Biology subjects

O'Connor, K.

Publications and source records attributed to O'Connor, K..

11 recordsLinked to original sources

bioRxiv2026

In vivo reconstruction of the cell lineage history of a developing mouse with DNA Typewriter, from zygote to late organogenesis

The complete cell lineage of C. elegans, mapped over four decades ago, was tractable because the animal is small, transparent, and lineage invariant1. Most animals are none of these, having orders of magnitude more cells, being opaque, and developing with substantial stochasticity2. Since 2016, genome editing-based lineage tracing has opened the door to dense cell lineage reconstruction in such organisms3-8, but delivering on that promise has proven technically challenging. Here we apply DNA Typewriter9-11, a prime editing-based recorder that writes stochastic symbolic insertions to an engineered genomic TAPE in strictly sequential order, to trace mouse development from zygote (E0) to late organogenesis (E13.5). We introduce constructs encoding a prime editor, engineered prime editing guide RNAs (epegRNAs), and Pol-III-driven circularized TAPE RNA (circTAPE) into wildtype zygotes by pronuclear injection (PNI), then assay embryos by single-nucleus transcriptional profiling (sci-RNA-seq3)12 with paired circTAPE recovery. From one E13.5 embryo bearing [~]7 integrations of a constitutively expressed prime editor and 11 integrations of 6-unit circTAPE, we recover [~]1.75M single-nucleus transcriptomes and reconstruct a time-calibrated phylogeny of 1,340,794 annotated cells with parsimony-based node support. The first cell division is marked unequivocally, and although the resulting blastomeres contribute asymmetrically to the embryo proper, they are fate-neutral and serve as internal replicates that reproduce every finding. A modest cohort of pre-gastrulation founders dominates the embryo, with inequality exceeding neutral expectation within one to two cell cycles of founder allocation, yet these founders remain broadly multipotent; a second phase of clonal dominance arises in specific lineages during organogenesis. At the finest scale, sibling cells share cell type 9-fold in excess of chance, reaching 68- to 107-fold for cell types arising from spatially restricted founder pools, while the recent differentiations of organogenesis are legible in the heterotypic structure of terminal clades. From clade co-occurrence alone, we recover germ-layer organization and a dated hierarchy of cell-type couplings with branch points from E8.5 onwards. Finally, by integrating these data with our single-cell time-series of mouse development13, we impute transcriptional states and annotations for the majority of internal nodes and recover established state histories for diverse cell types. All data are made freely available, together with NextCell, an interactive browser for this annotated cellular phylogeny of mouse development from zygote to late organogenesis.

developmental biology↗

bioRxiv2026

Self-Interaction Nanoparticle Spectroscopy Predicts High-Concentration Viscosity of Therapeutic IgG1 Antibodies

Predicting high-concentration viscosity of monoclonal antibodies such as IgG1 is crucial for their development as therapeutics for subcutaneous delivery. Unfortunately, traditional experimental rheometry methods for assessing viscosity are low-throughput. This study evaluates Self-Interaction Nanoparticle Spectroscopy (SINS) assays--specifically charge-stabilized SINS (CS-SINS) and PEG-stabilized SINS (PS-SINS)--for high-throughput viscosity prediction. We characterized 96 IgG1 antibodies, assessing SINS against in silico descriptors and dynamic light scattering (DLS) data. CS-SINS showed strong correlation with charge, offering limited additional utility. In contrast, PS-SINS provided orthogonal information; integrating it with in silico data and DLS significantly improved random forest model accuracy for binary viscosity classification. PS-SINS measurements in multiple buffers captured complementary information, achieving comparable accuracy without DLS. Importantly, PS-SINS scores exhibited a strong logarithmic relationship (r=0.98) with high-concentration viscosity in Fc variants of clinical antibodies, suggesting a direct mechanistic link. Furthermore, PS-SINS performed reliably with one column purified (protein A) samples, supporting its early-stage application. These findings establish PS-SINS as a high-throughput tool to accelerate the developability assessment of antibody candidates.

