Biology subjects

Kelly, R.

Publications and source records attributed to Kelly, R..

At least 19 recordsLinked to original sources

bioRxiv2026

The function of human PIF1 in G quadruplex formation and replication stress response at ALT telomeres

Cancers maintain their telomeres through two telomere maintenance mechanisms: 85-90% of cancers rely on telomerase (TEL+), while 10-15% of cancers adopt the Alternative Lengthening of Telomeres (ALT) pathway. The Break-Induced Replication (BIR) pathway plays a critical role in maintaining telomere length in the ALT+ cells. In both yeast and human, PIF1, a 5' to 3' helicase, is required for the robust activity of BIR. However, the extent of human PIF1 (hPIF1) involvement in the ALT pathway remains unknown. Here we showed that hPIF1 can be recruited to damaged telomeres in ALT+ cells. In addition, we demonstrated that inhibition of hPIF1 induced DNA damage and G quadruplex (G4) accumulation at ALT telomeres, leading to a moderate reduction of the mean telomere length. Most interestingly, we demonstrated that inhibition of hPIF1 also attenuates checkpoint activation, BLM recruitment, single-stranded DNA (ssDNA) formation, DNA damage, and G4s at telomeres in the FANCM deficient ALT+ cells. Finally, we showed that inactivation of hPIF1 affects the viability of both ALT+ and TEL+ cancers, suggesting that hPIF1 is a potential drug target for cancer therapy.

molecular biology↗

bioRxiv2026

Defining Quality Control Standards for Single-Cell Proteomics by Inter-Laboratory Benchmarking

Single-cell proteomics can quantify thousands of proteins from individual mammalian cells, yet the absence of community-wide quality control limits biological interpretability. Here, the HUPO Single Cell Initiative presents the first inter-laboratory single-cell proteomics benchmarking study across seven laboratories using standardized 384-well plates acquired on Orbitrap Astral and timsTOF Ultra2 instruments. Centralized analysis across six DIA software tools revealed that software choice impacts identification depth and quantitative accuracy more than instrument vendor. Multi-layered quality control enabled the detection of cell-leakage during sorting, LC misconfiguration, column degradation and site-specific pipetting failures. Inter-lab quantitative correlations were strongest between instruments of the same vendor relative to cross-platform comparisons. Sequential correction for plate identity and well position recovered clean cell-type separation for confident downstream differential expression analysis. This study provides a data-driven quality control framework spanning plate design to batch correction for reproducible single-cell proteomics across laboratories and platforms.

biochemistry↗

bioRxiv2026

NeuroFLAME: A Scalable, Privacy-Preserving Federated Framework for Secure, Reproducible, and Multi-Site Neuroimaging Analysis

Federated analysis offers a scalable approach to multi-site neuroimaging research by enabling distributed statistical modeling, machine learning, decomposition, harmonization, and validation without exchanging sensitive individual-level data. However, a significant implementation gap exists, as the majority of federated clinical neuroimaging studies remain technical proofs of concept, struggling to fully navigate the rigorous data-sharing regulations and security constraints of real-world clinical and research environments. Compounding this, existing federated analysis frameworks are typically exposed as command-line tools and configuration files, imposing a considerable setup burden that demands specialized technical expertise from neuroscience researchers. To address these barriers, we present NeuroFLAME, an enterprise-grade, open-source federated neuroimaging platform built upon the NVIDIA FLARE (NVFlare) framework. NeuroFLAME couples a client-outbound-only communication framework with a graphical user interface specifically tailored for neuroscientists. Using certificate-based trust and containerized execution, the platform ensures reproducible analyses with baseline privacy guarantees through data localization and mTLS encryption; advanced protections such as differential privacy and homomorphic encryption are available as optional configurations. By ensuring that clients only need outbound communication and that raw data never leaves each site, NeuroFLAME is designed to support institutional data governance requirements and regulatory frameworks such as HIPAA and GDPR. We demonstrate the platform's utility through three federated analysis workflows, each implemented as a NeuroFLAME computation: multi-site federated closed form voxel-wise regression, decentralized constrained joint independent component analysis (dcjICA) and federated label-based dimensional prediction. Empirical validation of both workflows shows that NeuroFLAME achieves high consistency with established centralized approaches, effectively bridging the gap between experimental federated learning prototypes and production-ready collaborative tools for privacy-preserving, large-scale federated neuroimaging analysis.

