Biology subjects

Ryan, J.

Publications and source records attributed to Ryan, J..

At least 19 recordsLinked to original sources

bioRxiv2026

Unconventional presentation of an immunodominant HLA-DR15-restricted nephritogenic proteinase 3 epitope implicated in vasculitis

Proteinase 3 (PR3) anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis is associated with HLA-DR15, implicating HLA-DR15-restricted CD4 T cell autoimmunity in the disease. However, the PR3 epitopes presented by HLA-DR15 and the autoreactive CD4 T cell responses they elicit are undefined. Using humanized hPR3.DR15+ mice, we identified PR3216-231 as an immunodominant human PR3-derived CD4 T cell epitope presented by HLA-DR15. Immunopeptidomic profiling confirmed the natural processing and HLA-DR15-restricted presentation of PR3216-231. Structural analyses of HLA-DR15-PR3216-231 complexes revealed an unconventional mode of HLA class II antigen presentation, in which a 10 amino acid peptide core occupies the 9 amino acid binding groove, causing a central kink in the bound peptide. PR3216-231-specific CD4 T cells from both hPR3.DR15+ mice and patients with PR3-ANCA-associated vasculitis exhibited convergent T cell receptor features, and patients with active vasculitis displayed a clonally expanded, PR3216-231-specific CD4 TCR repertoire. Immunization of hPR3.DR15 mice with PR3216-231 induced cell-mediated glomerulonephritis characterized by increased renal infiltration of CD4 T cells and macrophages together with segmental glomerular necrosis. These findings identify a nephritogenic PR3-derived CD4 T cell epitope presented by HLA-DR15 in ANCA-associated vasculitis and define a new extended core binding pattern, broadening our understanding of HLA class II peptide presentation.

immunology↗

bioRxiv2025

Dynamic BH3 profiling predicts clinical outcomes in acute myeloid leukemia

Predictive biomarkers can potentially meet the need for improved drug assignment in acute myeloid leukemia (AML). Fewer than half of AML patients have actionable mutations: consequently, targeted therapy achieves remission in only a fraction of those who have them. Dynamic BH3 Profiling (DBP), a functional assay, can measure changes in ex vivo drug-induced apoptotic priming in multiple cancers. To assess the feasibility and predictive capacity of DBP in AML, we prospectively tested DBP using a fixed-drug panel in myeloblasts from 92 patients. We generated a database combining genetic and functional annotation. Established AML clinical and genetic prognostic characteristics were associated with drug-induced apoptotic priming. We observed distinct interpatient sensitivities to single drugs or combinations with the BCL2-inhibitor venetoclax, and intrapatient apoptotic priming differences based on CD123-expression within distinct cell subpopulations. DBP further predicted the likelihood of remission to chemotherapy and targeted agents, supporting its use to identify optimal personalized therapy. Statement of significanceDynamic BH3 profiling provides patient-specific drug vulnerability data in real-time to inform prognosis and therapy selection. Key takeawaysO_LIDynamic BH3 profiling can be performed on bone marrow and leukemic blood from AML patients in 48 hours. C_LIO_LIKnown clinical prognostic factors associate with drug-induced apoptotic priming in AML. C_LIO_LIDrug-induced apoptotic priming identifies drug vulnerabilities in individual patients and predicts clinical response to chemotherapy and small molecule inhibitors. C_LI

cancer biology↗

bioRxiv2025

Big Data, Sound Science, Lasting Impact: a framework for passive acoustic monitoring

Marine passive acoustic monitoring (PAM) has produced petabytes of data that are used by researchers, resource managers, industry, and regulators to understand how marine animals use sound and the impacts of anthropogenic noise on species and ecosystems throughout the global ocean. These big data provide unprecedented opportunities to study underwater soundscapes and marine ecology but also enormous challenges to efficiently extract information. To address these challenges, a U.S. federally funded and led Sound Cooperative (SoundCoop) project built community-focused cyberinfrastructure to promote improved, scalable and sustainable processing and access of marine PAM data for management, science, industry and military applications. Driven by cross-institutional participation representing a diversity of data collection methods and conditions, the SoundCoop project established guidance for standardized processing of ocean sound level metrics using freeware software toolkits and developed core tools and processes that support open science. Four examples of comparative analyses that connect disparate PAM monitoring efforts, and integrate non-acoustic data illustrate how comparable, interoperable sound level metrics support a more coherent and synoptic perspective on global ocean soundscapes using methods that current and future PAM projects can leverage. Such a framework around PAM big data offers the opportunity to revolutionize large-scale marine ecology and oceanography in similar ways to other transformative approaches for understanding environmental or ecological patterns and processes at global scales.

