Biology subjects

Chen, V.

Publications and source records attributed to Chen, V..

At least 19 recordsLinked to original sources

bioRxiv2026

Antibody treatment targeting nitrated alpha-synuclein counteracts protein spreading pathology

-Synuclein nitration is a prominent post-translational modification in Parkinsons disease, but whether nitrated -synuclein merely reflects oxidative stress or actively contributes to pathology remains unclear. Here, we generated and characterized 6G6, an antibody selective for Tyr39-nitrated -synuclein, and tested whether targeting this modified -synuclein species affected pathology in different mouse models of -synuclein aggregation and spread. In two models of -synuclein overexpression targeting medullary vagal neurons, oxidative stress was induced by either exposure to the herbicide paraquat or transgenic heterozygous expression of the Gba1-L444P mutation. Both conditions were characterized by robust -synuclein spreading that was markedly counteracted by 6G6 administration. A third model consisted of an injection of -synuclein fibrils into the striatum of -synuclein-overexpressing mice. In this model, treatment with 6G6 protected against fibril-induced aggregate pathology and ensuing degeneration of nigral dopaminergic neurons. In a pilot human study, CSF levels of Tyr39-nitrated -synuclein were measured and found increased in Parkinson patients as compared to controls. These findings identify Tyr39-nitrated -synuclein as a pathogenic, therapeutically targetable -synuclein species linking oxidative/nitrative stress to PD pathological processes.

neuroscience↗

bioRxiv2026

Circulating miR-4532 is associated with loss of ambulation in dysferlinopathy

BackgroundLimb-girdle muscular dystrophies (LGMDs) are inherited myopathies characterized mainly by progressive weakness of the proximal muscles of the shoulder and pelvic girdle areas, leading to functional decline and eventual loss of independent ambulation. Dysferlinopathy (LGMD2B) is an autosomal recessive LGMD subtype, is caused by mutations in the DYSF gene that lead to lack of dysferlin which results in muscle death and chronic muscle fiber degeneration. Preservation of ambulation is a key clinical milestone, as loss of independent gait markedly reduces quality of life and complicates care management. Although patients often perceive functional decline before their initial clinical presentation, current clinical assessments typically detect disease progression after substantial muscle damage has occurred. MethodsIn this multigroup case-control study, we profiled plasma miRNAs from 49 genetically confirmed dysferlinopathy patients (24 ambulatory, 25 non-ambulatory) and 25 age- and sex-matched healthy controls. Total RNA was extracted from blood samples and hybridized to Affymetrix GeneChip miRNA 3.1 arrays. After quality control and filtering, differential expression analysis was performed using linear models for microarrays, adjusting for age and sex, with a false discovery rate cutoff of 10%. Results14 miRNAs were significantly altered between dysferlinopathy patients and controls. Notably, miR-4532 was upregulated in ambulatory patients relative to controls, whereas it was downregulated in non-ambulatory patients compared with ambulatory patients, although expression levels remained higher than in controls. Levels of miR-4532 were positively associated with circulating monocyte levels in ambulatory patients only. ConclusionThese results suggest that miR-4532 may be a circulating marker associated with ambulatory status in dysferlinopathy. Its known involvement in inflammatory signaling and muscle regeneration pathways underscores its potential as an early indicator for disease activity.

