Biology subjects

Hall, A.

Publications and source records attributed to Hall, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Regulatory architecture tunes gene expression noise in the plant heat shock response

Cells within the same tissue can respond differently to the same stress, yet how gene regulatory networks generate and control this variability remains unclear. Here, we show that cell-to-cell variation in the Arabidopsis heat shock response is coordinated across the heat shock regulon and that the distribution of responses is tuned by the underlying regulatory network. Imaging of an HSP70 promoter reporter revealed basal activity in a subset of root cells and heterogeneous induction after heat shock, with some cells remaining near pre-stress expression levels. Dual reporters showed strong coordination between independently integrated HSP70 reporter loci, consistent with substantial shared upstream regulatory variation. Single-nucleus RNA sequencing confirmed heterogeneous but coordinated activation of heat shock genes across root cell types. Stronger heat shock states at both ambient and heat stress temperatures were associated with a recurrent thermoprotective programme across root cell populations, whereas weak responders after heat shock were associated with distinct cell-type specific transcriptional programmes, including defence- and jasmonate-associated states in some root cell types. Stochastic modelling suggested that HSFA2-dependent amplification could broaden the distribution of HSP70 expression across cells. Consistent with this prediction, experiments showed that loss of HSFA2 reduced HSP70 induction and lowered cell-to-cell variability after controlling for differences in mean expression. Together, these results show that the plant heat shock network controls both the magnitude and distribution of stress-responsive gene expression across cells.

plant biology↗

bioRxiv2026

Structural Determinants of Synaptic Vesicle Protein 2C Ligand Selectivity and Their Impact on Dopamine Release

The synaptic vesicle protein 2 (SV2) family is a highly conserved group of transmembrane glycoproteins sharing approximately 70% sequence identity. SV2A and SV2B are broadly distributed throughout the brain, while SV2C, the evolutionarily oldest member of the family, is primarily found in dopaminergic brain regions in the basal ganglia. Genome-wide association studies have linked SV2C to Parkinson's disease, and SV2C appears to enhance dopamine storage in synaptic vesicles, but the basis for this effect is unknown. SV2s are the target of the racetam class of antiseizure medications, and SV2A-specific ligands are widely used to treat seizures. Recently several SV2C-specific ligands have been developed, but the structural basis for ligand specificity is unclear. A better understanding of SV2C structure, function, and pharmacology could lead to better targeted therapies for epilepsy, Parkinson's disease, and other dopamine-related conditions. Here we present cryo-EM structures of apo SV2C, SV2C bound to the high affinity non-selective SV2 ligand padsevonil, SV2C bound to the SV2C-selective ligand UCB-F, and SV2A bound to the SV2A-selective ligand plosaracetam (also known as ABBV-552/SDI-118). We find that SV2C has a wider luminal opening than SV2A and SV2B, which allows for UCB-F to bind to the primary site and form favorable interactions that are not possible in the narrower primary binding site of SV2A and SV2B. We also demonstrate that UCB-F and padsevonil, but not plosaracetam, reduce dopamine release in striatal sections of mouse brain. Our biochemical experiments and structures provide insights into SV2 ligand specificity and offer a template for the rational development of therapeutics targeting SV2C.

biochemistry↗

bioRxiv2026

Functional disruption of vgsc reveals haplosufficiency with implications for insecticide resistance and genetic control in Anopheles gambiae

Pyrethroid insecticides on bednets have been the mainstay of malaria control since the millennium by killing mosquitoes. The target of these pyrethroids is the voltage-gated sodium channel (VGSC). Resistance mutations at the pyrethroid binding site (L995F/S) have spread widely, but functional tools to dissect vgsc itself have lagged behind the population-genetic surveillance data. Manipulation of vgsc via knockout or functional mutation would be a vital step towards understanding its contribution to insecticide resistance, as well as exploring its utility as a genetic control target. Here we generate and characterise two CRISPR-edited An. gambiae lines: an exonic knockout (vgscKO) and an intronic CRISPR-mediated cassette exchange intermediate (vgscInt) sited near SNPs linked to L995F. Both insertions are viable and fertile in heterozygosity but homozygous lethal, demonstrating that vgsc is haplosufficient and therefore the locus represents a viable target for population suppression genetic control strategies. These insertions can also be used as balancers for studying vgsc mutations without the confounding effects of wild type alleles. We investigate the insecticide resistance phenotype of vgscKO and demonstrate that knocking out one copy of vgsc produces reduced susceptibility to deltamethrin and DDT at sub-discriminating doses, with implications for vgsc-mediated mechanisms of insecticide resistance.

