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Biology subjects

Wells, J.

Publications and source records attributed to Wells, J..

At least 19 recordsLinked to original sources

Projected Gradient Descent for Hallucination-Based Protein Binder Design

Gradient-based hallucination is a popular approach to protein binder design that backpropagates a loss defined on a structure predictor's output to guide optimisation in a continuous relaxation of sequence space, yet the choice of optimiser for this task has received little systematic evaluation. In this work we evaluate simplex-projected gradient descent (PGD) as an alternative to the commonly used gradient descent on softmax-reparameterised sequence logits. By updating amino-acid probabilities directly, PGD avoids softmax-induced gradient attenuation and allows amino acids suppressed early in optimisation to re-enter the design. Using an ESMFold2-based pipeline on ten targets, we find that PGD minimises the design loss in fewer iterations and produces designs with higher predicted interface confidence compared to its softmax counterpart. Simulating selection in real campaigns by considering the upper tail of the ipSAE distribution across a pool of 1,024 designs per target, we show that PGD significantly improves on six of the ten targets, with no significant degradation on any, and produces more structurally diverse designs on every target. Additionally, we find that pairing shorter PGD runs with candidate screening raises the total number of in silico hits per GPU-hour by 29\% relative to default-length trajectories. These results support PGD as a simple and effective alternative for hallucination-based binder design. Code and design data are publicly available.

bioinformatics↗

Coronavirus spherules are supported by a novel triple-layer protein lattice

Coronaviruses (CoVs) cause significant disease in humans and animals, with a track-record of cross-species transmission. Despite their global significance, available control strategies are limited, especially for emerging CoVs. A deeper fundamental understanding of the virus life cycle is essential to underpin development of control measures. A critical step of the CoV life cycle is the rearrangement of host cell membranes into replication organelles (ROs). This is conserved across all CoVs and ROs comprise double membrane vesicles (DMVs), the site of viral RNA synthesis, and double membrane spherules. CoV spherules are complex structures requiring both positive and negative curvature of paired membranes so are predicted to have a high energy cost to maintain. To date they have an entirely unknown function. Interestingly, although always present, abundance of spherules in CoV infected cells varies with Betacoronavirus Middle East Respiratory Syndrome coronavirus, Gammacoronavirus infectious bronchitis virus (IBV) and porcine deltacoronavirus in particular producing a considerable number. Here, using cryo-bioimaging we identified for the first time that CoV spherules are embedded with a complex and organised triple-layered protein lattice and that spherules always contain a cargo that appears arranged as a shell inside the vesicle. In addition, we confirmed the presence of a double membrane spanning molecular pore in Gammacoronavirus DMVs and further identified a population of potential DMV formation intermediates. Together, this work provides a foundation for understanding a fundamental yet unresolved aspect of coronavirus replication.

microbiology↗

Ontological Analysis of Brain Proteostasis Highlights the Sex-Dependent Trajectory of ApoE Isoform-Specific Regulation

Apolipoprotein E (ApoE) is the strongest genetic predictor of Alzheimers disease (AD) risk, with ApoE4 increasing and ApoE2 decreasing risk relative to ApoE3. Using a global LC-MS proteomic approach, we integrated protein abundance and kinetics in Human-APOE knock-in mice for young (3-month) and aged (18-month) cohorts to quantify the changes in steady-state proteostasis. By mapping 6,052 identified proteins and 3,986 associated turnover rates into ontological groups, we observed that vesicle trafficking and mitochondrial dysregulation occur as early as 3 months in ApoE4 mice accompanied by hyperactive metabolism that eventually reduces with age. In contrast, young and old ApoE2 mice retain similar signatures to ApoE3 mice in metabolic, mitochondrial, cellular regulation, and membrane trafficking ontologies. We found that females had more isoform-induced ontological changes relative to ApoE3, providing insight into sex-dependent vulnerabilities. Our global proteomic approach for ApoE proteostasis crucially unifies independent literature observations while providing turnover kinetics to uncover the underlying mechanism behind abundance changes. Data are available via ProteomeXchange with identifier PXD079261. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=189 SRC="FIGDIR/small/735293v1_ufig2.gif" ALT="Figure 1000"> View larger version (45K): org.highwire.dtl.DTLVardef@28ebfforg.highwire.dtl.DTLVardef@9dc6f2org.highwire.dtl.DTLVardef@704577org.highwire.dtl.DTLVardef@e0b0e2_HPS_FORMAT_FIGEXP M_FIG C_FIG O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/735293v1_ufig1.gif" ALT="Figure 1001"> View larger version (48K): org.highwire.dtl.DTLVardef@17d0474org.highwire.dtl.DTLVardef@af771borg.highwire.dtl.DTLVardef@1b49f67org.highwire.dtl.DTLVardef@14d3aa2_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

