Biology subjects

Smith, D. F.

Publications and source records attributed to Smith, D. F..

14 recordsLinked to original sources

bioRxiv2026

Antigen Stimulation Reactivates HIV-1 Proviruses Despite Integration in Repressive Chromatin

Intact HIV-1 proviruses become progressively enriched in transcriptionally repressive genomic regions during long-term antiretroviral therapy (ART) and in elite controllers, raising questions about their capacity for reactivation in vivo. We used an antigen-restricted quantitative viral outgrowth assay (ag qVOA) to test whether cognate antigen stimulation can reverse latency of proviruses integrated within repressive chromatin. Using cells from two people with HIV (PWH) on ART, one on long-term treatment and one an elite controller, we show that antigen-specific stimulation induces viral outgrowth from intact proviruses integrated into a pericentromeric transition region and a zinc finger gene, respectively. These findings demonstrate that antigen recognition can overcome epigenetic constraints to reactivate proviruses with low inducibility and suggest that proviruses in so-called "deeper latency" may contribute to residual viremia and viral rebound following treatment interruption.

immunology↗

bioRxiv2026

Fung-AI: An AI/ML-driven pipeline for antifungal peptide discovery

Emerging fungal pathogens represent a concerning threat to both global health and food security. In this study, we aimed to address our rising vulnerability to fungal pathogens through the development of the Fung-AI pipeline: an AI/ML-driven approach for antifungal discovery. A generative adversarial network (GAN) was trained to generate novel candidate antifungal peptide sequences. Next, in silico antifungal and hemolytic classifiers were built to further prioritize AI-generated peptides for experimental validation. From a pool of [~]10,000 candidates, thirteen peptides were selected for testing over two-stages of experimentation. Five peptides were found to display mild antifungal activity against the wheat pathogen, Fusarium graminearum, with minimal inhibitory concentrations (MICs) ranging from 250 {micro}g/mL to 500 {micro}g/mL. Four of the five peptides also showed activity against the human pathogen, Candida albicans (MIC: 500 {micro}g/mL). Two of our AI-generated antifungal peptides additionally demonstrated low cytotoxicity in HepG2 human liver carcinoma cells (LC50 > 704.2 {micro}g/mL) indicating that they may be useful as scaffolds for future optimization for therapeutic applications. None of our peptides were found to considerably inhibit the emerging pathogen C. auris, suggesting the need for pathogen-specific down-selection of candidate peptides. Overall, we present a proof-of-principle, generative-AI-based approach for the rapid design of de novo antifungal peptides.

synthetic biology↗

bioRxiv2025

Peroxisomes regulate virulence and cell density sensing in Cryptococcus neoformans

Cryptococcus neoformans, a ubiquitous environmental fungus that causes cryptococcosis, survives in diverse environments including human hosts due to metabolic flexibility. Consequently, identifying how C. neoformans connects diverse metabolic pathways and virulence factor expression is important for understanding fungal pathogenesis. Peroxisomes play an essential role in metabolic homeostasis and regulation of carbon and lipid metabolism. In this article, we report a link between nickel exposure, a known hypoxia-mimetic and mitochondrial respiration inhibitor in yeast, and peroxisomal {beta}-oxidation. Loss of the last two genes involved in the peroxisomal {beta}-oxidation pathway, MFE2 (CNAG_05721) and POT1 (CNAG_00490), resulted in cell density-dependent virulence factor defects and growth inhibition attributed to a metabolic state involving large peroxisomes. We found that increasing cell density rescued virulence factor phenotypes and growth. Our results implicate mitochondrial retrograde signaling (RTG), a previously uncharacterized pathway in C. neoformans, in cell density sensing, peroxisomal {beta}-oxidation pathway expression, and virulence, thus highlighting a critical role for metabolism in cryptococcal virulence.

microbiology↗

bioRxiv2024

Tissue-specific and spatially dependent metabolic signatures perturbed by injury in skeletally mature male and female C57Bl/6J mice

