Biology subjects

Woodward, J.

Publications and source records attributed to Woodward, J..

6 recordsLinked to original sources

bioRxiv2026

Prime editing enables high-throughput identification and characterization of selective MutSβ inhibitors

DNA mismatch repair (MMR) maintains genome stability but paradoxically drives expansion of pathogenic repeats responsible for trinucleotide repeat disorders. Attenuating repeat instability through targeted inhibition of MMR is therefore a therapeutic strategy of interest; however, progress is constrained by a lack of robust methods for defining and isolating MMR subpathways. We exploited features of prime editing to profile the effects of core MMR genes on thousands of programmed DNA mispairs in cells and resolved distinct MMR processes, including a noncanonical, loop-directed mechanism. Guided by these results, we built a set of scalable reporters that detect loss of separable MMR processes and screened ~72,000 small molecules for activity against MutS{beta}-directed MMR. This effort identified a novel series of MutS{beta} inhibitors that reduced (CAG)n expansion in a cell model. Altogether, this study establishes several methods for systematically interrogating MMR, with demonstrated capacity to identify inhibitors of therapeutic value.

bioengineering↗

bioRxiv2026

Yersinia pseudotuberculosis employs a multifaceted strategy to survive antimicrobials

Microbes have evolved a variety of strategies to survive exposure to naturally occurring and synthetic antimicrobials. These strategies have been investigated extensively in model bacterial organisms, whereas less is known about under explored pathogenic bacteria such as bacteria within the Yersinia genus. In this study we investigated the inhibitory effect and bactericidal activity of antibiotics from five different classes and of the disinfectant hydrogen peroxide against Yersinia pseudotuberculosis, the ancestral species from which Yersinia pestis and Yersinia enterocolitica have emerged. We found that Y. pseudotuberculosis is able to survive exposure to clinical antibiotics and disinfectants by employing a variety of strategies, with persisters and the Eagle effect playing a role in survival to quinolones, tolerance playing a role in survival to ceftriaxone and overexpression of catalases and peroxidases playing a role in survival to hydrogen peroxide. Our findings suggest that future research should focus on informing new, effective ways to treat infections caused by Yersinia species. IMPORTANCEAntimicrobial resistance is routinely investigated by measuring the minimum inhibitory concentration of antimicrobials needed to stop microbial growth. Here we show that the bacterial pathogen Yersinia pseudotuberculosis is not killed when antibiotics and disinfectants are used at these concentrations and that, in some cases, increasing antibiotic concentrations decreases their activity against this bacterium, therefore posing a potential risk to human and animal health.

microbiology↗

bioRxiv2023

Adaptor protein complex 2 in the orbitofrontal cortexpredicts alcohol use disorder

Alcohol use disorder (AUD) is a life-threatening disease characterized by compulsive drinking, cognitive deficits, and social impairment that continue despite negative consequences. The inability of individuals with AUD to regulate drinking may involve functional deficits in cortical areas that normally balance actions that have aspects of both reward and risk. Among these, the orbitofrontal cortex (OFC) is critically involved in goal-directed behavior and is thought to maintain a representation of reward value that guides decision making. In the present study, we analyzed post-mortem OFC brain samples collected from age- and sex-matched control subjects and those with AUD using proteomics, bioinformatics, machine learning, and reverse genetics approaches. Of the 4,500+ total unique proteins identified in the proteomics screen, there were 47 proteins that differed significantly by sex that were enriched in processes regulating extracellular matrix and axonal structure. Gene ontology enrichment analysis revealed that proteins differentially expressed in AUD cases were involved in synaptic and mitochondrial function, as well as transmembrane transporter activity. Alcohol-sensitive OFC proteins also mapped to abnormal social behaviors and social interactions. Machine learning analysis of the post-mortem OFC proteome revealed dysregulation of presynaptic (e.g., AP2A1) and mitochondrial proteins that predicted the occurrence and severity of AUD. Using a reverse genetics approach to validate a target protein, we found that prefrontal Ap2a1 expression significantly correlated with voluntary alcohol drinking in male and female genetically diverse mouse strains. Moreover, recombinant inbred strains that inherited the C57BL/6J allele at the Ap2a1 interval consumed higher amounts of alcohol than those that inherited the DBA/2J allele. Together, these findings highlight the impact of excessive alcohol consumption on the human OFC proteome and identify important cross-species cortical mechanisms and proteins that control drinking in individuals with AUD.

