Biology subjects

Long, T.

Publications and source records attributed to Long, T..

At least 19 recordsLinked to original sources

bioRxiv2026

Defining nutrient dependencies of intestinal Klebsiella pneumoniae guides rational probiotic engineering

Klebsiella pneumoniae is a major cause of antibiotic-resistant infections, yet the ecological determinants enabling its intestinal expansion remain poorly defined. Here, we combined in vivo transcriptomics and metabolomics to identify carbon sources exploited by K. pneumoniae during colonization of the antibiotic-disrupted gut across strains and dietary contexts. Despite its metabolic breadth, K. pneumoniae consistently exploited a restricted set of substrates, including arabinose, raffinose, oligogalacturonate, arabitol, xylose, and gluconate, that contributed to intestinal fitness, with related metabolic signatures also observed in antibiotic-treated human samples. Guided by these findings, we engineered Escherichia coli Nissle to enhance consumption of these nutrients and assembled a defined consortium, NICHE, designed to reinforce colonization resistance through nutrient competition. NICHE restricted intestinal expansion of multidrug-resistant and hypervirulent K. pneumoniae in preventive and therapeutic settings, with efficacy influenced by dietary nutrient availability. Together, this work defines exploitable K. pneumoniae nutrient dependencies and a framework for rational probiotic engineering.

microbiology↗

bioRxiv2026

Biology Undergraduates Use of AI-Enabled Feedback to Revise Scientific Writing: Recipience and Barriers

Background: Instructors and education researchers are increasingly developing feedback with the assistance of generative AI, which we refer to as AI-enabled feedback, yet we do not know whether established understandings of feedback uptake and effectiveness apply similarly in AI-mediated contexts. This makes the recipience of AI-enabled feedback a central concern to understanding how to best support students in developing their scientific writing skills. This study examined how two teams of undergraduate biology students used AI-enabled feedback to revise their scientific writing, and the extent to which their revisions addressed that feedback. The teams revised their research proposal drafts after receiving AI-enabled feedback developed by their instructor with the aid of a custom AI tool. Results: Our findings show that AI-enabled feedback prompted actionable revision across both teams. Each team addressed approximately half of the feedback comments, indicating comparable levels of uptake despite their differing approaches to revision. We observed that both teams were inconsistent in providing the additional information and reasoning requested in the feedback, and they struggled to address comments requiring self-evaluation. Taken together, we see that students often addressed the more easily actionable parts of AI-enabled feedback while omitting more demanding revisions. Conclusions: These findings provide behavioral evidence of how students used AI-enabled feedback to revise their scientific writing. The observed patterns suggest that the content of AI-enabled feedback supported meaningful revision, while limitations in students agency, strategies, and volition likely shaped their implementation of comments. Although the degree of uptake observed in this study is encouraging, it is unlikely that a single application of generative AI within the feedback process will resolve all the challenges associated with feedback uptake.

scientific communication and education↗

bioRxiv2026

Large Language Models Predict Human Social Behavior via Interpretable Mechanisms

The development of large language models (LLMs) offers promising opportunities for predicting human behavior across diverse contexts. However, most prior work has emphasized behavioral imitation, with limited attention to transparent or interpretable models of the cognitive mechanisms underlying human decisions. In this study, we introduce MindEvolve, an autonomous workflow designed to predict behavior in social interactions by generating interpretable symbolic models of cognition. We systematically evaluate the modeling capabilities of multiple LLMs across a battery of socioeconomic games covering four core domains of social cognition: economic preferences, social preferences, social reasoning (theory of mind), and recursive planning. The symbolic models proposed by the LLMs are assessed by expert human evaluators for interpretability and theoretical coherence. Our results show that while most LLMs robustly capture economic and social preferences in relatively simple strategic settings, and in some cases generate novel models that integrate broader knowledge than those proposed by human experts, their capacity to model more complex psychological processes remains limited. However, a subset of state-of-the-art models demonstrates promising performance in capturing higher-order reasoning processes such as theory of mind and recursive planning. Together, these findings highlight the emerging potential of LLMs not only to imitate behavior, but to generate interpretable, mechanistic accounts of human social decision-making. Our results provide a roadmap for advancing LLM-based cognitive modeling toward human-expert-level theory construction.

neuroscience↗

bioRxiv2026

High resolution spatial transcriptomics decodes the microenvironmental determinants of response to Nr-CWS therapy in cervical precancerous lesions

