Biology subjects

Roy, T.

Publications and source records attributed to Roy, T..

At least 19 recordsLinked to original sources

bioRxiv2026

Interleukin-4 suppresses TIMP-2 transcription during endometriosis: Allele-specific regulation of the TIMP-2 -418G/C promoter by SP1

Abstract Tissue inhibitor of metalloproteinase-2 (TIMP-2) is a critical regulator of extracellular matrix (ECM) homeostasis in inflammatory disorder, but the mechanisms underlying its dysregulation in endometriosis remain poorly understood. Furthermore, whether inflammatory cytokine signaling and functional genetic variants within the TIMP-2 promoter contribute to its altered expression during disease progression is unknown. This study investigated the regulation of TIMP-2 expression by interleukin-4 (IL-4) signaling and the functional significance of the TIMP-2 -418G/C promoter polymorphism in endometriosis. We found that TIMP-2 expression was significantly reduced in ectopic lesions and serum samples from endometriosis patients in comparison to normal. Functional promoter analyses revealed that the -418C allele exhibited higher basal transcriptional activity than the G allele while displayed greater sensitivity to IL-4-mediated repression. IL-4 activated STAT6 signaling concomitant with SP1 and TIMP-2 downregulation, whereas SP1 overexpression restored TIMP-2 expression and SP1 silencing further suppressed its transcription, identifying SP1 as a positive regulator of TIMP-2. Single-cell transcriptomic analyses identified fibroblasts and mesenchymal populations as the major TIMP-2-expressing cell types and revealed enhanced inflammatory signaling and ECM remodeling networks in endometriosis. Collectively, these findings identify an IL-4-SP1-TIMP-2 regulatory axis that contributes to aberrant ECM turnover in endometriosis. Furthermore, the TIMP-2 -418G/C promoter polymorphism influences cytokine-mediated transcriptional regulation of TIMP-2, providing new mechanistic insights into the interplay between inflammatory signaling, genetic variation, and ECM homeostasis during endometriosis progression.

molecular biology↗

bioRxiv2026

Cohesin–axis interaction via a conserved Red1 motif promotes domain-specific DSB formation and Mek1 activation

Faithful chromosome segregation during meiosis I requires tight control of interhomolog recombination. In budding yeast, the meiotic chromosome axis, built on Rec8-containing cohesin together with Red1 and Hop1, acts as a central platform regulating meiotic recombination from programmed DNA double-strand break (DSB) formation to checkpoint signaling and chromosome segregation, yet how cohesin recruits axis proteins remains unclear. Here, we identified a conserved cohesin-interacting motif (CIM) in Red1 that directly binds Rec8. AlphaFold3 modeling predicted that Red1-CIM forms a short -helix that docks into a conserved hydrophobic pocket within the Rec8 C-terminal winged-helix domain, which we confirmed biochemically. Disruption of the Red1-CIM preferentially impaired Red1 recruitment to Rec8-dependent chromosomal regions, while relative enrichment in Rec8-independent domains was preserved, leading to reduced DSB formation in Rec8-dependent domains. The Red1-CIM mutation also reduced crossover formation, increased chromosome missegregation, and reduced spore viability. Notably, this spore lethality exceeded that predicted by the reduction in DSB formation. Consistently, red1-CIM mutants failed to activate the meiotic checkpoint kinase Mek1. Finally, we provide evolutionary, structural, and biochemical evidence that this Red1-Rec8 interaction is conserved across fungi and plants. Together, these findings define a direct molecular bridge linking cohesin to chromosome-axis organization, spatial DSB regulation, and checkpoint signaling during meiosis.

molecular biology↗

bioRxiv2026

Mite-induced migratory skin-like CD103+ ILC2s establish lung residency and impact immunity

Tissue-resident immune cells are critical for lung homeostasis and protection against inflammation. Here, we describe a novel population of migratory inflammatory CD103+ skin ILC2s (skILC2s) that accumulate in the lung vasculature following transient infestation with ubiquitous Demodex mites, hair follicle commensals that drive local skILC2 proliferation and entry into blood. With time, ex-skILC2s become tissue resident in the lung, occupy previously described immune cell adventitial niches and respond earlier to lung perturbations than endogenous lung ST2+ ILC2s to alter the trajectories of a subsequent immune response. Thus, like transiting ex-gut ILC2s described after small intestinal infection by helminths and protists, ex-skILC2s migrate post-birth in response to pathobionts to change the inflammatory milieu of the lung, revealing a common paradigm by which internal mucosal responses become shaped by ILC2s from barrier epithelia colonized post-birth. HighlightsO_LIDemodex mites infestation drives local expansion of CD103+ skin ILC2s that egress and enter the circulation C_LIO_LIMigratory CD103+ skin-like ILC2s infiltrate into lung, adapt to the local environment and establish long-term residency C_LIO_LIPrior Demodex mite exposure re-organizes the lung ILC2 landscape C_LIO_LIThe distinct cytokine and receptor repertoire of ex-skILC2 alters lung immunity C_LI