genetics↗

bioRxiv2026

Contemporary hybridization and localized genomic differentiation between grey seal subspecies

Grey seals are divided into two subspecies, Halichoerus grypus grypus in the Baltic Sea and H. grypus atlantica in the North Atlantic Ocean. Historically, intense hunting caused local extinctions of grey seals across their range and promoted geographical isolation of the subspecies. However, recent population recovery and recolonization have renewed their overlap in a contact zone in Danish and Swedish waters. Here, using whole-genome sequencing data from 119 individuals, we investigate the demographic history of grey seals, the divergence of the subspecies, and genomic signatures of local adaptation and potential hybridization in the contact zone. We estimate that divergence began approximately 10,000 years ago, with gene flow ceasing 2000 years ago. We detect peaks of high genetic differentiation near genes with putative functions in osmo- and thermoregulation, consistent with salinity and temperature differences between the Baltic Sea and the North Atlantic. As evidence of the geographic isolation breaking up, we report a hybrid individual in the southwest Baltic contact zone, with Baltic maternal and Atlantic paternal ancestry, and identify a migrant of Baltic origin in the North Sea. Our study illustrates how local environmental variation and long-term hunting pressure have contributed to divergence between mammal subspecies over a short evolutionary timescale, with recent population recovery, recolonization and hybridization reshaping gene flow dynamics. Broadly, our work emphasizes the importance of studying evolutionary processes amid anthropogenic influences, including both disturbances and conservation successes, where demographic fluctuations, range shifts, altered gene flow, and adaptation to changing environments interact in complex, potentially consequential ways.

evolutionary biology↗

bioRxiv2026

Long-read sequencing of Mycobacterial tuberculosis is comparable to short-read sequencing for antimicrobial resistance prediction and epidemiological studies.