Immunotherapy with Nocardia rubra cell wall skeleton (Nr-CWS) can clear human papillomavirus (HPV) and induce regression of cervical precancerous lesions, yet many patients do not respond. The cellular and molecular basis for this heterogeneous clinical outcome remains unknown. Using high-resolution spatial transcriptomics, we profiled squamous intraepithelial lesions (SIL) from patients stratified by their subsequent response to Nr-CWS therapy. We discovered that non-responders were characterized by an immunosuppressive epithelial phenotype, defined by aberrant expression of the pro-inflammatory alarmin S100A9 in squamous epithelial cells (SECs), which was associated with poorer overall survival in cervical cancer. Non-responding lesions were enriched in proliferative (MKI67+) and pro-angiogenic (VEGFA+) SEC subpopulations and spatially organized into a pro-tumorigenic cellular neighborhood (CN-10) overexpressing S100A9 and ANXA1. In contrast, responders exhibited protective glandular-cell-enriched neighborhoods (CN-13) and supportive spatial interactions between lymphatic endothelial cells and fibroblasts. The unfavorable microenvironment was further defined by an immunosuppressive cloak of VCAN+ stromal cells and CD163+ M2 macrophages encircling SECs. Thus, response to Nr-CWS is determined by a pre-existing spatial network of epithelial-stromal-immune interactions, providing a roadmap for patient stratification and rational combination therapies.

cancer biology↗

bioRxiv2026

Intrinsic resistance networks shape cefiderocol susceptibility in ST258 Klebsiella pneumoniae

Cefiderocol (CFDC) is a siderophore-conjugated cephalosporin developed to overcome multidrug resistance in Gram-negative bacteria. Despite its unique iron-dependent entry mechanism, CFDC resistance has emerged in Klebsiella pneumoniae, primarily driven by alterations in siderophore transport and {beta}-lactamase evolution; however, the broader intrinsic resistome that supports CFDC tolerance remains incompletely defined. Here, we performed high-density transposon mutagenesis (Tn-Seq) in the epidemic ST258 K. pneumoniae to map the functional genetic landscape of CFDC susceptibility. Tn-Seq identified siderophore uptake components (tonB and cirA) as the dominant determinants of CFDC resistance. In contrast, disruption of genes involved in peptidoglycan recycling (ampG, ldcA), synthesis (mrcB, lpoB) and enterobacterial common antigen (ECA) biosynthesis (wzxE, wzyE), as well as deletion of plasmid-encoded blaKPC-3, increased CFDC susceptibility. In a CFDC-resistant {Delta}tonB strain, targeting these envelope homeostasis pathways yielded only limited resensitization relative to the siderophore-competent parental strain. Deletion of blaKPC-3 produced the greatest increase in susceptibility, reducing the CFDC MIC four-fold. This pattern is consistent with a model in which reduced CFDC influx in the {Delta}tonB background lowers intracellular drug exposure to levels at which the otherwise limited anti-CFDC activity of KPC {beta}-lactamase becomes sufficient to drive resistance. Together, these data define a hierarchical genetic architecture for CFDC resistance in ST258 K. pneumoniae, in which iron-dependent drug uptake is primary, {beta}-lactamase activity is secondary, and intrinsic envelope stress buffering shapes bacterial fitness once CFDC enters the cell.

microbiology↗

bioRxiv2025

Distinct and Shared Impacts of Virulence Plasmids on the Phenotype and Transcriptome in Convergent Carbapenem-Resistant and Hypervirulent Klebsiella pneumoniae

