Biology subjects

Allen, G.

Publications and source records attributed to Allen, G..

15 recordsLinked to original sources

bioRxiv2026

Botrytis cinerea infection reshapes the grape berry microbiome during noble rot

Noble rot, caused by Botrytis cinerea, profoundly alters grape berry physiology and is essential to produce botrytized wines. In this study, we profiled bacterial and fungal communities associated with Vitis vinifera cv. Semillon berries across four stages of noble rot development and four consecutive vintages using 16S rRNA gene and ITS1 amplicon sequencing. Noble rot stage significantly impacted the structure of bacterial communities across vintages, while fungal communities showed more variable vintage-dependent responses. Bacterial alpha diversity increased consistently at advanced stages of infection (S3), coinciding with a marked shift from Pseudomonas-dominated communities toward acetic acid bacteria, particularly Gluconobacter, which was significantly enriched at S3 in all vintages. Fungal communities remained dominated by Sclerotiniaceae throughout infection, consistent with pervasive B. cinerea colonization, while non-Botrytis fungal taxa shifted from filamentous fungi such as Cladosporium and Alternaria toward fermentative yeasts including Hanseniaspora and Lachancea. Co-occurrence network analysis revealed a positive association between Gluconobacter and these fermentative yeasts, suggesting coordinated enrichment of oxidative and fermentative microorganisms at advanced noble rot stages. Together, these results reveal a reproducible stage-associated microbial succession during noble rot progression and identify acetic acid bacteria as consistent markers of advanced infection.

microbiology↗

bioRxiv2026

Ultra-large targeted DNA integrations in primary human cells

Genetic engineering experiments and therapies are constrained by the size of DNA integrations into human cells genomes. Existing AAV, lentiviral, and non-viral methods rapidly decrease in integration efficiency beyond [~]5kb of sequence. Through systematic evaluation of non-viral DNA template formats, we identified circular ssDNA and dsDNA as capable of mediating >5kb integrations. Large circular DNA delivery efficiency and its impacts on cell viability and payload expression could be significantly improved with small DNA "helper" plasmids, mRNA-encoded nucleases, and sequence design optimizations. Collectively, these modifications enabled ultra-large--up to 10 kb DNA--integrations at >20% efficiency in primary human T cells at the TRAC locus and at >60% efficiency in human iPSCs at the AAVS1 locus. Finally, we demonstrate that GMP clinical-manufactured T cells with ultra-large integrations are functional in vitro and in vivo. Overall, we identified optimal template architectures, delivery modes, and sequence design rules for ultra-large DNA integrations in both research and clinical settings to accelerate basic genetic research and next-generation cellular therapies.

genetics↗

bioRxiv2025

In vivo genome-wide CRISPR screens in human T cells to enhance T cell therapy for solid tumors

Large-scale CRISPR screening in human T cells holds significant promise for identifying genetic modifications that can enhance cellular immunotherapy. However, many genetic regulators of T cell performance in solid tumors may not be readily revealed in vitro. In vivo screening in tumor-bearing mice offers greater physiological relevance, but has historically been limited by low intratumoral T cell recovery. Here, we developed a new model system that achieves significantly higher human T cell recovery from tumors, enabling genome-wide in vivo screens with small numbers of mice. Tumor-infiltrating T cells in this model exhibit hallmarks of dysfunction compared to matched splenic T cells, creating an ideal context for screening for genetic modifiers of T cell activity in the tumor microenvironment. Using this platform, we performed two genome-wide CRISPR knockout screens to identify genes regulating T cell intratumoral abundance and effector function (e.g., IFN-{gamma} production). The intratumoral abundance screen uncovered the P2RY8-G13 GPCR signaling pathway as a negative regulator of human T cell infiltration into tumors. The effector function screen identified GNAS (Gs), a central signaling mediator downstream of multiple GPCRs that sense different suppressive ligands, as a key regulator of T cell dysfunction in tumors. Targeted GNAS knockout rendered T cells resistant to multiple suppressive cues and significantly improved therapeutic performance across diverse solid tumor models. Moreover, combinatorial knockout of P2RY8 (trafficking) and GNAS (effector function) further enhanced overall tumor control, demonstrating that genetic modifications targeting distinct T cell phenotypes can be combined to improve therapeutic potency. This flexible and scalable in vivo screening platform can be adapted to diverse tumor models and pooled CRISPR libraries, enabling future discovery of genetic strategies that equip T cell therapies to overcome barriers imposed by solid tumors.