High-resolution photocatalytic crosslinking of protein neighborhoods using contact-dependent antibody mapping (contactMAP)

Photo-proximity labeling proteomics (PLP) has emerged as a powerful method for rapid and temporal mapping of transient and fragile protein-protein interactions, especially those in membranes. Numerous catalysts can trigger highly reactive diffusive biotinylated probes to label protein neighborhoods at various length scales. Photocatalysts can also trigger protein crosslinking, predominantly between neighboring tyrosines or between histidine and lysine residues. We have exploited this sidechain directed crosslinking for photo-PLP, in a method we call contactMAP. Using biotinylated antibody binders to photocrosslink Her2 or EGFR neighborhoods, contactMAP enriched high resolution maps of these cancer-associated protein neighborhoods in a manner that rivals or outperforms established photo-PLP methods. ContactMAP is an extraordinarily simple and democratic photo-PLP labeling method with broad applications for probing biomolecular interactions in complex mixtures.

Cancer Biology↗

Cell-type Specific Alteration of Dicer1 Accelerates Tumor Progression in Mouse Models of KRAS-driven Lung Adenocarcinoma

MicroRNAs (miRNAs) have been widely implicated in cancer initiation and progression, yet examination of the effects of global miRNA disruption on these processes has been limited. We developed novel genetically engineered mouse models of Kras-driven pulmonary adenocarcinoma (LUAD) with cell-type-specific disruption of miRNA biosynthesis via Dicer1 allele deletion, which exhibit significant differences in tumor progression rates and expected survival. Dicer1 is an RNase III enzyme that is required for the biogenesis of mature, functional miRNAs. Lung tumor progression was accelerated, and expected survival was decreased only when we initiated tumors and deleted one allele of Dicer1 in club cells and mutated Dicer1 in alveolar type 2 (AT2) cells. Reversing the cell types by inducing tumorigenesis, deleting one Dicer1 allele in AT2 cells, and mutating Dicer1 in club cells modestly accelerated tumor progression and had no effect on expected survival. Collectively, our results demonstrate that Dicer1 disruption accelerates lung cancer progression in a cell-type-dependent and non-cell-autonomous manner, and our mice represent tools for investigating the roles of miRNAs and miRNA-mediated intercellular communication in tumor progression. SummaryKras-driven mouse models show that Dicer1 mutations accelerate lung adenocarcinoma (LUAD) progression in a cell-type-dependent manner and suggest that the influence of miRNA-mediated intercellular communication is unidirectional and non-cell-autonomous.

cancer biology↗

Advanced in High-Resolution Cryo Volume Electron Microscopy (cvEM) Imaging for Unicellular and Multicellular Organisms

Cryo-Focused Ion Beam Scanning Electron Microscopy (cryoFIB-SEM) using samples fixed by high-pressure freezing uniquely enables high resolution cryo-volume Electron Microscope (cvEM) images of cell ultrastructure to be obtained from whole cells and complex tissues in their near native state. As the freezing process also preserves fluorescence, the link between three-dimensional (3D) ultrastructure and biological process is also enabled by targeted cryo-Correlative Light and Electron Microscopy (CLEM). However, the overall viability of cvEM is challenged by sample preparation, charge balance during imaging, sample sensitivity to beam damage, contamination, and very long acquisition times. Here we detail new experimental workflows to significantly reduce each of these effects and demonstrate the improvement in resolution possible with results from the nematode Caenorhabditis elegans and the ciliated protozoon Paramecium bursaria containing many endosymbiotic algae. These results demonstrate the versatility and potential wide-ranging utility of cvEM for 3D ultrastructural imaging of whole multicellular and unicellular organisms.