BackgroundShort-read genetic sequencing technologies (mainly Illumina) have been extensively used for around a decade for Mycobacterium tuberculosis complex (MTBC) outbreak analysis and genomic drug susceptibility testing (gDST) with the result that Illumina has become the de facto gold standard. Long-read sequencing, as exemplified by Oxford Nanopore Technologies (ONT), offer the prospect of faster, simpler, and portable sequencing. In this work, we carry out the largest to date comparison of how well Illumina and ONT technologies sequence MTBC samples, making use of R10.4 ONT flowcells, updated basecalling models and deep-learning variant calling. MethodsA total of 508 samples were sequenced using both short and long-read platforms. All samples originated from South Africa or Vietnam and were over-selected for drug resistance and also included several local outbreaks and a range of lineages. The South African and Vietnamese samples had already been Illumina sequenced. Samples with [≥]50 read depth by Illumina were selected for sequencing by ONT using one of the GridION or PromethION platforms. Bioinformatics processing was done using a modified online cloud platform which included reference-based variant calling, catalogue-based gDST and identified related samples via SNP counting to inform outbreak detection. The lineages and gDST predictions obtained by short-and long-sequencing were compared for all samples as were all putative clusters identified via SNP counting. For convenience Illumina was used as the reference method. FindingsOf the 508 samples, 425 (83.7%) had sufficient read depths to permit comparison between the two sequencing technologies. The assigned lineages were identical for 407/425 (95.8%) samples and all discordances were due to mixed lineages being identified by one technology. Evidence of non-tuberculous mycobacterium (NTM) subpopulations were found in nine samples. Using Illumina as the reference method, the very major error (VME) rate of ONT for predicting resistance to all 15 drugs is 1.0% (0.6-1.5%) whilst the major error (ME) rate is 1.7% (1.3-2.2%) with an unclassified rate of 6.9% (6.3-7.5%). This is below the thresholds specified by the CLSI. Considering each of the 15 drugs individually they had VME and ME point estimates below [≤]3% in 29/30 cases; and most 25/30 below [≤]1.5%. Filtering out all samples containing mixtures left 382 isolates. By appropriate masking of the reference genome we were able to obtain a mean SNP distance between the two platforms of 0.13 (median of zero) for the same sample and for 376/382 samples (98.4%, CI:96.6-99.4%) the difference was [≤]1 SNPs. The high concordance in SNP identification ensured that few differences in the 43 putative clusters among 172 isolates were observed. InterpretationThe differences between the two sequencing platforms for the key clinical outputs is so small that it is now within the tolerances set by regulatory agencies. Provided the sequencing is of sufficient quality, we have therefore reached a threshold whereby sequencing data from long-and short-read platforms can be aggregated. This will enable large scale analyses by national and international public health agencies whilst allowing the MTBC community to take advantage of the portability and speed of long-read sequencing. FundingThe NIHR Health Protection Research Unit: Healthcare Associated Infections and Antimicrobial Resistance at University of Oxford (NIHR200915), a partnership between the UK Health Security Agency (UKHSA) and the University of Oxford, the National Institute for Health and Care Research Biomedical Research Centre: Oxford (BRC) and the Ellison Institute of Technology, Oxford Ltd. The CRyPTIC project was funded by Wellcome [214560/Z/18/Z], a Wellcome Trust/Newton Fund-MRC Collaborative Award (200205/Z/15/Z); and the Bill & Melinda Gates Foundation Trust (OPP1133541). Research in contextO_ST_ABSEvidence before this studyC_ST_ABSWe conducted a PubMed Central full text search for "tuberculosis" AND ("drug resistance prediction" OR "drug susceptibility prediction") AND ("genome" OR "genomic" OR "geno-typic") AND ("ont" OR "oxford nanopore") between 2022 and 2026 (conducted 1 April 2026). This returned 62 papers; of which, six used both Illumina and ONT sequencing. One of these, published in 2023, directly compared the performance of the two platforms on 151 M. tuberculosis isolates oversampled for resistance. The investigation yielded comparative results for the earlier generation ONT flow cell (R9{middle dot}4{middle dot}1) and base-caller (guppy version 5{middle dot}0{middle dot}16). Another, published in 2026, investigated a targeted next-generation sequencing panel of 20 amplicons using ONT sequencing on R10.4.1 flow cells with guppy 6{middle dot}4{middle dot}6. They compared the results on 71 isolates against phenotypic data and Illumina whole genome sequencing (for 53 isolates) but had low rates of resistance, with all drugs but isoniazid being limited to under five resistant isolates. Two other small studies (10 and 13 samples, respectively) conducted feasibility studies comparing ONT with Illumina, also using earlier generation flow cells and base-calling technology from ONT. Two further studies compared Illumina with ONT for direct sputum sequencing and did not investigate the comparative performance of the two platforms for variant call accuracy, resistance prediction, and outbreak detection. Illumina sequencing technology is widely used for genomic sequence analysis in research, and clinical and public health contexts. Consequently, it has become the de facto reference standard for generating whole genome sequence data. Whilst previous studies established the promise and limitations of long-read (ONT) sequencing as an alternative to short-read sequencing (mainly Illumina), the enhanced performance arising from newer flowcells (e.g. R10.4.1), V14 chemistry, and the latest basecallers (dorado v4.3.0/5.0.0) has not been analysed. Neither has any ONT analysis incorporating the new deep-learning variant callers been evaluated in a large-scale comparative study. Thus, it is currently unclear whether data generated by either platform can be used safely in aggregated analyses for research and clinical or public health service. Added value of this studyWe compared how well short-(Illumina) and long-read (ONT) sequencing platforms identify the genetic variants in M. tuberculosis, predict antituberculous drug resistance and recog-nise outbreaks. The long-reads were generated using the latest generation ONT R10.4.1 flows cells, V14 chemistry, super high accuracy basecalling (dorado v4.3.0/5.0.0) and a bioinformatics analysis pipeline built using the Clair3 deep-learning based variant caller. A total of 508 clinical samples were sequenced using both technologies, substantially more than previous studies. The sampling frame was much larger than previously investigations and included a large proportion of isolates with resistance to first-line and second-line antibiotics as well as bedaquiline. Thus, providing greater statistical power for resistance prediction than before. In particular, the inclusion of bedaquiline resistance provided evidence useful for predicting resistance to this newly deployed drug for treating multi-drug resistant (MDR) TB. We find that the differences between technologies are small meaning that either technology can be used alone safely, and services using both technologies can confidently aggregate the data for analysis. Implications of all the available evidenceThis will be a benefit to local, regional and international organisations, particularly public health agencies, which often have a mix of the two main sequencing technologies for characterising TB whole genome sequences. It also opens up the sequence based diagnostic market to greater competition, particularly if the observed performance can be replicated for other pathogen species.

