Biology subjects

Murphy, T.

Publications and source records attributed to Murphy, T..

15 recordsLinked to original sources

bioRxiv2026

HIV broadly neutralizing antibodies with enhanced Fc-CD16 affinity increase NK cell ADCC even with limited envelope binding

The persistence of latent HIV-1 reservoirs remains the primary barrier to a cure. Shock and kill strategies aim to reactivate these reservoirs and eliminate them via effector cells, such as Natural Killer (NK) cells. However, chronic infection leaves NK cells exhausted. In this study, we investigated the interplay between cytokine-mediated NK cell activation and antibody-dependent cellular cytotoxicity (ADCC) across diverse HIV-1 subtypes. We demonstrated that while cytokine stimulation enhanced natural cytotoxicity, it simultaneously induces shedding of the Fc receptor CD16 via the metalloproteinase enzyme ADAM17. However, restoring CD16 expression through ADAM17 inhibition (TAPI-1) did not improve ADCC, suggesting that CD16 surface levels in NK cells is not the only limiting factor. On the other hand, Fc-engineered broadly neutralizing antibodies (bNAbs) with increased CD16 affinity, in particular LPLIL and GASDALIE, significantly enhance ADCC across the six different HIV-1 subtypes, regardless of NK cells activation, CD16 downregulation or limited surface Envelope expression in CD4T cells. These findings suggest that enhancing receptor affinity of bNAbs can bypass viral immune evasion and NK cell exhaustion, supporting their potential incorporation into HIV cure strategies. ImportanceEradicating latent HIV reservoirs remains a global health priority, as it would liberate millions of people living with HIV (PLWH) from the economic and physiological burdens of lifelong antiretroviral therapy (ART). By utilizing a primary human cell model that resembles in vivo conditions across diverse viral strains, we identified that simply preventing CD16 receptor shedding from NK cells is insufficient to improve HIV-1 clearance. Instead, we demonstrated that enhancing the binding of antibodies to the CD16 receptor enables NK cells to overcome viral immune evasion and the loss of CD16 expression. This allows for potent elimination of infected cells through antibody-dependent cellular cytotoxicity (ADCC). These findings provide a clear strategy for designing more effective "kill" components in future therapeutic strategies.

immunology↗

bioRxiv2026

Organotypic artery-graft culture enables label-free multiphoton tracking of remodeling that links to long-term graft microarchitecture

The long-term performance of tissue-engineered scaffolds, particularly small-diameter vascular grafts, is shaped by remodeling events at the tissue-graft interface, yet these processes remain difficult to resolve longitudinally and at microstructural resolution in conventional implantation models. Here we develop an organotypic artery-graft model that preserves cylindrical vessel geometry and enables non-destructive label-free multiphoton monitoring of interface remodeling. Using second harmonic generation and two-photon excited fluorescence, we capture evolving fibrillar collagen architecture and cellularization over time, demonstrate compatibility with multiple biomaterial classes, and show integration with rat and mouse explants, live-cell dyes, and fluorescent reporter tissues. The platform resolved distinct remodeling responses to transforming growth factor-{beta} isoforms (TGF-{beta}1, -{beta}2, and -{beta}3), with differential shifts in collagen-fiber distributions, accompanied by changes in matrix-remodeling and contractile gene expression. Across two graft designs, culture-derived remodeling phenotypes, collagen fiber distributions, and initial trajectories agreed with those observed in long-term 6-month interpositional explants. Together, these results establish an accessible intermediate platform for interrogating artery-graft remodeling, tracking these trajectories, and prioritizing graft designs through interface-resolved outcomes before and alongside animal implantation studies.

bioengineering↗

bioRxiv2025

The complete genome of a songbird

Bird genomes are the smallest among amniotes, but remain challenging to assemble due to their structural complexity. This study presents the first fully phased, diploid, telomere-to-telomere (T2T) reference genome for the zebra finch (Taeniopygia guttata), a model organism for neuroscience and evolutionary genomics. Combining multiple sequencing strategies resulted in closing nearly all gaps, adding [~]90 Mbp of previously missing sequence (7.8%). This includes T2T assemblies for all microchromosomes, including dot chromosomes, and the previously almost entirely missing chr16. The T2T genome is comprehensively annotated for genes, repeats, structural variants, and long-read methylation calls. Complete centromeric structures were assembled and annotated along with kinetochore binding sites. Relative to the previous high-quality reference of the Vertebrate Genomes Project, 2,778 (8.51%) previously unassembled or unannotated genes were identified, of which 9% overlap with segmental duplications. This first complete genome of a songbird, now the new public reference, illuminates avian genome architecture and function.