biophysics↗

Advancing Vibrio genetics: A platform for efficient genomic manipulation

Most non-model Vibrio species lack the genetic tools needed for targeted mutagenesis, which limits the ability to functionally characterize newly identified pathways. To address this challenge, we present here efficient, robust methods for genetically manipulating Vibrio species that rely on RecA-mediated homologous recombination and two well-characterized counterselection methods, galactokinase (galK) 2-Deoxy-D-galactose (DOG-2) toxicity and the rpsLR/rpsLS streptomycin susceptibility system, both of which are active across a broad range of Vibrio species. We further characterized two genus-specific conserved promoters capable of driving high-level ectopic expression across all tested species. These promoters were incorporated into two broadly applicable, conjugatively transferable suicide backbones designed to facilitate double homologous recombination. Using these systems, we successfully disrupted polar flagellar motility in multiple Vibrio species and introduced extensive modifications to both flagellar and secretory pathways in V. diazotrophicus. Notably, although the galK system exhibited broader applicability, the rpsL system proved to be more efficient in cases where a streptomycin resistant strain could be generated. We also developed two mobilizable replicative backbones that express pH-stable fluorescent proteins for use within the genus. Collectively, these tools expand the genetic toolkit available for both gene disruption and heterologous gene expression in non-model members of the Vibrionaceae. ImportanceMembers of the Vibrionaceae are not only among the most abundant and ecologically influential microorganisms in marine ecosystems, but they also represent major drivers of disease across a wide range of hosts, including humans. However, identifying the genetic determinants of Vibrionaceae pathogenesis has remained challenging due to their halophilic growth requirements, restriction enzyme profiles, and resistance to expressing foreign proteins. Canonical counterselection pathways are largely ineffective in these species, which underscores the need for novel and efficient genetic tools to advance functional studies. This study adapts two strategies previously successful in other non-model organisms for use within the Vibrio genus and its close relatives. These methods therefore represent an essential step toward overcoming long-standing genetic barriers in Vibrio species and provide a framework to expand our understanding of biology in this important bacterial genus.

microbiology↗

An Energy Landscape Approach to Miniaturizing Enzymes using Protein Language Model Embeddings

AO_SCPLOWBSTRACTC_SCPLOWWe present a general approach to find amino acid sequences corresponding to the most compact enzyme likely to retain the structure of a given catalytic site. Our approach is based on using Monte Carlo (MC) simulations to sample an energy landscape where minima correspond, by construction, to sequences with the aforementioned properties. Building on previous work (Wu et al., 2025) and with the BAGEL package (Lala et al., 2025), we implement a route to achieve this goal using only the information extracted from a protein language model (PLM), without structural information. After generating a set of candidate sequences with this PLM-guided BAGEL optimization, we further filter potential candidates for downstream experimental validation using a two-stage protocol. First, deep-learning-based structure prediction models (ESMFold, Chai-1, Boltz-2) are used to identify a structural consensus among designs with highly conserved active-site geometries, yielding many candidates with active-site RMSD below a few angstroms relative to the wild-type and pLDDT scores above 80. Second, molecular dynamics simulations are performed on a filtered subset of sequences (based on active-site RMSD and SolubleMPNN log-likelihoods) to evaluate active-site stability when including thermal fluctuations. For the most promising enzymes, these yield RMSF values in the active site below 1.0 [A] and an active-site RMSD drift between 0.5 and 1.5 [A], making these mini-variants comparable to the wild type, though outcomes vary across enzymes. Given the protocols generality, we believe these results represent a step forward in AI-guided enzyme design. To facilitate rapid experimental validation by the broader community, we open-source all sequences generated by our computational pipeline. These include designs for four representative enzymes of this study: PETase, subtilisin Carlsberg (serine protease), Taq DNA polymerase, and VioA.

