set of 285 ClinVar mutations in VHL, we developed a multiparametric scoring algorithm to evaluate the overall clinical severity of missense mutations in pVHL. The mutations were assessed according to eight weighted parameters as a comprehensive evaluation of protein misfolding and malfunction. Higher mutation scores were strongly associated with pathogenicity. Our approach establishes a novel in silico method by which VHL-specific mutations can be assessed for their severity and effect on the biophysical functions of the VHL protein.View Full Text

pathology↗