ecology↗

bioRxiv2025

Drug tolerant persisters and immunotherapy persister cells exhibit cross-resistance and share common survival mechanisms

Persisters are a rare sub-population of tumor cells that survive anti-cancer therapy and are thought to be a major cause of recurrence. These cells have been identified following both drug- and immune-therapy but are generally considered to be distinct entities. Since both pharmacological agents and immune cells often kill via apoptosis, we tested a hypothesis that both types of cells survive based on reduced mitochondrial apoptotic sensitivity, which in turn would yield a similar and reciprocal multi-agent resistant phenotype. Supporting this hypothesis, we indeed observed that IPCs acquired a reduced sensitivity to multiple drug classes and radiotherapy, suggesting non-immune mechanisms are important in the survival of cancer cells after immunotherapy. Likewise, DTPs developed not only a reduced sensitivity to multiple drug classes and radiotherapy, but also acquired a reduced sensitivity to T cell killing. Both IPCs and DTPs developed a decreased sensitivity to mitochondrial apoptosis. A sub-population of IPCs downregulated antigen and upregulated PD-L1. Intriguingly, in the IPCs that didnt employ these mechanisms of resistance, a greater decrease in sensitivity to mitochondrial apoptosis was observed, suggesting that the presence or absence of a resistance mechanism can exert selective pressures over the emergence of others. Targeting anti-apoptotic dependencies in persisters increased sensitivity to chemotherapy or CAR T therapy. These results suggest that common biological mechanisms underly survival of persisters, whether derived from immune or drug therapy, and offer an explanation for the acquired cross-resistance to these two types of therapies often observed in the clinic. HighlightsO_LIImmunotherapy persister cells (IPCs) are less sensitive to drugs and radiation. C_LIO_LIDrug tolerant persisters (DTPs) are less sensitive to radiation and CAR T cell attack. C_LIO_LIIPCs and DTPs are less sensitive to mitochondrial apoptosis. C_LIO_LITargeting anti-apoptotic dependencies helps eliminate IPCs/DTPs. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/641492v2_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@fe98b4org.highwire.dtl.DTLVardef@db7bfdorg.highwire.dtl.DTLVardef@1a94469org.highwire.dtl.DTLVardef@1ca6b2c_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2023

Assessing the performance of commercial reagent antibodies

Antibodies are critical reagents to detect and characterize proteins. It is commonly understood that many commercial antibodies do not recognize their intended targets, but information on the scope of the problem remains largely anecdotal, and as such, feasibility of the goal of at least one potent and specific antibody targeting each protein in a proteome cannot be assessed. Focusing on antibodies for human proteins, we have scaled a standardized characterization approach using parental and knockout cell lines (Laflamme et al., 2019) to assess the performance of 614 commercial antibodies for 65 neuroscience-related proteins. Side-by-side comparisons of all antibodies against each target, obtained from multiple commercial partners, demonstrates that: i) more than 50% of all antibodies failed in one or more tests, ii) yet, [~]50-75% of the protein set was covered by at least one high-performing antibody, depending on application, suggesting that coverage of human proteins by commercial antibodies is significant; and iii) recombinant antibodies performed better than monoclonal or polyclonal antibodies. The hundreds of underperforming antibodies identified in this study were found to have been used in a large number of published articles, which should raise alarm. Encouragingly, more than half of the underperforming commercial antibodies were reassessed by the manufacturers, and many had alterations to their recommended usage or were removed from the market. This first such study helps demonstrate the scale of the antibody specificity problem but also suggests an efficient strategy toward achieving coverage of the human proteome; mine the existing commercial antibody repertoire, and use the data to focus new renewable antibody generation efforts.

molecular biology↗

bioRxiv2022

Stathmin-2 loss leads to neurofilament-dependent axonal collapse driving motor and sensory denervation

