Biology subjects

Brown, A. N.

Publications and source records attributed to Brown, A. N..

9 recordsLinked to original sources

bioRxiv2025

Late treatment initiation leads to reduced antiviral potency

The timing of initiation is critical in antiviral treatment and viral dynamic (VD) modeling is a powerful tool to study the within-host viral load changes and evaluate antiviral treatment effects using mathematical equations. Previous simulation studies have shown that early treatment initiation is critical to maximize the therapeutic response in antiviral treatment in an acute viral infection such as influenza and SARS-CoV-2. A recent experimental study demonstrated that late therapy initiation can lead to diminished antiviral potency. However, most VD model simulations with varying treatment initiation time accounted only for the effect of initiation condition (i.e., state of different cell populations when the therapy started), the loss of drug potency has been under-investigated. This may overestimate the antiviral effect, potentially resulting in suboptimal dose selection. To this end, we aimed to characterize relationship between the drug potency (EC50) and the timing of drug addition, using nirmatrelvir and GS-441524 against SARS-CoV-2 as an example. Viral load data were obtained from in vitro experiments with various drug concentrations and treatment initiated between 0 to 3 days post infection. EC50 values were fitted for each treatment initiation group and were found to vary with the timing of treatment initiation in both drugs. Also, a VD model with time-varying EC50 provided better fits than a constant EC50 model (BIC = 1667.90 vs. 1677.84). Further simulations also indicated that a constant EC50 model overestimated the antiviral efficacy when treatment started late. These findings highlighted the importance of considering EC50 shift when optimizing dosage regimens for patients presenting late.

pharmacology and toxicology↗

bioRxiv2025

SFPQ Promotes Homologous Recombination via mRNA Stabilization of RAD51 and Its Paralogs

Double-strand break (DSB) repair occurs through non-homologous end joining (NHEJ) or homologous recombination (HR). To identify non-canonical factors that influence DSB repair outcomes, we parsed data from pooled genetic screens. Through this approach, we identified the splicing factor SFPQ, which has been previously reported to associate with DSBs and promote repair. Here, we show that SFPQ depletion alters DSB repair via HR. However, in contrast to other published work, we find that SFPQ does not localize to DSBs but instead stabilizes the expression of RAD51 and its paralogs independently of p53 activation or DNA damage. Our findings suggest that SFPQ contributes to constitutive DSB repair by maintaining RAD51 paralog mRNA stability rather than through direct interaction with DSBs or RAD51 protein and highlight indirect mechanisms by which RNA-binding proteins can influence genome stability.

molecular biology↗

bioRxiv2025

The FANCD2-FANCI heterodimer coordinates chromatin openness and cell cycle progression throughout DNA double-strand break repair.

The FANCD2-FANCI heterodimer contributes to DNA repair at interstrand crosslinks and sites of replication stress. This complex has been physically and mechanistically linked to double-strand break (DSB) repair, but its role in that process remains undefined. Here we show that the FANCD2-FANCI heterodimer dynamically interacts with open chromatin regions, including transient, DSB-induced open chromatin, where it can be stabilized by co-activation by the DNA repair kinase ATM and the Fanconi anemia core ubiquitin ligase. The loaded FANCD2-FANCI heterodimer stabilizes open chromatin and promotes resection and loading of RPA through increased association of BRCA1 and BLM. Chromatin-loaded FANCD2-FANCI has a second distinct function promoting a G2 arrest that is dependent on the ATR-CHK1-WEE1 axis. Our results support a two-step genome surveillance model in which FANCD2-FANCI monitors open chromatin sites and is stably loaded to coordinate DNA repair activities in response to signaling from a DNA repair kinase.

cell biology↗

bioRxiv2025

Transcription can be sufficient, but is not necessary, to advance replication timing