biochemistry↗

bioRxiv2026

Biophysical Constraints Dictate the Stability of Social Traits in Pseudomonas aeruginosa Aggregates

The maintenance of cooperative behaviors within microbial populations remains an evolutionary puzzle, particularly in spatially structured environments like those found in chronic infection. In Pseudomonas aeruginosa, quorum sensing (QS) directs the production of costly public goods that are vulnerable to exploitation by non-producing cheaters. However, the biophysical mechanisms that stabilize this cooperation in complex environments remain poorly understood. Here, we elucidate how the biophysical properties of the bacterial cell surface govern the micron-scale architecture of bacterial aggregates to promote or constrain the stability of cooperation. Using a polymer-structured growth environment, we demonstrate that cell surface hydrophobicity acts as a primary determinant of spatial organization, where hydrophilic wild-type cells assemble into "stacked" aggregates, whereas hydrophobic O-specific antigen-deficient cells form dense "clumps". In mixed populations, distinct surface properties exhibit phase-separation-like immiscibility, segregating the cells at the micron scale. We show that this segregation directly suppresses cheater fitness by physically sequestering cooperative clusters. Specifically, hydrophobic QS-deficient cells were effectively excluded by hydrophilic QS cooperators, limiting their access to public goods. Furthermore, invasion experiments revealed that hydrophilic cells are inherently fitter, capable of unidirectionally invading populations with a hydrophobic cell surface and independent of social dynamics. Conversely, the need to gain access to nutrient resources can fine-tune these barriers, enabling hydrophilic QS-deficient cells to cluster proximally to hydrophobic cooperators. These findings establish that bacterial cell surface traits impact strongly on microbial social interactions and offer new insights into the maintenance and loss of cooperative traits in chronic infections such as those found in chronic lung infections. Significance StatementCooperative behaviors are essential for the survival of pathogenic bacteria like Pseudomonas aeruginosa, yet they are continually threatened by "cheater" cells that exploit shared public good resources. We demonstrate that the physical properties of the cell surface, specifically hydrophobicity, act as a key architect of microbial social structure. In polymer-rich environments, differences in surface hydrophobicity drive a distinct "oil-and-water" segregation between cell populations. This micron-scale phase separation physically sequesters cooperative cells, insulating them from exploitation by cheaters. Consequently, spatial structure serves as a biophysical stabilizer of cooperation, limiting the metabolic advantages typically held by cheaters. Our findings demonstrate that microbial social evolution is governed not only by genetic strategies, but by fundamental biophysical constraints that dictate the spatial limits of exploitation.

microbiology↗

bioRxiv2025

Cell-type-specific adaptations to mitochondrial stress underly the neurological presentations of MTRFR mutations

Mitochondrial diseases are a group of heterogeneous genetic disorders that exhibit striking tissue specificity. Neurological involvement is among the most consistent features, yet the mechanisms that determine why selective neuronal populations are particularly vulnerable to mitochondrial dysfunction remain poorly understood. Mutations in MTRFR, a mitochondrial ribosome rescue factor, cause a progressive neuromuscular phenotype, but no relevant disease model exists to explain its cell-type-specific pathology. Here, we established the first human iPSC-derived neuronal model of MTRFR loss and identified mechanisms driving differential vulnerability between cortical and motor neurons. Although knockdown led to comparable deficits in mitochondrial translation and OXPHOS across both subtypes, cortical neurons engaged adaptive programs, including dendritic mitochondrial remodelling and heat-shock response activation, that preserved survival. Motor neurons failed to mount these responses and instead displayed apoptotic and inflammatory priming. Pharmacological enhancement of stress adaptation rescued motor neuron survival, indicating that resilience is programmable. These findings provide the first mechanistic evidence that neuronal susceptibility to mitochondrial translation defects is defined by the capacity to activate mitochondrial and cytoprotective stress-response pathways.

neuroscience↗

bioRxiv2025

Permissive central tolerance plus defective peripheral checkpoints licence pathogenic memory B cells in CASPR2-antibody encephalitis