immunology↗

bioRxiv2026

Spike antibodies targeting GRP78 predispose to cardiovascular complications compared to Dengue

One major aftermath of COVID-19 pandemic is cardiovascular consequences. SARS-CoV-2 binds to ACE2 and downregulates vasodilation. Dengue favors hypotension by weakening endothelial glycocalyx leading to plasma leakage. C1q levels, immune complexes (ICs), and proteomic profiles in serum samples from 52 COVID-19 and 19 pre-pandemic Dengue cases were studied. Unlike Dengue, COVID-19 serums showed elevated coagulation proteins promoting vaso-occlusion and peripheral artery diseases. The stress-induced chaperone and atherosclerosis marker, GRP78 (gene/ protein) was found upregulated upon SARS-CoV-2 spike expression in cardiac/ lung cell lines. Elevated GRP78 levels were also observed in serum samples from COVID-19-diagnosed individuals and subjects with myocardial infarction (MI) in post COVID-era. Surprisingly, spike antibodies (Abs) showed cross-binding to GRP78 and possibly contributed to the observed higher-level ICs in COVID-19 serums (cardiovascular embolism?). Co-localization studies showed that spike Abs (analogous to pro-atherosclerotic GRP78 auto-Abs) could directly bind to upregulated cellular GRP78 (type II hypersensitivity?). Both pathways could worsen vascular injury and atherosclerosis, leading to cardiac complications in COVID-19 cases with narrowed vessels.

microbiology↗

bioRxiv2026

Associations Between QuantiFERON-TB Gold Plus IFNγ Concentrations and Progression to Symptomatic Tuberculosis in Global High-Burden TB Settings

IntroductionPredictive biomarkers for symptomatic tuberculosis (TB) progression would transform targeted prevention efforts. Although interferon-gamma release assays (IGRAs), including QuantiFERON(R) TB-Gold Plus (QFT-Plus), have been studied for this purpose, systematic evaluation of the QFT-Plus TB1 and TB2 Interferon-Gamma (IFN{gamma}) concentrations remains limited, particularly in high-burden TB settings. MethodsBaseline TB1 and TB2 IFN{gamma} concentrations from 5,259 participants in TB-endemic regions were analyzed in relation to subsequent TB outcomes over a median of 525 days follow-up. Participants were categorized as controls (no TB), suspected TB (no microbiological confirmation), or laboratory-confirmed TB, including a subset meeting a stringent case definition ([≥]2 positive microbiologic tests). Associations between baseline IFN{gamma} concentrations and progression to symptomatic TB were assessed. ResultsIn the full cohort (IGRA+/-participants), baseline TB2 IFN{gamma} concentrations were significantly higher compared to controls among participants who developed suspected TB (p=0.01), laboratory-confirmed TB (p=0.01), or met the stringent case definition (p<0.0001). In IGRA+ participants, baseline TB2 concentrations were significantly higher than controls in suspected (p=0.01) and laboratory-confirmed (p=0.02) groups. Associations with baseline TB1 IFN{gamma} concentrations and TB progression were observed for participants meeting the stringent case definition within the full cohort (p=0.009). Among stringent definition cases, TB2 concentrations achieved an area under the Receiver Operating Characteristic curve of 0.84, with a sensitivity of 80% and specificity of 78%. ConclusionsQuantitative IFN{gamma} concentrations from QFT-Plus, particularly TB2, were associated with progression to symptomatic TB, met or exceeded WHO-recommended sensitivity and specificity thresholds for predictive biomarkers, and may support biomarker-based stratification in TB clinical research. SummaryIn an exploratory analysis from a global tuberculosis (TB) epidemiology study, higher QuantiFERON-TB Gold Plus IFN{gamma} concentrations were associated with progression to symptomatic TB and met WHO predictive biomarker thresholds, supporting their potential value for risk stratification in TB research.

immunology↗

bioRxiv2025

A shared genetic regulator of metabolism and addiction-related behavior in mice and humans

