Biology subjects

Murphy, J.

Publications and source records attributed to Murphy, J..

At least 19 recordsLinked to original sources

bioRxiv2026

tRNA-derived fragments restrict KSHV lytic replication through miRNA-like repression of host pro-viral signalling networks

Transfer RNA derived fragments (tRFs) are emerging regulators of gene expression. Despite mounting evidence of their functional relevance in cellular and pathological processes, their roles in virus infection remain poorly understood. Here, we show that host tRFs are dynamically reprogrammed during Kaposi sarcoma-associated herpesvirus (KSHV) lytic replication and function as potent antiviral effectors. Transcriptome-wide profiling revealed extensive remodelling of the tRF landscape following viral reactivation, identifying a subset of tRFs; tRFs (tRF-His-GTG, tRF-Gly-CCC, and tRF-Lys-CTT) that are robustly induced independently of parental tRNA abundance. Functional analyses demonstrate that these tRFs suppress viral gene expression and infectious virion production. Mechanistically, we show that tRF-His-GTG and tRF-Lys-CTT engage canonical RNA silencing pathways by associating with AGO2 complexes and mediating sequence-specific repression of host target genes. Among these targets, we identify SHF as a critical pro-viral factor that promotes PI3K/Akt signalling, cellular survival and efficient viral replication. Collectively, our findings define a previously uncharacterised host defence pathway in which tRFs restrict KSHV replication by targeting host factors required for efficient viral propagation, thus expanding the functional repertoire of non-coding RNAs in antiviral immunity.

microbiology↗

bioRxiv2025

StereoMapper: Clarifying Metabolite Identity Through Stereochemically Aware Relationship Assignment

Inaccuracies in metabolite cross-mapping frequently introduce errors into genome-scale metabolic models (GEMs). A key challenge lies in distinguishing truly identical metabolites from closely related structural variants, such as stereoisomers, across biochemical databases. StereoMapper addresses this issue by establishing stereochemically aware relationships that clarify metabolite identity. It operates entirely on structural information, using molecular structures to infer equivalence and define relationships. StereoMapper was implemented using RDKit (2025.3.3) and Open Babel-wheel (3.1.1.22) which provides the core functionality of the Open Babel software, and benchmarked across four curated control datasets to evaluate performance in relationship classification (enantiomer, diastereomer, stereo-resolution pairs, and protomer). The pipeline was subsequently applied to 1.3 million molecular structures from multiple metabolic databases to generate equivalent isomeric sets (groups of structures identified as equivalent by StereoMapper). These sets were compared against the human subset of MetaNetX, a context-aware reference, to assess concordance and investigate disagreements. On curated positive control sets containing only positive examples across multiple relationship classes, StereoMapper achieved mean precision, recall, and F1-scores of 92.6, 98.0, and 95.3, respectively; following optimisation, these improved to 98.9, 98.2, and 98.5. Application to the 1.3 million structures produced over 339,000 stereochemically defined relationships between molecular structures. Comparison with the human subset of MetaNetX assignments showed an overall grouping concordance of 93.5%. The remaining 6.5% of differences were mainly associated with protomer relationships (52.3%) and stereo-resolution pairs (37.4%), while 21.6% involved complex or ambiguous cases. Scientific contribution statementStereoMapper provides a stereochemically aware pipeline for metabolite relationship assignment that shows high performance across multiple benchmark datasets, resolving key ambiguities in metabolite identity and providing a complementary approach to existing context-aware frameworks. Its integration will enhance reaction-level cross-mapping and improve the accuracy of future GEM reconstructions.

systems biology↗

bioRxiv2025

Complete de novo assembly and re-annotation of the zebrafish genome

The zebrafish (Danio rerio) is widely used in vertebrate research, but its reference genome assembly has contained extensively unresolved regions across both euchromatic and heterochromatic compartments. The previous reference genome assembly, GRCz11, consisted of 19,725 contigs assembled into 1,917 scaffolds. Recent advances in both long-read sequencing technologies and genome assembly algorithms have made "complete" genome assemblies possible for the first time. We used homozygous fish from two lab strains, "Tubingen" and "AB," for de novo genome assemblies. The new assemblies incorporated 7% more genomic sequence than GRCz11 and an additional 130 million bases of previously unassembled sequence. RefSeq annotation incorporating newly generated Iso-Seq cDNA sequences have added notable increases in mRNAs (68%), lncRNAs (47%), and misc_RNAs (1099%). Two assemblies have been elevated to reference genome status (GRCz12tu and GRCz12ab). We generated an additional 40 draft haplotypes to create a zebrafish pangenome resource and demonstrate its utility for variant analysis.

