Biology subjects

Brubaker, D.

Publications and source records attributed to Brubaker, D..

7 recordsLinked to original sources

bioRxiv2025

Rethinking the Estrogen Receptor Beta Dominance Hypothesis in Endometriosis: Insights from Single Cell RNA Sequencing Meta-analysis

Structured AbstractO_ST_ABSBackgroundC_ST_ABSO_LIEndometriosis is a chronic, estrogen-dependent disease characterized by the presence of endometrial-like tissue growing outside the uterus. The molecular and clinical heterogeneity of endometriosis complicate diagnostic and treatment options -- diagnostic delays of seven to ten years are common and therapies often lack long-term efficacy. Estrogen signaling and estrogen receptor beta (ER{beta}) expression is thought to be increased in endometriosis, contributing to increased cell proliferation in lesions. The "ER{beta} dominance hypothesis" is a prevailing hypothesis in the field, setting ER{beta} as a high-priority therapeutic target. If effectively modulated, ER{beta} could be the first therapy to directly target lesion biology, rather than only managing symptoms. C_LI Objective(s)O_LIWe aimed to characterize ER{beta}s expression in endometriosis by cell type and evaluate its therapeutic relevance, primarily assessing the validity of the ER{beta} dominance hypothesis. C_LI Study DesignO_LIWe reanalyzed scRNAseq data from eight previously published studies. Our final filtered dataset included 557,061 cells, the largest endometriosis single cell atlas ever constructed. We quantified gene expression levels of ESR1 and ESR2, which encode ER[a] and ER{beta} respectively, across each tissue and cell type, to identify cell-type specific drivers of ESR2/ER{beta} expression across diseased and healthy tissues. To characterize the differences between cells that uniquely express ESR1 versus those that uniquely express ESR2, we performed differential gene expression and pathway enrichment analyses. C_LI ResultsO_LICount and distribution analyses revealed no significant ESR2/ER{beta} dominance in any cell or tissue type by Fishers Exact Tests and Wilcoxon Rank Sum Tests. Differential gene expression and pathway enrichment analyses suggest distinct roles of each estrogen receptor isoform. C_LI Conclusion(s)O_LIOverall, our results argue against a simplified model of ER{beta} dominance and instead propose a dual-isoform and cell and tissue-specific framework for understanding estrogen receptor signaling in endometriosis. These findings hold important implications for future therapeutic strategies. Specifically, treatments that target ER{beta} alone may fail to account for the functional role and relative abundance of ER. In the future, therapeutic approaches that consider isoform-specific, tissue-specific, and cell-specific expression patterns may prove most effective in reducing recurrence and improving outcomes for patients. C_LI Condensation pageO_ST_ABSTweetable statementC_ST_ABSSingle cell RNA Sequencing meta-analysis shows estrogen receptor beta is not dominantly expressed in most endometriosis tissues. Estrogen receptor alpha to estrogen receptor beta ratios vary by cell type and tissue type. Each isoform directs cell-type specific behavior in endometriosis and disease-free tissues. AJOG at a GlanceO_LIWhy was this study conducted? O_LIWe wanted to characterize estrogen receptor betas expression in endometriosis and evaluate its therapeutic relevance. C_LI C_LIO_LIWhat are the key findings? O_LIEstrogen receptor beta is not dominantly expressed in any tissue. Estrogen receptor alpha and estrogen receptor beta have disease- and cell-type specific behaviors. C_LI C_LIO_LIWhat does this study add to what is already known? O_LIIt characterizes estrogen receptor isoform expression and signaling by cell type. It also challenges the current estrogen receptor beta dominance hypothesis, meaning estrogen receptor beta may not be a key driver of endometriosis. C_LI C_LI

systems biology↗

bioRxiv2025

Identifying a Vaginal Microbiome-Derived Selective Antibiotic Metabolite via Microbiome Pharmacology Analysis