Substance use disorders and other mental health conditions often co-occur with metabolic disorders, suggesting shared biological underpinnings1. These heightened behavioral and physiological responses may have evolved to promote survival during resource scarcity but can become maladaptive in modern environments2. The genetic mechanisms linking these traits have remained elusive. Here, we show that a variable gene enhancer in mice jointly regulates genes encoding an epigenetic factor (Eed) and a mitochondrial enzyme (Me3) thereby influencing propensity to addiction-related behaviors and mitochondrial function. We further identify variation in a corresponding enhancer in humans regulating EED and ME3 associated with substance use, psychiatric and metabolic disorders. These findings reveal a convergent genetic regulatory network linking mitochondrial biology to behavioral and metabolic risk, offering insight into how genetic variation in beneficial regulatory pathways can predispose individuals to substance use disorders and related conditions.

genetics↗

bioRxiv2025

Investigating the Role of Exosome-associated Human Endogenous Retroviruses as Biomarkers in Motor Neuron Disease

Human endogenous retrovirus-K (HERV-K) reactivation is increasingly implicated in amyotrophic lateral sclerosis (ALS), with ongoing clinical trials investigating antiretroviral therapies. However, there is limited understanding of how HERV-K is trafficked in peripheral biofluids, and the role of exosomes, nano-sized extracellular vesicles, in this process remains largely unexplored. Exosomes offer a stable and cell-specific cargo reservoir that may reflect central pathogenic processes and serve as a minimally invasive biomarker source. In this study, we isolated plasma-derived exosomes from ALS patients (n = 21) and healthy controls (n = 16), and quantified exosomal HERV-K gag, env, and pol transcript levels using SYBR Green qPCR with RNase treatment and normalization to both traditional and exosome-enriched reference genes. HERV-K pol expression was significantly elevated in ALS, with fold-changes ranging from 1.59 to 1.85 (P = 0.037-0.051). env and gag also showed increased expression, though with greater variability. Normalization to the exosome-specific gene SOD2 provided the most consistent signal. These findings suggest that exosomal HERV-K transcripts, particularly pol, could serve as accessible biomarkers for patient stratification and treatment monitoring in HERV-K-targeted ALS trials. This work establishes proof-of-concept for using exosomal cargo to track endogenous retroviral activity in neurodegeneration and supports further investigation of liquid biopsy approaches in ALS precision medicine.

neuroscience↗

bioRxiv2025

Modeling collective cell migration in a heterogeneous environment: Drosophila border cell migration as a model paradigm

Collective cell migration underpins development and disease progression, yet the influence of heterogeneous microenvironments on intercellular forces and migration dynamics remains elusive. Focusing on the chemotactic migration of border cell clusters in Drosophila egg chambers, we present a computational framework that integrates intercellular force dynamics and cell shape changes during large-scale migration, validated with live imaging data. Our model replicates in vivo observations, such as rotations of the cell cluster and its tendency to follow a central path in the egg chamber. Using the model, we discover instantaneous acceleration/deceleration phases as the cell cluster migrates through junctions of other cell types. We also predict how environmental tissue topology and perturbations in chemoattractant gradients and adhesion impact cluster speed and rotation. By addressing the limitations of prior models of border cell migration, our approach offers broad applicability to other migration contexts involving interactions among different cell types in complex environments.

biophysics↗

bioRxiv2025

A synbiotic medical food improves gut barrier function, reduces immune responses, and inhibits osteoclast activity in models of postmenopausal bone loss aligned with clinical outcomes