bioinformatics↗

bioRxiv2025

Macrophage PIM1 Drives Atherosclerosis by Enhancing Foam Cell Formation Via CD36

BackgroundAtherosclerosis is characterized by the buildup of fatty plaques that thicken and stiffen arterial walls. Macrophages (M{varphi}s) significantly contribute to this process through their scavenger receptor CD36. PIM1 is a serine/threonine kinase known to modulate immune responses and cell metabolism. However, its role in M{varphi} lipid handling and atherogenesis is not well defined. This study examines the role of PIM1 in regulating CD36 expression and function in M{varphi}s during foam cell formation and atherosclerosis progression. MethodsWe performed in vitro studies by treating murine peritoneal M{varphi}s from Pim1-/- and wild-type (WT) mice with oxidized low-density lipoprotein (oxLDL). We measured CD36, PIM1, and plaque-associated proteins and mRNA levels, oxLDL binding and uptake rates, as well as foam cell formation. For in vivo studies, we fed M{varphi}-specific PIM1-deficient (Apoe-/- Lyz2Cre/+Pim1fl/fl) and their littermate control (Apoe-/-Pim1fl/fl) mice a high-fat diet for 12 weeks. We then evaluated the plaque formation in their aortic sinuses and arches. ResultsDeletion of Pim1 in M{varphi}s reduced CD36 protein expression by up to 96.7% compared to WT controls. This led to a 49.6% decrease in foam cell formation and a 25.5% reduction in cellular cholesterol after oxLDL treatment. Pharmacological inhibition of PIM kinase activity in WT M{varphi}s also impaired oxLDL handling, with a 64.5% reduction in binding and a 57.9% in uptake. Bulk RNA-seq revealed that Pim1 deficiency downregulated PPAR{gamma} signaling. Treatment with a PPAR{gamma} agonist restored CD36 levels in the PIM1 knockdown M{varphi}s, suggesting that PIM1 regulates CD36 through PPAR{gamma}. Moreover, PIM1 M{varphi}-specific deficiency caused a 69.4% reduction in atherosclerotic plaque formation. ConclusionPIM1 acts as a key upstream regulator of CD36 by enhancing PPAR{gamma} activity in M{varphi}s. The PIM1-CD36 axis promotes oxLDL binding, uptake, and foam cell formation. Targeting the PIM1/PPAR{gamma}/CD36 pathway could offer new ways to modulate M{varphi} lipid metabolism and reduce atherosclerotic plaque progression. Non-standard Abbreviations and AcronymsELISA: enzyme-linked immunosorbent assay; HFD: high-fat diet; M{varphi}s: macrophages; MCP-1: monocyte chemoattractant protein-1; ORO: oil red O; oxLDL: oxidized low-density lipoprotein; PBS: phosphate-buffered saline; PPAR{gamma}: peroxisome proliferator-activated receptor gamma; WT: wild type.

immunology↗

bioRxiv2025

Association of E. coli Nissle with insect-parasitic nematode Steinernema hermaphroditum

Steinernema species are soil-dwelling and insect-parasitic nematodes that associate with symbiotic Xenorhabdus bacteria. During the infective juvenile (IJ) stage, Steinernema nematodes package species-specific Xenorhabdus bacteria in the anterior intestinal pockets. The nematodes can survive in the soil for months as they seek insect prey. The mechanisms of how these nematodes associate with environmental microbes other than their Xenorhabdus symbionts is barely known. Here, we report a new mechanism of E. coli Nissle (EcN) association with the nematode Steinernema hermaphroditum. We show that EcN cells are enclosed and lysed in at least four pairs of coelomocytes, suggesting these immune cells respond to bacterial invasion. During the IJ stage of nematode development, EcN cells localize to posterior intestinal vacuoles and enter the inter-cuticular space, where they proliferate, aggregate, then lyse. EcN cells expressed proteins in the cell lysates were maintained in the nematode cuticle over eight weeks in non-sterile soil. These observations suggest sequential steps of EcN colonization in the host nematodes involving an immune response that is distinctive from interactions with mutualistic symbiont. Our work establishes a novel framework of nematode-bacteria interaction with potential applications in environmental bioengineering.