The global rise of convergent carbapenem-resistant and hypervirulent Klebsiella pneumoniae (CR-hvKp) represents a major clinical challenge, yet the role of virulence plasmids (pVirs) in shaping bacterial physiology and pathogenicity remains incompletely understood. Using a CRISPR-Cas9-based curing system, we precisely eliminated pVirs from two clinical CR-hvKp strains with distinct genetic backgrounds, ST23-KL1 (blaNDM-1) and ST11-KL64 (blaKPC-2). Loss of pVir conferred fitness advantages in vitro, reduced capsule production and hypermucoviscosity, and promoted biofilm formation, while markedly attenuating virulence in murine sepsis models. Despite this reduction, the pVir-cured ST23-KL1 strain retained higher virulence than the pVir-cured ST11-KL64 strain, underscoring the contribution of chromosomal background to pathogenic potential. Transcriptomic profiling revealed both shared and strain-specific transcriptional responses to pVir deletion, with broader perturbations observed in the ST11-KL64 strain. pVir removal had limited effects on antibiotic MICs. Complementation experiments further demonstrated differential regulatory roles of the rmpADC and rmpA2D2 operons in capsule expression and hypermucoviscosity across the two strains. Together, these findings establish pVirs as central determinants of CR-hvKp virulence and highlight complex host-plasmid interactions that influence bacterial adaptation and pathogenicity. IMPORTANCEThe emergence of carbapenem-resistant and hypervirulent Klebsiella pneumoniae (CR-hvKp) poses a critical threat to global health, yet the contribution of virulence plasmids (pVirs) to bacterial fitness and pathogenicity remains poorly defined. By employing a CRISPR-Cas9-based curing strategy, we dissected the role of pVirs in two genetically distinct CR-hvKp strains and uncovered their multifaceted impact on capsule production, hypermucoviscosity, biofilm formation, and virulence. Our findings reveal that pVir loss confers fitness advantages in vitro while attenuating virulence in vivo, with strain-specific transcriptional responses and differential regulation by rmp operons. These results underscore the complex interplay between plasmid-encoded and chromosomal determinants in shaping CR-hvKp pathogenicity and adaptation, offering mechanistic insights that may inform future therapeutic strategies targeting plasmid-mediated virulence.

microbiology↗

bioRxiv2025

Rapid Liquid Chromatography-Mass Spectrometry (rLC-MS) for Deep Metabolomics Analysis of Population Scale Studies

Mass spectrometry (MS)-based metabolomics is a key technology for the interrogation of exogenous and endogenous small molecule mediators that influence human health and disease. To date, however, low throughput of MS systems have largely precluded large-scale metabolomics studies of human populations, limiting power to discover physiological roles of metabolites. Here, we introduce a fully automated rapid liquid chromatography-mass spectrometry (rLC-MS) system coupled to an AI-enabled computational pipeline that enables high-throughput, reproducible, non-targeted metabolite measurements across tens of thousands of samples. This system captures thousands of polar, amphipathic and nonpolar (lipid) metabolites in a human plasma sample in 53 seconds of analytical time, enabling analysis of greater than 1,000 samples per day per instrument. To demonstrate the discovery power of the rLC-MS platform, a subset of samples from Sapients DynamiQ biorepository - comprised of 62,039 total plasma samples collected longitudinally from 11,045 individuals - were selected for deep analysis by rLC-MS to capture a rich, dynamic landscape of chemical variation that reflects both physiological processes and environmental influences. 26,042 plasma samples with matched real-world data (RWD) were chosen for the study, representing 6,935 individuals with diverse demographic backgrounds and disease profiles. Unbiased exploratory analysis revealed human metabotypes that correlate with heterogenous disease phenotypes, including key sub-populations of cardiometabolic and other human diseases. Moreover, a metabolic aging clock machine learning model trained on healthy individuals in this dataset accurately predicted accelerated aging in various chronic diseases, with dynamic reversal of metabolic aging following definitive therapy. These data demonstrate that the rLC-MS platform enables prediction of clinically relevant physiological states from plasma metabolomics at scale in human populations.

biochemistry↗

bioRxiv2025

CRISPR/Cas9 editing of the wheat iron sensor TaHRZ1 confirms its conserved role in iron homeostasis and allocation in grains

Plants rely on specialized sensing systems, including transcriptional regulators, to maintain iron (Fe) homeostasis. Among these, Hemerythrin RING Zinc finger (HRZ) proteins have emerged as key regulators of Fe homeostasis. In this study, six Triticum aestivum (wheat) HRZ homoeologs belonging to TaHRZ1 and TaHRZ2, were identified by BLAST search using rice (Oryza sativa) HRZ sequences and mapped to chromosomes 1 and 3. These encode proteins with conserved N-terminal Hemerythrin (HHE) domains and C-terminal CHY-RING and Zn-ribbon motifs. Phylogenetic analysis grouped these genes into distinct clades, while expression profiling revealed strong root-specific and Fe-responsive expression patterns, indicating roles in nutrient sensing. Functional conservation was demonstrated by complementation of the Arabidopsis thaliana bts-1 mutant, where both wheat genes restored normal Fe regulation. Full-length TaHRZ1 and TaHRZ2 interacted with members of wheat bHLH IVc transcription factors, while truncated versions lacking the RING domain did not, emphasizing their conserved role in protein interactions. CRISPR-Cas9 editing of the conserved HHE3 domain in all the TaHRZ1 homoeologs, coupled with GRF4-GIF1 chimeric protein, achieved 6.4-8.8% regeneration efficiency in wheat. Elemental analysis indicated enhanced Fe loading in the grains of the edited lines, particularly in the scutellum, suggesting improved iron partitioning compared to the wild type. Additionally, qRT-PCR revealed upregulation of TaFIT and TaIRO3, and downregulation of IDEF1 in edited lines, supporting an important regulatory role for TaHRZ1 in Fe homeostasis signalling. These findings position TaHRZ1 as a valuable target for biofortification strategies to enhance Fe content in wheat grains.