Over half of women above age 50 are affected by osteopenia or osteoporosis, boneloss conditions influenced by estrogen decline, inflammation, and the intestinal microbiota. Probiotic-based interventions have shown promise in preclinical osteoporosis models. In a recent randomized, double-blind, placebo-controlled clinical trial of postmenopausal women, dietary intervention with SBD111, a synbiotic medical food combining plant-derived probiotics and prebiotic fibers, reduced bone loss in women with osteopenia, elevated body mass index (BMI), and/or elevated body fat. To investigate potential mechanisms underlying these outcomes, we examined intestinal epithelial, immune, and osteoclast responses to SBD111 in vitro. SBD111 administration improved intestinal barrier integrity, reduced immune cell cytokine secretion, and inhibited osteoclast activity. These effects align with clinically observed reductions in severe gastrointestinal symptoms and bone resorption markers. Together, these findings suggest that SBD111 modulates the gut-bone axis via barrier, immune, and antiresorptive pathways, supporting its role in maintaining skeletal health in postmenopausal women. IRB StatementsThe study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board of MaineHealth under protocol 1689738-1 (approved 12/29/2020) and 958914 (approved 05/31/2005). Human peripheral blood mononuclear cells (PBMCs) were purchased from Charles River Laboratories and collected under their IRB-approved protocol with informed consent for commercial research use. Informed Consent Statements Informed consent was obtained from all participants involved in the study. HighlightsO_LISBD111 synbiotic medical food improves intestinal barrier function C_LIO_LISBD111 reduces cytokine release from inflamed immune cells C_LIO_LIThis synbiotic medical food inhibits osteoclast activity in vitro C_LIO_LIIn vitro SBD111 effects align with clinical reductions in bone loss and CTX-1 C_LI Graphical Abstract. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=128 SRC="FIGDIR/small/669142v1_ufig1.gif" ALT="Figure 1000"> View larger version (30K): org.highwire.dtl.DTLVardef@13b16dcorg.highwire.dtl.DTLVardef@59dfcaorg.highwire.dtl.DTLVardef@737e59org.highwire.dtl.DTLVardef@6c054a_HPS_FORMAT_FIGEXP M_FIG SBD111 synbiotic medical food reduces bone loss via barrier improvement, anti-inflammation, and osteoclast inhibition. (A) SBD111 improves barrier integrity in vitro. Visualized in the left panel, inflammation reduces intestinal epithelial barrier function, resulting in the influx of inflammatory mediators, thereby activating the immune system. The addition of SBD111, shown in purple (right), improves barrier function, reducing the ability of inflammatory mediators to cross the epithelial barrier. (B) SBD111 reduces inflammatory responses of inflamed immune cells in a concentration-dependent manner. Low concentrations of SBD111 induce inflammatory responses. At higher concentrations, fewer inflammatory cytokines are secreted. (C) SBD111-derived components/metabolites inhibit the activity of bone-resorbing osteoclasts and reduce their ability to degrade bone. Image created in https://BioRender.com C_FIG

immunology↗

bioRxiv2025

STUDIES ON INTERACTION OF MORIN WITH CURLI TO IDENTIFY AGGREGATION NATURE

Morin (3,5,7,2',4'-pentahydroxyflavone) is a flavonoid compound exhibited a range of biological functions, including anti-inflammatory, antioxidant, antiviral, and anti-allergic activities. It has the ability to disaggregate amyloid fibrils, making it a potential inhibitor of diseases caused by amyloid protein fibrillation. This study focuses on the interaction between Morin and Curli amyloid fibrils, which are produced by some Enterobacteriaceae, including Salmonella sp. And E. coli, as a major component of their biofilms. The Curli amyloid fibrils can lead to neurodegenerative diseases through a signalling cascade causing various human diseases, such as Alzheimers and Huntingtons diseases. Thioflavin T (ThT) fluorescence assays to purified Curli, and molecular docking between Morin and Curli indicates nature of binding at molecular level. The results showed that Morin quenched Curli fluorescence in a concentration-dependent manner, indicating dynamic quenching. Stern-Volmer analysis revealed that Morin binds to Curli with a binding constant of approximately 102 L/mol. This binding was found to be spontaneous, with hydrophobic interactions playing a significant role. ThT fluorescence assays confirmed that Morin inhibited Curli fibrillation, suggesting a potential therapeutic application. Molecular docking analysis revealed specific interactions between Morin and Curli, providing insights into their binding at the molecular level. In conclusion, this study demonstrates the dynamic quenching of Curli fluorescence by Morin and suggested that Morin may inhibit Curli fibrillation, making it a valuable candidate for the development of therapeutic drugs.

biochemistry↗

bioRxiv2024

CD4 Co-Receptor Regulates Sex-Specific NK Cell Responses to Acute Toxoplasma gondii Infection.