Autoimmunity affects 10% of the population. Within this umbrella, autoantibody-mediated diseases targeting one autoantigen provide a unique opportunity to comprehensively understand the developmental pathway of disease-causing B cells and autoantibodies. While such autoreactivities are believed to be generated during germinal centre reactions, the roles of earlier immune checkpoints in autoantigen-specific B cell tolerance are poorly understood. We address this concept in patients with CASPR2-autoantibody encephalitis and healthy controls. In both groups, comparable and high ([~]0.5%) frequencies of unmutated CASPR2-reactive naive B cells were identified. By contrast, CASPR2-reactive memory B cells were exclusive to patients, and their B cell receptors demonstrated affinity-enhancing somatic mutations with heterogenous binding kinetics. These effector molecules possessed epitope-dependent pathogenic effects in vitro neuronal cultures and in vivo. The unmutated common ancestors of these memory B cells showed a distinctive balance between strong CASPR2 reactivity and very limited binding across the remaining human proteome. Our results are the first to propose mechanisms underlying autoantigen-specific tolerance in humans. We identify permissive central tolerance, defective peripheral tolerance and heterogenous autoantibody binding properties as sequential pathogenic steps which licence CASPR2-directed pathology. By leveraging the basic immunobiology, we rationally direct tolerance-restoring approaches in CASPR2-antibody diseases. This paradigm is applicable across autoimmune conditions.

immunology↗

bioRxiv2024

Predicting purification process fit of monoclonal antibodies using machine learning

0In early stage development of therapeutic monoclonal antibodies, assessment of ease of purification process development typically requires extensive experimentation. However, upstream protein expression and downstream purification experiments are often in conflict with timeline pressures and material constraints, limiting the number of molecules and process conditions that can reasonably be assessed. Recently, high-throughput batch-binding screen data along with improved molecular descriptors have enabled development of robust quantitative structure-property relationship (QSPR) models which predict monoclonal antibody chromatographic binding behavior from the amino acid sequence. This work describes a QSPR strategy for in silico monoclonal antibody purification process fit assessment. Principal Component Analysis is applied to extract a one-dimensional basis for comparison of molecular chromatographic binding behavior from multi-dimensional high-throughput batch-binding screen data. Kernel Ridge Regression is employed to predict the first principal component for new molecular sequences. This workflow is demonstrated with a set of 97 monoclonal antibodies for five chromatography resins in two salt types across a range of pH and salt concentrations. Model development benchmarks four descriptor sets from biophysical structural models and protein language models. The investigation illustrates the value QSPR models can provide to purification process fit assessment, and selection of resins and operating conditions from sequence alone.

bioinformatics↗

bioRxiv2021

A new protocol for speed vernalisation of winter cereals

There are many challenges facing the development of high-yielding, nutritious crops for future environments. One limiting factor is generation time, which prolongs research and plant breeding timelines. Recent advances in speed breeding protocols have dramatically reduced generation time for many short-day and long-day species by optimising light and temperature conditions during plant growth. However, winter crops with a vernalisation requirement still require up to 6-10 weeks in low-temperature conditions before transition to reproductive development. Here, we tested a suite of environmental conditions and protocols to investigate if vernalisation can be satisfied more efficiently. We identified a vernalisation method consisting of exposing seeds at the soil surface to an extended photoperiod of 22 h day:2 h night at 10{degrees}C with transfer to speed breeding conditions that dramatically reduces generation time in both winter wheat (Triticum aestivum) and winter barley (Hordeum vulgare). Implementation of this protocol achieved up to five generations per year for winter wheat or barley, instead of the two typically obtained under standard vernalisation and plant growth conditions. The protocol has great potential to enhance training and to accelerate research, pre-breeding, and breeding outcomes focussed on winter crop improvement.

plant biology↗

bioRxiv2021

Frequency of quorum sensing mutations in Pseudomonas aeruginosa strains isolated from different environments

