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Williams, J. D.

Publications and source records attributed to Williams, J. D..

14 recordsLinked to original sources
bioRxiv2026

Fatty acid biosynthesis inhibitors fabimycin and triclosan trigger distinct resistance mutations in FabI and potently kill Gram-negative pathogens

In the effort to develop efficacious antibacterials that engage targets for which there is no pre-existing resistance, inhibition of the enoyl-acyl carrier protein reductase FabI has shown promise, with triclosan and fabimycin as representative members of the two major drug classes that show activity against important bacterial pathogens. Here, we use a morbidostat and whole genome sequencing to comprehensively evaluate the resistance profiles that arise in pathogenic bacteria in response to these FabI inhibitors. When assessed against E. coli, fabimycin and triclosan were found to induce primarily non-overlapping resistance profiles leading to minimal cross-resistance between the two compounds. Furthermore, in vivo evaluation of the prominent resistant mutants indicates poor fitness, with the most fit mutant still susceptible to fabimycin. Collectively, these results suggest the combination use of two antibiotics that engage different positions on the same target as a means to kill pathogenic bacteria and limit resistance.

microbiology↗
bioRxiv2026

A Data-Analysis Pipeline for High-Throughput Systematic Evolution of Ligands by Exponential Enrichment (HT-SELEX) in the Characterization of Telomeric Proteins

Telomeres are nucleoprotein structures at the ends of eukaryotic chromosomes that safeguard them from triggering inappropriate DNA damage signaling. POT1, a member of the mammalian shelterin complex, binds single-stranded (ss) telomeric DNA and blocks the activation of the ATR kinase-mediated DNA damage response at telomeres. Yet until recently, it was poorly understood how the double-stranded (ds)-ss telomeric junction was protected from DNA damage response factors. An initial study of the DNA-binding activity of human POT1 (hPOT1) using systematic evolution of ligands by exponential enrichment (SELEX) and subsequent investigation revealed that POT1 contains a binding pocket, known as the POT-hole, that binds the 5 phosphorylated dC of the telomeric ds-ss junction. The amino acid residues composing the POT-hole show full sequence identity with telomeric proteins from diverse eukaryotes, including Caenorhabditis elegans POT-1. The current study builds on this SELEX method, developing an extensive analysis pipeline for SELEX datasets sequenced by next-generation sequencing and achieving a deeper analysis of the resulting sequences. We validated our approach by applying it to the DNA-binding domain of hPOT1, yielding results consistent with a previous SELEX study. Furthermore, we employ our pipeline to characterize the DNA-binding activity of C. elegans proteins that are considered homologs of hPOT1: POT-1, POT-2, POT-3, and MRT-1. Our analysis suggests that all four proteins show a binding preference for G-enriched DNA sequences, with POT-1 additionally binding secondary structural elements. Overall, we present a bioinformatics pipeline that is accessible and applicable for determining the nucleic acid-binding properties of a variety of proteins.

biochemistry↗
bioRxiv2025

Enhanced PIEZO1 Function Contributes to the Pathogenesis of Sickle Cell Disease

Sickle cell disease (SCD), an inherited blood disorder caused by a mutation in the {beta}-globin gene, is characterized by sickle erythrocytes that are prone to hemolysis, causing anemia and vaso-occlusion crises. In sickle erythrocytes, hemoglobin aggregation is followed by altered cation permeability and subsequent dehydration. Interventions to restore cation permeability can decrease hemolysis and ameliorate the symptoms associated with SCD. PIEZO1 is a non-selective mechanosensitive cation channel that regulates erythrocyte volume. Gain-of-function (GOF) mutations in PIEZO1 cause hemolytic anemia by increasing cation permeability, leading to erythrocyte dehydration in humans and mice. Although PIEZO1 plays a key role in erythrocyte homeostasis, its role in SCD remains unknown. Here, we demonstrate that the function of the PIEZO1 channel is upregulated in sickle erythrocytes of humans and mice, and this enhancement can be restored through a dietary intervention. We found that PIEZO1 function in sickle erythrocytes resembles that of the GOF mutation causing hemolytic anemia. A diet enriched in the{omega} -3 fatty acid eicosapentaenoic (EPA) acid decreases PIEZO1 function in sickle erythrocytes and hemolysis in a mouse model of SCD. Furthermore, EPA decreases hemolysis and reduces inflammatory markers. We propose that PIEZO1 contributes to the increase in nonselective cationic conductance (i.e., Psickle), which leads to dehydration downstream of hemoglobin polymerization. Our results suggest that reducing PIEZO1 function is a promising therapeutic approach to reestablishing normal cation permeability in SCD.