genomics↗

bioRxiv2025

Single cell multi-omic whole genome sequencing and chromatin accessibility profiling reveals genome-epigenome coevolution in colorectal cancer

Epigenetic alterations co-evolve with genetic mutations to drive carcinogenesis and treatment response. Resolving genome-epigenome coevolution requires accurate multi-omic single cell measurement. Here we develop a new technology called "double ATAC" (dATAC) for high-throughput, high-quality, concurrent whole genome sequencing and chromatin accessibility profiling of individual somatic cells. dATAC is a "one pot" method that uses two rounds of tagmentation to sequentially label open chromatin regions and then the whole genome, and produces data of the same quality as current leading single-omic methods. Using colorectal cancer as a model system, we apply dATAC to reveal convergent reorganisation of the epigenome across expanding drug-resistant clones during 5-FU chemotherapy exposure, remarkable stoichiometry of chromatin accessibility at somatic copy number alterations, and the clonal expansion of copy-number altered T cells in the stroma of metastatic disease. dATAC is a robust single cell technology to accurately profile genome-epigenome coevolution across tissues, diseases and species.

molecular biology↗

bioRxiv2024

A necroptotic-to-apoptotic signaling axis underlies inflammatory bowel disease

Inflammatory bowel disease (IBD) is a chronic condition caused by altered cytokine signaling, maladaptive immunity, dysbiosis, and intestinal barrier dysfunction. Patients with IBD receive therapy to correct these imbalances and achieve remission. However, most patients relapse, suggesting that pathological mechanisms persist during remission. Here, we show that excess epithelial cell death is an underlying feature of IBD that arises in patients in remission and on advanced therapy. Mechanistically, nascent inflammation reprograms epithelial cells into a macrophage-like state that promotes RIPK1-independent necroptotic signaling, then triggers iNOS-mediated mitochondrial apoptosis of absorptive epithelial cells and PUMA-mediated intestinal stem cell death. These findings reveal aberrant epithelial cell death signaling as a hallmark of IBD that occurs early in mucosal lesion development and persists despite current therapeutic approaches. One-Sentence SummaryEpithelial cell death is dysregulated in patients with inflammatory bowel disease.

cell biology↗

bioRxiv2023

Towards a Brighter Constellation: Multi-Organ Neuroimaging of Neural and Vascular Dynamics in the Spinal Cord and Brain

SignificancePain is comprised of a complex interaction between motor action and somatosensation that is dependent on dynamic interactions between the brain and spinal cord. This makes understanding pain particularly challenging as it involves rich interactions between many circuits (e.g., neural and vascular) and signaling cascades throughout the body. As such, experimentation on a single region may lead to an incomplete and potentially incorrect understanding of crucial underlying mechanisms. AimHere, we aimed to develop and validate new tools to enable detailed and extended observation of neural and vascular activity in the brain and spinal cord. The first key set of innovations were targeted to developing novel imaging hardware that addresses the many challenges of multi-site imaging. The second key set of innovations were targeted to enabling bioluminescent imaging, as this approach can address limitations of fluorescent microscopy including photobleaching, phototoxicity and decreased resolution due to scattering of excitation signals. ApproachWe designed 3D-printed brain and spinal cord implants to enable effective surgical implantations and optical access with wearable miniscopes or an open window (e.g., for one-or two-photon microscopy or optogenetic stimulation). We also tested the viability for bioluminescent imaging, and developed a novel modified miniscope optimized for these signals (BLmini). ResultsHere, we describe novel universal implants for acute and chronic simultaneous brain-spinal cord imaging and optical stimulation. We further describe successful imaging of bioluminescent signals in both foci, and a new miniscope, the BLmini, which has reduced weight, cost and form-factor relative to standard wearable miniscopes. ConclusionsThe combination of 3D printed implants, advanced imaging tools, and bioluminescence imaging techniques offers a new coalition of methods for understanding spinal cord-brain interactions. This work has the potential for use in future research into neuropathic pain and other sensory disorders and motor behavior.