Pseudomonas aeruginosa uses quorum sensing (QS) to coordinate the expression of multiple genes necessary for establishing and maintaining infection. It has previously been shown that lasR QS mutations frequently arise in cystic fibrosis (CF) lung infections, however, there has been far less emphasis on determining whether other QS system mutations arise during infection or in other environments. To test this, we utilized 852 publicly available sequenced P. aeruginosa genomes from the Pseudomonas International Consortium Database (IPCD) to study P. aeruginosa QS mutational signatures. To study isolates by source, we focused on a subset of 654 isolates collected from CF, wounds, and non-infection environmental isolates, where we could clearly identify their source. We also worked a small collection of isolates in vitro to determine the impact of lasR and pqs mutations on isolate phenotypes. We found that lasR mutations are common across all environments and are not specific to infection nor a particular infection type. We also found that the pqs system proteins PqsA, PqsH, PqsL and MexT, a protein of increasing importance to the QS field, are highly variable. Conversely, RsaL, a negative transcriptional regulator of the las system, was found to be highly conserved, suggesting selective pressure to repress las system activity. Overall, our findings suggest that QS mutations in P. aeruginosa are common and not limited to the las system; however, LasR is unique in the frequency of putative loss-of-function mutations.

microbiology↗

bioRxiv2020

Experimentally-Determined Strengths of Atom-Atom (C, N, O) Interactions Responsible for Protein Self-Assembly in Water: Applications to Folding and Other Protein Processes

Folding and other protein self-assembly processes are driven by favorable interactions between O, N, and C unified atoms of the polypeptide backbone and sidechains. These processes are perturbed by solutes that interact with these atoms differently than water does. C=O{middle dot}{middle dot}{middle dot}HN hydrogen bonding and various {pi}-system interactions have been better-characterized structurally or by simulations than experimentally in water, and unfavorable interactions are relatively uncharacterized. To address this situation, we previously quantified interactions of alkylureas with amide and aromatic compounds, relative to interactions with water. Analysis yielded strengths of interaction of each alkylurea with unit areas of different hybridization states of unified O, N, C atoms of amide and aromatic compounds. Here, by osmometry, we quantify interactions of ten pairs of amides selected to complete this dataset. A novel analysis yields intrinsic strengths of six favorable and four unfavorable atom-atom interactions, expressed per unit area of each atom and relative to interactions with water. The most favorable interactions are sp2O - sp2C (lone pair-{pi}, presumably n-{pi}*), sp2C - sp2C ({pi}-{pi} and/or hydrophobic), sp2O-sp2N (hydrogen bonding) and sp3C-sp2C (CH-{pi} and/or hydrophobic). Interactions of sp3C with itself (hydrophobic) and with sp2N are modestly favorable, while sp2N interactions with sp2N and with amide/aromatic sp2C are modestly unfavorable. Amide sp2O-sp2O interactions and sp2O-sp3C interactions are more unfavorable, indicating the preference of amide sp2O to interact with water. These intrinsic interaction strengths are used to predict interactions of amides with proteins and chemical effects of amides (including urea, N-ethylpyrrolidone (NEP), and polyvinyl-pyrrolidone (PVP)) on protein stability. SignificanceQuantitative information about strengths of amide nitrogen-amide oxygen hydrogen bonds and {pi}-system and hydrophobic interactions involving amide-context sp2 and/or sp3 carbons is needed to assess their contributions to specificity and stability of protein folds and assemblies in water, as well as to predict or interpret how urea and other amides interact with proteins and affect protein processes. Here we obtain this information from thermodynamic measurements of interactions between small amide molecules in water and a novel analysis that determines intrinsic strengths of atom-atom interactions, relative to water and per unit area of each atom-type present in amide compounds. These findings allow prediction or interpretation of effects of any amide on protein processes from structure, and may be useful to analyze protein interfaces.

biochemistry↗

bioRxiv2019

Effective buprenorphine use and tapering strategies: Endorsements and insights by people in recovery from opioid use disorder on a Reddit forum