microbiology↗

bioRxiv2025

Mechanisms of maternal antibody interference to rotavirus vaccination

Maternal antibodies (MatAbs) are transferred transplacentally during pregnancy and through breast milk after birth to provide protection whilst the neonatal immune response is immature. However, MatAbs also suppress the development of neonatal B cell responses via mechanisms that are not well defined. MatAbs can therefore result in poor vaccine performance in the infant, placing them at risk against potentially life-threatening pathogens such as rotavirus. It is essential we understand the mechanisms by which MatAbs interact with neonatal immunity, so that strategies can be developed to overcome this major vaccine issue. To investigate mechanisms of interference we developed a mouse model of neonatal oral rotavirus vaccination in the presence and absence of MatAbs. Oral vaccination with attenuated murine rotavirus induced robust neonatal antibody responses, whereas vaccination failed to induce seroconversion in the presence of MatAbs. Vaccination with heterologous strains, reduced MatAb titers and vaccination in Fc{gamma}RIIB knockout mice did not overcome interference. However, live vaccine replication was blocked in the presence of MatAbs and more rapid waning of MatAbs was observed following vaccination. This is indicative of premature vaccine clearance and likely reduced antigen encounter by B cells. Single-cell RNA sequencing of mesenteric lymph nodes revealed diminished plasma and germinal center B cell subpopulations as well as global reduction of interferon-stimulated genes in the presence of MatAbs. Our model has also enabled identification of strategies to reduce the effects of interference. In summary, we have tested multiple hypotheses for MatAb-mediated interference to rotavirus vaccination in a mouse model, and demonstrated that premature Fc{gamma}RIIB signaling and epitope masking are not the primary mechanisms of vaccine failure. Rather our data supports the conclusion that MatAb-mediated vaccine clearance is a key mechanism of interference to oral rotavirus vaccine.

immunology↗

bioRxiv2024

CD47 predominates over CD24 as a macrophage immune checkpoint in cancer.

Macrophages hold tremendous promise as effectors of cancer immunotherapy, but the best strategies to provoke these cells to attack tumors remain unknown. Here, we evaluated the therapeutic potential of targeting two distinct macrophage immune checkpoints: CD47 and CD24. We found that antibodies targeting these antigens could elicit maximal levels of phagocytosis when combined together in vitro. However, to our surprise, via unbiased genome-wide CRISPR screens, we found that CD24 primarily acts as a target of opsonization rather than an immune checkpoint. In a series of in vitro and in vivo genetic validation studies, we found that CD24 was neither necessary nor sufficient to protect cancer cells from macrophage phagocytosis in most mouse and human tumor models. Instead, anti-CD24 antibodies exhibit robust Fc-dependent activity, and as a consequence, they cause significant on-target hematologic toxicity in mice. To overcome these challenges and leverage our findings for therapeutic purposes, we engineered a collection of 77 novel bispecific antibodies that bind to a tumor antigen with one arm and engage macrophages with the second arm. We discovered multiple novel bispecifics that maximally activate macrophage-mediated cytotoxicity and reduce binding to healthy blood cells, including bispecifics targeting macrophage immune checkpoint molecules in combination with EGFR, TROP2, and CD71. Overall, our findings indicate that CD47 predominates over CD24 as a macrophage immune checkpoint in cancer, and that the novel bispecifics we created may be optimal immunotherapies to direct myeloid cells to eradicate solid tumors.

cancer biology↗

bioRxiv2024

Severe traumatic brain injury temporally affects cerebral blood flow, endothelial cell phenotype, and cilia

BackgroundPrevious clinical work suggested that altered cerebral blood flow (CBF) in severe traumatic brain injury (sTBI) correlates with poor executive function and clinical outcome. However, the molecular consequences of altered CBF on endothelial cells (ECs) and their blood flow-sensor organelle called cilia are not known. MethodsWe performed laser speckle contrast imaging, single cell isolation, and single cell RNA sequencing (scRNAseq) after sTBI in a closed skull, linear impact mouse model. Validation of select ciliary target protein changes was performed using flow cytometry. Additionally, in vitro experiments modeled the post-injury hypoxic environment to evaluate the effect on cilia protein ARL13B in human brain microvascular ECs. ResultsWe detected immediate reductions in CBF that were sustained for at least 100 minutes in both impacted and non-impacted sides of the brain. Our scRNAseq data detected heterogeneity in the brain cortex-derived EC cluster and demonstrated that two of five unique EC sub-clusters changed their relative proportions post-sTBI. Consistent with flow changes, we identified multiple genes associated with the fluid shear stress pathway that were significantly differentially expressed in brain ECs post-injury. Also, ECs displayed activation of ischemic pathway as early as day 1 post-injury, and enrichment of hypoxia pathway at day 7 and 28 post- injury. Arl13b ciliary gene expression was lost on day 1 in ECs cluster and remained lost for the entire course of the injury. We validated the loss of cilia protein ARL13B specifically from brain ECs as early as day 1 post-injury and detected the protein in the peripheral blood of the injured mice. We also determined that hypoxia could induce loss of ARL13B protein from cultured ECs. ConclusionsIn severe TBI, blood flow is disrupted in both impacted and non-impacted regions of the brain, creating a hypoxic environment that may influence ciliary gene and protein expression on ECs.

