Biology subjects

Allen, J. E.

Publications and source records attributed to Allen, J. E..

At least 19 recordsLinked to original sources

bioRxiv2026

Human and mouse cerebellar inhibitory circuits in dystonic crisis and their modulation with therapeutic stimulation

Dystonia is a neurological movement disorder characterized by abnormal muscle contractions that, at their most severe, lead to a life-threatening condition - dystonic crisis. Yet, therapeutic options remain limited by our incomplete understanding of what neural circuits underly the condition. Although cerebellar nuclei neurons are implicated in the origin of baseline dystonic symptoms, it is unclear whether their activity drives dystonic crisis. To explore this role, we found that cerebellar abnormalities and neural inhibition were recurring targets in patients with dystonic crisis, implicating cerebellar inhibitory neurons in its development. We devised a mouse genetics approach to test whether inhibitory cerebellar nuclei neurons (iCNNs) induce dystonic crisis. Directional optogenetic modulation of iCNNs induced dystonic crises on-demand and alleviated spontaneous crises in mice that mimic spontaneous dystonic crises. To investigate whether iCNNs interact with other motor areas during dystonic crisis, we identified monosynaptic iCNN projections to the centrolateral nucleus of the thalamus (CL). Deep brain stimulation of the CL alleviated dystonic crises induced by iCNN photoactivation. Our data uncover a cell type-specific cerebellar origin of dystonic crisis and highlight its therapeutic potential.

neuroscience↗

bioRxiv2025

BATF3 controls the homeostatic maintenance and function of innate-like γδ T cells

{gamma}{delta} T cells compose an evolutionarily conserved lineage of lymphocytes, with both adaptive- and innate-like characteristics, contributing to tissue homeostasis, immune surveillance, and rapid responses to stress and infection. While their functional diversity and tissue-specific roles are tightly regulated by transcriptional networks, the underlying molecular mechanisms remain incompletely understood. The transcription factor basic leucine zipper ATF-like transcription factor 3 (BATF3) plays a central role in the development of conventional type 1 dendritic cells (cDC1s). Here, we unveil BATF3 as a critical cell-intrinsic regulator of the homeostasis, functional specialization, and tissue distribution of {gamma}{delta} T cells. Batf3-deficient mice display an altered composition of {gamma}{delta} T cell subsets, with a marked decrease in the numbers of innate-like {gamma}{delta} T cells across multiple organs when compared to their wild-type counterparts, independently of cDC1s. Loss of BATF3 impacts not only cell survival but also IL-17 production after {gamma}{delta} T cells complete their thymic development. Mechanistically, Batf3-deficient innate-like {gamma}{delta} T cells exhibit transcriptional changes that disrupt pathways governing actin cytoskeleton remodelling, immunological synapse organization and cellular identity. Notably, Batf3-deficient mice present decreased survival in a viral infection model highly dependent on innate-like {gamma}{delta} T cells. Together, our findings uncover a previously unrecognized BATF3-dependent pathway that controls {gamma}{delta} T cell morphology and function, profoundly impacting their biology.

immunology↗

bioRxiv2025

The hyaluronan receptor CD44 drives COVID-19 severity through its regulation of neutrophil migration

The novel respiratory disease COVID-19 caused by the coronavirus SARS-CoV-2 continues to be a public health emergency worldwide, and there is a need for more effective therapy for patients. The relationship between the extracellular matrix and the host immune response to infection is severely understudied. Deposition of the polysaccharide hyaluronan (HA) into the lungs is associated with more severe COVID-19 disease outcomes. HA is a major component of the extracellular matrix in connective tissues and is abundant in many parts of the body, including cartilage, skin, brain, and vitreous body. CD44 is the primary receptor for HA and is found on almost all immune cells in the lung. Known functions of CD44 include mediation of immune cell migration, activation, and differentiation. We hypothesized that increased HA deposition during COVID-19 increases CD44-mediated immune cell infiltration into lungs and results in more severe pathology. Here, we report that in mice infected with a mouse-adapted strain of SARS-CoV-2, treatment with a combination of two anti-CD44 monoclonal antibodies confers a significant survival benefit and reduces weight loss and clinical score of the mice on Day 4 post infection. We show that anti-CD44 treatment decreases many key cytokines and chemokines in the bronchoalveolar lavage fluid on Day 4. With flow cytometry, we show that anti-CD44 reduces the numbers of neutrophils in infected lungs. We also show through immunofluorescence that treatment with anti-CD44 antibodies reduces colocalization of HA and CD45 in lung sections, indicating that HAs interaction with immune cells contributes to pathology. Our findings demonstrate that disruption of HA-receptor interactions is a way to prevent inflammatory pathology in pulmonary infection.