physiology↗
bioRxiv2024

Ivory-billed Woodpecker and Blue Jay kent comparisons at spectrogram level reveal differences

A representative audio file for Blue Jay (Cyanocitta cristata), that is thought to be most similar to suspected kent sounds of the Ivory-billed Woodpecker (Campephilus principalis), was examined at the spectrogram level. A total of 136 other Blue Jay files were looked at and compared to kents (n>200) from six Ivory-billed Woodpecker expeditions. At this precision, differences are seen such that these two species cannot be mistaken for each other.

ecology↗
bioRxiv2024

Characterization of vaginal microbiomes in clinician-collected bacterial vaginosis diagnosed samples

Bacterial vaginosis (BV) is a type of vaginal inflammation caused by bacterial overgrowth, upsetting the healthy microbiome of the vagina. Existing clinical testing for BV is primarily based upon physical and microscopic examination of vaginal secretions, while more modern PCR-based clinical tests target panels of BV-associated microbes, such as the Labcorp NuSwab(R) test. Remnant clinician-collected NuSwab(R) vaginal swabs underwent DNA extraction and 16S V3-V4 rRNA gene sequencing to profile microbes in addition to those included in the Labcorp NuSwab(R) test. Community State Types (CSTs) were determined using the most abundant taxon detected in each sample. PCR results for NuSwab(R) panel microbial targets were compared against the corresponding microbiome profiles. Metabolic pathway abundances were characterized via metagenomic prediction from amplicon sequence variants (ASVs). Sequencing of 75 remnant vaginal swabs yielded 492 unique 16S V3-V4 ASVs, identifying 83 unique genera. NuSwab(R) assay microbe quantification was strongly concordant with quantification by sequencing (p << 0.01). Samples in CST-I (18 of 18, 100%), CST-II (3 of 3, 100%), CST-III (15 of 17, 88%), and CST-V (1 of 1, 100%) were largely categorized as BV-negative via the NuSwab(R) panel, while most CST-IV samples (28 of 36, 78%) were BV-positive or BV-indeterminate. BV-associated microbial and predicted metabolic signatures were shared across multiple CSTs. These findings show that 16S V3-V4 rRNA gene sequencing robustly reproduces PCR-based BV diagnostic testing results, accurately discriminates vaginal microbiome CSTs dominated by distinct Lactobacilli, and further elucidates BV-associated bacterial and metabolic signatures. ImportanceBacterial vaginosis (BV) poses a significant health burden for women during reproductive years and onward. Current BV diagnostics rely on either physical and microscopic evaluations by technicians or panels of select microbes. Here, we sequenced the microbiome profiles of samples previously diagnosed by the Labcorp NuSwab(R) test to better understand disruptions to the vaginal microbiome during BV. We show that microbial sequencing can reproduce targeted panel diagnostic results, while also broadly characterizing healthy and BV-associated microbial and metabolic biomarkers. This work highlights a robust, agnostic BV classification scheme with potential for future development of sequencing-based BV diagnostic tools.

microbiology↗
bioRxiv2024

Spectrograms of Ivory-billed Woodpecker (Campephilus principalis) kent sounds from seven expeditions show 587 Hertz as a pattern

Although the Ivory-billed Woodpecker is widely considered to be extinct, expeditions led by professional ornithologists have acquired visual, video, and audio evidence since 1968, with increases in data since 2000. Audio evidence for kent sounds (n=150) were examined for commonality; this was found to be true with a pattern at 587 Hz that suggests a morphological basis. Differences from other species were found on spectrograms. The patterns extend to different states and years and reinforces that the species persists.

animal behavior and cognition↗
bioRxiv2023

Metabolite Fingerprinting for Phenotypic Screening by Infrared Matrix-Assisted Laser Desorption Electrospray Ionization Mass Spectrometry