The vaginal microbiome plays a critical role in maintaining immune and epithelial homeostasis in the female reproductive tract. Bacterial Vaginosis (BV) is deleterious to female health, causing the loss of beneficial Lactobacillus species, overgrowth of anaerobic taxa, changes in vaginal pH, breakdown of protective mucins and epithelial barriers, and activation of the immune system. Treatment with gel-based antibiotics (Metronidazole or Clindamycin) resolves BV for 85% of patients, but 50% of those cases recur, indicating a need to identify strategies for overcoming antibiotic resistance and achieving a more durable response. Here, we developed a systems biology approach termed Microbiome Pharmacology Analysis to characterize the antibiotic potential of vaginal microbes, their metabolites and functions, via computational fusion of human cohort multi-omics and post-drug perturbation transcriptomic profiles. We focused on Clindamycin and Metronidazole as candidate drugs and screened 780 vaginal microbiome-drug mimicry candidates to identify candidate taxa and metabolites with antibiotic potential. We demonstrate experimentally that Lactobacillus crispatus-derived Hydroxyisocaproate (HICA) selectively kills Gardnerella vaginalis and that HICA enhances epithelial barrier integrity in a human vagina-on-a-chip system. Our work demonstrates the first use of Pharmacobiome Analysis, for discovering novel, selective antibiotic metabolites for BV with implications for charting the full pharmacologic potential of the vaginal microbiome.

microbiology↗

bioRxiv2025

A Ligand-Centered Framework for γδ T Cell Activation in Colorectal Cancer Revealed by Single-Cell and Transformer-Based Perturbation

Understanding the activation mechanisms of {gamma}{delta} T cells in colorectal cancer (CRC) is critical for harnessing their therapeutic potential. Here, using an atlas of human CRC-infiltrating {gamma}{delta} T cells that we built by integrating multiple single-cell RNA-seq datasets, we developed a {gamma}{delta} T cell-refined ligand inference pipeline by combining differential gene expression, gene regulatory network prediction, ligand inference, and in silico perturbation analysis. This approach identified IL-15 and TNFSF9 (4-1BBL) as candidate ligands promoting {gamma}{delta} T cell effector function, and highlighted NCR2 and KLRC3 (NKG2E), whose in silico overexpression was associated with {gamma}{delta} T cell activation. Ligand enrichment analyses further indicated that monocytes and dendritic cells are key contributors to {gamma}{delta} T cell activation within the tumor microenvironment. Together, our results offer a systems-level view of the signaling and transcriptional programs governing {gamma}{delta} T cell phenotypes in CRC and provide a foundation for {gamma}{delta} T cell-based immunotherapies with enhanced antitumor function.

immunology↗

bioRxiv2025

Comparative Metabolomic Analysis of Vaginal Microbiota in Planktonic and Biofilm States Unveils Species-Specific Metabolic Signatures

Bacterial vaginosis (BV) affects approximately 29% of women in the U.S., with higher rates among certain demographics and up to 50% recurrence within a year. Besides complications like increased risk of sexually transmitted infections (STIs), pregnancy-related issues, it can negatively impact psychological well-being, leading to discomfort and reduced quality of life. While previous studies have provided insights into the overall metabolomic profile of healthy and diseased vaginal environments, the elucidation of individual microbial metabolite signatures remains limited. Furthermore, given that biofilms exhibit distinct metabolic requirements compared to planktonic cultures, a differential analysis of metabolites in both growth conditions could reveal potential therapeutic targets. This study presents a comprehensive metabolomic analysis and comparison of significant vaginal microbes including Lactobacillus crispatus, Gardenerella vaginalis, and Lactobacillus iners in both planktonic and biofilm growth conditions. Our analysis revealed distinct metabolite production and consumption patterns among different microbes and growth modes. In biofilm cultures, metabolite consumption is influenced by nutrient availability, which in turn regulates the profile of produced metabolites. G. vaginalis demonstrated the ability to form biofilms in various media types. Limited shared metabolic pathways in both biofilm types of G. vaginalis, highlights the unique metabolic processes involved in their formation. Despite L. crispatus suspension and biofilm cultures sharing 142 consumed and 104 produced metabolites, the biofilm culture demonstrated a remarkable metabolic shift. While comparing suspension and biofilm cultures of L. crispatus, L. iners, and G. vaginalis, we found convergence in nutrient utilization, but divergence in metabolic outputs reflecting growth-specific adaptations and underscore the importance of considering the state of existence when studying the vaginal microbiome. This study provides valuable insights into the growth mode-specific metabolic requirements of key vaginal microbes. The findings underscore the potential for leveraging metabolite-mediated microbial cross-talk as a novel therapeutic approach against BV. This avenue of research warrants further investigation, as it could lead to the development of targeted interventions that modulate the vaginal microbiome through metabolic manipulation, potentially offering more effective and personalized treatments for BV.

microbiology↗

bioRxiv2025

Single-Cell Analysis Reveals Tissue-Specific T Cell Adaptation and Clonal Distribution Across the Human Gut-Liver-Blood Axis