Joint injury is a risk factor for post-traumatic osteoarthritis. However, metabolic and microarchitectural changes within the joint post-injury in both sexes remain unexplored. This study identified tissue-specific and spatially-dependent metabolic signatures in male and female mice using matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI) and LC-MS metabolomics. Male and female C57Bl/6J mice were subjected to non-invasive joint injury. Eight days post-injury, serum, synovial fluid, and whole joints were collected for metabolomics. Analyses compared between injured, contralateral, and naive mice, revealing local and systemic responses. Data indicate sex influences metabolic profiles across all tissues, particularly amino acid, purine, and pyrimidine metabolism. MALDI-MSI generated 2D ion images of bone, the joint interface, and bone marrow, highlighting increased lipid species in injured limbs, suggesting physiological changes across injured joints at metabolic and spatial levels. Together, these findings reveal significant metabolic changes after injury, with notable sex differences. Significance statementOsteoarthritis, the leading cause of disability worldwide, disproportionately affects females with sex being one of the strongest predictors of disease. This disparity is partly driven by sex-specific differences in injury susceptibility, increasing the likelihood of traumatic injury to the anterior cruciate ligament (ACL), other ligaments, and menisci. Using a non-invasive injury model, we demonstrate that injury perturbs the local joint environment and has systemic effects in a sex-specific manner. Furthermore, by leveraging matrix-assisted laser desorption ionization-mass spectrometry imaging of the joint, we provide new insight into the composition of osteochondral tissue at the metabolite level. These sexually dimorphic metabolic responses to joint injury advance current understanding of the complex sexual dimorphism in OA pathogenesis providing a foundation for targeted therapeutic strategies and improved patient outcomes for female patients.

biochemistry↗

bioRxiv2024

Glutathione metabolism impacts fungal virulence by modulating the redox environment

Pathogens must overcome the hostile conditions of their hosts to survive, proliferate and cause disease. The fungal pathogen Cryptococcus neoformans is particularly adept at mitigating challenges in the host environment and has developed an arsenal of defense mechanisms to evade oxidative and nitrosative agents released by phagocytic cells during infection. Among these mechanisms, melanin production is crucially linked to both fungal virulence and defense against harmful free radicals that facilitate host innate immunity and clearance of invading pathogens. Here, we employed comparative global metabolomics to demonstrate that metabolism of the antioxidant glutathione (GSH) is inextricably linked to redox-active processes that facilitate melanin production, and that genetic perturbations in GSH biosynthesis affect fungal growth and virulence in a murine model of cryptococcosis. Furthermore, we show that disruption of GSH biosynthesis leads to overaccumulation of reducing and acidic compounds in the extracellular environment of mutant cells. These changes not only impacted melanin formation but also influenced titan cell and urease production as well as survival in macrophages. Overall, these findings highlight the importance of redox homeostasis and metabolic compensation in pathogen adaptation to the host environment and suggest new avenues for antifungal drug development.

microbiology↗

bioRxiv2022

High-resolution promoter interaction analysis in Type 3 Innate Lymphoid Cells implicates Batten Disease gene CLN3 in Crohn's Disease aetiology

Innate lymphoid cells (ILCs) are rare, tissue-resident innate lymphocytes that functionally mirror CD4+ T helper cell lineages but lack antigen receptors. Type 3 ILCs (ILC3s) are enriched in the gut, airways, and mucosal lymphoid tissues, where they regulate inflammation and promote barrier integrity. To define the regulatory architecture of primary human ILC3s, we map promoter-anchored chromosomal contacts using high-resolution, low-input Promoter Capture Hi-C (PCHi-C) in these cells alongside CD4+ T cells. By combining statistical detection with a PCHi-C-adapted Activity-by-Contact approach, we link promoters to distal regulatory elements, identifying hundreds of ILC3-specific contacts. We use these maps to connect genome-wide association study (GWAS) risk variants for Crohns disease to target genes using multiCOGS, a Bayesian framework that integrates PCHi-C with summary-statistic imputation and multivariate fine-mapping. This analysis highlights both known and unanticipated candidates, including CLN3, a causal gene for the neurodevelopmental Batten disease. Using a mouse ILC3-like cell line, we show that Cln3 is downregulated upon cytokine stimulation, and Cln3 overexpression alters stimulation-induced transcriptional programmes and cytokine secretion. Extending this approach, we generate a catalogue of ILC3-linked risk genes for five additional autoimmune conditions and show that they are enriched for regulators of the ILC3 inflammatory response identified in a CRISPR interference screen. Together, these findings illuminate long-range gene control in ILC3s and prioritise known and newly implicated autoimmune risk genes with potential roles in this clinically important cell type.

genetics↗

bioRxiv2021

Cryptococcus neoformans releases proteins during intracellular residence that affect the outcome of the fungal-macrophage interaction