immunology↗

bioRxiv2025

ONC206 demonstrates potent anti-tumorigenic activity and is a potential novel therapeutic strategy for high-risk medulloblastoma

BackgroundMedulloblastoma is the most common malignant pediatric brain tumor, and has an urgent need for novel treatment approaches. Dordaviprone (ONC201) and its chemical derivative with nanomolar potency, ONC206, induce apoptosis of cancer cells by activation of the mitochondrial caseinolytic protease P (ClpP). ONC206 is currently in Phase I clinical trials for pediatric patients with primary brain tumors. MethodsIn this study, we evaluated the preclinical therapeutic effects of ONC206 in medulloblastoma and investigated its mechanism of action. ResultsWe found evidence for high expression of ClpP at both the RNA and protein level in medulloblastoma tumors, compared to very low expression in normal brain tissue. In addition, we saw a pronounced reduction in cell viability of human Group 3 and Group 4 and murine SHH-driven and Group 3 medulloblastoma cells treated with ONC206 with low IC-50s. After treatment with ONC206, we observed an induction of integrated stress response and mitochondrial damage. To test the efficacy of ONC206 in vivo, we used murine models of SHH-driven and Group 3 medulloblastoma as well as Group 3 and Group 4 patient-derived xenografts (PDXs). ONC206 led to a significant prolongation of survival in both murine models, with the SHH mice demonstrating survival extension from 70 to 140 days. PDX-bearing mice also responded to ONC206, which led to a significant survival benefit. ConclusionOur results highlight ONC206 as a novel therapeutic option for patients with high-risk medulloblastoma and provide strong rationale for testing the efficacy of ONC206 in the treatment of these patients. Key points (2-3)ONC206 potently kills medulloblastoma cells by inducing integrated stress response and mitochondrial damage. ONC206 prolongs survival of medulloblastoma-bearing mice in both murine and patient-derived xenograft models. Importance of studyThere is an unmet need for better therapies for high-risk medulloblastoma patients. ONC201 has shown promising responses and recently received FDA approval for diffuse midline glioma. ONC206 is a chemical derivative with higher potency and better brain penetrance. In this study, we analyzed the therapeutic potential of ONC206 for high-risk medulloblastoma and found that the drug effectively killed mouse and human medulloblastoma cells with high nanomolar potency. We also saw that ONC206 very significantly prolonged survival of medulloblastoma-bearing mice, both in genetically engineered mouse models and patient-derived xenografts. Our study provides a strong rationale for testing the efficacy of ONC206 in the treatment of patients with medulloblastoma and has set the stage for a clinical trial with this agent in pediatric patients with recurrent malignant brain tumors, including medulloblastoma (NCT04732065).

cancer biology↗

bioRxiv2025

SLAB: Simultaneous Labeling And Binding affinity prediction for protein-ligand structures

Machine learning models are often used as scoring functions to predict the binding affinity of a protein-ligand complex. These models are trained with limited amounts of data with experimentally measured binding affinity values. A large number of compounds are labeled inactive throughs single-concentration screens without measuring binding affinities. These inactive compounds, along with the active ones, can be used to train binary classification models, while regression models are trained using compounds with binding affinities only. However, the classification and regression tasks are often handled separately, without sharing the learned feature representations. In this paper, we propose a novel model architecture that jointly performs regression and classification objectives, aiming to maximize data utilization and improve predictive performance by leveraging two complementary tasks. In our setup, the regression yields the binding affinity, whereas the classification task yields the label as active or inactive. We demonstrate our method using PDBbind, the standard 3D structure database, as well as a dataset of flavivirus protease compounds with binding affinity data. Our experiments show that the new joint training strategy improves the accuracy of the model, increasing applicability in various practical drug screening scenarios.