immunology↗

bioRxiv2025

Engineering lung-sensing T cells using synthetic receptors targeting RAGE

Our goal was to leverage synthetic biology approaches to engineer lung-sensing T cells that trigger synthetic transcriptional programs only when in the lung. First, we identified a lung-specific cell surface protein, receptor for advanced glycation endproducts (RAGE), expressed at high levels exclusively in the lung. Then, we engineered chimeric antigen receptors (CAR) and synthetic notch receptors (SynNotch) to bind this protein. We showed that anti-RAGE CAR-T cells traffic to and proliferate in the lung exclusively. Anti-RAGE SynNotch receptors activate transcription of a fluorescent reporter only when co-cultured with RAGE+ cell lines or primary lung cells. Finally, we tested an anti-RAGE SynNotch to anti-CD19 CAR circuit in vivo in mice implanted with lung and flank tumors and found that this approach cleared CD19-expressing lung tumors without affecting CD19-expressing flank tumors. Thus, we demonstrate that RAGE is a lung-specific target and that T cells expressing anti-RAGE receptors can sense and activate lung-specific therapeutic transcriptional programs. This approach could be extended to allow cell-based therapies that target and deliver genetically encoded payloads to treat lung diseases while avoiding systemic toxicity.

synthetic biology↗

bioRxiv2025

Ontology-aware DNA methylation classification with a curated atlas ofhuman tissues and cell types

AO_SCPLOWBSTRACTC_SCPLOWDNA methylation (DNAm) is a core gene regulatory mechanism that captures cellular responses to short- and long-term stimuli such as environmental exposures, aging, and cellular differentiation. Although DNAm has proven valuable as a baseline biomarker for aging by enabling robust characterization of disease-associated methylation shifts associated with age, its potential to reveal analogous shifts in the context of tissue remains underex-plored. A major obstacle has been the absence of comprehensive, curated reference atlases spanning diverse normal human tissues, limiting most existing work to disease-subtype differentiation or localized tissue comparisons. To bridge this gap, we assemble the largest and most diverse atlas of exclusively healthy human tissue and cell samples profiled by 450K arrays, comprising of 16,959 samples across 86 tissues and cell types. Leveraging this resource, we introduce an ontology-aware classification framework that identifies robust CpG features associated with tissue and cell identity while integrating known anatomical and functional relationships (e.g., prefrontal cortex in the brain, leukocytes in blood). Our novel application of Minipatch learning distills a set of 190 CpG sites that can accurately support multi-label classification. We further validate our approach through an ontology-based label transfer task, demonstrating the effectiveness of ontology-informed learning to accurately predict relevant labels for 31 tissues and cell types not seen during training. These findings underscore the potential of our framework to enhance our understanding of healthy methylation landscapes and facilitate future applications in disease detection and personalized medicine.

bioinformatics↗

bioRxiv2025

Vitronectin binding affinity and cell viability effect of novel mechanotherapy drugs for neuroblastoma