plant biology↗

bioRxiv2025

Bringing the Genetically Minimal Cell to Life on a Computer in 4D

We present a whole-cell spatial and kinetic model for the 100 minute cell cycle of the genetically minimal bacterium, JCVI-syn3A. This is the first simulation of a complete cell cycle in 4D including all genetic information processes, metabolic networks, growth, and cell division. Integrating hybrid computational methods, dynamics of the morphological transformations were achieved. Growth is driven by synthesis of lipids and membrane proteins and constrained by new fluorescence imaging data. Chromosome replication and segregation is controlled by essential SMC and topoisomerase proteins in Brownian dynamics simulations with replication rates responding to dNTP pools from metabolism. The model captures the origin to terminus ratio measured in our DNA sequencing and recovers other experimental measurements like doubling time, mRNA half-lives, protein distributions, and ribosome counts. Because of stochasticity, each replicate cell is unique. Not only do we predict average behavior for partitioning to daughter cells, we predict the heterogeneity among them.

biophysics↗

bioRxiv2025

Global motor system suppression as the primary mechanism of human action stopping: challenging the pause-then-cancel model

The ability to stop a planned or ongoing action is fundamental to inhibitory control. A recent theory proposes that stopping involves two distinct phases: an initial global suppression of motor activity ("pause") followed by a selective cancellation of the targeted action. However, the necessity of a second "cancel" stage remains debated. We tested whether global suppression alone is sufficient to stop movement by analysing electromyography from task-relevant agonist and antagonist muscles, alongside transcranial magnetic stimulation measures of global motor suppression from task-irrelevant muscles, during a stop-signal task in adult human participants of both sexes. In Experiment 1, reanalysis of ballistic finger movements revealed that agonist muscle offset consistently preceded behavioural stopping, aligning with the time course of global suppression. In Experiment 2, we extended these findings to whole-arm reaching movements, demonstrating that global motor suppression persisted beyond the termination of muscle activity when stopping prevented movement initiation, but disengaged in time for antagonist activation used to interrupt movements once they had begun. These findings challenge the pause-then-cancel model, instead supporting a single-stage global suppression framework. They also suggest that the global suppression mechanism is not a rigid, top-down stopping mechanism but rather part of a broader motor control system that flexibly adjusts movement commands based on task demands. Significance StatementA recent theory suggests that stopping an action involves a two-stage process: first pausing movement execution, then selectively cancelling specific motor commands. Our study challenges this view, demonstrating that global motor suppression alone is sufficient to terminate motor commands. Using electromyography and transcranial magnetic stimulation during two distinct motor tasks, we show that suppression fully curtails agonist motor commands before behavioural stopping, without requiring a distinct cancellation step. These findings reshape our understanding of action stopping and provide a foundation for exploring the pathophysiology of movement and neuropsychiatric disorders associated with impaired behavioural suppression.

neuroscience↗

bioRxiv2025

miR-486-3p ameliorates aging-related impaired angiogenesis in adult moyamoya disease by targeting Forkhead Box O4