bioengineering↗

bioRxiv2023

CaBLAM! A high-contrast bioluminescent Ca2+ indicator derived from an engineered Oplophorus gracilirostris luciferase

Measuring ongoing cellular activity is essential to understanding the dynamic functions of biological organisms. The most popular current approach is imaging fluorescence-based genetically encoded Ca2+ indicators (GECIs). While fluorescent probes are useful in many contexts, bioluminescence-based GECIs--probes that generate light through oxidation of a small-molecule by a luciferase or photoprotein--have several distinct advantages. Because bioluminescent (BL) GECIs do not use the bright extrinsic excitation light required for fluorescence, BL GECIs do not photobleach, do not suffer from nonspecific autofluorescent background, and do not cause phototoxicity. Further, BL GECIs can be applied in contexts where directly shining photons on an imaging target is not possible. Despite these advantages, the use of BL GECIs has to date been limited by their small changes in bioluminescence intensity, high baseline signal at resting Ca2+ concentrations, and suboptimal Ca2+ affinities. Here, we describe a new BL GECI, CaBLAM (Ca2+ BioLuminescence Activity Monitor), that displays much higher dynamic range than previous BL GECIs and has a Ca2+ affinity suitable for capturing physiological changes in cytosolic Ca2+ concentration. With these improvements, CaBLAM captures single-cell and subcellular resolution activity at high frame rates in cultured neurons and in vivo, and allows multi-hour recordings in mice and behaving zebrafish. This new advance provides a robust alternative to traditional fluorescent GECIs that can enable or enhance imaging across many experimental conditions.

neuroscience↗

bioRxiv2023

A FRET-based excitatory luminopsin with improved efficacy of bioluminescence-driven activation

SignificanceBioluminescent optogenetics (BL-OG) offers a unique and powerful approach to manipulate neural activity both opto- and chemogenetically using a single actuator molecule (a LuMinOpsin, LMO). AimTo further enhance the utility of BL-OG by improving the efficacy of chemogenetic (bioluminescence- driven) LMO activation. ApproachWe developed novel luciferases optimized for Forster resonance energy transfer (FRET) when fused to the fluorescent protein mNeonGreen, generating bright bioluminescent (BL) emitters spectrally tuned to Volvox Channelrhodopsin 1 (VChR1). ResultsA new LMO generated from this approach (LMO7) showed significantly stronger BL-driven opsin activation compared to previous and other new variants. We extensively benchmarked LMO7 against LMO3 (current standard), and found significantly stronger neuronal activity modulation ex vivo and in vivo, and efficient modulation of behavior. ConclusionsWe report a robust new option for achieving multiple modes of control in a single actuator, and a promising engineering strategy for continued improvement of BL-OG.

neuroscience↗

bioRxiv2023

Surveillance of Vermont wildlife in 2021-2022 reveals no detected SARS-CoV-2 viral RNA

Previous studies have documented natural infections of SARS-CoV-2 in various domestic and wild animals. More recently, studies have been published noting the susceptibility of members of the Cervidae family, and infections in both wild and captive cervid populations. In this study, we investigated the presence of SARS-CoV-2 in mammalian wildlife within the state of Vermont. 739 nasal or throat samples were collected from wildlife throughout the state during the 2021 and 2022 harvest season. Data was collected from red and gray foxes (Vulpes vulples and Urocyon cineroargentus, respectively), fishers (Martes pennati), river otters (Lutra canadensis), coyotes (Canis lantrans), bobcats (Lynx rufus rufus), black bears (Ursus americanus), and white-tailed deer (Odocoileus virginianus). Samples were tested for the presence of SARS-CoV-2 via quantitative RT-qPCR using the CDC N1/N2 primer set and/or the WHO-E gene primer set. Our results indicate that no sampled wildlife were positive for SARS-CoV-2. This finding is surprising, given that most published North America studies have found SARS-CoV-2 within their deer populations. The absence of SARS-CoV-2 RNA in populations sampled here may provide insights in to the various environmental and anthropogenic factors that reduce spillover and spread in North Americans wildlife populations.