High-risk neuroblastoma (HR-NB) is an aggressive form of childhood cancer with a five-year survival rate of under 50%, underscoring the need for more efficacious and less toxic treatments. The glycoprotein Vitronectin (VN) has been linked to poor prognosis in patients with HR-NB, and thus inhibitors of its function represent a promising avenue for molecular mechanotherapy. The present study sought to investigate the binding affinity between the somatomedin B (SMB) domain of VN and natural compounds derived from medicinal plants. The therapeutic potential of -amyrin (AMY), lupeol (LUP), and Olax chalcone A (Olax CHA) was tested in combination with an integrin antagonist of VN, cilengitide (CLG), using the SK-N-BE(2) HR-NB cell line as a model. Molecular docking studies indicated a potential for protein-ligand interactions for all selected compounds, of which CLG demonstrated the most favorable binding free energy (kcal/mol), followed by LUP, AMY and Olax CHA. Molecular dynamics simulations demonstrated that the SMB domain of VN initially exhibited flexibility, with alpha carbon-root mean square deviation (RMSD) stabilizing at approximately 1.8-2.1 [A]. While all compounds demonstrated a dose-dependent decrease in SK-N-BE(2) cell viability, CLG exhibited higher IC50 values. Although the combination of AMY and LUP with CLG did not result in enhanced efficacy, Olax CHA exhibited a superior antiproliferative effect with higher IC50 values than AMY and LUP, and additionally showed potential synergism with CLG, suggesting a more effective therapeutic approach. This work provides valuable insights into the potential use of mechanotherapy drugs and natural products to enhance HR-NB treatment that can be expanded in future studies centered on Olax CHA.

cancer biology↗

bioRxiv2025

A bioprinting approach for high-throughput production of micropatterned neuroepithelial tissues and modeling TSC2-deficient brain malformations

In vitro human pluripotent stem cell-derived models have been crucial in advancing our understanding of the mechanisms underlying neurodevelopment, though knowledge of the earliest stages of brain formation is lacking. Micropatterning of cell populations as they transition from pluripotency through the process of neurulation can produce self-assembled neuroepithelial tissues (NETs) with precise spatio-temporal control, enhancing the fidelity of hPSC models to the early developing human brain and their use in phenotypic assessments. Here, we introduce an accessible, customizable and scalable method to produce self-assembled NETs using bioprinting to rapidly deposit reproducibly sized extracellular matrix droplets. Matrix addition to the media provides a scaffold that promotes 3D tissue folding, reflecting neural tube development. We demonstrate that these scaffolded NETs (scNETs) exhibit key architectural and biological features of the human brain during normal and abnormal development, notably hyperproliferation and structural malformations induced by TSC2-deficiency, and provide a robust drug screening tool.

neuroscience↗

bioRxiv2025

Phenological turnover matters when making trait-based predictions of plant-pollinator interactions

1. Understanding the processes determining species interactions is key to predicting and safeguarding ecological networks under rapid environmental change. One approach to estimating interactions is to use morphologies of taxa interacting across trophic levels to reveal suites of traits they are more likely to interact with (i.e. a trait niche). 2. Previous work studying these morphological trait niches has typically used interactions between species that are pooled in space and time. However, species assemblages, and the traits of individuals within species, can change across even small landscapes over a season, leading to morphological trait space being dynamically reshaped. Therefore, it is unclear how morphological trait turnover affects our inferences of trait niches, and our ability to answer this is in part limited by a lack of individual-level trait data. 3. Here, we directly address this by studying a montane Arctic plant-pollinator community over five growing seasons (>1,300 hours of fieldwork). Specifically, we linked every recorded plant-bumblebee interaction with the traits of the bee individual involved (n = 1,150), to investigate 1) whether plant taxa (n = 10) exhibited bee trait niches by interacting with specific regions of multidimensional trait space of visiting bumblebees, and 2) how our inference of these trait niches was affected by considering bumblebee trait turnover and plant taxon turnover. 4. When not considering turnover (interactions in space and time are pooled), plant taxa demonstrated bee trait niches. However, next we considered how bee trait space is reshaped over the elevational and seasonal gradient (especially with the emergence of different castes), and how this reshaping co-occurs with different spatiotemporal ranges of the plant taxa. From this we found the degree to which plant taxa exhibited trait niches declined significantly, and that seasonal reshaping of bee trait space was the primary driver of this trend. 5. Overall, in highly dynamic systems, like the Arctic, overlooking community turnover could mask and even overestimate the ability of morphology to explain interactions. Hence, determining how morphological traits of individual interaction partners are in phenological synchrony at localised scales will be fundamental to understanding the role morphology plays in underpinning plant-pollinator interactions.

ecology↗

bioRxiv2022

Breathe Easy, an automated respiratory data pipeline for waveform characteristic analysis