microbiology↗

bioRxiv2026

Evaluation of an Oxford Nanopore sequencing workflow for mycobacteria from primary MGIT culture

Illumina sequencing of primary MGIT cultures is an established workflow in several reference mycobacteriology laboratories. Oxford Nanopore Technologies (ONT) provides real-time genetic sequencing yielding long reads which help resolve repetitive genomes and is being explored for in-house implementation within diagnostic laboratories. However, low DNA yields from primary MGIT cultures frequently limit the application of ONT workflows, due to high minimum DNA input requirements for library preparation. We validated a modified ONT workflow combining rapid, semi-automated DNA extraction from MGIT cultures with Rapid PCR-Barcoding for whole-genome amplification, and compared its performance with Illumina sequencing for species identification and Mycobacterium tuberculosis complex (MTBC) single-nucleotide polymorphism (SNP) detection. A platform-agnostic analysis pipeline enabled consistent human read removal, taxonomic assignment, and MTBC genomic characterisation. ONT sequencing data was subsampled at 1, 6, and 72 hours to determine the earliest time point for reliable species identification. The concordance between sequencing platforms on species classification was 98.3% (95.8-99.5%) with all differences arising from potential mixed infections. SNP agreement was high, with a mean of 0.3 and a median of 0 SNP differences between sequencing platforms after masking. These findings demonstrate the feasibility of PCR-amplified ONT sequencing as a reliable alternative for routine genomic characterisation of MGIT cultures. IMPORTANCERapid identification of mycobacterial infections is essential for timely patient care and infection control. Many clinical laboratories currently rely on outsourcing sequencing to external reference centres, which adds time and delays the return of clinically actionable results. These services commonly use Illumina sequencing, which, while highly accurate, involves complex workflows and longer turnaround times. Newer technologies offer the potential to generate results in real time, but their use has been limited by the low amount of DNA available from routine culture samples. In this study, we developed an improved workflow that increases the amount of usable DNA and enables reliable, rapid sequencing directly from these samples. This approach allows multiple samples to be processed together and reduces the time needed to obtain results. Importantly, it could enable clinical laboratories to perform sequencing in-house, reducing reliance on external services and improving turnaround times.

molecular biology↗

bioRxiv2025

BCL-XL Dependence is a Subtype Agnostic Actionable Feature of Difficult-to-Treat Kidney Cancers

The BCL-XL anti-apoptotic protein is a clear cell Renal Cell Carcinoma (ccRCC) dependency; however, the mechanism of this dependence and its relevance in other aggressive kidney cancer contexts, including metastatic and/or rare RCC subtypes [e.g., Fumarate Hydratase (FH)-deficient and sarcomatoid RCCs], is unknown. Computational predictions, using a machine learning model trained on the human RCC TCGA dataset, and cell-based validations, confirmed BCL-XL dependence in all RCC subtypes. Remarkably, cell state changes, anoikis programs, inflammatory state, and metabolic perturbations (e.g., fumarate production in FH-deficient RCCs) independently conferred increased BCL-XL dependence. Correlation studies revealed that increased AMPK isoform 2 (PRKAA2) expression is a kidney-specific biomarker of BCL-XL dependence. Indeed, pharmacological AMPK activation sensitized RCCs to BCL-XL blockade. Finally, using functional studies, we developed a multivariate model that accurately predicted BCL-XL dependence in RCC. Our studies offer biomarkers for patient stratification and credential BCL-XL as a subtype agnostic vulnerability in difficult-to-treat RCCs.

cancer biology↗

bioRxiv2025

Human hepatic stellate cells orchestrate the accumulation and function of CD103+ tissue-resident CD8+ T-cells in liver fibrosis