The adoption of mass spectrometry for high-throughput screening in drug discovery has become increasingly prevalent and has enabled label-free screening against diverse targets. Cellular assays for phenotypic screening, however, are primarily conducted by microscopy as there remain many challenges associated with conducting phenotypic screens via ultra-high throughput mass spectrometry. Following a simple on-plate extraction, infrared matrix assisted laser desorption electrospray ionization (IR-MALDESI) was employed to directly sample the cell lysate at a speed of one second per sample. A549 cells were treated with compounds identified as hits in literature including a recently reported glutaminase cellular screen. Among the test compounds, there were confirmed glutaminase inhibitors, proposed nuisance compounds, and cell-active but enzyme-inactive compounds. Filtered data were further processed in R for dimensionality reduction and unsupervised clustering. Though it was observed that all compounds affected the intracellular conversion of glutamine to glutamate, there were clear metabolic differences between the biochemically active compounds and the off-target hits. Moreover, two nuisance compounds were observed to cluster separately from the confirmed glutaminase inhibitors in the observed metabolite fingerprints. This proof-of-concept work establishes a complete workflow that enables highthroughput mass spectrometry-based phenotypic screening. The methods proposed herein, at the throughput enabled by IR-MALDESI, could offer a new avenue for discovery of novel drugs.

cell biology↗
bioRxiv2023

A New Platform for Label-Free, Proximal Cellular Pharmacodynamic Assays: Identification of Glutaminase Inhibitors Using Infrared Matrix-Assisted Laser Desorption Electrospray Ionization Mass Spectrometry

Cellular pharmacodynamic assays are crucial aspects of lead optimization programs in drug discovery. These assays are sometimes difficult to develop, oftentimes distal from the target and frequently low throughput which necessitates their incorporation in the drug discovery funnel later than desired. The earlier direct pharmacodynamic modulation of a target can be established, the less resources are wasted on compounds that are acting via an off-target mechanism. Mass spectrometry is a versatile tool that is often used for direct, proximal cellular pharmacodynamic assay analysis but liquid chromatography-mass spectrometry methods are low throughput and unable to fully support structure-activity relationships efforts in early medicinal chemistry programs. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) is an ambient ionization method amenable to high throughput cellular assays, capable of diverse analyte detection, ambient and rapid laser sampling process, and low cross contamination. Here we demonstrate the capability of IR-MALDESI for detection of diverse analytes directly from cells and report the development of a high throughput label free, proximal cellular pharmacodynamic assay using IR-MALDESI for discovery of glutaminase inhibitors and a biochemical assay for hit confirmation. We demonstrate the throughput with a [~]100,000 compound cellular screen. Hits from the screening were confirmed by retesting in dose-response with mass spectrometry-based cellular and biochemical assays. A similar workflow can be applied to other targets with minimal modifications, which will speed up discovery of cell active lead series and minimize wasted chemistry resources on off-target mechanisms.

biochemistry↗
bioRxiv2022

Genome-wide analysis of heat stress-stimulated transposon mobility in the human fungal pathogen Cryptococcus deneoformans