The human mRNA most affected by TDP-43 loss-of-function is transcribed from the STMN2 gene and encodes stathmin-2 (also known as SCG10), whose loss is a neurodegenerative disease hallmark. Here using multiple in vivo approaches, including transient antisense oligonucleotide (ASO)-mediated suppression, chronic shRNA-mediated depletion in aging mice, and germline deletion, we establish stathmin-2 to be essential for acquisition and maintenance of neurofilament-dependent structuring of axoplasm critical for maintaining diameter and conduction velocity of large-myelinated axons. Sustained stathmin-2 loss from an otherwise mature adult nervous system is demonstrated over a time course of eight months to initiate and drive motor neuron disease that includes 1) shrinkage in inter-neurofilament spacing that is required to produce a three-dimensional space filling array that defines axonal caliber, 2) collapse of mature axonal caliber with tearing of outer myelin layers, 3) reduced conduction velocity, 4) progressive motor and sensory deficits (including reduction of the pain transducing neuropeptide CGRP), and 5) muscle denervation. Demonstration that chronic stathmin-2 reduction is itself sufficient to trigger motor neuron disease reinforces restoration of stathmin-2 as an attractive therapeutic approach for TDP-43-dependent neurodegeneration, including the fatal adult motor neuron disease ALS.

cell biology↗

bioRxiv2022

Mitotic chromosomes scale to nucleo-cytoplasmic ratio and cell size in Xenopus

During the rapid and reductive cleavage divisions of early embryogenesis, subcellular structures such as the nucleus and mitotic spindle scale to decreasing cell size. Mitotic chromosomes also decrease in size during development, presumably to coordinately scale with mitotic spindles, but underlying mechanisms are unclear. Here we combine in vivo and in vitro approaches using eggs and embryos from the frog Xenopus laevis to show that mitotic chromosome scaling is mechanistically distinct from other forms of subcellular scaling. We found that mitotic chromosomes scale continuously with cell, spindle and nuclear size in vivo. However, unlike for spindles and nuclei, mitotic chromosome size cannot be re-set by cytoplasmic factors from earlier developmental stages. In vitro, increasing nucleo-cytoplasmic (N/C) ratio is sufficient to recapitulate mitotic chromosome scaling, but not nuclear or spindle scaling, through differential loading of maternal factors during interphase. An additional pathway involving importin scales mitotic chromosomes to cell surface area/volume (SA/V) during metaphase. Finally, single-chromosome immunofluorescence and analysis of Hi-C data suggest that mitotic chromosomes scale through decreased recruitment of condensin I, resulting in major rearrangements of DNA loop architecture to accommodate the same amount of DNA on a shorter axis. Together, our findings demonstrate how mitotic chromosome size is set by spatially and temporally distinct developmental cues in the early embryo.

cell biology↗

bioRxiv2022

Reprograming human fibroblasts into Sertoli cells: a tool for personalized medicine

Disorders/Differences of Sex Development (DSD) are congenital conditions in which the development of chromosomal, gonadal, or anatomical sex is atypical. With overlapping phenotypes and multiple genes involved, poor diagnostic yields are achieved for many of these conditions. The current DSD diagnostic regimen can be augmented by investigating transcriptome/proteome in vivo, but it is hampered by the unavailability of affected gonadal tissue at the relevant developmental stage. We try to mitigate this limitation by reprogramming readily available skin tissue-derived dermal fibroblasts into Sertoli cells (SC), which could then be deployed for different diagnostic strategies. SCs form the target cell type of choice because they act like an organizing center of embryonic gonadal development and many DSD arise when these developmental processes go awry. We employed a computational predictive algorithm for cell conversions called Mogrify to predict the transcription factors (TFs) required for direct reprogramming of human dermal fibroblasts into SCs. We established trans-differentiation culture conditions where stable transgenic expression of these TFs was achieved in 46, XY adult dermal fibroblasts using lentiviral vectors. The resulting Sertoli like cells (SLCs) were validated for SC phenotype using several approaches. SLCs exhibited Sertoli-like morphological and cellular properties as revealed by morphometry and xCelligence cell behavior assays. They also showed Sertoli-specific expression of molecular markers such as SOX9, PTGDS, BMP4, or DMRT1 as revealed by IF imaging, RNAseq and qPCR. The SLC transcriptome shared about two thirds of its differentially expressed genes with a human adult SC transcriptome and expressed markers typical of embryonic SCs. Notably, SLCs lacked expression of markers of other gonadal cell types such as Leydig, germ, peritubular myoid or granulosa cells. The trans-differentiation method was applied to a variety of commercially available 46, XY fibroblasts derived from patients with DSD and to a 46, XX cell line. The DSD SLCs displayed altered levels of trans-differentiation in comparison to normal 46, XY-derived SLCs, thus showcasing the robustness of this new trans-differentiation model.