pharmacology and toxicology↗

bioRxiv2025

Generation of a Ym1 Deficient Mouse utilising CRISPR-Cas9 in CB6 Embryos

Chitinase-like proteins (CLPs) are of wide interest due to their significant roles during both biological and pathological processes. Human CLPs such as YKL-40 have been suggested as biomarkers of disease severity in many conditions. Murine CLPs include Brp39, Ym1, and Ym2 and these are similarly upregulated in multiple mouse models of pathology. Investigation of these molecules, particularly Ym1 and Ym2, is plagued by complexity in the genomic locus due to recent gene duplication events in the C57BL/6 strain. Using a novel CRISPR-Cas9 targeting approach involving CB6 mixed background embryos, we generated a Ym1 deficient mouse. Validation using flow cytometry, ELISA, and immunofluorescence confirmed no expression of mature Ym1 protein with no alteration in the expression of related chitinases/CLP genes including Chia and Chil4. This new transgenic mouse line will be key for investigating CLP functions and the genetic approach utilised may provide a useful strategy for other genes which show differences between inbred mouse strains.

genetics↗

bioRxiv2025

Migratory dermal gamma delta T cells determine the balance between lung immunity and tissue damage during Nippostrongylus brasiliensis infection

Many helminth parasites migrate through multiple host organs during infection but how immune responses are relayed across these tissues is still poorly understood. To investigate the cellular and molecular aspects of inter-tissue communication during infection we established a percutaneous infection model with the tissue-migrating nematode Nippostrongylus brasiliensis. High-dimension profiling of the initial cutaneous immune response revealed that dermal {gamma}{delta} T cells become activated via an IL-1R-dependent mechanism, engage cell motility-associated transcriptional pathways and leave the skin after parasite invasion. Chemical and genetic inhibition of leukocyte migration prevents the accumulation of IL-17-producing {gamma}{delta} T cells in the lungs. Notably, bypassing the skin phase of infection, and therefore preventing dermal {gamma}{delta} T cell migration, dampens the increase in early IL-17 production in the lungs, and, instead, leads to enhanced IFN-{gamma} responses together with increased lung damage. Collectively, our data highlights a critical skin-lung axis regulating host-parasite interactions and safeguarding lung health.

immunology↗

bioRxiv2024

The IL-33/ST2 signaling axis drives pathogenesis in acute SARS-CoV-2 infection

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), remains a significant threat to global public health. Immunopathological damage plays a role in driving pneumonia, acute respiratory distress syndrome (ARDS), and multiorgan failure in severe COVID-19. Therefore, dissecting the pulmonary immune response to SARS-CoV-2 infection is critical to understand disease pathogenesis and identify immune pathways targetable by therapeutic intervention. Considering that the type 2 cytokine IL-13 enhances COVID-19 disease severity, therapeutic targeting of upstream signals that drive type 2 immunity may confer further protection. In this study, we investigate the role of the IL-33/ST2 signaling axis, a potent inducer of type 2 immunity in the lung, in a mouse model of COVID-19. Upon infection with mouse-adapted SARS-CoV-2 MA10, ST2-/- mice had significantly improved weight loss and survival (69.2% vs 13.3% survival; P = 0.0005), as compared to wild-type mice. In a complementary pharmacologic approach, IL-33/ST2 signaling was inhibited using HpBARI_Hom2, a helminth derived protein that binds to mouse ST2 and blocks IL-33 signaling. In SARS-CoV-2 MA10 infection, HpBARI_Hom2-treated mice had significantly improved weight loss and survival (60% vs 10% survival; P = 0.0035), as compared to inert control-treated mice. These data demonstrate that loss of IL-33/ST2 signaling confers protection during acute SARS-CoV-2 MA10 infection, implicating the IL-33/ST2 signaling axis as an enhancer of COVID-19 disease severity. The protection conferred by pharmacologic blockade of IL-33/ST2 signaling was independent of viral control, as HpBARI_Hom2-treated mice had no reduction in viral titers. This finding suggests an immunopathogenic role for IL-33/ST2 signaling. One potential mechanism through which IL-33/ST2 signaling may drive severe disease is through enhancement of type 2 immune pathways including IL-5 production, as pulmonary IL-5 concentrations were found to depend on IL-33/ST2 signaling in acute SARS-CoV-2 MA10 infection.