We recently reported transposon mutagenesis as a significant driver of spontaneous mutations in the human fungal pathogen Cryptococcus deneoformans during murine infection. Mutations caused by transposable element (TE) insertion into reporter genes were dramatically elevated at high temperature (37{degrees} versus 30{degrees}) in vitro, suggesting that heat stress stimulates TE mobility in the Cryptococcus genome. To explore the genome-wide impact of TE mobilization, we generated transposon accumulation lines by in vitro passage of C. deneoformans strain XL280 for multiple generations at both 30{degrees} and at the host-relevant temperature of 37{degrees}. Utilizing whole-genome sequencing, we identified native TE copies and mapped multiple de novo TE insertions in these lines. Movements of the T1 DNA transposon occurred at both temperatures with a strong bias for insertion between gene-coding regions. By contrast, the Tcn12 retrotransposon integrated primarily within genes and movement occurred exclusively at 37{degrees}. In addition, we observed a dramatic amplification in copy number of the Cnl1 (C. neoformans LINE-1) retrotransposon in sub-telomeric regions under heat-stress conditions. Comparing TE mutations to other sequence variations detected in passaged lines, the increase in genomic changes at elevated temperature was primarily due to mobilization of the retroelements Tcn12 and Cnl1. Finally, we found multiple TE movements (T1, Tcn12 and Cnl1) in the genomes of single C. deneoformans isolates recovered from infected mice, providing evidence that mobile elements are likely to facilitate microevolution and rapid adaptation during infection. Significance StatementRising global temperatures and climate change are predicted to increase fungal diseases in plants and mammals. However, the impact of heat stress on genetic changes in environmental fungi is largely unexplored. Environmental stressors can stimulate the movement of mobile DNA elements (transposons) within the genome to alter the genetic landscape. This report provides a genome-wide assessment of heat stress-induced transposon mobilization in the human fungal pathogen Cryptococcus. Transposon copies accumulated in genomes more rapidly following growth at the higher, host-relevant temperature. Additionally, movements of multiple elements were detected in the genomes of cryptococci recovered from infected mice. These findings suggest that heat stress-stimulated transposon mobility contributes to rapid adaptive changes in fungi both in the environment and during infection.

microbiology↗
bioRxiv2021

High Throughput Intact Protein Analysis Using Infrared Matrix-Assisted Laser Desorption Electrospray Ionization Mass Spectrometry

Mass spectrometry (MS) is the primary analytical tool used to characterize proteins within the biopharmaceutical industry. Electrospray ionization (ESI) coupled to liquid chromatography (LC) is the current gold standard for intact protein analysis. However, inherent speed limitations of LC/MS prevent analysis of large sample numbers (>1000) in a day. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI-MS), an ambient ionization MS technology, has recently been established as a platform for high throughput small molecule analysis. Here we report the applications of such a system for the analysis of intact proteins commonly performed within the drug discovery process. A wide molecular weight range of proteins 10 - 150 kDa was detected on the system with improved tolerance to salts and buffers compared to ESI. With high concentrations and model proteins, a sample rate up to 22 Hz was obtained. For proteins at low concentrations and in buffers used in commonly employed assays, robust data at a sample rate of 1.5 Hz was achieved, which is [~] 22x faster than current technologies used for high throughput ESI-MS-based protein assays. In addition, two multiplexed plate-based high throughput sample cleanup methods were coupled to IR-MALDESI-MS to enable analysis of samples containing excessive amounts of salts and buffers without fully compromising productivity. Example experiments, which leverage the speed of the IR-MALDESI-MS system to monitor NISTmAb reduction, protein autophosphorylation and compound binding kinetics in near real-time, are demonstrated. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=71 SRC="FIGDIR/small/467755v3_ufig1.gif" ALT="Figure 1"> View larger version (13K): org.highwire.dtl.DTLVardef@afa091org.highwire.dtl.DTLVardef@56b6ecorg.highwire.dtl.DTLVardef@1034f83org.highwire.dtl.DTLVardef@5949fb_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗
bioRxiv2021

Ultrahigh-Throughput Direct Sampling MS: Sampling at 22 Hz by Infrared Matrix-Assisted Desorption Electrospray Ionization Mass Spectrometry

Infrared Matrix-Assisted Laser Desorption Electrospray Ionization (IR-MALDESI) mass spectrometry is an ambient-direct sampling method being developed for high-throughput, label-free, biochemical screening of large-scale compound libraries. Here, we report the development of an ultrahigh-throughput continuous motion IR-MALDESI sampling approach capable of acquiring data at rates up to 22.7 samples per second in a 384-well microtiter plate. At top speed, less than 1% analyte carryover is observed from well-to-well and signal intensity relative standard deviations (RSD) of 11.5% and 20.9% for 3 M 1-hydroxymidazolam and 12 M dextrorphan, respectively, are achieved. The ability to perform parallel kinetics studies on 384 samples with ~30s time resolution using an isocitrate dehydrogenase 1 (IDH1) enzyme assay is shown. Finally, we demonstrate the repeatability and throughput of our approach by measuring 115,200 samples from 300 microtiter plate reads consecutively over 5.54 hours with RSDs under 8.14% for each freshly introduced plate. Taken together, these results demonstrate the use of IR-MALDESI at sample acquisition rates that surpass other currently reported direct sampling mass spectrometry approaches used for high throughput compound screening. For Table of Contents Only O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=125 SRC="FIGDIR/small/465730v3_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@5f916corg.highwire.dtl.DTLVardef@4473b4org.highwire.dtl.DTLVardef@82e8forg.highwire.dtl.DTLVardef@128b57b_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗
bioRxiv2020