neuroscience↗

bioRxiv2025

Property Enhancer - a data efficient multi-objective approach for functional antibody optimization

In-silico antibody lead optimization remains challenging due to scarce high-quality data, costly experimental validation, and the need to jointly optimize multiple developability properties. Discovery workflows often rely on high-throughput phage, ribosome or yeast display experiments, which yield large but noisy datasets; as leads emerge, strategies shift to low-throughput assays which are precise, yet unscalable. Deep-learning and language-model approaches are hindered by such limited, unreliable measurements. We introduce Property Enhancer (PropEn), a data-efficient framework for low-data, heterogeneous regimes that can simultaneously optimize multiple antibody properties. PropEn proposes a matching-based augmentation that expands the training data with sequence pairs differing by only a few mutations; within each pair the second sequence improves the target value, providing an implicit optimization signal. Extensive in silico and in vitro tests show 10-39x affinity gains across four targets and nine leads, and enable joint multi-property optimization, positioning PropEn as a scalable, general solution.

molecular biology↗

bioRxiv2025

IRF2 degradation tunes the innate immune response

The transcription factor IRF2 protects against skin inflammation in mice and humans but, paradoxically, promotes pyroptosis by inducing Gsdmd. How IRF2 activates some proinflammatory genes, but suppresses others is unclear. We show that skin inflammation in Irf2-deficient mice is driven by IRF1 activation of interferon-stimulated genes (ISGs). Chromatin profiling revealed that IRF1 and IRF2 occupy the same ISG regulatory sites, but as a weaker transcriptional activator, IRF2 limited ISG transcription by IRF1. Toll-like receptor signaling favored IRF1-driven transcription by inducing Irf1. In addition, IRF1 recruited the ubiquitin ligase SPOP to ISG sites, resulting in proteasomal degradation of IRF2. This shift from IRF2 to IRF1 occupancy enhanced ISG transcription. Collectively, these findings define a hierarchical transcriptional circuit in which IRF2 limits IRF1 activity under homeostatic conditions but is displaced during an immune response, allowing IRF1-dependent gene programs central to innate immunity and autoinflammation.

immunology↗

bioRxiv2025

Deep Evolutionary Fitness Inference for Variant Nomination from Directed Evolution

Iterative screening techniques, such as directed evolution, enable high-throughput affinity maturation to optimize binders to molecular interfaces. However, the decision problem of selecting variants from rich, evolved populations to enter low-throughput follow-up methods remains a significant bottleneck. Here, we present evolutionary fitness inference (EVFI) and DeepEVFI, two machine learning methods that model directed evolution from time-series sequencing data, and infer fitness, a variants ability to enrich under selection pressure. Our methods flexibly handle mutation mechanisms and starting populations that may be partially unknown - settings relevant to drug discovery - and achieve strong performance on a diverse set of experimental data. We conducted two experimental directed evolution campaigns, using antibodies and macrocyclic peptides libraries to identify and optimize binders to therapeutically relevant targets. EVFI and DeepEVFI identified tighter binders that were missed by human experts using conventional frequency-based approaches, including "rising stars" with low frequency. Beyond initial hit discovery, EVFI and Deep-EVFI enables labeling large-scale sequence-fitness datasets and identifying variants of initial binders with diverse properties.

bioinformatics↗

bioRxiv2025

Rational design of oxidation-resistant antibodies through local electrostatic modulation