immunology↗

bioRxiv2024

Addressing the dynamic nature of reference data: a new nt database for robust metagenomic classification

BackgroundAccurate metagenomic classification relies on comprehensive, up-to-date, and validated reference databases. While the NCBI BLAST Nucleotide (nt) database, encompassing a vast collection of sequences from all domains of life, represents an invaluable resource, its massive size --currently exceeding 1012 nucleotides-- and exponential growth pose significant challenges for researchers seeking to maintain current nt-based indices for metagenomic classification. Recognizing that no current nt-based indices exist for the widely used Centrifuge classifier, and the last public version was released in 2018, we addressed this critical gap by leveraging advanced high-performance computing resources. ResultsWe present new Centrifuge-compatible nt databases, meticulously constructed using a novel pipeline incorporating different quality control measures, including reference decontamination and filtering. These measures demonstrably reduce spurious classifications, and through temporal comparisons, we reveal how this approach minimizes inconsistencies in taxonomic assignments stemming from asynchronous updates between public sequence and taxonomy databases. These discrepancies are particularly evident in taxa such as Listeria monocytogenes and Naegleria fowleri, where classification accuracy varied significantly across database versions. ConclusionsThese new databases, made available as pre-built Centrifuge indexes, respond to the need for an open, robust, nt-based pipeline for taxonomic classification in metagenomics. Applications such as environmental metagenomics, forensics, and clinical metagenomics, which require comprehensive taxonomic coverage, will benefit from this resource. Our new nt-based index highlights the importance of treating reference databases as dynamic entities, subject to ongoing quality control and validation akin to software development best practices. This dynamic update approach is crucial for ensuring the accuracy and reliability of metagenomic analysis, especially as databases continue to expand in size and complexity.

bioinformatics↗

bioRxiv2023

Mgl2+ cDC2s coordinate fungal allergic airway type 2, but not type 17, inflammation.

Fungal spores are abundant in the environment and a major cause of asthma. Originally characterised as a type 2 inflammatory disease, allergic airway inflammation that underpins asthma can also involve type 17 inflammation, which can exacerbate disease causing failure of treatments tailored to inhibit type 2 factors. However, the mechanisms that determine the host response to fungi, which can trigger both type 2 and type 17 inflammation in allergic airway disease, remain unclear. We found that CD11c+ DCs and CD4+ T cells are essential for development of both type 2 and type 17 airway inflammation in mice when repeatedly exposed to inhaled spores. Single cell RNA-sequencing enabled the development of multi-parameter cytometry that identified allergic inflammation dramatically altered the proportion of numerous DC clusters in the lung, but that only two of these (Mgl2+ cDC2s and CCR7+ DCs) migrated to the dLNs. Targeted removal of several DC subsets revealed that only Mgl2+ cDC2 depletion dramatically reduced type 2, but not type 17, anti-fungal allergic airway inflammation. These data highlight distinct DC subsets are potential therapeutic targets for the treatment of pulmonary fungal disease.

immunology↗

bioRxiv2023

GENTANGLE: integrated computational design of gene entanglements

SummaryThe design of two overlapping genes in a microbial genome is an emerging technique for adding more reliable control mechanisms in engineered organisms for increased safety. The design of functional gene pairs is a challenging procedure and computational design tools are used to improve the efficiency to deploy successful designs in genetically engineered systems. GENTANGLE (Gene Tuples ArraNGed in overLapping Elements) is a high performance containerized pipeline for the computational design of two overlapping genes translated in different reading frames of the genome. This new software package can be used to design and test gene entanglements for microbial engineering projects using arbitrary sets of user specified gene pairs. Availability and ImplementationThe GENTANGLE source code and its submodules are freely available on GitHub at https://github.com/BiosecSFA/gentangle. The DATANGLE (DATA for genTANGLE) repository contains related data and results, and is freely available on GitHub at https://github.com/BiosecSFA/datangle. The GENTANGLE repository wiki contains detailed instructions on how to use the container and the different components of software and data, including reproducing the results. The code is licensed under the GNU Affero General Public License version 3 (https://www.gnu.org/licenses/agpl.html). Contactmartimartine1@llnl.gov and allen99@llnl.gov

bioinformatics↗

bioRxiv2023

Cutaneous T cell lymphoma atlas reveals malignant Th2 cells supported by a B cell-rich microenvironment