Opioid use disorder (OUD) is a public health emergency in the United States. Over 47,000 overdose-related deaths in 2017 involved opioids. Medication-assisted treatment (MAT), in particular, buprenorphine and buprenorphine combination products such as Suboxone(R), is the most effective, evidence-based treatment for OUD. However, there are a limited number of conclusive scientific studies that provide guidance to medical professionals about strategies for using buprenorphine to achieve stable recovery. In this study, we used data-driven natural language processing methods to mine a total of 16,146 posts about buprenorphine from 1933 unique users on the anonymous social network Reddit. Analysis of a sample of these posts showed that 74% of the posts described users personal experiences and that the top three topics included advice on using Suboxone(R) (55.0%), Suboxone(R) dosage information (35.5%) and information about Suboxone(R) tapering (32.0%). Based on two models, one that incorporated upvoting by other members and one that did not, we found that Reddit users reported more successful recovery with longer tapering schedules, particularly from 2.0 mg to 0.0 mg (median: 93 days; mean: 95 days), as compared to shorter tapering schedules investigated in past clinical trials. Diarrhea, insomnia, restlessness, and fatigue were commonly reported adverse events. Physical exercise, clonidine, and Imodium(R) were frequently reported to help during the recovery process. Due to the difficulties of conducting longer-term clinical trials involving patients with OUD, clinicians should consider other information sources including peer discussions from the abundant, real-time information available on Reddit. Significance StatementOpioid use disorder (OUD) is a national crisis in the United States and buprenorphine is one of the most effective evidence-based treatments. However, few studies have explored successful strategies for using and tapering buprenorphine to achieve stable recovery, particularly due to the difficulties of conducting long-term studies involving patients with OUD. In this study, we show that discussions on the anonymous social network Reddit may be leveraged, via automatic text mining methods, to discover successful buprenorphine use and tapering strategies. We discovered that longer tapering schedules, compared to those investigated in past clinical trials, may lead to (self-reported) sustained recovery. Furthermore, Reddit posts also provide key information regarding buprenorphine withdrawal, cravings, adjunct medications for withdrawal symptoms and relapse prevention strategies.

pharmacology and toxicology↗

bioRxiv2019

Integrin α6β4 is downregulated in mutant IDH1 oligodendrogliomas, promotes glioma growth, and associates with a worse outcome in glioma patients

The integrin 6{beta}4 is a laminin receptor that associates with aggressive behavior in many cancers, though its significance in gliomas has not been established. Mutations in isocitrate dehydrogenase 1 and 2 (IDHmut) are common in gliomas, especially 1p/19q co-deleted oligodendrogliomas, and correlate with improved patient survival. We sought to determine whether there is a link between integrin {beta}4 and malignant behavior in gliomas. An inverse relationship was identified between IDHmut and integrin {beta}4 expression in a glioma TMA (P < 0.0001) and in TCGA grade II-IV gliomas (P < 0.0001). Methylation of CpG sites in the ITGB4 promoter is significantly higher in IDHmut gliomas when compared to wild-type tumors, and both ITGB4 methylation and reduced ITGB4 mRNA are not only most prominent in the 1p/19q co-deleted subset of IDHmut tumors, but fully account for the lower ITGB4 expression observed in IDHmut gliomas. In fact, ITGB4 is one of the most downregulated genes in IDHmut 1p/19q co-deleted gliomas compared to IDHmut 1p/19q intact gliomas (P = 3.2x10-39). In patient-derived glioma cells, we found that integrin {beta}4 is enriched in the stem-like population, and that knockdown of integrin {beta}4 slows glioma growth in vitro (P < 0.001) and in vivo (P = 0.01). Lower ITGB4 mRNA levels were also associated with longer overall survival in multiple patient cohorts (P = 0.0005; P < 0.0001). These data suggest that integrin {beta}4 increases glioma malignancy, and that its relative paucity may contribute to the better prognosis in IDH1/2 mutant, 1p/19q co-deleted oligodendrogliomas.

cancer biology↗