genomics↗

bioRxiv2025

Dual Role of Glycosylation in Resistance to CD4-binding Site Broadly Neutralizing Antibodies

Broadly neutralizing antibodies (bNAbs) provide a useful tool for HIV cure strategies because of their ability to target conserved regions on the envelope (Env) protein in the context of both virions and infected cells. One of the most well studied bNAbs is the CD4 binding site (CD4bs) antibody, VRC01 and others in its class. A major obstacle to effective cure strategies with bNAbs is viral immune escape. A deeper understanding of escape pathways from VRC01-class antibodies in genetically diverse samples is needed. Using an in vitro viral escape assay where infected CD4+ T cells were cultured in the presence of increasing VRC01 concentrations, complete resistance to VRC01 was detected in Env 246.F3 by day 42. We determined that resistance was due to a mutation at position N276 that resulted in elimination of the glycan-attachment site. As the loss of a glycan at this site is known to increase virus sensitivity to CD4bs antibodies, we explored this finding further. Specifically, we introduced N276D mutation into the 12-virus global Env panel and measured neutralization susceptibility to a panel of CD4bs bNAbs. This N276D mutation increased resistance or sensitivity depending on the Env and bNAb in question, emphasizing a dual role of glycan N276 in VRC01-class bNAb neutralization. The role of this glycan in escape was demonstrated to be dependent on the context of the Env, the bNAb and the glycosylation complexity of the virus due to producer cells. These findings underscore the complexity of glycosylation in genetically diverse HIV escape from antibodies. ImportanceDespite decades of research, an HIV cure remains elusive, largely due to the viruss immense genetic variability and ability to evade immune clearance. While antiretroviral therapy (ART) suppresses viral replication, it does not eradicate the virus and presents long-term challenges related to toxicity, access, and adherence. Broadly neutralizing antibodies (bNAbs), particularly those targeting the conserved CD4 binding site such as VRC01 and others in its class, offer promise for durable control or cure by targeting both circulating virus and infected cells. However, viral escape from bNAbs remains a critical hurdle. In this study, we demonstrate that glycan-mediated escape from VRC01-class bNAbs is highly context-dependent--shaped by Env, bNAb, and the glycosylation patterns introduced by the producer cell. These findings emphasize the dual role of glycans in affecting antibody sensitivity and underscore the importance of viral and host factors in shaping effective bNAb-based cure strategies across diverse HIV-1 strains.

immunology↗

bioRxiv2025

Microtubule curling as an efficient readout to uncover fundamental concepts of axonal cell biology

Nerve fibres (aka axons) are the slender, up-to-meter-long processes of nerve cells that wire nervous systems. These delicate structures must survive for an organisms lifetime, making them prime lesion sites in neurodegeneration. Their long-term maintenance requires the homeostasis of complex local cell biology upheld by motor protein-driven transport along microtubule bundles that run uninterrupted along axons. To gain an understanding of axonal homeostasis, we report and discuss here the functional loss of 105 genes from a wide range of cell biological processes using a standardised Drosophila primary neuron system. [~]40% of these gene deficiencies caused microtubule bundle disturbances referred to as microtubule-curling, which we use as an indicator of axonal atrophy. Our live imaging showed that microtubule-curling initiates in areas where axons widen, such as areas close to the soma, branch points or growth cones. In wild-type neurons, any initiated curling was contained, but it persisted as growing footprints in mutant curl-promoting conditions. Closer analyses of 20 curl-promoting conditions suggested a general classification into two groups: mutations affecting axonal physiology cause ROS-mediated microtubule-curling, whereas mutations causing motor hyperactivation or affecting microtubule-regulating proteins cause structurally induced ROS-independent curling. As will be discussed, our data provide consistent support for the previously proposed dependency cycle of local axon homeostasis model which can explain the long-standing conundrum that genes from a wide range of cell biological processes often have mutational links to the same class of inherited neurodegenerative disease.

cell biology↗

bioRxiv2025

Blood-based Epigenetic Instability Linked to Human Aging and Disease

The abundance, dynamics, and context-dependent heterogeneity of DNA methylation--where a pattern considered abnormal in one cell type may be normal in another--poses challenges in identifying methylation abnormalities linked to disease risk. Through genome-wide analyses, we identified CpG sites with remarkably consistent methylation profiles in healthy whole blood, predominantly existing in an unmethylated state. We examined alterations at these epigenetically stable loci in diverse cohorts, including those with cardiovascular disease and hematological cancers. Our findings reveal methylation pattern disruption in myeloid and lymphoid malignancies, correlating with clonal burden fluctuations during leukemia treatment. In non-cancer cohorts, we observed that normally stable CpG sites exhibited progressive instability with advancing age, which was also associated with the onset of cardiovascular disease and decreased survival rates. This study links DNA methylation instability with the expansion of risk-prone blood cells and highlights its role as a biomarker for both cancer and cardiovascular disease.