neuroscience↗

bioRxiv2024

Analyzing Biodiversity in Protected vs. Unprotected Tide Pools

This study assessed the efficacy of marine protected areas (MPAs) by comparing tide pool biodiversity in Half Moon Bay, California between a protected site (Fitzgerald Marine Reserve) and an unprotected site (Mavericks Beach). Samples were collected through random quadrat surveys. ImageJ and RStudio were used to analyze data through three methods utilizing the Simpsons Diversity Index along with three metrics. The unprotected Mavericks Beach displayed greater biodiversity through two metrics while the third did not find significant difference in biodiversity between sites. 30 unique species were present at each site with 43 species between both. These mixed findings prevent us from drawing definitive conclusions on the effectiveness of Half Moon Bays tide pool MPAs but suggest the MPA is less biodiverse. Potential explanations include the Intermediate Disturbance Hypothesis, lack of sample size, or that the MPA is not effective in maintaining biodiversity.

ecology↗

bioRxiv2024

IL-27 Stablizes Myc-Mediated Transcription In Memory-Fated, Vaccine-Elicited CD8+ T Cells

Protection from pathogens relies on both humoral (antibody-mediated) and cellular (T cell-mediated) responses. While infections robustly elicit both types of immunity, currently approved vaccine adjuvants largely fail to induce T cell responses on par with that instigated by infections. Our goal was to investigate the transcriptional programming that supports the formation of CD8+ T cells elicited by subunit vaccines compared to those elicited by infections. Our data show that vaccine-elicited T cells represent a transcriptionally unique subset of activated T cells with high proliferative capacity and a memory cell fate. This relies on IL-27 signaling, which stabilizes c-Myc and thereby supports the biomass acquisition necessary for clonal expansion. Collectively, our findings reveal that subunit vaccine-elicited T cells uniquely combine aspects of both memory and effector T cell subsets, and selectively utilize IL-27 signaling to sustain the clonal expansion of cells dedicated to a memory fate. One Sentence SummaryIn contrast to infection, subunit vaccines induce a distinct population of CD8+ T cells with memory fate characteristics which maintain their proliferative capacity during the expansion phase through IL-27 receptor signaling.

immunology↗

bioRxiv2023

Short-term social isolation acts on hypothalamic neurons to promote social behavior in a sex- and context-dependent manner

Social animals, including both humans and mice, are highly motivated to engage in social interactions. Short-term social isolation promotes social behavior, but the neural circuits through which it does so remain incompletely understood. Here, we sought to identify neurons that promote social behavior in single-housed female mice, which exhibit increased rates of social investigation, social ultrasonic vocalizations (USVs), and mounting during same-sex interactions that follow a period of short-term (3-day) isolation. We first used immunostaining for the immediate early gene Fos to identify a population of neurons in the preoptic hypothalamus (POA) that increase their activity in single-housed females following same-sex interactions (POAsocial neurons) but not in single-housed females that did not engage in social interactions. TRAP2-mediated chemogenetic silencing of POAsocial neurons in single-housed females significantly attenuates the effects of short-term isolation on social investigation, USV production, and mounting. In contrast, caspase-mediated ablation of POAsocial neurons in single-housed females robustly attenuates mounting but does not decrease social investigation or USV production. Optogenetic activation of POAsocial neurons in group-housed females promotes social investigation and USV production but does not recapitulate the effects of short-term isolation on mounting. To understand whether a similar population of POAsocial neurons promotes social behavior in single-housed males, we performed Fos immunostaining in single-housed males following either same-sex or opposite-sex social interactions. These experiments revealed a population of POA neurons that increase Fos expression in single-housed males following opposite-sex, but not same-sex, interactions. Chemogenetic silencing of POAsocial neurons in single-housed males during interactions with females reduces mounting but does not affect social investigation or USV production. These experiments identify a population of hypothalamic neurons that promote social behavior following short-term isolation in a sex- and social context-dependent manner.

neuroscience↗

bioRxiv2023

Post-Transplant Administration of G-CSF Impedes Engraftment of Gene Edited Human Hematopoietic Stem Cells by Exacerbating the p53-Mediated DNA Damage Response