cell biology↗

bioRxiv2021

Co-deletion of ATAD1 with PTEN primes cells for BIM-mediated apoptosis

PTEN is a potent tumor suppressor gene that is frequently mutated or deleted in human cancers. Such deletions often include portions of the 10q23 locus beyond the bounds of PTEN itself, in many cases resulting in the disruption of additional genes. Coincidental loss of PTEN-adjacent genes might impose vulnerabilities that could either affect patient outcome basally or be exploited therapeutically. Here we describe how the loss of ATAD1, which is adjacent to and frequently co-deleted with PTEN, predisposes cancer cells to apoptosis and correlates with improved survival in cancer patients. ATAD1 directly and specifically extracts the pro-apoptotic BIM protein from mitochondria to inactivate it. Cells lacking ATAD1 are hypersensitive to clinically used proteasome inhibitors, which increase BIM and trigger apoptosis. Thus, we demonstrate that mitochondrial protein quality control interfaces with cell death in a clinically actionable manner.

cancer biology↗

bioRxiv2021

MethodsJ2: A Software Tool to Improve Microscopy Methods Reporting

Proper reporting of metadata is essential to reproduce microscopy experiments, interpret results and share images. Experimental scientists can report details about sample preparation and imaging conditions while imaging scientists have the expertise required to collect and report the image acquisition, hardware and software metadata information. MethodsJ2 is an ImageJ/Fiji based software tool that gathers metadata and automatically generates text for the methods section of publications.

bioinformatics↗

bioRxiv2021

Micro-Meta App: an interactive software tool to facilitate the collection of microscopy metadata based on community-driven specifications

For the information content of microscopy images to be appropriately interpreted, reproduced, and meet FAIR (Findable Accessible Interoperable and Reusable) principles, they should be accompanied by detailed descriptions of microscope hardware, image acquisition settings, image pixel and dimensional structure, and instrument performance. Nonetheless, the thorough documentation of imaging experiments is significantly impaired by the lack of community-sanctioned easy-to-use software tools to facilitate the extraction and collection of relevant microscopy metadata. Here we present Micro-Meta App, an intuitive open-source software designed to tackle these issues that was developed in the context of nascent global bioimaging community organizations, including BioImaging North America (BINA) and QUAlity Assessment and REProducibility in Light Microscopy (QUAREP-LiMi), whose goal is to improve reproducibility, data quality and sharing value for imaging experiments. The App provides a user-friendly interface for building comprehensive descriptions of the conditions utilized to produce individual microscopy datasets as specified by the recently proposed 4DN-BINA-OME tiered-system of Microscopy Metadata model. To achieve this goal the App provides a visual guide for a microscope-user to: 1) interactively build diagrammatic representations of hardware configurations of given microscopes that can be easily reused and shared with colleagues needing to document similar instruments. 2) Automatically extracts relevant metadata from image files and facilitates the collection of missing image acquisition settings and calibration metrics associated with a given experiment. 3) Output all collected Microscopy Metadata to interoperable files that can be used for documenting imaging experiments and shared with the community. In addition to significantly lowering the burden of quality assurance, the visual nature of Micro-Meta App makes it particularly suited for training users that have limited knowledge of the intricacies of light microscopy experiments. To ensure wide-adoption by microscope-users with different needs Micro-Meta App closely interoperates with MethodsJ2 and OMERO.mde, two complementary tools described in parallel manuscripts.

bioinformatics↗

bioRxiv2021

Epiblast morphogenesis is controlled by selective mRNA decay triggered by LIN28A relocation