genetics↗

bioRxiv2025

Analysis of blood-based DNA Methylation signatures of aging and disease progression in IBD patients

BackgroundInflammatory bowel diseases (IBDs) are chronic inflammatory disorders with a dysregulated immune response partly influenced by environmental factors. DNA methylation (DNAm), a key epigenetic mechanism, is implicated in the etiology of complex diseases, including IBD. Epigenetic clocks, which use DNAm patterns to estimate biological aging, have been increasingly linked to various health and disease states. Previous studies have associated DNAm with IBD, and first- and second-generation epigenetic clocks with IBD subtypes. ResultsIn a discovery IBD cohort (n=149) with 8-year clinical follow-up data, we explored the relationship between DNAm variation, second- and third-generation epigenetic clocks, and IBD clinicopathological outcomes, including disease subtype, activity, and recurrence. One CpG site was significantly differentially methylated (Benjamini-Hochberg adjusted p-value<0.05) in patients with clinical recurrence of disease over the long term (i.e., after the first year of study) compared to non-recurrence (no treatment escalation after 8 years). Next, we assessed DNAm aging signatures and IBD outcomes using logistic regression. Individuals with IBD exhibited significantly increased epigenetic aging, as measured by GrimAge, GrimAge2, and DunedinPACE, compared with controls. These associations were replicated in two independent IBD cohorts (GSE87648 (n=377) and GSE112611 (n=238)). Additionally, in UC patients, the active disease group was associated with higher age acceleration (GrimAge (U=669, p=0.003)) and higher pace of aging (DunedinPACE (t=3.233, 0.002)) compared to the inactive group. In the discovery cohort, DunedinPACE outperforms CRP measures in discriminating activity in UC patients with an AUC, sensitivity and specificity of 0.71, 69.5% and 68.7% respectively, highlighting its potential as a useful biomarker of activity in UC. ConclusionsOverall, we present strong evidence that dynamic age-related DNAm changes can be used to differentiate between IBD (including separately by subtype) and controls. Furthermore, our study provides important new evidence that DunedinPACE may have utility as a biomarker for monitoring disease recurrence in IBD patients and may be a strong marker of disease activity in UC patients. Overall, this suggests that blood-based DNAm signatures could serve as biomarkers for detection and monitoring of IBD.

bioinformatics↗

bioRxiv2025

Different mechanisms link gain and loss of kinesin functions to axonal degeneration

Axons are the slender, often meter-long projections of neurons that form the biological cables wiring our bodies. Most of these delicate structures must survive for an organisms lifetime, meaning up to a century in humans. Long-term maintenance and sustained functionality of axons requires motor protein-driven transport distributing life-sustaining materials and organelles to places of need. It seems therefore plausible that loss of motor function would cause axon degeneration; however, also gain-of-function conditions were linked to disorders including motor neuron disease or spastic paraplegia. To understand this phenomenon, we studied [~]40 genetic manipulations of motor proteins, cargo linkers and regulators of reactive oxygen species in one standardised Drosophila primary neuron system. Using axonal microtubule bundle organisation as a relevant readout reflecting the state of axon integrity, we found that losses of Dynein heavy chain, KIF1A/Unc-104 and KIF5/Kinesin heavy chain (Khc) all cause bundle disintegration in the form of chaotically curled microtubules. Detailed functional studies of Khc and its adaptor proteins revealed that losses of mitochondrial or lysosomal transport cause ROS dyshomeostasis, which is a microtubule-curl-inducing condition in fly and mouse neurons alike. We find that hyper-activated Khc induces the same microtubule curling phenotype, not through ROS but likely more directly through enhanced mechanical forces. Studies with loss of Unc-104 or KIFBP and expression of an ALS-linked mutant form of the human Khc orthologue KIF5A suggest that loss or hyperactivation of different types of transport motors cause MT curling as a shared feature. We discuss a model which can explain our findings and their relevance for understanding motor-linked neurodegeneration.

neuroscience↗

bioRxiv2023

Traumatic brain injury disrupts state-dependent functional cortical connectivity in a mouse model