Granulocyte colony stimulating factor (G-CSF) is commonly used as adjunct treatment to hasten recovery from neutropenia following chemotherapy and autologous transplantation of hematopoietic stem and progenitor cells (HSPCs) for malignant disorders. However, the utility of G-CSF administration after ex vivo gene therapy procedures targeting human HSPCs has not been thoroughly evaluated. Here, we provide evidence that post-transplant administration of G-CSF impedes engraftment of CRISPR-Cas9 gene edited human HSPCs in xenograft models. G-CSF acts by exacerbating the p53-mediated DNA damage response triggered by Cas9- mediated DNA double-stranded breaks. Transient p53 inhibition in culture attenuates the negative impact of G-CSF on gene edited HSPC function. In contrast, post-transplant administration of G-CSF does not impair the repopulating properties of unmanipulated human HSPCs or HSPCs genetically engineered by transduction with lentiviral vectors. The potential for post-transplant G-CSF administration to aggravate HSPC toxicity associated with CRISPR-Cas9 gene editing should be considered in the design of ex vivo autologous HSPC gene editing clinical trials. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/547089v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1dae519org.highwire.dtl.DTLVardef@1f6ddacorg.highwire.dtl.DTLVardef@535cbaorg.highwire.dtl.DTLVardef@ecbeaf_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2022

Small Molecule Screen Identifies Pyrimethamine as an Inhibitor of NRF2-driven Esophageal Hyperplasia

ObjectiveNRF2 is a master transcription factor that regulates the stress response. NRF2 is frequently mutated and activated in human esophageal squamous cell carcinoma (ESCC), which drives resistance to chemotherapy and radiation therapy. Therefore, a great need exists for NRF2 inhibitors for targeted therapy of NRF2high ESCC. DesignWe performed high-throughput screening of two compound libraries from which hit compounds were further validated in human ESCC cells and a genetically modified mouse model. The mechanism of action of one compound was explored by biochemical assays. ResultsUsing high-throughput screening of two small molecule compound libraries, we identified 11 hit compounds as potential NRF2 inhibitors with minimal cytotoxicity at specified concentrations. We then validated two of these compounds, pyrimethamine and mitoxantrone, by demonstrating their dose- and time-dependent inhibitory effects on the expression of NRF2 and its target genes in two NRF2Mut human ESCC cells (KYSE70 and KYSE180). RNAseq and qPCR confirmed the suppression of global NRF2 signaling by these two compounds. Mechanistically, pyrimethamine reduced NRF2 half-life by promoting NRF2 ubiquitination and degradation in KYSE70 and KYSE180 cells. Expression of an Nrf2E79Q allele in mouse esophageal epithelium (Sox2CreER;LSL-Nrf2E79Q/+) resulted in an NRF2high phenotype, which included squamous hyperplasia, hyperkeratinization, and hyperactive glycolysis. Treatment with pyrimethamine (30mg/kg/day, p.o.) suppressed the NRF2high esophageal phenotype with no observed toxicity. ConclusionWe have identified and validated pyrimethamine as an NRF2 inhibitor that may be rapidly tested in the clinic as a radiation and chemotherapy sensitizer for NRF2high ESCC. SummaryO_ST_ABSWhat is already known on this topic - summarise the state of scientific knowledge on this subject before you did your study and why this study needed to be doneC_ST_ABSO_LIMutational activation of the NRF2 transcription factor drives ESCC progression and therapeutic resistance. Targeted therapies to block NRF2 have not yet been realized, despite great needs. C_LI What this study adds - summarise what we now know as a result of this study that we did not know beforeO_LIA screen of >35,000 small molecules identified eleven potential NRF2 inhibitors. Pyrimethamine and mitoxantrone were validated to inhibit the expression of NRF2 and NQO1 in human ESCC cells in both dose- and time-dependent manners. C_LIO_LIPyrimethamine enhanced NRF2 protein ubiquitination and degradation, resulting a decreased half-life. C_LIO_LIA genetically modified mouse model was established to express the Nrf2E79Q mutant allele in the mouse esophageal epithelium upon tamoxifen induction. Pyrimethamine suppressed the NRF2high esophageal phenotype induced by the mutant allele. C_LI How this study might affect research, practice or policy - summarise the implications of this studyO_LIAs an FDA-approved drug, Pyrimethamine has the potential for immediate translation to a clinical trial on NRF2high ESCC in humans. C_LIO_LIFurther exploration of its mechanisms of action may lead to more potent NRF2 inhibitors for future use. C_LI