ObjectiveInvestigate miR-486-3ps role in alleviating age-related angiogenic decline in Moyamoya disease (MMD) by regulating senescent endothelial cells. MethodsClinical analysis of 151 MMD patients correlated age with postoperative angiogenesis (DSA grading). Senescent HUVECs (SA-{beta}-gal>80%) exhibited elevated SASP factors (IL-6/IL-8/MCP-1). Functional assays (EdU/Transwell/Matrigel) and bioinformatics identified miR-486-3p targets, validated via luciferase/Western blot. ResultsPatients [≥]35 years had 3.67-fold higher risk of poor angiogenesis (OR=3.67). Senescent HUVECs showed 10-16-fold higher SASP secretion (p<0.01). miR-486-3p overexpression increased proliferation (EdU+25%), migration (1.7-fold), and tube formation (+40% branches) in senescent cells (p<0.01) and enhanced angiogenesis in vivo (p<0.001). miR-486-3p directly targeted FOXO4, reducing its luciferase activity (-35%) and protein levels (-37%) (p<0.01), suppressing SASP. ConclusionmiR-486-3p enhances post-revascularization angiogenesis in MMD by inhibiting FOXO4-mediated SASP, offering a therapeutic target and predictive biomarker.

molecular biology↗

bioRxiv2025

A method for the detection and enrichment of endogenous cereblon substrates

C-Terminal cyclic imides are posttranslational modifications on proteins that are recognized and removed by the E3 ligase substrate adapter cereblon (CRBN). Despite the observation of these modifications across the proteome by mass spectrometry-based proteomics, an orthogonal and generalizable method to visualize the C-terminal cyclic imide would enhance detection, sensitivity, and throughput of endogenous CRBN substrate characterization. Here we develop an antibody-like reagent, termed "cerebody," for visualizing and enriching C-terminal cyclic imide-modified proteins. We describe the engineering of CRBN derivatives to produce cerebody and use it to identify CRBN substrates by Western blot and enrichment from whole cell and tissue lysates. CRBN substrates identified by cerebody enrichment are mapped, validated, and further characterized for dependence on the C-terminal cyclic imide modification. These methods will accelerate the characterization of endogenous CRBN substrates and their regulation.

biochemistry↗

bioRxiv2025

Integrative spatial transcriptomic analysis pinpoints the role of TaMCO3 encoding ferroxidase in wheat root tip iron mobilization

Roots play an critical role in the sensing and absorption of essential minerals from the rhizosphere. Iron (Fe) deficiency, for example, triggers a well-known series of physiological and molecular responses within roots that facilitate uptake, which differs between monocots and dicots. In monocots, little is known about molecular responses that occur within specific root development zones in response to iron deprivation, and how these differences results in overall nutrient uptake. Here, we conducted a transcriptome analysis of wheat root tips under Fe deficiency (-Fe) and performed a comparative transcriptome analysis with the previous datasets generated from the whole root. Gene ontology analysis of differentially expressed genes highlighted the significance of oxidoreductase activity and metal/ion transport in the root tip, which are critical for Fe mobilisation. Interestingly, wheat, an allohexaploid species consisting of three different genomes (A, B, and D) displayed varying gene expression levels arising from the three genomes that contributed to similar molecular functions. Detailed analysis of oxidoreductase function at the root tip revealed multiple multi-copper oxidase (MCO) proteins, such as Fe-responsive TaMCO3, that likely contribute to the overall ferroxidase activity. Detailed characterisation of TaMCO3 shows that it complements the yeast FET3 mutant and rescues the -Fe sensitivity phenotype of Arabidopsis atmco3 mutants by enhancing vascular Fe loading. Transgenic wheat lines overexpressing TaMCO3 exhibited increased root Fe accumulation and improved tolerance to -Fe by augmenting the expression of Fe-mobilizing genes. Our findings highlight the role of spatially resolved gene expression in -Fe responses, suggesting strategies to reprogram cells for improved nutrient stress tolerance.

plant biology↗

bioRxiv2024

The transcription factor ZmbZIP75 promotes both grain filling and kernel dehydration in maize