neuroscience↗

bioRxiv2019

SLC19A1 is a cyclic dinucleotide transporter

The accumulation of DNA in the cytosol serves as a key immunostimulatory signal associated with infections, cancer and genomic damage1,2. Cytosolic DNA triggers immune responses by activating the cGAS/STING pathway3. The binding of DNA to the cytosolic enzyme cGAMP synthase (cGAS), activates its enzymatic activity, leading to the synthesis of a second messenger, cyclic[G(2,5)pA(3,5)] (23-cGAMP)4-8. 23-cGAMP, a cyclic dinucleotide (CDN), activates the protein stimulator of interferon genes (STING)9, which in turn activates the transcription factors IRF3 and NF-{kappa}B promoting the transcription of genes encoding type I interferons and other cytokines and mediators that stimulate a broader immune response. Exogenous 23-cGAMP and other CDNs, including CDNs produced by bacteria and synthetic CDNs used in cancer immunotherapy, must traverse the cell membrane to activate STING in target cells. How these charged CDNs pass through the lipid bilayer is unknown. Here we used a genome-wide CRISPR interference screen to identify the reduced folate carrier SLC19A1 as the major CDN transporter for uptake of synthetic and naturally occurring CDNs. CDN uptake and functional responses are inhibited by depleting SLC19A1 from cells and enhanced by overexpressing SLC19A1. In both cell lines and primary cells ex vivo, CDN uptake is inhibited competitively by folate and blocked by the SLC19A1 inhibitor sulfasalazine, a medication approved for the treatment of inflammatory diseases. The identification of SLC19A1 as the major transporter of CDNs into cells has far reaching implications for the immunotherapeutic treatment of cancer10, transport of 23-cGAMP from tumor cells to other immune cells to trigger the anti-tumor immune response11, host responsiveness to CDN-producing pathogenic microorganisms12, and potentially in certain inflammatory diseases.

immunology↗

bioRxiv2018

Monitoring Neural Activity During Exposure to Drugs of Abuse With In Vivo Fiber Photometry

Drugs of abuse are known to alter activity in areas of brain associated with reward, cognition and decision making. Changes in neural activity in these regions that follow repeated exposures to abused substances may underlie the development of addictive behaviors and contribute to the high rates of relapse associated with drug use. Measuring real-time changes in neural activity during drug seeking and taking is important for correlating changes in behavior with alterations in neuronal signaling typically measured using ex vivo electrophysiological recordings. In this study, C57BL/6J mice or Sprague-Dawley rats were injected in different brain areas with adeno-associated viruses (AAV) encoding the calcium sensor GCaMP6f along with an optical fiber. Calcium-dependent fluorescence was monitored in the nucleus accumbens core or mPFC during and following exposure to toluene vapor and in the medial prefrontal cortex (mPFC) and orbitofrontal cortex (OFC) during ethanol drinking. Toluene vapor, at concentrations previously shown to induce conditioned place preference, produced a rapid decrease in the frequency of calcium transients in the NAc core of rats that recovered following washout of the toluene vapor. In a probabilistic risk task, GCaMP6 signals in rat mPFC increased just prior to lever pressing and showed decreases during the reward phase that were proportional to reward size. Toluene pretreatment elevated the signal during the decision-making period while post-lever responses were independent of reward size. Using the drinking in the dark (DID) protocol in mice, we observed a consistent increase in GCaMP6 fluorescence during the period leading up to an ethanol drinking bout, a decrease during consumption and a rebound increase following the bout. The initial increase in signal prior to consumption was greater for ethanol and sucrose than water. GCaMP6 signals in the lateral OFC also decreased during ethanol consumption and increased following bout completion while no increase in activity was noted prior to bout initiation. Following repeated cycles of chronic intermittent ethanol (CIE) exposure that enhanced ethanol consumption, OFC calcium signals during and after ethanol drinking were similar to those in air-treated animals. Addition of quinine to the ethanol solution augmented the decrease in signal during consumption in both air and CIE mice while having no effect on the magnitude of the rebound in activity. Conversely, when sucrose was added to the ethanol solution, air exposed mice showed blunted changes in GCaMP6 signals while those in CIE mice were enhanced. Overall, the results from these experiments complement and extend data from prior behavioral and electrophysiological studies and support the use of in vivo fiber photometry in the study of effects of abused substances on brain function.

neuroscience↗

bioRxiv2018

The CRF-R1 regulation of VTA-GABAergic plasticity is suppressed by CB1 receptor inhibition following chronic exposure to alcohol

Dopamine neurons in the ventral tegmental area (VTA) influence learned behaviors and neuropsychiatric diseases including addiction. The stress peptide corticotrophin-releasing factor (CRF) contributes to relapse to drug and alcohol seeking following withdrawal, although the cellular actions are poorly understood. In this study, we show that presynaptic CRF type 1 receptors (CRF-R1) potentiate GABA release onto mouse VTA dopamine neurons via a PKC-Ca2+ signaling mechanism. In na1ve animals, activation of CRF-R1 by bath application of CRF or ethanol enhanced GABAA inhibitory postsynaptic currents (IPSCs). Following three days of withdrawal from four weekly cycles of chronic intermittent ethanol (CIE) vapor exposure, spontaneous IPSC frequency was enhanced while CRF- and ethanol-potentiation of IPSCs was intact. However, withdrawal for 3 weeks or more was associated with reduced spontaneous IPSC frequency and diminished CRF and ethanol responses. Long-term withdrawal was also accompanied by decreased sensitivity to the CB1 receptor agonist WIN55212 as well as greatly enhanced sensitivity to the CB1 antagonist AM-251. Inclusion of BAPTA in the internal recording solution restored the responsiveness to CRF or ethanol and reduced the potentiating actions of AM251. Together, these data suggest that GABAA inhibition of VTA dopamine neurons is regulated by presynaptic actions of CRF and endocannabinoids and that long-term withdrawal from CIE treatment enhances endocannabinoid mediated inhibition, thereby suppressing CRF facilitation of GABA release. Such findings have implications for understanding the impact of chronic alcohol on stress-related, dopamine-mediated alcohol seeking behaviors.

neuroscience↗