microbiology↗

bioRxiv2022

Dual Palmitoylation of PRCD, a Photoreceptor-Specific Protein Linked to RP, Alters Protein Stability and Subcellular Localization

Progressive rod-cone degeneration (PRCD) is a photoreceptor outer segment (POS) disc-specific protein essential for maintaining outer segment (OS) structures, while also contributing to rhodopsin packaging densities and distribution in the disc membranes. Previously, we showed PRCD undergoing palmitoylation at the sole cysteine (Cys2), where a mutation is found linked with retinitis pigmentosa (RP) that is crucial for protein stability and trafficking to POS. PRCD has several predicted structural domains with unknown significance, such as the polybasic region (PBR) where an Arg17Cys (R17C) mutation is linked with RP. In this study, we demonstrate that a mutation in the PBR augments additional palmitoyl lipid modification observed through acyl-RAC in the mutant cysteine (R17C). Immunolocalization of transiently expressed R17C protein in hRPE1 cells depicts similar characteristics to wild type (WT); however, a double mutant lacking endogenous palmitoylation at the Cys2 position is comparable to the C2Y protein as both are likely aggregated and mislocalized in the mitochondria. Subretinal injection of C2Y, R17C, and R17C/C2Y mutants followed by electroporation in murine retina exhibit mislocalization in the inner segment compared to WT PRCD. Our results in the R17C mutant show palmitoylation transpires at two different locations. Despite being dually palmitoylated and demonstrating membrane association, the mutation in the PBR affects protein stability and trafficking to the OS. Moreover, palmitoylation within the PBR alone does not compensate for protein stability or trafficking, revealing the PBR domain is indispensable and any defects likely lead to dysregulation of PRCD protein associated with blinding diseases.

biochemistry↗

bioRxiv2022

Neural representations of vicarious rewards are linked to interoception and prosocial behaviour

Every day we constantly observe other people receiving rewards. Theoretical accounts posit that vicarious reward processing might be linked to peoples sensitivity to internal body states (interoception) and facilitates a tendency to act prosocially. However, the neural processes underlying the links between vicarious reward processing, interoception and prosocial behaviour are poorly understood. Previous research has linked vicarious reward processing to the anterior cingulate gyrus (ACCg) and the anterior insula (AI). Can we predict someones propensity to be prosocial or to be aware of interoceptive signals from variability in how the ACCg and AI process rewards? Here, participants monitored rewards being delivered to themselves or a stranger during functional magnetic resonance imaging. Later, they performed a task measuring their willingness to exert effort to obtain rewards for others, and a task measuring their propensity to be aware and use interoceptive signals. Using multivariate similarity analysis, we show that peoples willingness to be prosocial is predicted by greater similarity between self and other representations in the ACCg. Moreover, greater dissimilarity in self-other representations in the AI is linked to interoceptive propensity. These findings highlight that vicarious reward is linked to bodily signals in AI, and foster prosocial tendencies through the ACCg.

neuroscience↗

bioRxiv2022

Genetic and immune landscape evolution defines subtypes of MMR deficient colorectal cancer

Mismatch repair deficient colorectal cancers have high mutation loads and many respond to immune checkpoint-inhibitors. We investigated how genetic and immune landscapes co-evolve in these tumors. All cases had high truncal mutation loads. Driver aberrations showed a clear hierarchy despite pervasive intratumor heterogeneity: Those in WNT/{beta}Catenin, mitogen-activated protein kinase and TGF{beta} receptor family genes were almost always truncal. Immune evasion drivers were predominantly subclonal and showed parallel evolution. Pan-tumor evolution, subclonal evolution, and evolutionary stasis of genetic immune evasion drivers defined three MMRd CRC subtypes with distinct T-cell infiltrates. These immune evasion drivers have been implicated in checkpoint-inhibitor resistance. Clonality and subtype assessments are hence critical for predictive immunotherapy biomarker development. Cancer cell PD-L1 expression was conditional on loss of the intestinal homeobox transcription factor CDX2. This explains infrequent PD-L1 expression by cancer cells and likely contributes to the high recurrence risk of MMRd CRCs with impaired CDX2 expression.