Cutaneous T-cell lymphoma (CTCL) is a potentially fatal clonal malignancy of T cells primarily affecting the skin. The most common form of CTCL, mycosis fungoides (MF), can be difficult to diagnose resulting in treatment delay. The pathogenesis of CTCL is not fully understood due to limited data from patient studies. We performed single-cell RNA sequencing and spatial transcriptomics profiling of skin from patients with MF-type CTCL, and an integrated comparative analysis with human skin cell atlas datasets from healthy skin, atopic dermatitis and psoriasis. We reveal the co-optation of Th2-immune gene programmes by malignant CTCL cells and modelling of the tumour microenvironment to support their survival. We identify MHC-II+ fibroblast subsets reminiscent of lymph node T-zone reticular cells and monocyte-derived dendritic cells that can maintain Th2-like tumour cells. CTCL Th2-like tumour cells are spatially associated with B cells, forming aggregates reminiscent of tertiary lymphoid structures which are more prominent with progressive disease. Finally, we validated the enrichment of B cells in CTCL skin infiltrates and its association with disease progression across three independent patient cohorts. Our findings provide diagnostic aids, potential biomarkers for disease staging and therapeutic strategies for CTCL.

genomics↗

bioRxiv2023

Allergen-induced airway matrix remodelling in mice can be prevented or reversed by targeting chitinase-like proteins.

Chitinase-like proteins (CLPs) are biomarkers of inflammation and airway remodelling in asthma, yet their direct contribution towards disease pathogenesis is unknown. Using a mouse model of allergen-induced type 2/type 17 airway inflammation we sought to directly investigate the role of the murine CLPs Ym1 and Ym2 during chronic lung pathology. Data demonstrated distinct chronic inflammatory roles for Ym2, IL-13, and IL-17a signalling pathways. Notably, only CLPs were key for initiating the pathogenic accumulation and re-organisation of the pulmonary extracellular matrix (ECM) environment. Furthermore, inhibition of CLPs after chronic pathology developed, reversed airway remodelling independently of chronic inflammation. These studies disentangle chronic IL-13 and IL-17a signalling from the development of allergic airway remodelling and instead highlight a central role for CLPs, which provides new avenues to therapeutically target aberrant ECM accumulation.

immunology↗

bioRxiv2023

Clustering Protein Binding Pockets and Identifying Potential Drug Interactions: A Novel Ligand-based Featurization Method

Protein-ligand interactions are essential to drug discovery and drug development efforts. Desirable on-target or multi-target interactions are a first step in finding an effective therapeutic; undesirable off-target interactions are a first step in assessing safety. In this work, we introduce a novel ligand-based featurization and mapping of human protein pockets to identify closely related protein targets, and to project novel drugs into a hybrid protein-ligand feature space to identify their likely protein interactions. Using structure-based template matches from PDB, protein pockets are featurized by the ligands which bind to their best co-complex template matches. The simplicity and interpretability of this approach provides a granular characterization of the human proteome at the protein pocket level instead of the traditional protein-level characterization by family, function, or pathway. We demonstrate the power of this featurization method by clustering a subset of the human proteome and evaluating the predicted cluster associations of over 7,000 compounds.

synthetic biology↗

bioRxiv2023

Generative Molecular Design and Experimental Validation of Selective Histamine H1 Inhibitors

Generative molecular design (GMD) is an increasingly popular strategy for drug discovery, using machine learning models to propose, evaluate and optimize chemical structures against a set of target design criteria. We present the ATOM-GMD platform, a scalable multiprocessing framework to optimize many parameters simultaneously over large populations of proposed molecules. ATOM-GMD uses a junction tree variational autoencoder mapping structures to latent vectors, along with a genetic algorithm operating on latent vector elements, to search a diverse molecular space for compounds that meet the design criteria. We used the ATOM-GMD framework in a lead optimization case study to develop potent and selective histamine H1 receptor antagonists. We synthesized 103 of the top scoring compounds and measured their properties experimentally. Six of the tested compounds bind H1 with Kis between 10 and 100 nM and are at least 100-fold selective relative to muscarinic M2 receptors, validating the effectiveness of our GMD approach.