The embryonic progression from naive to primed pluripotency is accompanied by the rapid decay of pluripotency-associated mRNAs and a concomitant radical morphogenetic sequence of epiblast polarization, rosette formation and lumenogenesis. The mechanisms triggering and linking these events remain poorly understood. Guided by machine learning and metabolic RNA sequencing, we identified RNA binding proteins (RBPs), especially LIN28A, as primary mRNA decay factors. Using mRNA-RBP interactome capture, we revealed a dramatic increase in LIN28A mRNA binding during the naive-rosette-primed pluripotency transition, driven by its nucleolar-to-cytoplasmic translocation. Cytoplasmic LIN28A binds to 3UTRs of pluripotency-associated mRNAs to directly stimulate their decay and drive lumenogenesis. Accordingly, forced nuclear retention of LIN28A impeded lumenogenesis, impaired gastrulation, and caused an unforeseen embryonic multiplication. Selective mRNA decay, driven by nucleo-cytoplasmic RBP translocation, therefore acts as an intrinsic mechanism linking cell identity switches to the control of embryonic growth and morphogenesis.

developmental biology↗

bioRxiv2020

An in vivo CRISPR screening platform for prioritizing therapeutic targets in AML

CRISPR-Cas9-based genetic screens have successfully identified cell type-dependent liabilities in cancers, including acute myeloid leukemia (AML), a devastating hematologic malignancy with poor overall survival. Because most of these screens have been performed in vitro, evaluating the physiological relevance of these targets is critical. We have established a CRISPR screening approach using orthotopic xenograft models to prioritize AML-enriched dependencies in vivo, complemented by the validation in CRISPR-competent AML patient-derived xenograft (PDX) models tractable for genome editing. Our integrated pipeline has revealed several targets with translational value, including SLC5A3 as a metabolic vulnerability for AML addicted to exogenous myo-inositol and MARCH5 as a critical guardian to prevent apoptosis in AML. MARCH5 repression enhanced the efficacy of BCL2 inhibitors such as venetoclax, highlighting the clinical potential of targeting MARCH5 in AML. Our study provides a valuable strategy for discovery and prioritization of new candidate AML therapeutic targets. Statement of significanceThere is an unmet need to improve the clinical outcome of AML. We developed an integrated in vivo screening approach to prioritize and validate AML dependencies with high translational potential. We identified SLC5A3 as a metabolic vulnerability and MARCH5 as a critical apoptosis regulator in AML, representing novel therapeutic opportunities.

cancer biology↗

bioRxiv2020

Dynamics of bacterial cell division: Z ring condensation is essential for cytokinesis

How proteins in the bacterial cell division complex (the divisome) coordinate to divide bacteria remains unknown. To explore how these proteins collectively function, we conducted a complete dynamic characterization of the proteins involved, and then examined the function of FtsZ binding proteins (ZBPs) and their role in cytokinesis. We find that the divisome consists of two dynamically distinct subcomplexes: stationary ZBPs that transiently bind to treadmilling FtsZ filaments, and a directionally-moving complex that includes cell wall synthases. FtsZ filaments treadmill at steady state and the ZBPs have no effect on filament dynamics. Rather, ZBPs bundle FtsZ filaments, condensing them into Z rings. Z ring condensation increases the recruitment of cell wall synthesis enzymes to the division site, and this condensation is necessary for cytokinesis.

microbiology↗

bioRxiv2020

Metaproteomic responses of in vitro gut microbiomes to resistant starches: the role of resistant starch type and inter-individual variations

Resistant starches (RS) are dietary compounds processed by the gut microbiota into metabolites, such as butyrate, that are beneficial to the host. The production of butyrate by the microbiome appears to be affected by the plant source and type of RS as well as the individuals microbiota. In this study, we used in vitro culture and metaproteomic methods to explore the consistency and variations in individual microbiomes functional responses to three types of RS - RS2(Hi Maize 260), RS3(Novelose 330) and RS4(Fibersym RW). Results showed that RS2 and RS3 significantly altered the levels of protein expression in the individual gut microbiomes, while RS4 did not result in significant protein changes. Significantly elevated protein groups were enriched in carbohydrate metabolism and transport functions of families Eubacteriaceae, Lachnospiraceae and Ruminococcaceae. In addition, Bifidobacteriaceae was significantly increased in response to RS3. We also observed taxon-specific enrichments of starch metabolism and pentose phosphate pathways corresponding to this family. Functions related to starch utilization, ABC transporters and pyruvate metabolism pathways were consistently increased in the individual microbiomes in response to RS2 and RS3; in contrast, the downstream butyrate producing pathway response varied. Our study confirm that different types of RS have markedly variable functional effects on the human gut microbiome, and also found considerable inter-individual differences in microbiome pathway responses.