Traumatic brain injury (TBI) is the leading cause of death in young people and can cause cognitive and motor dysfunction and disruptions in functional connectivity between brain regions. In human TBI patients and rodent models of TBI, functional connectivity is decreased after injury. Recovery of connectivity after TBI is associated with improved cognition and memory, suggesting an important link between connectivity and functional outcome. We examined widespread alterations in functional connectivity following TBI using simultaneous widefield mesoscale GCaMP7c calcium imaging and electrocorticography (ECoG) in mice injured using the controlled cortical impact (CCI) model of TBI. Combining CCI with widefield cortical imaging provides us with unprecedented access to characterize network connectivity changes throughout the entire injured cortex over time. Our data demonstrate that CCI profoundly disrupts functional connectivity immediately after injury, followed by partial recovery over 3 weeks. Examining discrete periods of locomotion and stillness reveals that CCI alters functional connectivity and reduces theta power only during periods of behavioral stillness. Together, these findings demonstrate that TBI causes dynamic, behavioral state-dependent changes in functional connectivity and ECoG activity across the cortex.

neuroscience↗

bioRxiv2023

Cell-extrinsic effects of Irf2 on cDC2 development

Characterization of the functional effects of cDC2s in vivo requires model systems in which cDC2s are depleted. Previous literature has reported a loss of cDC2s in mice lacking the transcription factor IRF21,2. We sought to further characterize the cDC2 defect in these animals. Here, we find that the requirement for IRF2 in cDC2 development and survival is cell-extrinsic and correlated to the development of dermatitis in the Irf2-/- model system. We also find that Flt3L-mediated in vitro development of cDC1s and cDC2s, but not pDCs, is abrogated in Irf2-/- bone marrow, as well as in wild-type bone marrow cultured with IFN. Loss of interferon (IFN) signaling in Irf2-/- mice restored cDC2 development in vivo and cDC1 and cDC2 development in vitro. We therefore conclude that IRF2 is required for cDC2 development in a cell-extrinsic manner dependent on IFN signaling.

immunology↗

bioRxiv2022

Single-nuclei transcriptomics of mammalian prion diseases identifies dynamic gene signatures shared between species

Mammalian prion diseases are fatal and transmissible neurological conditions caused by the propagation of prions, self-replicating multimeric assemblies of misfolded forms of host cellular prion protein (PrP). The most common human form of the disease, sporadic Creutzfeldt-Jakob disease (sCJD), typically presents as a rapidly progressive dementia and has no effective treatments. Prion diseases are transmissible to laboratory rodents affording unprecedented opportunities to understand neurodegeneration in its evolving stages. Murine models are especially useful in prion research as they develop bona fide prion disease and recapitulate all biochemical and neuropathological hallmarks of human prion disease. Despite extensive studies investigating the changes in transcriptional profiles in prion diseases the mechanisms by which prion diseases induce cellular toxicity, including changes in gene expression profiles are yet to be fully characterized. This is at least in part because confounding effects related to brain cellular heterogeneity have not been resolved. Here, we took advantage of the recent developments in single-cell technologies and performed an unbiased whole-transcriptome single-nucleus transcriptomic analysis in prion disease.

neuroscience↗

bioRxiv2022

WDFY4 deficiency in NOD mice abrogates autoimmune diabetes and insulitis

The events that initiate autoimmune diabetes in NOD mice remain poorly understood. CD4 and CD8 T cells are both required but whether either cell initiates disease is unclear. To test whether CD4 T cell infiltration into islet required damage to {beta} cells induced by autoreactive CD8 T cells, we selectively inactivated Wdfy4 in NOD mice (NOD.Wdfy4-/-) using CRISPR/Cas9 targeting. Similar to C57BL/6 Wdfy4-/- mice NOD.Wdfy4-/- mice develop type 1 conventional dendritic cells (cDC1) that are unable to cross-present cell-associated antigens required to activate CD8 T cells. By contrast, cDC1 from heterozygous Wdfy4+/- mice can cross-present normally. Heterozygous NOD.Wdfy4+/- mice develop diabetes similar to NOD mice, but NOD.Wdfy4-/- mice neither develop diabetes nor prime autoreactive CD8 T cells in vivo. By contrast, NOD.Wdfy4-/- mice can process and present MHC-II-restricted autoantigens and can activate {beta} cell specific CD4 T cells in lymph nodes, and yet do not develop CD4 T cell infiltration in islets. These results indicate that the priming of autoreactive CD8 T cells in NOD mice requires cross-presentation by cDC1. Further, autoreactive CD8 T cells are required not only to develop diabetes, but to recruit autoreactive CD4 T cells into islets of NOD mice, perhaps in response to progressive {beta} cell damage.