Tissue-resident memory cells (TRM) contribute to protective and pathogenic responses in the liver, yet precisely how hepatic TRM adapt and integrate cues from the underlying stroma and extracellular matrix (ECM) in chronic liver disease (CLD) has yet to be fully defined. Here we describe a role for activated myofibroblast-like hepatic stellate cells (HSCs) in the accumulation and in situ localisation of CD8+ TRM in the CLD liver. Activated HSCs drive a program of tissue residence in activated, tissue-infiltrating CD8+ T-cells in a TGF{beta}-dependent manner. We show upregulation of CD103, ECM-binding integrins and adhesion molecules driven by TGF{beta} which together contribute to the sequestration of TRM within the ECM-rich fibrotic niche. Ex vivo, hepatic CD103+ TRM correlate with the extent of ECM deposited, express an altered repertoire of co-stimulatory and co-inhibitory receptors, transcriptional regulators of cellular exhaustion and produce less proinflammatory mediators upon TCR engagement in CLD than in health. Through expression of several co-inhibitory ligands, we further demonstrate the potential for activated HSCs to acquire an immunomodulatory phenotype and limit the capacity of CD103+ TRM to produce anti-viral and anti-tumour mediators upon antigen encounter. Finally, we demonstrate that strategies to block such regulatory pathways, including the PD1:PD-L1/PD-L2 axis, have the potential to restore the antigen-specific effector function of tissue-compartmentalised CD103+ TRM and thus contribute to improving the effectiveness of local immunosurveillance in CLD. One Sentence Summary: Activated hepatic stellate cells characteristic of liver fibrosis orchestrate an accumulation of a CD103+ TRM population with a reduced capacity for antigen-specific effector function in human CLD.

immunology↗

bioRxiv2025

Altered striatal dopamine dynamics and behavior in Grin2a mutant mice, a genetic mouse model of schizophrenia

Schizophrenia (SCZ), a complex psychiatric disorder with a strong genetic basis, is thought to involve, at least in part, dopamine dysregulation in the striatum. Recent large-scale exome sequencing has identified multiple SCZ risk genes, including GRIN2A (encoding an NMDA (N-methyl-D-aspartate) receptor subunit) and AKAP11 (encoding a protein kinase A binding protein), which is also a risk gene for bipolar disorder (BD). However, the mechanisms by which these genetic risk factors perturb dopamine circuits and cause psychotic symptoms remain poorly understood. We asked how behavior, dorsomedial striatal dopamine (DA) dynamics and the activity of D1-/D2-spiny projection neurons (SPNs) were altered in Grin2a and Akap11 mutants. In the open field, Grin2a knockout mice display hyperlocomotion and an abnormal organization of naturalistic behaviors. They also displayed heightened behavioral responses to objects and auditory stimuli. Grin2a heterozygous animals mostly showed intermediate phenotypes, suggesting a dose-dependent effect of Grin2a loss. Further, Grin2a knockout mutants exhibited aberrant dopamine events, altered coupling of dopamine with locomotor features, and exaggerated stimuli-evoked dopamine responses. Notably, heterozygous animals also showed altered striatal SPN events in the open field reflecting dysregulated neural activity in both dSPNs and iSPNs. Compared to Grin2a mutants, Akap11 mutants displayed opposite phenotypes, showing reduced locomotion with a shift from high- to low-velocity movement and distinct alterations in striatal neural activity. Together, GRIN2A (SCZ risk) and AKAP11 (BD/SCZ risk) mutations induce different, often opposite, effects on striatal dopamine signaling and behavior, suggesting that these two risk genes act through distinct mechanisms.

neuroscience↗

bioRxiv2025

Insect Identification with ESEM Imaging and DNA Barcoding for Forensic Use

Flies are among the first insects to arrive at a corpse to lay eggs. The developing larvae can be used to determine post-mortem interval (PMI) using species specific growth models. The larvae can be difficult to identify due to minute morphological characteristics and rearing larvae into adults is time consuming and may be unsuccessful. We investigated the potential of coupling Environmental Scanning Electron Microscopy (ESEM) and DNA barcoding to enable the acquisition of high resolution images while obtaining DNA in a minimally destructive manner. This study focused on the widespread and forensically relevant species Chrysomya rufifacies as a model. Specimens were prepared in three different drying treatments: unaltered, dried, dried and imaged. Following the imaging process, three different DNA extraction methods with different levels of sample destruction were tested. ESEM imaging showed that there were no visual differences between specimens that underwent drying treatments and ones that were not dried. No significant differences in DNA yield were observed for drying treatments and extraction methods post extraction. However, extraction methods demonstrated significant differences in yield of PCR products. Sequenced PCR products aligned with 100% identity to the C. rufifacies mitochondrial genome reference sequence. Lower nucleotide identities between the sequenced PCR products and other forensically relevant fly species demonstrated that combining ESEM and DNA barcoding is a promising approach to rapid and reliable identification of insect larvae recovered from forensic samples.