Selecting both high-yield and low-kernel-moisture varieties is essential for modern maize production, but relevant breeding efforts are hindered by a lack of valuable regulatory genes. Here, we demonstrate that the transcription factor (TF) basic leucine zipper 75 (ZmbZIP75) promotes grain yield and reduces kernel moisture in maize. Knockout of ZmbZIP75 results in defective grain filling and kernel dehydration, whereas ZmbZIP75 overexpression confers increased grain yield per plant and decreased kernel moisture without altering plant architecture. Mechanistically, during the grain filling stage, ZmbZIP75 is transcriptionally induced by maternal-derived basal abscisic acid (ABA) and directly activates multiple core starch synthesis-related genes and key TFs, thereby promoting grain filling and final yield. In the late stage of kernel development, high concentrations of zygotic ABA enhance ZmbZIP75 phosphorylation through SnRK2.10. The phosphorylated ZmbZIP75 subsequently transactivates and interacts with TF VP1 to synergistically promote kernel dehydration. This study thus highlights the potential of ZmbZIP75 for engineering both high-yield and low-kernel-moisture varieties to meet the demands of high-efficient maize production. IN A NUTSHELLO_ST_ABSBackgroundC_ST_ABSHigh grain yield in maize is generally associated with elevated kernel moisture at harvest, which is the main limiting factor for modern maize production. Therefore, it is of importance to select varieties with both high-yield and low-kernel-moisture. These traits are largely controlled by grain filling and kernel dehydration, two tightly connected processes during maize kernel development. Abscisic acid (ABA) is well-documented for its vital role in grain filling and dehydration maturation. However, the molecular mechanisms by which ABA coordinates these two processes remain unclear. QuestionWhat are the regulatory factors involved, and how do they mediate ABA signaling to coordinate grain filling and dehydration maturation in maize? FindingsZmbZIP75 is transcriptionally induced by ABA and directly activates multiple core starch synthesis-related genes and key TFs in developing maize endosperm, thereby promoting grain filling. In developing embryo, ABA enhances ZmbZIP75 phosphorylation via SnRK2.10. The phosphorylated ZmbZIP75 then transactivates and interacts with VP1 to synergistically promote kernel dehydration. Moreover, ZmbZIP75 overexpression confers increased grain yield and reduced kernel moisture in maize. Next stepsWhile ZmbZIP75 is directly phosphorylated to mediate ABA signaling during dehydration maturation, ZmbZIP75 is transcriptionally induced by ABA during grain filling. We plan to identify upstream factors that mediate ABA signaling to regulate ZmbZIP75 expression, thereby enhancing our understanding of ABA-promoted grain filling in maize.

plant biology↗

bioRxiv2024

Optimizing single-cell RNA sequencing methods for human colon biopsies: droplet-based vs. picowell-based platforms

Background & AimsSingle-cell RNA sequencing (scRNA) has empowered many insights into gastrointestinal microenvironments. However, profiling human biopsies using droplet-based scRNA (D-scRNA) is challenging since it requires immediate processing to minimize epithelial cell damage. In contrast, picowell-based (P-scRNA) platforms permit short-term frozen storage before sequencing. We compared P- and D-scRNA platforms on cells derived from human colon biopsies. MethodsEndoscopic rectosigmoid mucosal biopsies were obtained from two adults and conducted D-scRNA (10X Chromium) and P-scRNA (Honeycomb HIVE) in parallel using an individuals pool of single cells (> 10,000 cells/participant). Three experiments were performed to evaluate 1) P-scRNA with cells under specific storage conditions (immediately processed [fresh], vs. frozen at -20C vs. -80C [2 weeks]); 2) fresh P-scRNA versus fresh D-scRNA; and 3) P-scRNA stored at -80C with fresh D-scRNA. ResultsSignificant recovery of loaded cells was achieved for fresh (80.9%) and -80C (48.5%) P-scRNA and D-scRNA (76.6%), but not -20C P-scRNA (3.7%). However, D-scRNA captures more typeable cells among recovered cells (71.5% vs. 15.8% Fresh and 18.4% -80C P-scRNA), and these cells exhibit higher gene coverage at the expense of higher mitochondrial read fractions across most cell types. Cells profiled using D-scRNA demonstrated more consistent gene expression profiles among the same cell type than those profiled using P-scRNA. Significant intra-cell-type differences were observed in profiled gene classes across platforms. ConclusionsOur results highlight non-overlapping advantages of P-scRNA and D-scRNA and underscore the need for innovation to enable high-fidelity capture of colonic epithelial cells. The platform-specific variation highlights the challenges of maintaining rigor and reproducibility across studies that use different platforms.