cancer biology↗

bioRxiv2021

Selective Control of Synaptically-Connected Circuit Elements by All-Optical Synapses

Understanding percepts, engrams and actions requires methods for selectively modulating synaptic communication between specific subsets of interconnected cells. Here, we develop an approach to control synaptically connected elements using bioluminescent light: Luciferase-generated light, originating from a presynaptic axon terminal, modulates an opsin in its postsynaptic target. Vesicular-localized luciferase is released into the synaptic cleft in response to presynaptic activity, creating a real-time Optical Synapse. Light production is under experimenter-control by introduction of the small molecule luciferin. Signal transmission across this optical synapse is temporally defined by the presence of both the luciferin and presynaptic activity. We validate synaptic Interluminescence by multi-electrode recording in cultured neurons and in mice in vivo. Interluminescence represents a powerful approach to achieve synapse-specific and activity-dependent circuit control during behavior in vivo.

neuroscience↗

bioRxiv2021

The central nucleus of the amygdala and gustatory cortex assess affective valence during CTA learning and expression in male and female rats

This study evaluated behavior (Boost(R) intake, LiCl-induced behaviors, ultrasonic vocalizations (USVs), task performance) and c-Fos activation during conditioned taste aversion (CTA), the reinforced task (Boost(R) task) and control task (cage only) to understand how male and female rats balance the relative danger or safety of a stimulus in learning and performing a task. Females drank more Boost(R) than males but showed similar aversive behaviors after LiCl treatment. Males produced 55 kHz USVs (indicating positive valence) when anticipating Boost(R) and inhibited these calls after pairing with LiCl. Females produced 55 kHz USVs based on their estrous cycle but were more likely to make 22 kHz USVs than males (indicating negative valence) after pairing with LiCl. c-Fos responses were similar in males and females after Boost(R) or LiCl. Females engaged the gustatory cortex and ventral tegmental area more than males during the Boost(R) task and males engaged the amygdala more than females in both the reinforcing and devalued tasks. Network analysis of correlated c-Fos responses across brain regions identified two unique networks characterizing the Boost(R) and LiCl (CTA) tasks, in both of which the VTA played a central role. RNAscope identified a population of D1-receptor expressing cells in the CeA that responded to Boost(R) and D2 receptor-expressing cells that responded to LiCl. The present study suggests that males and females differentially process the affective valence of a stimulus to produce the same goal-directed behavior.

animal behavior and cognition↗

bioRxiv2021

Motor unit recruitment patterns of the quadriceps differ between continuous high- and low-torque isometric knee extension to momentary failure

The size principle is a theory of motor unit (MU) recruitment that suggests MUs are recruited in an orderly manner from the smallest (lower threshold) to the largest (higher threshold) MUs. A consequence of this biophysical theory is that, for isometric contractions, recruitment is dependent on the intensity of actual effort required to meet task demands. This concept has been supported by modelling work demonstrating that, in tasks performed to momentary failure, full MU recruitment will have occurred upon reaching failure irrespective of the force requirements of the task. However, in vivo studies examining this are limited. Therefore, the aim of the current study was to examine MU recruitment of the quadriceps under both higher- and lower-torque (70% and 30% of MVC, respectively) isometric knee extension, performed to momentary failure. Specifically, we compared surface electromyography (sEMG) frequency characteristics, determined by wavelet analysis, across the two continuous isometric knee extension tasks to identify potential differences in recruitment patterns. A convenience sample of 10 recreationally active adult males (height: 179.6{+/-}6.0 cm; mass: 76.8{+/-}7.3 kg; age: 26{+/-}7 years) with previous resistance training experience (6{+/-}3 years) were recruited. Using a within-session, repeated-measures, randomised crossover design participants performed the knee extension tasks whilst sEMG was collected from the vastus medialis (VM), rectus femoris (RF) and vastus lateralis (VL). Myoelectric signals were decomposed into intensities as a function of time and frequency using an EMG-specific wavelet transformation. Our first analysis compared the mean frequency at momentary failure; second, we investigated the effects of load on relative changes in wavelet intensities; finally, we quantified the degree of wavelet similarity over time. Wavelet-based calculation of the mean signal frequency appeared to show similar mean frequency characteristics occurring when reaching momentary failure. However, individual wavelets revealed that different changes in frequency components occurred between the two tasks, suggesting that patterns of recruitment differed. Low-torque conditions resulted in an increase in intensity of all frequency components across the trials for each muscle whereas high-torque conditions resulted in a wider range of frequency components contained within the myoelectric signals at the beginning of the trials. However, as the low-torque trial neared momentary failure there was an increased agreement between conditions across wavelets. Our results corroborate modelling studies as well as recent biopsy evidence, suggesting overall MU recruitment may largely be similar for isometric tasks performed to momentary failure with the highest threshold MUs likely recruited, despite being achieved with differences in the pattern of recruitment over time utilised.

