Biology subjects

Hewitt, F. C.

Publications and source records attributed to Hewitt, F. C..

3 recordsLinked to original sources

bioRxiv2026

Predicting neutron radiation exposure characteristics from an in vitro human skin model using RNA-seq

Accurate assessment of low-dose neutron radiation exposure remains a central challenge in biodosimetry, particularly for applications requiring non-invasive sample types such as skin. Here, we characterized the transcriptional response of a three-dimensional in vitro human skin model (EpiDermFT) to neutron irradiation at doses up to 0.75 Gy, measured from pre-exposure through a 14-day post-exposure period. RNA sequencing revealed greater than 800 significantly altered genes, including upregulation of FOS, FOSB, CDKN1A, MDM2, and GADD45A, and downregulation of NRG1, H3C11, and CENPX. Gene ontology enrichment indicated activation of DNA damage checkpoint signaling, cell cycle arrest, and stress-response pathways, alongside suppression of nucleosome assembly and DNA replication processes. Machine learning models trained on transcriptomic features exhibited strong predictive performance across biodosimetric endpoints. Classification models accurately distinguished irradiated from sham samples (AUC > 0.99), and regression models achieved high accuracy for estimating both absorbed dose (R2 = 0.97) and days post-exposure (R2 = 0.99). The latter, while highly predictive, may partially reflect transcriptional shifts associated with progressive degradation of the in vitro tissue model over time. Collectively, these findings demonstrate that RNA-based molecular signatures from human skin tissue provide a robust framework for quantitative estimation of neutron radiation exposure and temporal response dynamics.

genomics↗

bioRxiv2021

Detection of ESKAPE pathogens and Clostridioides difficile in Simulated Skin Transmission Events with Metagenomic and Metatranscriptomic Sequencing

1BackgroundAntimicrobial resistance is a significant global threat, posing major public health risks and economic costs to healthcare systems. Bacterial cultures are typically used to diagnose healthcare-acquired infections (HAI); however, culture-dependent methods provide limited presence/absence information and are not applicable to all pathogens. Next generation sequencing (NGS) has the capacity to detect a wide variety of pathogens, virulence elements, and antimicrobial resistance (AMR) signatures in healthcare settings without the need for culturing, but few research studies have explored how NGS could be used to detect viable human pathogen transmission events under different HAI-relevant scenarios. MethodsThe objective of this project was to assess the capability of NGS-based methods to detect the direct and indirect transmission of high priority healthcare-related pathogens. DNA was extracted and sequenced from a previously published study exploring pathogen transfer with simulated skin containing background microorganisms, which allowed for complementary culture and metagenomic analysis comparisons. RNA was also isolated from an additional set of samples to evaluate metatranscriptomic analysis methods at different concentrations. ResultsUsing various analysis methods and custom reference databases, both pathogenic and non-pathogenic members of the microbial community were taxonomically identified. Virulence and AMR genes known to reside within the community were also routinely detected. Ultimately, pathogen abundance within the overall microbial community played the largest role in successful taxonomic classification and gene identification. ConclusionsThese results illustrate the utility of metagenomic analysis in clinical settings or for epidemiological studies, but also highlight the limits associated with the detection and characterization of pathogens at low abundance in a microbial community.

genomics↗

bioRxiv2017

Toward Rapid Sequenced-Based Detection and Characterization of Causative Agents of Bacteremia

Rapid pathogen diagnosis and characterization performed by metagenomic DNA sequencing may permit physicians to better target therapies in order to improve patient outcomes. To this end, a novel sample-to-answer workflow was assembled to enable rapid clinical detection of causative pathogens of bacteremia in whole blood utilizing metagenomic sequence data captured by the MinION. Rapid lysis, nucleic acid purification, host depletion, and genomic DNA library preparation permitted the detection of multiple bacterial and fungal agents spiked into whole blood, with sequencing commencing within 40 minutes of sample receipt. A hybrid detection strategy utilizing targeted PCR detection of specific pathogens of concern was adopted to improve overall sensitivity. As a proof of concept, primers for relatively long amplicons (~ 1800 bp) were selected to enable the specific detection of Yersinia pestis. The resulting amplicon library was spiked onto the same sequencing flow cell used to perform genomic sequencing, permitting simultaneous pathogen detection via both targeted and untargeted sequencing workflows. Sensitivities on the order of 1x10^6 cells/mL and 1x10^5 cells/mL were achieved for untargeted and targeted detection, respectively, of Y. pestis genomes spiked into whole blood. Bacterial and fungal species present in the ZymoBIOMICS Microbial Community Standard were also detected when spiked at similar levels. Variable quality of sequence reads was observed between the transposase-based and ligation-based library preparation methods, demonstrating that the more time consuming ligation-based approach may be more appropriate for the workflow described herein. Overall, this approach provides a foundation from which future point of care platforms could be developed to permit characterization of bacteremia within hours of admittance into a clinical environment.\n\nAuthor SummaryCases of bacteremia in the U.S. present a significant clinical challenge, especially due to rising rates of antimicrobial resistant strains. Rapid diagnosis of the etiologic pathogen and underlying drug resistance genetic signatures between the first and second antibiotic dose should improve patient outcomes and may permit physicians to better target antibiotic therapies without turning to broad spectrum antibiotics, which may further propagate resistant strains. The methods described herein have been developed to enhance the real time nature of the MinION sequencer. DNA sequencing and real time analysis begin within 40 minutes of sample receipt (as opposed to hours or days for common clinical nucleic acid extraction or blood culture techniques). The incorporation of sensitivity enhancements, such as methylation-based pulldown of human DNA or PCR targeted for pathogens of interest, ensures that this assay can detect bacterial blood infections at clinically relevant levels. The pathogen-agnostic aspect of the assay could one day allow clinicians to identify any unknown bacterial, fungal, or viral DNA in a sample. Ultimately, this study serves as an important step toward establishing a pipeline to rapidly detect and characterize pathogens present in whole blood.

genomics↗