Immunity to Toxoplasma gondii (T. gondii) is sexually dimorphic in humans and mice, with females having higher morbidity and mortality during immune dysfunction and HIV-AIDS. The mechanisms underlying these sex differences are unclear. We investigated how a lack of CD4+ T cells (CD4 co-receptor KO) impacted T. gondii survival in mice. Female CD4 co-receptor KO mice succumbed to T. gondii much faster than males. To dissect why female CD4 co-receptor KO mice died faster, we tested their NK cell responses to acute T. gondii infection compared to males. Although in wild-type (WT) animals, both sexes had similar increases in total NK cells and IFN{gamma} + NK cells, infected CD4 co-receptor KO female mice had 50% fewer IFN{gamma}+ NK cells than infected WT female mice. Infected male CD4 co-receptor KO had a similar increase in IFN{gamma}+ NK cells as WT male mice. Since CD4 co-receptor deficient mice still have functional helper T cells that are CD4-, we next tested survival and NK cell responses in female and male MHCII deficient (MHCIIKO) animals, which completely lack helper CD4+T cells. Surprisingly, survival, NK cell numbers, and IFN{gamma}+ NK cells were not significantly different between WT or MHCIIKO female and male mice. These results suggest CD4 co-receptor expression is required for survival via optimal NK cell responses during acute T. gondii infection only in female mice and not in male mice. Our findings reveal an unappreciated sexual dimorphic role of CD4 co-receptor expression in regulating NK cell responses to acute T. gondii infection.

immunology↗

bioRxiv2024

The impact of ERUPR on mitochondrial integrity mediated by PDK4

ER and mitochondrial stress are often interconnected and considered as major contributors to aging as well as neurodegeneration. Co-ordinated induction of ERUPR and mitoUPR has been observed in diabetes and pulmonary disorders. However, in the context of aging and neurodegeneration, regulation of this intra-organellar crosstalk has remained relatively elusive. Here, we demonstrate that pyruvate dehydrogenase kinase 4 (PDK4), a mitochondrial protein accumulates at the ER-mitochondrial contact sites (MAMs) during ER stress. Classically, PDK4 is known to phosphorylate PDHA1 (pyruvate dehydrogenase E1 subunit alpha 1), and plays a significant role in regulating the oxidative phosphorylation driven ATP production. In this study, we propose a non-canonical function of PDK4; we show that it acts as a connecting link between ERUPR and mitoUPR with significance in aging and neurodegeneration. Transcriptomics analyses show increased PDK4 levels upon drug induced ER stress. We detect elevated PDK4 levels in brain lysates of Alzheimers disease (AD) patients, as well as in in vivo and ex vivo AD models. Additionally, exogenous expression of PDK4 was found to refine ER-mitochondrial communication, significantly altering mitochondrial morphology and function. Further, we also observe defective autophagic clearance of mitochondria under such conditions. It is prudent to suggest that elevated PDK4 levels could be one of the key factors connecting ERUPR with mitoUPR, a phenotypic contributor in aging and at least some neurodegenerative diseases.

molecular biology↗

bioRxiv2024

Robust inducible gene expression in intracellular Listeria monocytogenes in vivo

Attenuated strains of the intracellular pathogen Listeria monocytogenes can deliver genetically encoded payloads inside tumor cells. L. monocytogenes preferentially accumulates and propagates inside immune-suppressed tumor microenvironments. To maximize the payload impact in tumors and minimize damage to healthy tissues, it is desirable to induce payload synthesis when bacteria are eliminated from the healthy tissues but are grown to high numbers intratumorally. Here, we have engineered a tightly controlled gene expression system for intracellular L. monocytogenes inducible with a cumin derivative, cumate. Upon cumate addition, expression of a reporter gene is increased in L. monocytogenes growing in vitro by 80-fold, and in intracellular L. monocytogenes in murine tumors by 10-fold. This study demonstrates the feasibility of activating gene expression in intracellular bacteria in live animals using an edible inducer. The system is expected to enhance the efficacy and safety of the attenuated L. monocytogenes strains as antitumor payload delivery bacterial drones.

synthetic biology↗

bioRxiv2024

CD44/Integrin β1 association drives fast motility on HA substrates

In addition to proteins such as collagen (Col) and fibronectin, the extracellular matrix (ECM) is enriched with bulky proteoglycan molecules such as hyaluronic acid (HA). However, how ECM proteins and proteoglycans collectively regulate cellular processes has not been adequately explored. Here, we address this question by studying cytoskeletal and focal adhesion organization and dynamics on cells cultured on polyacrylamide hydrogels functionalized with Col, HA and a combination of Col and HA (Col/HA). We show that fastest migration on HA substrates is attributed to the presence of smaller and weaker focal adhesions. Integrin {beta}1 co-localization and its association with CD44--which is the receptor for HA, and insensitivity of cell spreading to RGD on HA substrates suggests that focal adhesions on HA substrates are formed via integrin association with HA bound CD44. Consistent with this, adhesion formation and cell motility were inhibited when CD44 was knocked out. Collectively, our results suggest that association of integrin {beta}1 with CD44 drives fast motility on HA substrates.