physiology↗

bioRxiv2021

Egocentric Biases are Determined by the Precision of Self-related Predictions.

According to predictive processing theories, emotional inference involves simultaneously minimising discrepancies between predictions and sensory data relating to both ones own and others states, achievable by altering either ones own state (empathy) or perception of anothers state (egocentric bias) so they are more congruent. We tested a key hypothesis of these accounts, that predictions are weighted in inference according to their precision (inverse variance). If correct, more precise self-related predictions should bias perception of anothers emotional expression to a greater extent than less precise predictions. We manipulated predictions about upcoming own-pain (low or high magnitude) using cues that afforded either precise (a narrow range of possible magnitudes) or imprecise (a wide range) predictions. Participants judged pained facial expressions presented concurrently with own-pain to be more intense when own-pain was greater, and precise cues increased this biasing effect. Implications of conceptualising interpersonal influence in terms of predictive processing are discussed.

neuroscience↗

bioRxiv2021

Chemoproteomics Enabled Discovery of Selective Probes for NuA4 Factor BRD8

Bromodomain-containing proteins frequently reside in multisubunit chromatin complexes with tissue or cell state-specific compositions. Recent studies have revealed tumor-specific dependencies on the BAF complex bromodomain subunit BRD9 that are a result of recurrent mutations afflicting the structure and composition of associated complex members. To enable the study of ligand engaged complex assemblies, we established a chemoproteomics approach using a functionalized derivative of the BRD9 ligand BI-9564 as an affinity matrix. Unexpectedly, in addition to known interactions with BRD9 and associated BAF complex proteins, we identify a previously unreported interaction with members of the NuA4 complex through the bromodomain-containing subunit BRD8. We apply this finding, alongside homology model guided design, to develop chemical biology approaches for the study of BRD8 inhibition, and to arrive at first-in-class selective and cellularly active probes for BRD8. These tools will empower further pharmacological studies of BRD9 and BRD8 within respective BAF and NuA4 complexes.

biochemistry↗

bioRxiv2020

Accessible Analysis of Longitudinal Data with Linear Mixed Effects Models

Longitudinal mouse models are commonly used to study possible causal factors associated with human health and disease. However, the statistical models, such as two-way ANOVA, often applied in these studies do not appropriately model the experimental design, resulting in biased and imprecise results. Here, we describe the linear mixed effects (LME) model and how to use it for longitudinal mice studies. We re-analyze a dataset published by Blanton et al (Science 2016) that modeled mice growth trajectories after microbiome implantation from nourished or malnourished children. We compare the fit and stability of different parameterizations of ANOVA and LME models; most models found the nourished vs. malnourished growth trajectories differed significantly. We show through simulation that the results from the two-way ANOVA and LME models are not always consistent. Incorrectly modeling correlated data can result in increased rates of false positives or false negatives, supporting the need to model correlated data correctly. We provide an interactive Shiny App to enable accessible and appropriate analysis of longitudinal data using LME models.

bioinformatics↗