cancer biology↗

bioRxiv2022

Best Practices for Interpretable Machine Learning in Computational Biology

Advances in machine learning (ML) have enabled the development of next-generation prediction models for complex computational biology problems. These developments have spurred the use of interpretable machine learning (IML) to unveil fundamental biological insights through data-driven knowledge discovery. However, in general, standards and guidelines for IML usage in computational biology have not been well-characterized, representing a major gap toward fully realizing the potential of IML. Here, we introduce a workflow on the best practices for using IML methods to perform knowledge discovery which covers verification strategies that bridge data, prediction model, and explanation. We outline a workflow incorporating these verification strategies to increase an IML methods accountability, reliability, and generalizability. We contextualize our proposed workflow in a series of widely applicable computational biology problems. Together, we provide an extensive workflow with important principles for the appropriate use of IML in computational biology, paving the way for a better mechanistic understanding of ML models and advancing the ability to discover novel biological phenomena.

bioinformatics↗

bioRxiv2022

A Chloroplast Protein Atlas Reveals Novel Structures and Spatial Organization of Biosynthetic Pathways

Chloroplasts are eukaryotic photosynthetic organelles that drive the global carbon cycle. Despite their importance, our understanding of their protein composition, function, and spatial organization remains limited. Here, we determined the localizations of 1,032 candidate chloroplast proteins by using fluorescent protein tagging in the model alga Chlamydomonas reinhardtii. The localizations provide insights into the functions of hundreds of poorly-characterized proteins, including identifying novel components of nucleoids, plastoglobules, and the pyrenoid. We discovered and further characterized novel organizational features, including eleven chloroplast punctate structures, cytosolic crescent structures, and diverse unexpected spatial distributions of enzymes within the chloroplast. We observed widespread protein targeting to multiple organelles, identifying proteins that likely function in multiple compartments. We also used machine learning to predict the localizations of all Chlamydomonas proteins. The strains and localization atlas developed here will serve as a resource to enable studies of chloroplast architecture and functions. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/493820v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@19c871eorg.highwire.dtl.DTLVardef@16ac46corg.highwire.dtl.DTLVardef@859b7dorg.highwire.dtl.DTLVardef@1a3446e_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LI1,032 candidate chloroplast proteins localized by fluorescent tagging. C_LIO_LIThis protein atlas reveals novel chloroplast structures, functional regions, and components. C_LIO_LIPrevalent dual-organelle localization suggests extensive cross-compartment coordination. C_LIO_LIAtlas-trained machine learning predicts localizations of all C. reinhardtii proteins. C_LI

plant biology↗

bioRxiv2022

Adaptation delay causes a burst of mutations in bacteria responding to oxidative stress

Understanding the interplay between phenotypic plasticity and genetic adaptation is a long-standing focus of evolutionary biology. In bacteria, the oxidative stress response limits the formation of mutagenic reactive oxygen species (ROS) under diverse stress conditions. This suggests that the dynamics of the oxidative stress response are closely tied to the timing of the mutation supply that fuels genetic adaptation to stress. Here, we explored how mutation rates change in real-time in Escherichia coli cells during continuous hydrogen peroxide treatment in microfluidic channels. By visualising nascent DNA replication errors, we uncovered that sudden oxidative stress causes a burst of mutations. We developed a range of single-molecule and single-cell microscopy assays to determine how these mutation dynamics arise from phenotypic adaptation mechanisms. Signalling of peroxide stress by the transcription factor OxyR rapidly induces ROS scavenging enzymes. However, an adaptation delay leaves cells vulnerable to the mutagenic and toxic effects of hydroxyl radicals generated by the Fenton reaction. Resulting DNA damage is counteracted by a spike in DNA repair activities during the adaptation delay. Prior stress exposure or constitutive OxyR induction allowed cells to avoid the burst of mutations. Similar observations for alkylation stress show that mutation bursts are a general phenomenon associated with adaptation delays.