microbiology↗

bioRxiv2020

Acute Stress Reduces Reward-Related Neural Activity: Evidence from the Reward Positivity

Stress and blunted reward processing are risk factors for mood disorders, including Major Depressive Disorder (MDD). The experience of acute stress reduces fMRI correlates of reward-related neural activity; however, few studies have examined how acute stress impacts measures of reward derived from event-related potentials (ERPs). The current study examined the impact of an acute stressor on the Reward Positivity (RewP), an ERP that indexes reward sensitivity, in twenty-seven college students. Participants completed a monetary reward task while they placed their left hand in cold water set at 13 degrees Celsius (i.e., acute stress condition) and again while their hand was placed in room temperature water (i.e., control condition). These conditions were separated by one week and performed in a counter-balanced order across participants. The results revealed that the RewP amplitude was blunted in the acute stress condition compared to the control condition. Moreover, there was a trend toward this effect interacting with self-reported depressive symptoms: the RewP was only reduced among individuals who reported low depressive symptoms. The current study suggests that an acute stressor reduces the RewP, and that this effect might be moderated by current depressive symptoms. Future studies might examine the temporal association between reward processing and stress --and how they interact to predict depressive symptoms.

neuroscience↗

bioRxiv2019

Process noise distinguishes between indistinguishable population dynamics

Model selection is becoming increasingly important in mathematical biology. Model selection often involves comparing a set of observations with predictions from a suite of continuum mathematical models and selecting the model that provides the best explanation of the data. In this work we consider the more challenging problem of model selection in a stochastic setting. We consider five different stochastic models describing population growth. Through simulation we show that all five stochastic models gives rise to classical logistic growth in the limit where we consider a large number of identically prepared realisations. Therefore, comparing mean data from each of the models gives indistinguishable predictions and model selection based on population-level information is impossible. To overcome this challenge we extract process noise from individual realisations of each model and identify properties in the process noise that differ between the various stochastic models. Using a Bayesian framework, we show how process noise can be used successfully to make a probabilistic distinction between the various stochastic models. The relative success of this approach depends upon the identification of appropriate summary statistics and we illustrate how increasingly sophisticated summary statistics can lead to improved model selection, but this improvement comes at the cost of requiring more detailed summary statistics.

synthetic biology↗

bioRxiv2019

Noninvasive sleep monitoring in large-scale screening of knock-out mice reveals novel sleep-related genes

Sleep is a critical process that is well-conserved across mammalian species, and perhaps most animals, yet its functions and underlying mechanisms remain poorly understood. Identification of genes and pathways that can influence sleep may shed new light on these functions. Genomic screens enable the detection of previously unsuspected molecular processes that influence sleep. In this study, we report results from a large-scale phenotyping study of sleep-wake parameters for a population of single-gene knockout mice. Sleep-wake parameters were measured using a high throughput, non-invasive piezoelectric system called PiezoSleep. Knockout mice generated on a C57BL6/N (B6N) background were monitored for sleep and wake parameters for five days. By analyzing data from over 6000 mice representing 343 single gene knockout lines, we identified 122 genes influencing traits like sleep duration and bout length that have not been previously implicated in sleep, including those that affect sleep only during a specific circadian phase. PiezoSleep also allows assessment of breath rates during sleep and this was integrated as a supplemental tool in identifying aberrant physiology in these knockout lines. Sex differences were evident in both normal and altered sleep behavior. Through a combination of genetic and phenotypic associations, and known QTLs for sleep, we propose a set of candidate genes playing specific roles in sleep. The high \"hit rate\" demonstrates that many genes can alter normal sleep behaviors through a variety of mechanisms. Further investigation of these genes may provide insight into the pathways regulating sleep, functional aspects of sleep, or indirect potentially pathological processes that alter normal sleep.

neuroscience↗