DNA replication timing (RT) is correlated with transcription during cell fate changes but there are many exceptions and our understanding of this relationship suffers from a paucity of reductionist approaches. Here, we manipulated length and strength of transcription in hybrid-genome mouse embryonic stem cells (mESCs) at a single locus upstream of the silent, late replicating, Pleiotrophin (Ptn) gene, directly comparing RT to nascent transcription rates at engineered vs. wild-type alleles. First, we inserted four reporter genes that differ only in their promoter. Two promoters transcribed the reporter gene at high rates and advanced RT. The other two transcribed at lower rates and did not advance RT. Since these promoters may prove useful in applications where effects on RT are undesirable, we confirmed the inability of one of them to advance RT at numerous ectopic sites. We next juxtaposed these same four promoters upstream of the Ptn transcription start site where they all transcribed the 96kb Ptn gene and advanced RT to different extents correlated with transcription rates. Indeed, a doxycycline-responsive promoter, which could not advance RT when induced as a small reporter gene, elicited a rapid and reversible RT advance proportional to the rate of transcription, providing direct evidence that transcription itself can advance RT. However, deletion of the Ptn promoter and enhancer, followed by directed differentiation to neural precursors, eliminated induction of transcription throughout the entire Ptn replication domain, without preventing the switch to early replication. Our results provide a solid empirical base with which to re-evaluate many decades of literature, demonstrating that length and strength of transcription is sufficient but not necessary to advance RT. Our results also provide a robust system in which to rapidly effect an RT change, permitting mechanistic studies of the role of transcription in RT and the consequences of RT changes to epigenomic remodeling.

genomics↗

bioRxiv2024

A novel method for integrating genomic and Tn-Seq data to identify common in vivo fitness mechanisms across multiple bacterial species

Sepsis is life-threatening organ dysfunction due to an unregulated immune response to infection. Bacteremia is a leading cause of sepsis, and members of the Enterobacterales cause nearly half of bacteremia cases annually. While previous Tn-Seq studies to identify novel bacteremia-fitness genes have provided valuable insight into virulence mechanisms, evidence for common pathways across species is lacking. To identify common fitness pathways in five bacteremia- caused Enterobacterales species, we utilized the JCVI pan-genome pipeline to integrate Tn-Seq fitness data with multiple available functional data types. Core genes from species pan-genomes were used to construct a multi-species core pan-genome, producing 2,850 core gene clusters found in four out of the five species. Integration of Tn-Seq fitness data enabled identification of 373 protein clusters that were conserved in all five species. A scoring rubric and filter was applied to these clusters, which incorporated Tn-Seq fitness defects, operon localization, and antibiotic susceptibility data, which reduced the number of bacteremia-fitness genes and identified seven common fitness mechanisms. Independent mutational validation of one prioritized fitness gene, tatC, showed reduced fitness in vivo and increased susceptibility to beta- lactams that were restored following tatC complementation in trans. By integrating known operon structures and antibiotic susceptibility with Tn-Seq fitness data, common genes within the core pan-genome emerged and revealed mechanisms that are essential for colonization of, or survival in, the mammalian bloodstream. Our prediction and validation of tatC as a common bacteremia fitness factor and contributor of antibiotic resistance supports the utility of this bioinformatic approach. This study represents a major step forward to prioritize potentially novel targets for therapy against these deadly widespread sepsis infections. Author SummaryBacteremia is a leading cause of sepsis, a life-threatening condition where an unregulated immune response to infection causes systemic organ failure. Nearly half of bacteremia cases are caused by members of the Gram-negative bacterial taxonomic order Enterobacterales. Given the public health impact of bacteremia and the reduction of existing antibiotic treatment options, novel strategies are needed to combat these infections. Pan-genome software was used to predict seven shared fitness pathways in these bacteria that may serve as novel targets for treatment of bacteremia. Briefly, a scoring rubric was applied to shared pan-genome clusters, which incorporated multiple data types, including Tn-Seq fitness defects, operon localization, and antibiotic susceptibility data to rank and prioritize fitness genes. To validate one of our predictions, mutations were constructed in tatC, which showed both reduced fitness in mice and increased susceptibility to beta-lactam antibiotics; complementation restored fitness and antibiotic susceptibility to wild type levels. This study takes a novel bioinformatics approach to build a core pan-genome across multiple distantly related bacteria to integrate computational and experimental data to predict important shared fitness genes and represents a major step forward toward identifying novel targets of therapy against these deadly widespread life-threatening infections.