Oxidation is a significant degradation pathway in proteins, particularly therapeutic antibodies, that can impair function, efficacy, and stability. Understanding the sequence and structural factors that drive oxidation susceptibility is critical for incorporating chemical stability into early-stage antibody design. Here, we present a machine learning classifier trained on expert-guided structural features to assess tryptophan (Trp) oxidation risks in the complementarity-determining regions (CDR) of 187 antibodies produced internally at Genentech. The model reveals a strong correlation between local negative electrostatic potential and Trp oxidation susceptibility. A simplified two-parameter model derived from these insights achieves 79% accuracy in classifying oxidation risk, compared to 84% accuracy with the full-feature classifier. Beyond our internal dataset, the two-parameter model successfully predicted oxidation risk for all CDR Trp sites in a blind subset of eight clinical-stage antibodies. In addition, we show that modulating the electrostatic potential around the Trp side-chains through distant charge-altering mutations can significantly reduce oxidation rates. In four out of five re-engineered antibodies, oxidation rates decreased by approximately 50%, while half of these maintained binding affinity. Finally, we demonstrate that this approach can guide multi-property optimization by balancing oxidation resistance and affinity in an anti-CD33 antibody. These results establish a strong link between local electrostatic environments and Trp oxidation susceptibility, and provide a practical framework for designing oxidation resistant biotherapeutics.

biochemistry↗

bioRxiv2025

Sp Transcription Factors Establish the Signaling Environment in the Neuromesodermal Progenitor Niche During Axial Elongation

Neuromesodermal competent progenitors (NMCs) are located in the caudal epiblast near the node and primitive streak and give rise to spinal cord and somitic mesoderm during trunk and tail elongation. Their self-renewal depends on an autoregulatory loop involving Wnt3a and Fgf signaling, and the Tbxt and Cdx transcription factors, but the mechanisms underlying loop formation and the establishment of the niche are poorly understood. Here, we identify the zinc-finger transcription factors Sp5 and Sp8 (Sp5/8) as essential regulators of NMC maintenance. Sp5/8 expression is controlled by Wnt, Fgf and retinoic acid signaling, and they cooperate with Tbxt, Tcf7 and Cdx2 to sustain a robust autoregulatory network that promotes high Wnt/Fgf and low retinoic acid activity in the niche. These factors bind a novel enhancer essential for Wnt3a expression and feedback-loop integrity. Mechanistically, Sp5/8 regulate the dynamic exchange of activating and repressive Tcf complexes at Wnt-responsive enhancers. Our findings define a transcriptional module centered on Sp5/8 that stabilizes niche signaling and transcriptional circuitry essential for NMC fate decisions and trunk development.

developmental biology↗

bioRxiv2025

Quantification of individual dataset contributions to prediction accuracy in cooperative learning

We consider cooperative learning, a recently proposed technique to leverage the predictive power of several datasets (also called data views) to improve the prediction accuracy of an outcome of interest. Cooperative learning uses a Lasso-type penalty to fit several datasets to a given outcome, while a so-called agreement penalty enforces that the predictions made by the individual datasets agree. We are interested in the question of whether the predictive power of each individual dataset can be quantified using cooperative learning. In this work, we demonstrate that certain trace plots, analogously to the ones for the classic Lasso, allow one to quantify the predictive power of each individual dataset. Importantly, this allows one to detect datasets which do not carry any predictive power on the outcome. In an experimental study, we quantify the predictive power of three real datasets in the context of the Childhood Asthma Management Program (CAMP), with the three datasets containing information on epidemiological variables, metabolites, and clinical data, respectively.

bioinformatics↗

bioRxiv2024

CD4 T cells and CD8α+ lymphocytes are necessary for intravenous BCG-induced protection against tuberculosis in macaques