Cryptococcus neoformans is a facultative intracellular pathogen that can replicate and disseminate in mammalian macrophages. In this study, we analyzed fungal proteins identified in murine macrophage-like cells after infection with C. neoformans. To accomplish this, we developed a protocol to identify proteins released from cryptococcal cells inside macrophage-like cells; we identified 127 proteins of fungal origin in infected macrophage-like cells. Among the proteins identified was urease, a known virulence factor, and others such as transaldolase and phospholipase D, which have catalytic activities that could contribute to virulence. This method provides a straightforward methodology to study host-pathogen interactions. We chose to study further Yor1, a relatively uncharacterized protein belonging to the large family of ATP binding cassette transporter (ABC transporters). These transporters belong to a large and ancient protein family found in all extant phyla. While ABC transporters have an enormous diversity of functions across varied species, in pathogenic fungi they are better studied as drug efflux pumps. Analysis of C. neoformans yor1{Delta} strains revealed defects in non-lytic exocytosis and capsule size, when compared to wild-type strains. We detected no difference in growth rates, cell body size and vesicle secretion. Our results indicate that C. neoformans releases a large suite of proteins during macrophage infection, some of which can modulate fungal virulence and are likely to affect the fungal-macrophage interaction.

microbiology↗

bioRxiv2021

Machine learning approaches to identify sleep genes

Genetics impacts sleep, yet, the molecular mechanisms underlying sleep regulation remain elusive. We built machine learning (ML) models to predict genes based on their similarity to known sleep genes. Using a manually curated list of 109 labeled sleep genes, we trained a prediction model on thousands of published datasets, representing circadian, immune, sleep deprivation, and many other processes. Our predictions fit with prior knowledge of sleep regulation and also identify several key genes/pathways to pursue in follow-up studies. We tested one of our findings, the NF-{kappa}B pathway, and showed that its genetic alteration affects sleep duration in mice. Our study highlights the power of ML to integrate prior knowledge and genome-wide data to study genetic regulation of sleep and other complex behaviors.

genomics↗

bioRxiv2020

Short-term exposure to intermittent hypoxia in mice leads to changes in gene expression seen in chronic pulmonary disease

Obstructive sleep apnea (OSA) results from episodes of airway collapse and intermittent hypoxia and is associated with a host of health complications including dementia, diabetes, heart failure, and stroke. Although the lung is the first organ to sense changes in inspired oxygen levels, little is known about the consequences of IH to the lung hypoxia-inducible factor (HIF)-responsive pathways. Furthermore, cellular mechanisms causing disease progression across multiple systems in OSA are unknown. We hypothesized that exposure to IH would lead to up- and down-regulation of diverse expression pathways and that individual cell populations would show distinctive responses to IH. We identify changes in circadian and immune pathways in lungs from mice exposed to IH. Among all cell types, endothelial cells show the most prominent transcriptional changes. Interestingly, up-regulated genes in endothelial, fibroblast, and myofibroblast cells were enriched for genes associated with pulmonary fibrosis and pulmonary hypertension. These genes include targets of several drugs currently used to treat chronic pulmonary diseases. Our results reveal potential candidates for cell-targeted therapy seeking to minimize pulmonary effects of OSA. A better understanding of the pathophysiologic mechanisms underlying diseases associated with OSA could improve our therapeutic approaches, directing therapies to the most relevant cells and molecular pathways.

pathology↗

bioRxiv2019

A single-sample circadian biomarker that performs across populations and platforms

BackgroundFor circadian medicine to influence health, such as when to take a drug or undergo a procedure, a practical way to measure body time is needed. Recent machine learning algorithms show that gene expression data from blood and skin can provide reliable estimates of body time. However, for clinical viability, a biomarker must be easily measured and generalizable to a broad population. It is not clear that any circadian biomarker yet satisfies these criteria.\n\nResultsWe analyzed 24 h molecular rhythms in human dermis and epidermis at three distinct body sites, leveraging both longitudinal and population data. Circadian clock function was strongest in the epidermis, regardless of body site. We identified a 12-gene biomarker set that reported circadian phase to within 3 hours from a single sample of epidermis--the skins most superficial layer. This set performed well across body sites, ages, sexes, and detection platforms.\n\nConclusionsThis research shows that the clock in epidermis is more robust than dermis regardless of body site. To encourage ongoing validation of this biomarker in diverse populations, diseases, and experimental designs, we developed SkinPhaser--a user-friendly app to test biomarker performance in datasets (https://github.com/gangwug/SkinPhaser).