immunology↗

bioRxiv2021

New cell biological explanations for kinesin-linked axon degeneration

Axons are the slender, up to meter-long projections of neurons that form the biological cables wiring our bodies. Most of these delicate structures must survive for an organisms lifetime, meaning up to a century in humans. Axon maintenance requires life-sustaining motor protein-driven transport distributing materials and organelles from the distant cell body. It seems logic that impairing this transport causes systemic deprivation linking to axon degeneration. But the key steps underlying these pathological processes are little understood. To investigate mechanisms triggered by motor protein aberrations, we studied more than 40 loss- and gain-of-function conditions of motor proteins, cargo linkers or further genes involved in related processes of cellular physiology. We used one standardised Drosophila primary neuron system and focussed on the organisation of axonal microtubule bundles as an easy to assess readout reflecting axon integrity. We found that bundle disintegration into curled microtubules is caused by the losses of Dynein heavy chain and the Kif1 and Kif5 homologues Unc-104 and Kinesin heavy chain (Khc). Using point mutations of Khc and functional loss of its linker proteins, we studied which of Khcs sub-functions might link to microtubule curling. One cause was emergence of harmful reactive oxygen species through loss of Milton/Miro-mediated mitochondrial transport. In contrast, loss of the Kinesin light chain linker caused microtubule curling through an entirely different mechanism appearing to involve increased mechanical challenge to microtubule bundles through de-inhibition of Khc. The wider implications of our findings for the understanding of axon maintenance and pathology are discussed.

cell biology↗

bioRxiv2019

A YoeB toxin from A. tumefaciens has metal-dependent DNA cleaving activity

Toxin-antitoxin (TA) systems, including YoeB-YefM, are important mediators of bacterial physiological changes. Agrobacterium tumefaciens YoeB and YefM are similar to that from E. coli, and interact as a tight heterotetramer with a KD of 653 pM. We have verified that AtYoeB can perform both ribosome-dependent and -independent RNA cleavage. We have also characterized a newly described metal-dependent and pH-sensitive DNA cleaving ability. We note that this DNA cleaving ability is observed at toxin concentrations as low as 150 nM. The dose-dependence of in vitro ribosome-independent RNA and metal-dependent DNA cleavage is equivalent, and requires a ten-fold increase in toxin concentration as opposed to in the presence of the ribosome. The toxin concentration inside bacterial cells is unknown and according to current models, should increase upon activation of YoeB through degradation of the YefM antitoxin. The discovery of general nuclease activity by AtYoeB, and perhaps other YoeB toxins, offers an opportunity to explore the plasticity of this protein fold and its potential role in the evolution of nucleases.

biochemistry↗

bioRxiv2017

Cellular phenotyping of hippocampal progenitors exposed to patient serum predicts conversion to Alzheimer’s Disease

The generation of new neurons persists into adulthood in the human hippocampus and can be modulated by the circulatory systemic environment. Hippocampal neurogenesis is important for learning and memory and is altered in Alzheimers Disease (AD). Evaluating the hippocampal neurogenic process during disease progression could therefore identify neurogenesis as an important target for AD prevention and intervention as well as a biomarker for early disease detection. In this study, we used a human hippocampal progenitor cell line to design an in vitro assay evaluating over time the neurogenic impact of the systemic milieu (i.e. serum) of individuals with mild cognitive impairment (MCI) as they either converted to AD or remained cognitively stable. Cells were exposed to serum collected over several years from the same patients. Cellular phenotyping and linear mixed effects models for repeated measures revealed that decreased proliferation, increased apoptotic hippocampal progenitor cell death and increased hippocampal neurogenesis characterized progression from MCI to AD. Using stepwise logistic regression and machine learning we show that these cellular readouts for the baseline serum sample and years of education of the patient are significant predictors of conversion from MCI to AD, already 3.5 years before AD clinical diagnosis. Finally, serum proteomic analyses indicated pathways linked to the cellular readouts distinguishing MCI to AD converters from non-converters. The proposed assay is thus not only promising for AD pre-clinical diagnosis, but it also provides a proxy into temporal changes of the hippocampal neurogenic process during disease progression.\n\nOne Sentence SummaryIn this study, we demonstrate for the first time that the systemic environment (i.e. blood serum) of mild cognitively impaired patients differentially alters human hippocampal progenitor cell fate to predict conversion to Alzheimers Disease up to 3.5 years before clinical diagnosis.

neuroscience↗