bioinformatics↗

ProFam: Open-Source Protein Family Language Modelling for Fitness Prediction and Design

Protein language models have become essential tools for engineering novel functional proteins. The emerging paradigm of family-based language models makes use of homologous sequences to steer protein design and enhance zero-shot fitness prediction, by imbuing models with an ability to explicitly reason over evolutionary context. To provide an open foundation for this modelling approach, we introduce ProFam-1, a 251M-parameter autoregressive protein family language model (pfLM) trained with next-token prediction on millions of protein families represented as concatenated, unaligned sets of sequences. ProFam-1 is competitive with state-of-the-art models on the ProteinGym zero-shot fitness prediction benchmark, achieving Spearman correlations of 0.47 for substitutions and 0.53 for indels. For homology-guided generation, ProFam-1 generates diverse sequences with predicted structural similarity, while preserving residue conservation and covariance patterns. All of ProFams training and inference pipelines, together with our curated, large-scale training dataset ProFam Atlas, are released fully open source, lowering the barrier to future method development.

bioinformatics↗

Human pancreatic organoids derived from pluripotent stem cells recapitulate pancreatic organogenesis

Pancreas organogenesis relies on sequential interactions between the pancreatic epithelium and surrounding mesodermal cell types that initiate epithelial budding and branching to form a complex ductal network with terminal acini. Despite recent advances with pluripotent stem cell-based approaches, there are no models that robustly recapitulate pancreas morphogenesis or the spatial organization of ductal, exocrine, endocrine and mesenchymal cells seen in the native organ. Here, we introduce a new pluripotent stem cell-based pancreatic organoid that captures the complexity seen during pancreatic development, with budding and stratification of multipotent progenitors followed by formation of a ductal network that give rise to peripheral acini. We identify a critical role for mesenchyme-derived factors to robustly promote pancreatic organoid formation and morphogenesis. Human pancreatic organoids are correctly patterned, with functional exocrine acini secreting digestive enzymes into a ductal network. Comparative analysis confirms that the pancreatic organoids are similar to early second trimester human pancreas, with potential to further mature upon transplantation in mice. Finally, we show that endocrinogenesis can be reproduced in organoids, generating functional islet-like clusters interspersed within the ductal network. Together, this represents an exciting new platform to study human pancreas development and a broad array of pancreatic diseases.

developmental biology↗

Steering Vector Fields for Property-Controlled Molecular Generation with Chemical Language Models

Chemical language models have recently become a powerful tool for the de novo generation of drug-like molecules represented as SMILES strings. A central challenge is steering generation toward compounds with favorable properties such as solubility and absorption. To this end, we investigate inference time control of generative chemical language models using activation steering. Using contrastive activation addition, we seek to improve three relevant properties: molecular size, aqueous solubility (log S), and lipophilicity (log P) without changing the model weights. We compare two interventions: a single global vector which is added to the activation in the last transformer layer, and a novel vector field where the addition vector is computed as a function of the current hidden state. Across multiple protein targets and two pre-trained models, the global steering vector yields desired results in just over half of our experiments, while the vector field achieves larger shifts at the expense of a decrease in the validity rate.

molecular biology↗

Using targeted therapy to promote a pro-inflammatory tumour microenvironment and anti-tumour immune response in high grade serous ovarian cancer