Comprehensive and accurate analysis of respiratory and metabolic data is crucial to modelling congenital, pathogenic, and degenerative diseases converging on autonomic control failure. A lack of tools for high-throughput analysis of respiratory datasets remains a major challenge. We present Breathe Easy, a novel open-source pipeline for processing raw recordings and associated metadata into operative outcomes, publication-worthy graphs, and robust statistical analyses including QQ and residual plots for assumption queries and data transformations. This pipeline uses a facile graphical user interface for uploading data files, setting waveform feature thresholds, and defining experimental variables. Breathe Easy was validated against manual selection by experts, which represents the current standard in the field. We demonstrate Breathe Easys utility by examining a 2-year longitudinal study of an Alzheimers Disease mouse model to assess contributions of forebrain pathology in disordered breathing. Whole body plethysmography has become an important experimental outcome measure for a variety of diseases with primary and secondary respiratory indications. Respiratory dysfunction, while not an initial symptom in many of these disorders, often drives disability or death in patient outcomes. Breathe Easy provides an open-source respiratory analysis tool for all respiratory datasets and represents a necessary improvement upon current analytical methods in the field.

physiology↗

bioRxiv2022

Not4-dependent targeting of MMF1 mRNA to mitochondria limits its expression via ribosome pausing, Egd1 ubiquitination, Caf130, No-Go-Decay and autophagy

The Ccr4-Not complex is a conserved multi protein complex with diverse roles in the mRNA life cycle. Recently we determined that the Not1 and Not4 subunits of Ccr4-Not inversely regulate mRNA solubility and thereby impact dynamics of co-translation events. One mRNA whose solubility is limited by Not4 is MMF1 encoding a mitochondrial matrix protein. In this work we determine that Not4 promotes the co-translational docking of MMF1 mRNA to mitochondria via the mitochondrial targeting sequence of the Mmf1 nascent chain, the Egd1 chaperone, the Om14 mitochondrial outer membrane protein and the co-translational import machinery. We observe that MMF1 mRNA is translated with ribosome pausing and uncover a mechanism that depends upon its targeting to the mitochondria and limits its overexpression. We have named this mechanism Mito-ENCay. It relies on Egd1 ubiquitination by Not4, the Caf130 subunit of the Ccr4-Not complex, the mitochondrial outer membrane protein Cis1, No-Go-Decay as well as autophagy. We propose that in fermenting yeast, mRNAs whose encoded proteins depend upon co-translational folding and/or assembly are regulated by Caf130-dependent quality control mechanisms similar to Mito-ENCay.

molecular biology↗

bioRxiv2022

Distinct bile salt hydrolase substrate preferences dictate C. difficile pathogenesis

Summary paragraphBile acids (BAs) mediate the crosstalk between human and microbial cells and influence intestinal diseases including Clostridioides difficile infection (CDI). While bile salt hydrolases (BSHs) shape the BA pool by deconjugating conjugated BAs, the basis for their substrate preferences and impact on C. difficile remain elusive. Here, we survey the diversity of Lactobacillus BSHs and unravel the structural basis of their substrate preference. We show that leveraging BSH activity and specificity is an effective strategy to prevent C. difficile growth in clinically relevant CDI models. A range of non-canonical conjugated BAs is also identified, comprising unique BSH substrates that also inhibit C. difficile spore germination. These findings establish BSHs as intestinal enzymes essential to BA homeostasis and colonization resistance against C. difficile. One sentence summaryBile salt hydrolase activity inhibits C. difficile by shaping the conventional and non-canonical conjugated bile acid pools

microbiology↗

bioRxiv2022

Not1 and Not4 inversely determine mRNA solubility that sets the dynamics of co-translational events