microbiology↗

bioRxiv2021

Chemoproteomics of microbiota metabolites reveals small-molecule agonists for orphan receptor GPRC5A

The microbiota generates diverse metabolites that can engage multiple pathways to modulate host physiology and disease, but their protein targets and mechanism(s) of action have not been fully elucidated. To address this challenge, we focused on indole-3-acetic acid (IAA), a prominent microbiota metabolite, and developed IAA-based chemical reporters for proteomic studies. We discovered that IAA interacts with many proteins in host cells, including small-molecule transporters, receptors and metabolic enzymes. Notably, our functional studies revealed that IAA binds to orphan G protein-coupled receptors such as GPRC5A, but only aromatic monoamines were capable of inducing GPRC5A signaling. Functional profiling of microbiota uncovered specific bacterial species and enzymes that generate GPRC5A agonists. Finally, biochemical characterization of GPRC5A activation identified more potent synthetic agonists as well as key amino acid residues involved in ligand binding. These studies highlight the utility of chemoproteomics to dissect protein targets and mechanisms of action for microbiota metabolites.

biochemistry↗

bioRxiv2020

Cancer cells are sensitive to wild-type IDH1 inhibition under nutrient limitation

Pancreatic cancer cells alter their metabolism to survive cancer-associated stress (1-4). For example, cancer cells must adapt to steep nutrient gradients that characterize the natural tumor microenvironment (TME) (5-7). In the absence of adaptive strategies, harsh metabolic conditions promote the generation of free radicals (8) and impair energy production in tumor cells. Towards this end, wild-type isocitrate dehydrogenase 1 (IDH1) activity is a metabolic requirement for cancer cells living in a harsh metabolic milieu. The cytosolic enzyme interconverts isocitrate and alpha-ketoglutarate, and uses NADP(H) as a cofactor. We show that under low nutrient conditions, the enzymatic reaction favors oxidative decarboxylation to yield NADPH and alpha-ketoglutarate. Metabolic studies showed that the IDH1 products directly support antioxidant defense and mitochondrial function in stressed cancer cells. Genetic IDH1 suppression reduced growth of pancreatic cancer cells in vitro under low nutrient conditions and in mouse models of pancreatic cancer. Surprisingly, allosteric inhibitors of mutant IDH1 proved to be potent wild-type IDH1 inhibitors under conditions specific to the TME, highlighting a natural therapeutic window. The presence of low magnesium enhanced allosteric inhibition by the drug, and ambient low glucose levels enhanced cancer cells dependence on wild-type IDH1. Thus, intrinsic TME conditions sensitized wild-type IDH1 to FDA-approved AG-120 (ivosidenib), and revealed the drug to be a potent single-agent therapeutic in cell culture and diverse in vivo cancer models. This work identified a potentially new repertoire of safe cancer therapies, including a clinically available compound, for the treatment of multiple wild-type IDH1 cancers (e.g., pancreatic).

cancer biology↗

bioRxiv2020

Urine single cell RNA-sequencing in focal segmental glomerulosclerosis reveals inflammatory signatures in immune cells and podocytes

The diagnosis of focal segmental glomerulosclerosis (FSGS) requires a renal biopsy, which is invasive and can be problematic in children and in some adults. We used single cell RNA-sequencing to explore disease-related cellular signatures in 23 urine samples from 12 FSGS subjects. We identified immune cells, predominantly monocytes, and renal epithelial cells, including podocytes. Analysis revealed M1 and M2 monocyte subsets, and podocytes showing high expression of genes for epithelial-to-mesenchymal transition (EMT). We confirmed M1 and M2 gene signatures using published monocyte/macrophage data from lupus nephritis and cancer. Using renal transcriptomic data from the Nephrotic Syndrome Study Network (NEPTUNE), we found that urine cell immune and EMT signature genes showed higher expression in FSGS biopsies compared to minimal change disease biopsies. These results suggest that urine cell profiling may serve as a diagnostic and prognostic tool in nephrotic syndrome and aid in identifying novel biomarkers and developing personalized therapeutic strategies.

immunology↗