bioinformatics↗

bioRxiv2024

Fitness Factor Genes Conserved within the Multi-species Core Genome of Gram-negative Enterobacterales Species Contribute to Bacteremia Pathogenesis

There is a critical gap in knowledge about how Gram-negative bacterial pathogens, using survival strategies developed for other niches, cause lethal bacteremia. Facultative anaerobic species of the Enterobacterales order are the most common cause of Gram-negative bacteremia, including Escherichia coli, Klebsiella pneumoniae, Serratia marcescens, Citrobacter freundii, and Enterobacter hormaechei. Bacteremia often leads to sepsis, a life-threatening organ dysfunction resulting from an unregulated immune response to infection. Despite a lack of specialization for this host environment, Gram-negative pathogens cause nearly half of bacteremia cases annually. Based on our existing Tn-Seq fitness factor data from a murine model of bacteremia combined with comparative genomics of the five Enterobacterales species above, we prioritized 18 conserved fitness genes or operons for further characterization. Each mutant in each species was used to cochallenge C57BL/6 mice via tail vein injection along with the respective wild-type strain to determine competitive indices for each fitness gene or operon. Among the five species, we found three fitness factor genes, that when mutated, attenuated the mutant for all species in the spleen and liver (tatC, ruvA, gmhB). Nine additional fitness factor genes or operons were validated as outcompeted by wild-type in three or four bacterial species in the spleen (xerC, wzxE, arcA, prc, apaGH, atpG, lpdA, ubiH, aroC). Overall, 17 of 18 fitness factor mutants were attenuated in at least one species in the spleen or liver. Together, these findings allow for the development of a model of bacteremia pathogenesis that may include future targets of therapy against bloodstream infections. >Author SummaryFrequent cases of bacteremia plague our ICUs, bone marrow transplant units, and inpatient facilities. Nearly half of these infections are caused by Gram-negative bacteria. The Enterobacterales order including E. coli, K. pneumoniae, S. marcescens, C. freundii, and E. hormaechei are leading causes of bacteremia. An alarming proportion of these are due to antibiotic-resistant isolates, which are four times more likely to kill than antibiotic-susceptible isolates. Clearly, we need new therapeutic targets to treat cases of bacteremia and sepsis. Previously, it has been unclear what genes contribute to their ability to survive in this hostile host environment. We have previously undertaken unbiased genetic screens to identify 18 genes shared by all five bacterial genera that are required for survival in blood and blood-filtering organs. These include genes that encode proteins that maintain proton motive force, resist antimicrobial peptides and complement, mediate genome maintenance, transport key metabolites and proteins, avoid oxidative stress, acquire iron, and regulate key pathways. Mutants, constructed in these shared genes in the five species, were validated for a high proportion of genes as critical for infection in the mouse model of bacteremia.

microbiology↗

bioRxiv2023

Master transcription factor binding sites are necessary for early replication control element activity

Eukaryotic genomes replicate in a defined temporal order called the replication timing (RT) program. RT is developmentally regulated with potential to drive cell fate transitions, but mechanisms controlling RT remain elusive. We previously identified "Early Replication Control Elements" (ERCEs) necessary for early RT, domain-wide transcription, 3D chromatin architecture and compartmentalization in mouse embryonic stem cells (mESCs) but, deletions identifying ERCEs were large and encompassed many putative regulatory elements. Here, we show that ERCEs are compound elements whose RT activity can largely be accounted for by multiple sites of diverse master transcription factor binding (subERCEs), distinguished from other such sites by their long-range interactions. While deletion of subERCEs had large effects on both transcription and RT, deleting transcription start sites eliminated nearly all transcription with moderate effects on RT. Our results suggest a model in which subERCEs respond to diverse master transcription factors by functioning both as transcription enhancers and as elements that organize chromatin domains structurally and support early RT, potentially providing a feed-forward loop to drive robust epigenomic change during cell fate transitions.