cell biology↗

bioRxiv2024

Multiple molecular links between the circadian clock and memory centers in honey bees

Time and memory are intimately linked: the capability to learn and recall varies over the day and humans and many animals can associate important events with the time of day. However, how the circadian clock and memory centers are connected is not well understood. We time-trained honey bee foragers and used RNA-sequencing and RNAscope imaging to analyze gene expression changes in focal populations of mushroom body neurons. Thus, we identified three candidate functional modules of time-memory: synchronized peak-level expression of memory-related genes during training time, anticipatory activation of transcription in pdfr-expressing neurons, and cry2 and per co-expressing neurons that might represent local clocks. The complex interactions between the clock and memory centers, which appear to be more similar to mammals than other insects, might have been facilitated to optimize social foraging in honey bees.

neuroscience↗

bioRxiv2024

Nuclear compression-mediated DNA damage drives ATR-dependent Lamin expression and mouse ESC differentiation

Embryonic stem cells (ESCs) which are susceptible to DNA damage depend on a robust and highly efficient DNA damage response (DDR) mechanism for their survival. However, the implications of physical force-mediated DNA damage on ESC fate remains unclear. We show that stiffness-dependent spreading of mouse ESCs (mESCs) induces DNA damage through nuclear compression, with DNA damage causing differentiation through early induction of Lamin A/C expression. Interestingly, differentiation is associated with rescue of DNA damage and activation of the DDR factor ATR. While ATR is typically known to play roles in DDR pathway, its role during stiffness-mediated nuclear compression and mESC differentiation is unknown. Nuclear enrichment of activated ATR on stiff substrates and reduction of Lamin A/C expression upon ATR inhibition suggests that mESC differentiation is driven by nuclear compression-mediated DNA damage and involves ATR-dependent modulation of Lamin A/C.

cell biology↗

bioRxiv2023

Measuring RBC deformability and its heterogeneity using a fast microfluidic device

We report a high-throughput microfluidic device to determine the Youngs modulus of single red blood cells (RBCs). Our device consists of a single channel opening into a funnel, with a semi-circular obstacle placed at the mouth of the funnel. As a RBC passes the obstacle, it deflects from its original path. Using populations of artificially-stiffened RBCs, we show that the stiffer RBCs deflect more compared to the healthy RBCs. We then generate a calibration curve that maps each RBC trajectory to its Youngs modulus obtained using an atomic force microscope. Finally, we sort a mixed population of RBCs based on their deformability alone. Our device could potentially be further miniaturized to sort and obtain the elastic constants of nanoscale objects, such exosomes, whose shape change is difficult to monitor by optical microscopy.

bioengineering↗

bioRxiv2023

DISCOVERY AND VALIDATION OF GENES DRIVING DRUG-INTAKE AND RELATED BEHAVIORAL TRAITS IN MICE

Substance use disorders (SUDs) are heritable disorders characterized by compulsive drug use, but the biological mechanisms driving addiction remain largely unknown. Genetic correlations reveal that predisposing drug-naive phenotypes, including anxiety, depression, novelty preference, and sensation seeking, are predictive of drug-use phenotypes, implicating shared genetic mechanisms. Because of this relationship, high-throughput behavioral screening of predictive phenotypes in knockout (KO) mice allows efficient discovery of genes likely to be involved in drug use. We used this strategy in two rounds of screening in which we identified 33 drug-use candidate genes and ultimately validated the perturbation of 22 of these genes as causal drivers of substance intake. In our initial round of screening, we employed the two-bottle-choice paradigms to assess alcohol, methamphetamine, and nicotine intake. We identified 19 KO strains that were extreme responders on at least one predictive phenotype. Thirteen of the 19 gene deletions (68%) significantly affected alcohol use three methamphetamine use, and two both. In the second round of screening, we employed a multivariate approach to identify outliers and performed validation using methamphetamine two-bottle choice and ethanol drinking-in-the-dark protocols. We identified 15 KO strains that were extreme responders across the predisposing drug-naive phenotypes. Eight of the 15 gene deletions (53%) significantly affected intake or preference for three alcohol, eight methamphetamine or three both (3). We observed multiple relations between predisposing behaviors and drug intake, revealing many distinct biobehavioral processes underlying these relationships. The set of mouse models identified in this study can be used to characterize these addiction-related processes further.

genetics↗