Tuberculosis (TB) is a major cause of morbidity and mortality worldwide despite widespread intradermal (ID) BCG vaccination in newborns. We previously demonstrated that changing the route and dose of BCG vaccination from 5{xi}105 CFU ID to 5{xi}107 CFU intravenous (IV) resulted in prevention of infection and disease in a rigorous, highly susceptible non-human primate model of TB. Identifying the immune mechanisms of protection for IV BCG will facilitate development of more effective vaccines against TB. Here, we depleted select lymphocyte subsets in IV BCG vaccinated macaques prior to Mtb challenge to determine the cell types necessary for that protection. Depletion of CD4 T cells or all CD8 expressing lymphoycytes (both innate and adaptive) resulted in loss of protection in most macaques, concomitant with increased bacterial burdens ([~]4-5 log10 thoracic CFU) and dissemination of infection. In contrast, depletion of only adaptive CD8{beta}+ T cells did not significantly reduce protection against disease. Our results demonstrate that CD4 T cells and innate CD8+ lymphocytes are critical for IV BCG-induced protection, supporting investigation of how eliciting these cells and their functions can improve future TB vaccines. One Sentence SummaryAntibody depletion of lymphocytes in rhesus macques demonstrates key roles for CD4 T cells and innate-like CD8+ lymphocytes in conferring sterilizing immunity against tuberculosis following intravenous BCG vaccination.

immunology↗

bioRxiv2024

Dramatic reduction in trypanosome motility occurs without large-scale changes to paraflagellar rod ultrastructure

Eukaryotic flagella - widely conserved structures involved in signalling, metabolism and motility - have a core microtubular axoneme that, in many organisms, is accompanied by prominent extra-axonemal structures. In kinetoplastids, including human parasites such as trypanosomes and Leishmania, a dense filamentous lattice called the paraflagellar rod (PFR) accompanies the axoneme for most of its length. While functional studies showed that the presence of the core PFR structure is required for normal motility, the evaluation of more subtle roles for the PFR in motility has been hampered by limited functional and localisation data, particularly on components not essential to form the core PFR, such as signalling and metabolism proteins. Here, we addressed these issues by using the genome-wide protein localisation database TrypTag to define a PFR proteome, which was used as a base for a subtler analysis of PFR structure and function. We combined the localisation of fluorescently tagged PFR proteins relative to other cellular components with electron microscopy data on the PFR ultrastructure to localise 81 proteins to specific subdomains of the PFR. Functional analysis of a subset of PFR proteins by gene deletion and RNAi demonstrated that a novel PFR component (PFC21) is required for correct assembly of the outer PFR domain. Importantly, in some single deletion mutants, cell motility was impaired without gross disruption to the core PFR ultrastructure. Thus, our study shows that the PFR has subtle, likely regulatory roles in motility unrelated to any physical constraints that the bulky PFR structure may impose on flagella function.

microbiology↗

bioRxiv2024

Woman the Hunter? Female foragers sometimes hunt, yet gendered divisions of labor are real

Gendered divisions of labor are a feature of every known contemporary hunter-gatherer (forager) society. While gender roles are certainly flexible, and prominent and well-studied cases of female hunting do exist, it is more often men who hunt. A new study (Anderson et al., 2023) surveyed ethnographically known foragers and found that women hunt in 79% of foraging societies, with big-game hunting occurring in 33%. Based on this single type of labor, which is one among dozens performed in foraging societies, the authors question the existence of gendered division of labor altogether. As a diverse group of hunter-gatherer experts, we find that claims that foraging societies lack or have weak gendered divisions of labor are contradicted by empirical evidence. We conducted an in-depth examination of Anderson et al. (2023) data and methods, finding evidence of sample selection bias and numerous coding errors undermining the papers conclusions. Anderson et al. (2023) have started a useful dialogue to ameliorate the popular misconception that women never hunt. However, their analysis does not contradict the wide body of empirical evidence for gendered divisions of labor in foraging societies. Furthermore, a myopic focus on hunting diminishes the value of contributions that take different forms and downplays the trade-offs foragers of both sexes routinely face. We caution against ethnographic revisionism that projects Westernized conceptions of labor and its value onto foraging societies.