BACKGROUNDHigh-grade serous ovarian cancer (HGSOC) is characterized by elevated replication stress and an immunosuppressive microenvironment. A synergistic combination of checkpoint kinase 1 inhibitor (CHK1i) with low-dose hydroxyurea (LDHU) promotes a unique ATR-independent moderate replication stress response with potent anti-tumour effects. The ability of this approach to reprogram the tumour immune microenvironment (TIME) to overcome the immunosuppression and promote an anti-tumour immune response in HGSOC is the focus of this study. METHODSWe investigated the therapeutic potential of CHK1i+LDHU in established HGSOC cell cultures, fresh tumour cell explants from HGSOC patient ascites, and syngeneic mouse models, assessing tumour cell killing, immunogenic cell death, pro-inflammatory cytokine/chemokine expression, and anti-tumour immune responses. RESULTSCHK1i+LDHU effectively killed ovarian cancer cells regardless of chemotherapy resistance, BRCA2 mutation and homologous recombination repair status in vitro. In vivo, treatment significantly reduced tumour burden and ascites accumulation. CHK1i+LDHU enhanced expression of pro-inflammatory cytokines/chemokines and triggered immunogenic cell death in tumour. In syngeneic models, treatment promoted CD8+ cytotoxic T cell-dependent anti-tumour responses and reduced immunosuppressive signalling within the TIME. CONCLUSIONSCHK1i+LDHU is a promising therapy for chemotherapy-resistant HGSOC, combining direct cytotoxic effects with reprogramming the TIME to reduce immunosuppression and activate a CD8+ T cell-dependent anti-tumour response.

cancer biology↗

Design in Voxel Space Decode in SMILES Space: Plixer Generates Drug-Like Molecules for Protein Pockets

We introduce Plixer, a two-stage generative model for de novo drug design that generates small-molecule ligand binders conditioned on an empty protein pocket. Plixer combines a conditional voxel inpainting network to generate 3D ligand hypotheses with an independently trained voxel-to-SMILES decoder that translates these voxel representations into valid chemical structures. By de-coupling the learning of spatial protein-ligand interactions from the learning of chemical grammar, our approach leverages large libraries of 3D ligand conformers to augment the limited data available for protein-ligand complexes. We show that this approach generates molecules with higher predicted binding affinity than recent methods.

molecular biology↗

Genome-Wide Identification of Conditionally Essential Genes Supporting Streptococcus suis Growth in Serum and Cerebrospinal Fluid

Streptococcus suis is a major cause of sepsis and meningitis in pigs, and zoonosis through the emergence of disease-associated lineages. The ability of S. suis to adapt and survive in host environments, such as blood and cerebrospinal fluid (CSF), is important for pathogenesis. Here, we used Tn-Seq coupled with Nanopore sequencing to identify conditionally essential genes for growth of S. suis P1/7 in active porcine serum (APS) and CSF derived from choroid plexus organoids (Zhao et al., 2025). Through comparative fitness analyses we identified 33 conditionally essential genes (CEGs) supporting growth in APS and 25 CEGs in CSF, highlighting the importance of pathways involved in amino acid transport, nucleotide metabolism, and cell envelope integrity. Notably, the LiaFSR regulatory system and multiple ABC transporters were important for proliferation. We also identified several genes of unknown function as essential for growth, pointing to previously unrecognized genetic factors involved in S. suis adaptation during infection. These findings provide new insights into the genetic requirements for S. suis survival in host-like environments and a deeper understanding of its ability to adapt to distinct physiological niches.

microbiology↗

Fetal Liver-like Organoids Recapitulate Blood-Liver Niche Development and Multipotent Hematopoiesis from Human Pluripotent Stem Cells

The fetal liver is a hematopoietic organ, hosting a diverse and evolving progenitor population. While human liver organoids derived from pluripotent stem cells (PSCs) mimic aspects of embryonic and fetal development, they typically lack the complex hematopoietic niche and the interaction between hepatic and hematopoietic development. We describe the generation of human Fetal Liver-like Organoids (FLOs), that model human hepato-hematopoietic interactions previously characterized in mouse models. Developing FLOs first integrate a yolk sac-like hemogenic endothelium into hepatic endoderm and mesoderm specification. As the hepatic and hematopoietic lineages differentiate, the FLO culture model establishes an autonomous niche capable of driving subsequent progenitor differentiation without exogenous factors. Consistent with yolk sac-derived waves, hematopoietic progenitor cells (HPCs) within FLOs exhibit multipotency with a preference for myeloid lineage commitment, while retaining fetal B and T cell differentiation potential. We reconstruct in FLOs the embryonic monocyte-to-macrophage and granulocyte immune trajectories within the FLO microenvironment and assess their functional responses in the liver niche. In vivo, FLOs demonstrate a liver engraftment bias of hematopoietic cells, recapitulating a key phenomenon of human hematopoietic ontogeny. Our findings highlight the intrinsic capacity of liver organoids to support hematopoietic development, establishing FLOs as a platform for modeling and manipulating human blood-liver niche interactions during critical stages of development and disease.