Understanding T cell clonal relationships and tissue-specific adaptations is crucial for deciphering human immune responses, particularly within the gut-liver axis. We performed paired single-cell RNA and T cell receptor sequencing on matched colon (epithelium, lamina propria), liver, and blood T cells from the same human donors. This approach tracked clones across sites and assessed microenvironmental impacts on T cell phenotype. While some clones were shared between blood and tissues, colonic intraepithelial lymphocytes (IELs) exhibited limited overlap with lamina propria T cells, suggesting a largely resident population. Furthermore, tissue-resident memory T cells (TRM) in the colon and liver displayed distinct transcriptional profiles. Notably, our analysis suggested that factors enriched in the liver microenvironment may influence the phenotype of colon lamina propria TRM. This integrated single-cell analysis maps T cell clonal distribution and adaptation across the gut-liver-blood axis, highlighting a potential liver role in shaping colonic immunity.

immunology↗

bioRxiv2024

Systematic Analysis of Human Colorectal Cancer scRNA-seq Revealed Limited Pro-tumoral IL-17 Production Potential in Gamma Delta T Cells

Gamma delta ({gamma}{delta}) T cells play a crucial role in anti-tumor immunity due to their cytotoxic properties. However, the role and extent of {gamma}{delta} T cells in production of pro-tumorigenic interleukin-17 (IL-17) within the tumor microenvironment (TME) of colorectal cancer (CRC) remains controversial. In this study, we re-analyzed nine published human CRC whole-tissue single-cell RNA sequencing (scRNA-seq) datasets, identifying 18,483 {gamma}{delta} T cells out of 951,785 total cells, in the neoplastic or adjacent normal tissue of 165 human CRC patients. Our results confirm that tumor-infiltrating {gamma}{delta} T cells exhibit high cytotoxicity-related transcription in both tumor and adjacent normal tissues, but critically, none of the {gamma}{delta} T cell clusters showed IL-17 production potential. We also identified various {gamma}{delta} T cell subsets, including Teff, TRM, Tpex, and Tex, and noted an increased expression of cytotoxic molecules in tumor-infiltrating {gamma}{delta} T cells compared to their normal area counterparts. Our work demonstrates that {gamma}{delta} T cells in CRC primarily function as cytotoxic effector cells rather than IL-17 producers, mitigating the concerns about their potential pro-tumorigenic roles in CRC, highlighting the importance of accurately characterizing these cells for cancer immunotherapy research and the unneglectable cross-species discrepancy between the mouse and human immune system in the study of cancer immunology.

immunology↗

bioRxiv2023

Detailed Survey of an in-vitro Intestinal Epithelium Model by Single-Cell Transcriptomics

The gut plays a critical role in maintaining human health by facilitating the absorption of nutrients, regulating metabolism, and interacting with the immune system and gut microbiota. The co-culture of two human colorectal cancer cell lines, Caco-2 and HT29, on Transwell is commonly used as an in vitro gut mimic in studies of intestinal absorption pharmacokinetics, gut mechanics, and gut-microbe interplay given the similar morphology, expression of transporters and enzymes, and barrier function. However, to sufficiently evaluate the translatability of insights from such a system to human physiological contexts, a detailed survey of cell type heterogeneity in the system and a holistic comparison with human physiology are needed to be conducted rather than by the presence of a few well-studied proteins. Single-cell RNA sequencing provides high-resolution expression profiles of cells in the co-culture, enabling the heterogeneity to be characterized and the similarity to human epithelial cells to be evaluated. Transcriptional profiles of 16019 genes in 13784 cells were acquired and compared to human epithelial cells (GSE185224). We identified the intestinal stem cell-, transit amplifying-, enterocyte-, goblet cell-, and enteroendocrine-like cells together with differentiating HT29 cells in the system based on the expression of canonical markers in healthy adult human epithelial cells. The epithelium-like co-culture was fetal intestine-like, with less variety of gene expression compared to the human gut. Transporters for major types of substance (lipid, amino acid, ion, water, etc.) were found transcribed in the majority of the enterocytes-like cells in the system. However, some of the well-studied transporters such as FATP4 and GLUT2 were absent. Toll-like receptors were not highly expressed in the sample, yet the treatment of lipopolysaccharide still caused a mild change in trans-epithelial electrical resistance and gene expression, possibly by the interaction with CD14, the co-receptor for TLRs. Overall, the Caco-2/HT29 co-culture is a cost-effective epithelium model for drug permeability testing or mechanical simulation, but its phenotypic discrepancy with the real epithelium is not negligible. As a result, its response to biological factors might not provide transferrable knowledge to the study of human gut physiology, especially the innate immune aspect.

cell biology↗