genetics↗

bioRxiv2025

Developmental temperature drives distinct transcriptomic responses to acute temperatures and correlated differences in thermal tolerance

Marine invertebrate populations exhibit varying capacities to withstand rising environmental temperatures, but the genetic basis of this differential tolerance remains an important area of investigation. Plasticity can be a valuable tool in the arsenal of an animal trying to maintain physiological function under rapidly changing conditions and can be an important contributor to thermal tolerance. In this work, we characterized the transcriptomic response to elevated developmental temperature, and subsequent acute exposure to two higher temperatures using the widespread and ecologically important copepod, Acartia tonsa. Using a split brood experimental design, we found that copepods that developed at 22{degrees}C had higher upper lethal temperatures compared to those reared at 18{degrees}C, demonstrating developmental plasticity in thermal tolerance. Transcriptomic analyses revealed that developmental temperature strongly influenced gene expression both at baseline and in response to acute thermal stress. Exposure to a moderate heat challenge (28{degrees}C) elicited divergent transcriptional responses between developmental treatments, suggesting developmental preconditioning, whereas exposure to extreme heat (33{degrees}C) triggered a more conserved stress response across groups. Weighted gene co-expression network analysis (WGCNA) identified gene modules associated with upper lethal temperature, highlighting stress response, cellular regulation, and metabolic pathways as key contributors to thermal tolerance. Together, our results reveal how developmental environments shape gene expression patterns and thermal phenotypes, providing insight into the molecular basis of plasticity and potential resilience to climate change.

genomics↗

bioRxiv2025

FOXO1 inhibition and FADD knockdown have opposing effects on anticancer drug-induced cytotoxicity and p21 expression in osteosarcoma

Forkhead box class O1 (FOXO1) and fas-associated death domain (FADD) regulate cell death pathways and homeostatic processes such as cell cycle progression and apoptosis. FADD phosphorylation promotes nuclear localization of FOXO1 and FOXO1 regulates FADD expression. Therefore, it is plausible that FOXO1 and FADD have synergistic or antagonistic effects on cell cycle regulation and the response to anticancer drug treatment in cancer cells. In the present study we report that AS1842856-mediated inhibition of FOXO1 reverses anticancer drug-induced cytotoxicity while FADD knockdown increases anticancer drug-induced cytotoxicity in osteosarcoma (OS). Reversed anticancer drug-induced cytotoxicity was accompanied with G2/M cell cycle arrest and increased expression of p21. The anti-cancer function of FOXO1 was further supported by the observation that OS cells that express higher basal levels of FOXO1 had increased sensitivity to camptothecin-induced cytotoxicity. FADD knockdown reversed FOXO1 inhibition-induced increase in p21 expression. The results presented in this study indicate that FOXO1 has a tumor suppressor function while FADD has a tumor promoting function in OS following anticancer drug treatment. The experimental approach used in this investigation also indicates that FADD antagonists the effect of FOXO1 on p21 expression in OS.

cancer biology↗

bioRxiv2025

Mechanisms Underlying Allosteric Modulation of Antiseizure Medication Binding to Synaptic Vesicle Protein 2A (SV2A)

Brivaracetam (BRV) and levetiracetam (LEV) are antiseizure medications (ASMs) that target synaptic vesicle protein 2A (SV2A), while UCB1244283 acts as a positive allosteric modulator of these medications. The SV2A-BRV-UCB1244283 complex reveals how UCB1244283 allosterically enhances BRV binding by occupying an allosteric site near the primary binding site, preventing BRV dissociation. This allosteric site, formed by hydrophobic and uncharged residues, is a novel small-molecule binding site in SV2A. Structural analysis and mutagenesis suggest that an allosteric network between the primary and allosteric sites governs high-affinity ASM binding. UCB1244283 selectively binds SV2A over SV2B and SV2C, with specific mutations disrupting binding. The structure explains why UCB1244283 binding to SV2A selectively allows interaction with specific ASMs but not others due to steric hinderance. Structural comparison reveals that distinct conformational differences between the SV2A-BRV-UCB1244283 and other SV2A-ligand complexes, particularly in the transmembrane domain, influence binding at both sites. Future research will explore potential therapeutics targeting the allosteric site and their impact on SV2A regulation. TeaserUCB1244283 enhances LEV and BRV binding to SV2A, revealing an allosteric site that could aid in developing targeted therapeutics.