developmental biology↗

Centromeric transposable elements and epigenetic status drive karyotypic variation in the eastern hoolock gibbon

Great apes have maintained a stable karyotype with few large-scale rearrangements; in contrast, gibbons have undergone a high rate of chromosomal rearrangements coincident with rapid centromere turnover. Here we characterize assembled centromeres in the Eastern hoolock gibbon, Hoolock leuconedys (HLE), finding a diverse group of transposable elements (TEs) that differ from the canonical alpha satellites found across centromeres of other apes. We find that HLE centromeres contain a CpG methylation centromere dip region, providing evidence this epigenetic feature is conserved in the absence of satellite arrays; nevertheless, we report a variety of atypical centromeric features, including protein-coding genes and mismatched replication timing. Further, large structural variations define HLE centromeres and distinguish them from other gibbons. Combined with differentially methylated TEs, topologically associated domain boundaries, and segmental duplications at chromosomal breakpoints, we propose that a "perfect storm" of multiple genomic attributes with propensities for chromosome instability shaped gibbon centromere evolution.

genomics↗

Differences in maternal diet fiber content influence patterns of gene expression and chromatin accessibility in fetuses and piglets

This study investigates the impact of maternal gestation diets with varying fiber contents on gene expression and chromatin accessibility in fetuses and piglets fed a low fiber diet post weaning. High-fiber maternal diets, enriched with sugar beet pulp or pea internal fiber, were compared to a low-fiber maternal diet to evaluate their effects on liver and muscle tissues. The findings demonstrate that maternal high-fiber diets significantly alter the chromatin accessibility, predicted transcription factor activity and transcriptional landscape in both fetuses and piglets. A gene set enrichment analysis revealed over-expression of gene ontology terms related to metabolic processes and under-expression of those linked to immune responses in piglets from sows given the high-fiber diets during gestation. This suggests better metabolic health and immune tolerance of the fetus and offspring, in line with the documented epigenetic effects of short chain fatty acids on immune and metabolic pathways. A deconvolution analysis of the bulk RNA-seq data was performed using cell-type specific markers from a single cell transcriptome atlas of adult pigs. These results confirmed that the transcriptomic and chromatin accessibility data do not reflect different cell type compositions between maternal diet groups but rather phenotypic changes triggered by the critical role of maternal nutrition in shaping the epigenetic and transcriptional environment of fetus and offspring. Our findings have implications for improving animal health and productivity as well as broader implications for human health, suggesting that optimizing maternal diet with high-fiber content could enhance metabolic health and immune function in the formative years after birth and potentially to adulthood.

genomics↗

CRISPRi-based screen of Autism Spectrum Disorder risk genes in microglia uncovers roles of ADNP in microglia endocytosis and uptake of synaptic material

Autism Spectrum Disorders (ASD) are a set of neurodevelopmental disorders with complex biology. The identification of ASD risk genes from exome-wide association studies and de novo variation analyses has enabled mechanistic investigations into how ASD-risk genes alter development. Most functional genomics studies have focused on the role of these genes in neurons and neural progenitor cells. However, roles for ASD risk genes in other cell types are largely uncharacterized. There is evidence from postmortem tissue that microglia, the resident immune cells of the brain, appear activated in ASD. Here, we used CRISPRi-based functional genomics to systematically assess the impact of ASD risk gene knockdown on microglia activation and phagocytosis. We developed an iPSC-derived microglia-neuron coculture system and high-throughput flow cytometry readout for synaptic pruning to enable parallel CRISPRi-based screening of phagocytosis of beads, synaptosomes, and synaptic pruning. Our screen identified ADNP, a high-confidence ASD risk genes, as a modifier of microglial synaptic pruning. We found that microglia with ADNP loss have altered endocytic trafficking, remodeled proteomes, and increased motility in coculture.

neuroscience↗