molecular biology↗

bioRxiv2023

Conserved metabolic regulator ArcA responds to oxygen availability, iron limitation, and cell envelope perturbations during bacteremia

Bacteremia, a systemic infection associated with severe clinical outcomes, is often caused by Gram-negative facultative anaerobes. ArcAB, a two-component regulatory system that represses aerobic respiration, is a key mediator of metabolic adaptation for such bacteria. Using targeted mutational analysis informed by global genetic screens, we identified the arcA gene as promoting fitness of Klebsiella pneumoniae, Citrobacter freundii, and Serratia marcescens but not Escherichia coli in a murine model of bacteremia. Engineered mutants lacking arcA exhibit a dysregulated response to changes in oxygen availability, iron limitation, and membrane perturbations, all of which bacterial cells experience during infection. The genetic response of the arcA mutants relative to wild-type strains to the cationic antimicrobial peptide polymyxin B demonstrates an expanded role for ArcA as an activator in response to membrane damage in addition to metabolic adaptation. ArcA function is furthermore linked to electron transport chain activity based on its response to uncoupling of proton motive force by carbonyl cyanide-m-chlorophenylhydrazone (CCCP). Differences in lactate and acetate levels as well as lactate dehydrogenase activity between arcA mutant and wild-type cells following CCCP treatment establish an ArcA-mediated shift to fermentation independent of oxygen availability. This study highlights the semi-conserved role of ArcA during bacteremia and consolidates infection phenotypes into a comprehensive model based on respiratory activity. AUTHOR SUMMARYInfections of the bloodstream are life-threatening and can result in sepsis, an overreaction of the host immune system that ultimately damages the body. Gram-negative bacteria are responsible for causing many cases of bloodstream infections, also referred to as bacteremia. The long-term goal of our work is to understand how these bacteria establish and maintain infection during bacteremia. We have previously identified the transcription factor ArcA, which promotes fermentation in bacteria, as a likely contributor to the growth and survival of bacteria in this environment. Here, we study ArcA in the Gram-negative species Citrobacter freundii, Klebsiella pneumoniae, and Serratia marcescens. Our findings aid in determining how these bacteria sense their environment, utilize nutrients, and generate energy while also countering attacks from the host immune system. This information is critical for developing better models of infection to inform future therapeutic development.

microbiology↗

bioRxiv2021

Diet-derived metabolites and mucus link the gut microbiome to fever after cytotoxic cancer treatment

Not all cancer patients with severe neutropenia develop fever, and the fecal microbiome may play a role. In neutropenic hematopoietic cell transplant patients (n=119), 63 (53%) developed a subsequent fever and had increased fecal Akkermansia muciniphila, a mucus-degrading bacteria (p=0.006, corrected for multiple comparisons). In mouse models, two therapies, irradiation and melphalan, similarly expanded A. muciniphila. Dietary restriction of unirradiated mice also expanded A. muciniphila and thinned the colonic mucus layer. Azithromycin treatment depleted A. muciniphila and preserved colonic mucus. Dietary restriction raised colonic luminal pH and reduced acetate, propionate, and butyrate. Culturing A. muciniphila with lower pH and increased propionate prevented utilization of mucin. Treating irradiated mice with azithromycin or propionate preserved the mucus layer, lessened hypothermia, and reduced inflammatory cytokines in the colon. These results suggest that diet, metabolites and colonic mucus link the microbiome to neutropenic fever, and could guide future microbiome-based preventive strategies.

microbiology↗