systems biology↗

bioRxiv2019

A Large-scale Study Reveals 24 hour Operational Rhythms in Hospital Treatment

Hospitals operate 24 hours a day, and it is assumed that critical decisions occur continuously around the clock. However, many aspects of hospital operation occur at particular times of day, including medical team rounding and shift changes. It is unclear if this impacts patient care, as an empirical account of 24 h treatment patterns is lacking. We analyzed the daily distribution of ~120K doses of 12 separate drugs in 1,486 inpatients at a major childrens hospital in the U.S. Treatment orders and administration were strongly time-of-day-dependent, marked by distinct morning time surges and overnight lulls. These 24 h rhythms in treatment were remarkably consistent across drugs, diagnoses, and hospital units. In sum, nearly one-third of all 116,975 orders for treatment were placed between 8 AM and 12 PM. This rhythm in hospital medicine coincided with medical team rounding time, not necessarily immediate medical need. Lastly, we show that the clinical response to hydralazine, an acute antihypertensive, is dosing time-dependent and greatest at night, when the fewest doses were administered. The prevailing dogma is that hospital treatment is administered as needed regardless of time of day. Our findings challenge this notion and reveal a potential operational barrier to best clinical care.\n\nSIGNIFICANCE STATEMENTThe order and administration of hospital treatment was characterized by morning time surges and overnight lulls, regardless of drug type, diagnosis or care unit. As the first large-scale account of 24 h rhythms in hospital medicine, this study identifies a potential operational barrier to best clinical care. Critical clinical decisions should be made around the clock; pain, infection, hypertensive crisis, and other conditions do not occur selectively in the morning. Systemic bias in the timing of medicine is also at odds with circadian biology, which can influence when certain treatments are most effective or safe. Prevailing dogma is that hospital treatment is administered as needed regardless of time of day. Our findings challenge this notion and suggest that time of day in hospital operations deserves further consideration.

epidemiology↗

bioRxiv2019

Dosing Time Matters

Trainees in medicine are taught to diagnose and administer treatment as needed; time-of-day is rarely considered. Yet accumulating evidence shows that [~]half of human genes and physiologic functions follow daily rhythms. Circadian medicine aims to incorporate knowledge of these rhythms to enhance diagnosis and treatment. Interest in this approach goes back at least six decades, but the path to the clinic has been marked by starts, stops, and ambiguity. How do we move the field forward to impact clinical practice? To gain insight into successful strategies, we studied the results of more than 100 human trials that evaluated time-of-administration of drugs.

epidemiology↗

bioRxiv2018

A population-based human enCYCLOPedia for circadian medicine

The discovery that half of the mammalian protein-coding genome is clock-regulated has clear implications for medicine. Indeed, recent studies demonstrate time-of-day impact on therapeutic outcomes in human heart disease and cancer. Yet biological time is rarely given clinical consideration. A key barrier is the absence of information on the what and where of molecular rhythms in the human body. Here, we have applied CYCLOPS, an algorithm designed to reconstruct sample order in the absence of time-of-day information, to the GTEx collection of 632 human donors contributing 4,292 RNA-seq samples from 13 distinct human tissue types. We identify rhythms in expression across the body that persist at the population-level. This includes a set of ubiquitous cyclers comprised of well-established circadian clock factors but also many genes without prior circadian context. Among thousands of tissue-divergent rhythms, we discover a set of genes robustly oscillating in cardiovascular tissue, including key drug targets relevant to heart disease. These results also have implications for genetic studies where circadian variability may have masked genetic influence. It is our hope that the human enCYCLOPedia helps drive the translation of circadian biology into prospective clinical trials in cardiology and many other therapeutic areas.\n\nOne Sentence SummaryBioinformatic analyses on thousands of human tissue samples reveals an enCYCLOPedia of rhythmic gene expression across the body and identifies key translational opportunities for circadian medicine in cardiovascular disease.

bioinformatics↗

bioRxiv2018

P-bodies and the miRNA pathway regulate translational repression of bicoid mRNA during Drosophila melanogaster oogenesis

Embryonic axis patterning in Drosophila melanogaster is partly achieved by mRNAs that are maternally localized to the oocyte; the spatio-temporal regulation of these transcripts stability and translation is a characteristic feature of oogenesis. While protein regulatory factors are necessary for the translational regulation of some maternal transcripts (e.g. oskar and gurken), small RNA pathways are also known to regulate mRNA stability and translation in eukaryotes. MicroRNAs (miRNAs) are small RNA regulators of gene expression, widely conserved throughout eukaryotic genomes and essential for animal development. The main D. melanogaster anterior determinant, bicoid, is maternally transcribed, but it is not translated until early embryogenesis. We investigated the possibility that its translational repression during oogenesis is mediated by miRNA activity. We found that the bicoid 3UTR contains a highly conserved, predicted binding site for miR-305. Our studies reveal that miR-305 regulates the translation of a reporter gene containing the bicoid 3UTR in cell culture, and that miR-305 only partially contributes to bicoid mRNA translational repression during oogenesis. We also found that Processing bodies (P-bodies) in the egg chamber may play a role in stabilizing bicoid and other maternal transcripts. Here, we offer insights into the possible role of P-bodies and the miRNA pathway in the translational repression of bicoid mRNA during oogenesis.

cell biology↗