BackgroundThe Ccr4-Not complex is most well known as the major eukaryotic deadenylase. However, several studies have uncovered roles of the complex, in particular of the Not subunits, unrelated to deadenylation and relevant for translation. In particular, the existence of Not condensates that regulate translation elongation dynamics have been reported. Typical studies that evaluate translation efficiency rely on soluble extracts obtained after disruption of cells and ribosome profiling. Yet cellular mRNAs in condensates can be actively translated and may not be present in such extracts. ResultsIn this work, by analyzing soluble and insoluble mRNA decay intermediates in yeast, we determine that insoluble mRNAs are enriched for ribosomes dwelling at non-optimal codons compared to soluble mRNAs. mRNA decay is higher for soluble RNAs, but the proportion of co-translational degradation relative to the overall mRNA decay is higher for insoluble mRNAs. We show that depletion of Not1 and Not4 inversely impact mRNA solubilities and, for soluble mRNAs, ribosome dwelling according to codon optimality. Depletion of Not4 solubilizes mRNAs with lower non-optimal codon content and higher expression that are rendered insoluble by Not1 depletion. By contrast, depletion of Not1 solubilizes mitochondrial mRNAs, which are rendered insoluble upon Not4 depletion. ConclusionOur results reveal that mRNA solubility defines dynamics of co-translation events and is oppositely regulated by Not1 and Not4, a mechanism that we additionally determine may already be set by Not1 promoter association in the nucleus.

molecular biology↗

bioRxiv2020

Modulating glial genes involved in synaptic function mitigates pathogenesis and behavioral deficits in a Drosophila model of Huntington's Disease

Most research on neurodegenerative diseases has focused on neurons, yet glia help form and maintain the synapses whose loss is so prominent in these conditions. To investigate the contributions of glia to Huntingtons disease (HD), we studied transcriptomic changes in HD human, HD mice, and Drosophila expressing human mutant Huntingtin (mHTT) in either glia, neurons or both. A large portion of conserved genes are concordantly dysregulated across the three species; we tested these genes in a high-throughput behavioral assay and found that downregulation of genes involved in synapse assembly mitigated pathogenesis and behavioral deficits. To our surprise, mitigating glial pathogenesis by dNRXN3 knockdown was sufficient to improve the phenotype of flies expressing mHTT in neurons, suggesting that mHTTs toxic effects in glia ramify throughout the brain. This supports a model in which dampening synaptic function is protective because it attenuates the excitotoxicity that characterizes HD.

systems biology↗

bioRxiv2020

Modeling cell turning by mechanics at the cell rear

In this study, we explore a simulation of a mechanical model of the keratocyte lamellipodium as previously tested and calibrated for straight steady-state motility [1] and for the process of polarization and motility initiation [2]. In brief, this model uses the balance of three essential forces (myosin contraction, adhesive drag and actin network viscosity) to determine the cells mechanical behavior. Cell shape is set by the balance between the actin polymerization-driven protrusion at the cell boundary and myosin-driven retraction of the actin-myosin network. In the model, myosin acts to generate contractile stress applied to a viscous actin network with viscous resistance to actin flow created by adhesion to the substrate. Previous study [3] demonstrated that similar simple model with uniform constant adhesion predicts a rotating behavior of the cell; however, this behavior is idealized, and does not mimic observed features of the keratocytes turning behavior. Our goal is to explore what are the consequences of introducing mechanosensitive adhesions to the model.

biophysics↗

bioRxiv2018

Relationships between Human Brain Structural Connectomes and Traits

Advanced brain imaging techniques make it possible to measure individuals structural connectomes in large cohort studies non-invasively. However, due to limitations in image resolution and pre-processing, questions remain about whether reconstructed connectomes are measured accurately enough to detect relationships with human traits and behaviors. Using a state-of-the-art structural connectome processing pipeline and a novel dimensionality reduction technique applied to data from the Human Connectome Project (HCP), we show strong relationships between connectome structure and various human traits. Our dimensionality reduction approach uses a tensor characterization of the connectomes and relies on a generalization of principal components analysis. We analyze over 1100 scans for 1076 subjects from the HCP and the Sherbrooke test-retest data set as well as 175 human traits that measure domains including cognition, substance use, motor, sensory and emotion. We find that brain connectomes are associated with many traits. Specifically, fluid intelligence, language comprehension, and motor skills are associated with increased cortical-cortical brain connectivity, while the use of alcohol, tobacco, and marijuana are associated with decreased cortical-cortical connectivity.

neuroscience↗