pharmacology and toxicology↗

bioRxiv2022

IL-17A promotes epithelial cell IL-33 production during nematode lung migration

The early migratory phase of pulmonary helminth infections is characterized by tissue injury leading to the release of the alarmin IL-33 and subsequent induction of type 2 immune responses. We recently described a role for IL-17A, through regulation of IFN{gamma}, as an important inducer of type 2 responses during infection with the lung-migrating rodent nematode Nippostrongylus brasiliensis. Here, we aimed to investigate the interaction between IL-17A and IL-33 during the early lung migratory stages of N. brasiliensis infection. In this brief report, we demonstrate that deficiency of IL-17A leads to impaired IL-33 expression and secretion early in infection, independent of IL-17A suppression of IFN{gamma}. Impaired IL-33 production was evident in lung epithelial cells, but not innate immune cells. Therefore, our results demonstrate that IL-17A can drive IL-33 during helminth infection, highlighting an additional mechanism through which IL-17A can regulate pulmonary type 2 immunity.

immunology↗

bioRxiv2022

Model Choice Metrics to Optimize Profile-QSAR Performance

BackgroundPredicting molecular activity against protein targets is difficult because of the paucity of experimental data. Approaches like multitask modeling and collaborative filtering seek to improve model accuracy by leveraging results from multiple targets, but are limited because different compounds are measured with different assays, leading to sparse data matrices. Profile-QSAR (pQSAR) 2.0 addresses this problem by fitting a series of partial least squares models for each target, using as features the predictions from single-task models on the remaining targets. This method has been shown to produce better results than single task and multitask models. However, the factors determining the success of pQSAR 2.0 have as yet not been characterized. In this paper we examine the experimental conditions that lead to better pQSAR models. We limit the amount of data available to the method by retraining with decreasing amounts of data and explore the models ability to generalize to compounds that have never been assayed. Finally, we look at the properties of training data needed to demonstrate pQSAR improvement. ResultsWe apply pQSAR 2.0 on a collection of GPCR and safety targets collected from Drug Target Commons, ExcapeDB, and ChEMBL. We found that pQSAR improved models on 34 of the 149 assays selected. In the other 115 assays, single task random forests offered better performance. There are many factors that contribute to an increase in performance, but the main factor is compound assay coverage. The pQSAR model improves when more compounds are measured in multiple assays. ConclusionIt is necessary to consider the available data before applying pQSAR. Successful pQSAR models require a profile made of correlated targets that share compounds with other assays. This technique is best used when experimental data is available as random forest regressors often do not generalize well enough for virtual drug search applications.

bioinformatics↗

bioRxiv2022

Monocyte-derived peritoneal macrophages protect C57BL/6 mice against surgery-induced adhesions

Peritoneal adhesions commonly occur after abdominal or pelvic surgery. These scars join internal organs to each other or to the cavity wall and can present with abdominal or pelvic pain, and bowel obstruction or female infertility. The mechanisms underlying adhesion formation remain unclear and thus, effective treatments are not forthcoming. Peritoneal macrophages accumulate after surgery and previous studies have attributed either pro- or anti-scarring properties to these cells. We propose that there are complex and nuanced responses after surgery with respect to both resident and also monocyte-derived peritoneal macrophage subpopulations. Moreover, we contend that differences in responses of specific macrophage subpopulations in part explain the risk of developing peritoneal scars. We characterised alterations in peritoneal macrophage subpopulations after surgery-induced injury using two strains of mice, BALB/c and C57BL/6, with known differences in macrophage response post-infection. At 14 days post-surgery, BALB/c mice displayed more adhesions compared with C57BL/6 mice. This increase in scarring correlated with a lower influx of monocyte-derived macrophages at day 3 post-surgery. Moreover, BALB/c mice showed distinct macrophage repopulation dynamics after surgery. To confirm a role for monocyte-derived macrophages, we used Ccr2-deficient mice as well as antibody-mediated depletion of CCR2 expressing cells during initial stages of adhesion formation. Both Ccr2-deficient and CCR2-depleted mice showed a significant increase in adhesion formation associated with the loss of peritoneal monocyte influx. These findings revealed an important protective role for monocyte-derived cells in reducing adhesion formation after surgery.

immunology↗