biochemistry↗

bioRxiv2024

The DNA Mismatch repair protein, MSH6 is a novel regulator of PD-L1 expression

Immune checkpoint inhibitors (ICIs) are extremely effective in a subgroup of mismatch repair-deficient (MMRd) cancers, but [~]50% remain resistant to treatment. We have shown for the first time that this may be due to the differential regulation of factors linked to response to ICIs upon loss of the different MMR genes. Here, we show that increased PD-L1 expression is observed upon loss of the MMR genes MLH1, MSH2 and PMS2. However, this is not true upon loss of MSH6. Here, we show that this is due to a novel role for MSH6 as a direct regulator of PD-L1 transcription, dependent on recruitment by the histone trimethyltransferase SETD2. Next-generation sequencing of MLH1 and MSH6 knockout (KO) cells revealed that MSH6 KO cells have significantly lower microsatellite instability in comparison to MLH1 KO cells, despite MSH6 KO cells having a higher mutational burden. These findings emphasise the need for gene-specific stratification in the MMRd cohort.

cancer biology↗

bioRxiv2024

The safety and efficacy of Ultrasound Histotripsy and human pluripotent stem cell-derived hepatic spheroid implantation as a potential therapy for treatment of congenital metabolic liver disease: assessment in an immunocompetent rodent model.

BackgroundLiver disease secondary to an inborn or genetic error of metabolism is a rare group of conditions often associated with chronic ill health and reduced survival. Curative treatment is mainly limited to liver transplantation with major long-term risks. Cell therapy is a promising alternative, but current approaches are ineffective. AimTo develop histotripsy, a non-invasive high intensity ultrasound procedure for liver tissue mechanical ablation, combined with hepatocyte stem cell implantation as a novel method of reversing liver failure from genetic disease. This study assessed the safety and feasibility of this approach in healthy rodents. MethodsUnder general anaesthesia, adult rats (n=12) underwent laparotomy and ultrasound histotripsy to the exposed liver. Around 1 million cells were injected into a single histotripsy cavity in each animal under direct vision (n=10) with two receiving only histotripsy without cell injection. On completion of cell implant haemostasis was secured, laparotomy incision closed, and the animals recovered. Groups of animals were terminated immediately and after 4 hrs, 8 hrs, 24hrs, 4 days and 7 days. Liver and vital organs were assessed for procedure related injuries and evidence of viable implanted cells by histology and immunohistochemistry. ResultsAll animals successfully recovered, and no complication was observed throughout the study. Created cavities were successfully identified in histological analysis of rat. The presence of human cells was verified using anti-human nuclei antibody confirming successful implantation of liver organoids into decellularized cavities. ConclusionsIn this feasibility study, we demonstrated suitability of histotripsy to create decellularized cavities in liver parenchyma. In addition, feasibility of direct transplantation of undissociated liver organoids into the created cavities was demonstrated as a potential approach to treat inborn liver disease by creating nodules of healthy cells capable of performing loss metabolic function. Therapeutic efficacy of this approach will be evaluated in an upcoming study.

bioengineering↗

bioRxiv2024

Enhancement and validation of the antibiotic resistance prediction performance of a cloud-based genetics processing platform for Mycobacteria