evolutionary biology↗

bioRxiv2023

Enhancing Collaborative Neuroimaging Research: Introducing COINSTAC Vaults for Federated Analysis and Reproducibility

Collaborative neuroimaging research is often hindered by technological, policy, administrative, and methodological barriers, despite the abundance of available data. COINSTAC is a platform that successfully tackles these challenges through federated analysis, allowing researchers to analyze datasets without publicly sharing their data. This paper presents a significant enhancement to the COINSTAC platform: COINSTAC Vaults (CVs). CVs are designed to further reduce barriers by hosting standardized, persistent, and highly-available datasets, while seamlessly integrating with COINSTACs federated analysis capabilities. CVs offer a user-friendly interface for self-service analysis, streamlining collaboration and eliminating the need for manual coordination with data owners. Importantly, CVs can also be used in conjunction with open data as well, by simply creating a CV hosting the open data one would like to include in the analysis, thus filling an important gap in the data sharing ecosystem. We demonstrate the impact of CVs through several functional and structural neuroimaging studies utilizing federated analysis showcasing their potential to improve the reproducibility of research and increase sample sizes in neuroimaging studies.

neuroscience↗

bioRxiv2023

Histone Deacetylases (HDACs) maintain expression of the pluripotent gene network via recruitment of RNA polymerase II to coding and non-coding loci

Histone acetylation is a dynamic modification regulated by the opposing actions of histone acetyltransferases (HATs) and histone deacetylases (HDACs). Deacetylation of histone tails results in chromatin tightening and therefore HDACs are generally regarded as transcriptional repressors. Counterintuitively, simultaneous deletion of Hdac1 and Hdac2 in embryonic stem cells (ESC) reduced expression of pluripotent transcription factors, Oct4, Sox2 and Nanog (OSN). By shaping global histone acetylation patterns, HDACs indirectly regulate the activity of acetyl-lysine readers, such as the transcriptional activator, BRD4. We used inhibitors of HDACs and BRD4 (LBH589 and JQ1 respectively) in combination with precision nuclear run-on and sequencing (PRO-seq) to examine their roles in defining the ESC transcriptome. Both LBH589 and JQ1 caused a marked reduction in the pluripotent network. However, while JQ1 treatment induced widespread transcriptional pausing, HDAC inhibition caused a reduction in both paused and elongating polymerase, suggesting an overall reduction in polymerase recruitment. Using enhancer RNA (eRNA) expression to measure enhancer activity we found that LBH589-sensitive eRNAs were preferentially associated with super-enhancers and OSN binding sites. These findings suggest that HDAC activity is required to maintain pluripotency by regulating the OSN enhancer network via the recruitment of RNA polymerase II.

molecular biology↗

bioRxiv2023

Alpaca Single B Cell Interrogation and Heavy-Chain-Only Antibody Discovery on an Optofluidic Platform

In vivo discovery approaches for single domain antibodies such as VHH have been limited by the lack of methodologies available for camelid B cell interrogation. Here, we report a novel application of the Berkeley Lights Beacon(R) optofluidic platform to the discovery of heavy-chain-only antibodies by screening single B cells from alpacas immunized with two different targets. Custom methods for alpaca B cell enrichment, culture, on-Beacon IgG2/3 detection, and sequencing were developed and used to discover target-specific VHH candidates from an alpaca immunized with either human prostate specific membrane antigen (PSMA) or a second blinded target, in a proof-of-concept study. PSMA-specific VHH hits discovered on the Beacon were recombinantly expressed as VHH-Fc, purified, and characterized using label-free techniques. All but one VHH-Fc bound PSMA with a single-digit nanomolar affinity, and four candidates were successfully humanized in silico using a rapid bulk humanization approach. In addition, next-generation repertoire sequencing was performed at longitudinal timepoints after immunization, uncovering additional variants within the clonal lineages of the validated hits discovered on the Beacon platform. The establishment of this single B cell VHH discovery workflow extends the powerful Beacon technology to enable rapid discovery of VHH directly from natural camelid immune repertoires.