genomics↗

bioRxiv2024

Pulmonary primary oxysterol and bile acid synthesis as a predictor of outcomesin pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a rare and fatal vascular disease with heterogeneous clinical manifestations. To date, molecular determinants underlying the development of PAH and related outcomes remain poorly understood. Herein, we identify pulmonary primary oxysterol and bile acid synthesis (PPOBAS) as a previously unrecognized pathway central to PAH pathophysiology. Mass spectrometry analysis of 2,756 individuals across five independent studies revealed 51 distinct circulating metabolites that predicted PAH-related mortality and were enriched within the PPOBAS pathway. Across independent single-center PAH studies, PPOBAS pathway metabolites were also associated with multiple cardiopulmonary measures of PAH-specific pathophysiology. Furthermore, PPOBAS metabolites were found to be increased in human and rodent PAH lung tissue and specifically produced by pulmonary endothelial cells, consistent with pulmonary origin. Finally, a poly-metabolite risk score comprising 13 PPOBAS molecules was found to not only predict PAH-related mortality but also outperform current clinical risk scores. This work identifies PPOBAS as specifically altered within PAH and establishes needed prognostic biomarkers for guiding therapy in PAH. One-Sentence SummaryThis work identifies pulmonary primary oxysterol and bile acid synthesis as altered in pulmonary arterial hypertension, thus establishing a new prognostic test for this disease.

systems biology↗

bioRxiv2023

LucFlow: A method to measure Luciferase reporter expression in single cells

Reporter assays, in which the expression of an inert protein is driven by gene regulatory elements such as promoters and enhancers, are a workhorse for investigating gene regulation. Techniques for measuring reporter gene expression vary from single-cell or single-molecule approaches having low throughput to bulk Luciferase assays that have high throughput. We developed a Luciferase Reporter Assay using Flow-Cytometry (LucFlow), which measures reporter expression in single cells immunostained for Luciferase. We optimized and tested LucFlow with a murine cell line that can be differentiated into neutrophils, into which promoter-reporter and enhancer-promoter-reporter constructs have been integrated in a site-specific manner. The single-cell measurements are comparable to bulk ones but we found that dead cells have no detectable Luciferase protein, so that bulk assays underestimate reporter expression. LucFlow is able to achieve a higher accuracy than bulk methods by excluding dead cells during flow cytometry. Prior to fixation and staining, the samples are spiked with stained cells that can be discriminated during flow cytometry and control for tube-to-tube variation in experimental conditions. Computing fold change relative to control cells allows LucFlow to achieve a high level of precision. LucFlow, therefore, enables the accurate and precise measurement of reporter expression in a high throughput manner.

molecular biology↗

bioRxiv2023

Binding-site resolution structure and dynamics of Cebpa regulatory regions during macrophage-neutrophil differentiation

The upregulation of gene expression by enhancers depends upon the interplay between the binding of sequence-specific transcription factors (TFs) and DNA accessibility. DNA accessibility is thought to limit the ability of TFs to bind to their sites, while TFs can increase accessibility to recruit additional factors that upregulate gene expression. Given this interplay, the causative regulatory events underlying the modulation of gene expression during cellular differentiation remain unknown for the vast majority of genes. We investigated the binding-site resolution dynamics of DNA accessibility and the expression dynamics of the enhancers of an important neutrophil gene, Cebpa, during macrophage-neutrophil differentiation. Reporter genes were integrated in a site-specific manner in PUER cells, which are progenitors that can be differentiated into neutrophils or macrophages in vitro by activating the pan-leukocyte TF PU.1. Time series data show that two enhancers upregulate reporter expression during the first 48 hours of neutrophil differentiation. Surprisingly, there is little or no increase in the total accessibility, measured by ATAC-Seq, of the enhancers during the same time period. Conversely, total accessibility peaks 96 hrs after PU.1 activation--consistent with its role as a pioneer--but the enhancers do not upregulate gene expression. Combining deeply sequenced ATAC-Seq data with a new bias-correction method allowed the profiling of acces-sibility at single-nucleotide resolution and revealed protected regions in the enhancers that match all previously characterized TF binding sites and ChIP-Seq data. Although the accessibility of most positions does not change during early differentiation, that of positions neighboring TF binding sites, an indicator of TF occupancy, did in-crease significantly. The localized accessibility changes are limited to nucleotides neighboring C/EBP-family TF binding sites, showing that the upregulation of enhancer activity during early differentiation is driven by C/EBP-family TF binding. These results show that increasing the total accessibility of enhancers is not sufficient for upregulating their activity and other events such as TF binding are necessary for upregulation. Also, TF binding can cause upregulation without a perceptible increase in total accessibility. Finally, this study demonstrates the feasibility of comprehensively mapping individual TF binding sites as footprints using high coverage ATAC-Seq and inferring the sequence of events in gene regulation by combining with time-series gene expression data.

genomics↗