Tuberculosis remains a global health problem. Making it easier and quicker to identify which antibiotics an infection is likely to be susceptible to will be a key part of the solution. Whilst whole-genome sequencing offers many advantages, the processing of the genetic reads to produce the relevant public health and clinical information is, surprisingly, often the responsibility of the end user which inhibits uptake. Here we characterise how well a freely-available tool we have developed, gnomonicus, predicts the antibiotic resistance profile of a sample (given its variant call file) using our implementation of the second edition of the WHO catalogue of resistance-associated variants (WHOv2). To facilitate this, we have constructed a Diverse Testset of 2,663 publicly-available M. tuberculosis samples which have both genetic and drug susceptibility testing (DST) data. We have chosen to apply the catalogue such that our tool will return a result of (i) Fail if there are insufficient reads at a genetic locus associated with resistance, (ii) Unknown if a genetic variant in a resistance gene not listed in the catalogue is encountered and (iii) Resistant if three or more short-reads support the presence of a resistance-associated variant. The last step increases the sensitivity for all 15 antibiotics but only reaches significance in a few in our testset. Comparing our results to those of TB-Profiler, an existing tool, highlights the different design choices and demonstrates the performance of both tools on our Diverse Testset is comparable. By only considering high confidence DST results we show that gnomonicus, in combination with our translation of WHOv2, achieves sensitivities and specificities in excess of 95% for both isoniazid and rifampicin. Impact StatementWhole genome sequencing clinical samples taken from patients with tuberculosis is a potentially fast and accurate method for determining to which antibiotics the infection will be susceptible. Two barriers need to be overcome; the first, which is knowing which mutations are associated with resistance (or not) to a range of antibiotics is well on the way to be solved thanks to the efforts of the World Health Organization (WHO) who have published extensive catalogues containing lists of such mutations. The second barrier is that the processing of the raw genetic files remains a largely manual process overseen by bioinformaticians. Here we describe gnomonicus, our open-source AMR prediction tool, and report the performance of our translation of the second edition of the WHO catalogue using a carefully designed publicly-available dataset of 2,663 M. tuberculosis samples. We hope that not only will this tool be useful but also that this dataset will be used by other researchers to facilitate comparisons between pipelines, approaches and tools. Data SummaryThe attendant GitHub repository1 allows gnomonicus to be rerun on all 2,663 samples in the Diverse Testset; it therefore includes instructions, the necessary input files (all the variant call files, the version of the WHOv2 catalogue and a link to the H37Rv GenBank file used in this study) and also the output JSON files. The ENA accession numbers for all 2,663 samples (including a bash script to download them) and their corresponding phenotypic drug susceptibility testing results are included with the intention that people can either reproduce our results, or use the same dataset for other analyses. The JSON files output by TB-Profiler have also been added for comparison. The repository contains a series of Juypter notebooks containing Python3 code that allows the user to discover and parse the output JSON files from either tool and save the results as data tables. Other notebooks allow the user to reproduce all the analysis underlying this work, including reproducing the figures and many of the tables.

microbiology↗

bioRxiv2024

Machine learning models reveal environmental and genetic factors associated with the plant circadian clock

The circadian clock of plants contributes to their survival and fitness. However, understanding clock function at the transcriptome level and its response to the environment requires assaying across high resolution time-course experiments. Generating these datasets is labour-intensive, costly and, in most cases, performed under tightly controlled laboratory conditions. To overcome this barrier, we have developed ChronoGauge: an ensemble model which can reliably estimate the endogenous circadian time of plants using the expression of a handful of time-indicating genes within a single time-pointed transcriptomic sample. ChronoGauge can predict a plants circadian time with high accuracy across unseen Arabidopsis bulk RNA-seq and microarray samples, and can be further applied across samples in non-model species, including field samples. Finally, we demonstrate how ChronoGauge can be applied to test hypotheses regarding the response of the circadian transcriptome to specific genotypes or environmental conditions.

plant biology↗

bioRxiv2024

Selective breeding for determinacy and photoperiod sensitivity in common bean (Phaseolus vulgaris L.)

Common bean (Phaseolus vulgaris L.) is a legume pulse crop that provides significant dietary and ecosystem benefits globally. We investigated two key traits, determinacy and photoperiod sensitivity, that are integral to its management and crop production, and that were early selected during the domestication of both Mesoamerican and Andean gene pools. Still, significant variation exists among common bean landraces for these traits. Since landraces form the basis for trait introgression in pre-breeding, understanding these traits genetic underpinnings and relation with population structure is vital for guiding breeding and genetic studies. We explored genetic admixture, principal component, and phylogenetic analyses to define subpopulations and gene pools, and genome-wide association mapping (GWAS) to identify marker-trait associations in a diversity panel of common bean landraces. We observed a clear correlation between these traits, gene pool and subpopulation structure. We found extensive admixture between the Andean and Mesoamerican gene pools in some regions. We identified 13 QTLs for determinacy and 10 QTLs for photoperiod sensitivity, and underlying causative genes. Most QTLs appear to be firstly described. Our study identified known and novel causative genes and a high proportion of pleiotropic effects for these traits in common bean, and likely translatable to other legume species. HighlightWe identified and explored QTLs for the domestication-related determinacy and photoperiod sensitivity traits, which are traits critically associated with population structure and management and crop production.

plant biology↗