immunology↗

bioRxiv2022

Group 3 Innate Lymphoid Cells are regulated by WASP in a microbiota-dependent manner

Wiskott-Aldrich syndrome protein (WASP) is a cytoskeletal regulator that is largely restricted to hematopoietic cells. While WASP expression in both lymphocytes and macrophages play a critical role in maintaining intestinal homeostasis, the function of WASP in innate lymphoid cells is unknown. Here we analyzed the role of WASP in the differentiation and function of group 3 innate lymphoid cells (ILC3s). WASP-deficient mice (Was-/-) have a marked reduction in ILC3s. Moreover, antimicrobial peptide expression in response to ILC3-derived IL-22 was also reduced in the absence of WASP. In Was-/- mice, we observed a reduction in CCR6+ ILC3s, cells known to restrict immune responses to commensal bacteria. WASP-deficient mice were more susceptible to Citrobacter rodentium, an enteric infection controlled by ILC3s. Interestingly, there was no reduction in ILC3s in Was-/- germ-free mice when compared to WT germ-free mice. ILC3s lacking WASP expression also demonstrated microbially-dependent alterations in gene expression associated with cell migration. Finally, ILC3-like (Rorgt+CD3-) cells were reduced in the GI tract of WASP-deficient patients. In conclusion, ILC3-specific expression of WASP is critical for the generation and function of ILC3s in the presence of commensal microflora. Aberrant ILC3 function in the setting of WASP-deficiency may contribute to underlying disease pathogenesis.

immunology↗

bioRxiv2022

Federated analysis in COINSTAC reveals functional network connectivity and spectral links to smoking and alcohol consumption in nearly 2,000 adolescent brains

With the growth of decentralized/federated analysis approaches in neuroimaging, the opportunities to study brain disorders using data from multiple sites has grown multi-fold. One such initiative is the Neuromark, a fully automated spatially constrained independent component analysis (ICA) that is used to link brain network abnormalities among different datasets, studies, and disorders while leveraging subject-specific networks. In this study, we implement the neuromark pipeline in COINSTAC, an open-source neuroimaging framework for collaborative/decentralized analysis. Decentralized analysis of nearly 2000 resting-state functional magnetic resonance imaging datasets collected at different sites across two cohorts and co-located in different countries was performed to study the resting brain functional network connectivity changes in adolescents who smoke and consume alcohol. Results showed hypoconnectivity across the majority of networks including sensory, default mode, and subcortical domains, more for alcohol than smoking, and decreased low frequency power. These findings suggest that global reduced synchronization is associated with both tobacco and alcohol use. This work demonstrates the utility and incentives associated with large-scale decentralized collaborations spanning multiple sites.

bioinformatics↗

bioRxiv2021

Nanotrap Particles Improve Nanopore Sequencing of SARS-CoV-2 and Other Respiratory Viruses

Presented here is a magnetic hydrogel particle enabled workflow for capturing and concentrating SARS-CoV-2 from diagnostic remnant swab samples that significantly improves sequencing results using the Oxford Nanopore Technologies MinION sequencing platform. Our approach utilizes a novel affinity-based magnetic hydrogel particle, circumventing low input sample volumes and allowing for both rapid manual and automated high throughput workflows that are compatible with nanopore sequencing. This approach enhances standard RNA extraction protocols, providing up to 40x improvements in viral mapped reads, and improves sequencing coverage by 20-80% from lower titer diagnostic remnant samples. Furthermore, we demonstrate that this approach works for contrived influenza virus and respiratory syncytial virus samples, suggesting that it can be used to identify and improve sequencing results of multiple viruses in VTM samples. These methods can be performed manually or on a KingFisher Apex system.

microbiology↗