Transposon mobilization during infection promotes drug resistance in the human fungal pathogen Cryptococcus deneoformans

When transitioning from the environment, pathogenic microorganisms must adapt rapidly to survive in hostile host conditions. This is especially true for environmental fungi that cause opportunistic infections in immunocompromised patients since these microbes are not well adapted human pathogens. Cryptococcus species are yeast-like fungi that cause lethal infections, especially in HIV-infected patients. Using Cryptococcus deneoformans in a murine model of infection, we examined contributors to drug resistance and demonstrated that transposon mutagenesis drives the development of 5-fluoroorotic acid (5FOA) resistance. Inactivation of target genes URA3 or URA5 primarily reflected the insertion of two transposable elements (TEs): the T1 DNA transposon and the TCN12 retrotransposon. Consistent with in vivo results, increased rates of mutagenesis and resistance to 5FOA and the antifungal drugs rapamycin/FK506 and 5-fluorocytosine (5FC) were found when Cryptococcus was incubated at 37{degrees} compared to 30{degrees} in vitro, a condition that mimics the temperature shift that occurs during the environment-to-host transition. Inactivation of the RNAi pathway, which suppresses TE movement in many organisms, was not sufficient to elevate TE movement at 30{degrees} to the level observed at 37{degrees}. We propose that temperature-dependent TE mobilization in Cryptococcus is an important mechanism that enhances microbial adaptation and promotes pathogenesis and drug resistance in the human host. SIGNIFICANCE STATEMENTThe incidence of infections due to fungal pathogens has dramatically increased in the past few decades with similar increases in human populations with weakened or suppressed immune systems. Understanding the mechanisms by which organisms rapidly adapt during human infection to enhance virulence and evolve drug resistance is important for developing effective treatments. We find that transposon mobilization in the human pathogen Cryptococcus causes genomic mutations in a murine model of infection and promotes resistance to antifungal drugs in vitro. Thermotolerance is a key virulence determinant for pathogenic fungi during the environment-to-host transition, and we demonstrate that a temperature increase is sufficient to trigger transposon mobilization in vitro. The link between temperature stress and transposon-associated mutations may significantly impact adaptation to the host during infection, including the acquisition of drug resistance.

microbiology↗
bioRxiv2019

Second Harmonic Generation Spectroscopy of Membrane Probe Dynamics in Gram-Positive Bacteria

Bacterial membranes are complex mixtures with dispersity that is dynamic over scales of both space and time. In order to capture adsorption onto and transport within these mixtures, we conduct simultaneous second harmonic generation (SHG) and two photon fluorescence measurements on two different gram-positive bacterial species as the cells uptake membrane-specific probe molecules. Our results show that SHG can not only monitor the movement of small molecules across membrane leaflets, but is also sensitive to higher-level ordering of the molecules within the membrane. Further, we show that the membranes of Staphylococcus aureus remain more dynamic after longer times at room temperature in comparison to Enterococcus faecalis. Our findings provide insight into the variability of activities seen between structurally similar molecules in gram-positive bacteria while also demonstrating the power of SHG to examine these dynamics.\n\nSTATEMENT OF SIGNIFICANCEBacterial membranes are highly adept at discerning and modifying their interactions with different small molecules in their environment. Here we show how second harmonic generation (SHG) spectroscopy can track the dynamics of structurally similar membrane probes in two gram-positive bacterial species. Our results reveal behavior that is dependent on both the probe molecule and the membrane composition. Specifically, we observe flip-flop between leaflets for one molecule, while the other molecule produces a signal indicative of larger scale ordering in the membrane. These phenomena can all be explained by considering potential differences in the membrane fluidity and surface charge between the two bacterial species. Overall, our work highlights the dynamic differences between bacterial membranes and SHGs sensitivity to probing these systems.

biophysics↗