Biology subjects

Humphries, M.

Publications and source records attributed to Humphries, M..

11 recordsLinked to original sources

bioRxiv2026

Manchester Proteome Profiler: A User-Friendly Platform for Quantitative Proteomic Analysis

Manchester Proteome Profiler (MPP) is an open-source R Shiny application that streamlines downstream analysis of quantitative proteomic data. Compatible with grouped protein intensities tables from MaxQuant, FragPipe, Proteome Discoverer and other custom layouts, MPP provides an integrated platform for filtering, normalisation, imputation, differential expression analysis and cluster analysis across user-chosen experimental conditions. MPP supports both single- and dual-dataset comparisons, incorporates SAINTexpress for affinity purification and proximity labelling experiments, and downstream analysis of the significant protein list clusters to functional enrichment and interaction networks via Gene Ontology, BioGRID and STRING. Benchmarking with a KRAS proximity biotinylation dataset demonstrated the ability of MPP to identify reproducible clusters of differentially expressed proteins and reveal biologically meaningful patterns, including enrichment of solute carrier transporters and adhesion molecules. With interactive visualisations, customisable reports, and support for complex experimental designs, MPP offers a novel, versatile and user-friendly environment for proteomic data exploration and hypothesis generation.

bioinformatics↗

bioRxiv2025

Oscillatory and elevated flow distinctly regulate gene expression in human coronary artery endothelial cells

BackgroundAtherosclerosis develops at arterial sites exposed to disturbed flow, while plaque rupture and plaque erosion predominantly occur in regions subjected to elevated flow. The impact of elevated flow on regulation of endothelial gene expression is less well studied; therefore, we undertook a comprehensive analysis of primary human coronary artery endothelial cell (HCAEC) gene expression under elevated flow, comparing it to gene expression induced by normal physiological and oscillatory flow. MethodsAnalysis of HCAEC mRNA, microRNA and protein expression cultured under oscillatory shear stress (OSS), physiological laminar shear stress (LSS), and elevated shear stress (ESS) for 72 hours. Identification of changes in RNA isoform expression and proximity of flow-responsive genes to established coronary artery disease (CAD) risk loci were also performed. Results2,175 shear-regulated genes were identified, with 665 uniquely responsive to ESS. Both ESS and OSS induced significant changes in RNA isoform selection, predicted to affect 848 and 580 genes respectively. Signalling pathways regulating CAD pathogenesis including HIPPO, TGF{beta}/BMP, and IRF, showed altered RNA isoform selection which may influence plaque development and plaque erosion. 65% of linkage disequilibrium (LD)-filtered CAD-associated genetic variants contained at least one OSS or ESS-regulated gene within 250Kb. Proteomic analysis identified 289 proteins differentially expressed under OSS and 171 under ESS, with notable discordance between mRNA and protein changes observed in 28.7% (OSS vs LSS) and 16.6% (ESS vs LSS) genes. Additionally, 40 shear-responsive microRNAs were identified. ConclusionElevated flow elicits a distinct gene expression programme in HCAECs, modulating pathways central to CAD pathogenesis. Research PerspectiveWhat New Question Does This Study Raise? O_LIPlaque erosion predominantly occurs on the upstream surface of the plaque, where the endothelium is exposed to elevated flow, which we show within this study to evoke a significant and largely unique regulation of the transcriptome, miRome and proteome within primary human coronary artery endothelial cells. C_LIO_LIIdentify shear stress as a significant regulator of alternative splicing, with elevated flow causing the greatest shift in alternative transcript selection. C_LIO_LIIdentify 135 oscillatory, and 101 elevated differentially expressed genes in proximity to CAD risk loci. C_LI What Question Should be Addressed Next? O_LIStudy of the response of the coronary endothelium directly in patients to understand how the risk factors involved in plaque erosion change endothelial function. C_LIO_LICreation of physiological 3D arterial models replicating in vivo observations to study the precipitating factors involved in plaque erosion. C_LI

cell biology↗

bioRxiv2025

Hypoxia-induced extracellular matrix changes are conserved in cancer and directly impact radiotherapy benefit

Our overarching aim was to determine how hypoxia affects the extracellular matrix (ECM). Transcriptomic analysis (21,941 patients; 10 cancer types) identified ECM remodelling as the predominant pathway affected by hypoxia. Multi-omics confirmed that hypoxia impacts ECM organisation and collagen degradation; 53 ECM genes were affected, of which 74% were HIF1/HIF2-regulated. Spatial transcriptomics highlighted different hypoxia remodelling processes in tumour and stroma. Five ECM genes commonly affected in tumour and in vitro constituted a signature. This signature was independently prognostic and independently predictive of radiotherapy benefit in multiple malignancies. Patients within either high or low hypoxic-ECM score tertiles benefited from radiotherapy versus surgery. Hypoxic ECMs generated in vitro increased adhesion and decreased migration of cancer cells, an effect enhanced by irradiation. Immunofluorescence demonstrated that hypoxia decreased collagen fibre number, and irradiation decreased cell-ECM interactions. Taken together, these findings demonstrate hypoxia induces pan-cancer ECM changes, directly impacting radiotherapy benefit.

cancer biology↗

bioRxiv2021

Talin mechanosensitivity is modulated by a direct interaction with cyclin-dependent kinase-1

Talin is a mechanosensitive component of adhesion complexes that directly couples integrins to the actin cytoskeleton. In response to force, talin undergoes switch-like behaviour of its multiple rod domains that modulate interactions with its binding partners. Cyclin-dependent kinase-1 (CDK1) is a key regulator of the cell cycle, exerting its effects through synchronised phosphorylation of a large number of protein targets. CDK1 activity also maintains adhesion during interphase, and its inhibition is a prerequisite for the tightly choreographed changes in cell shape and adhesiveness that are required for successful completion of mitosis. Using a combination of biochemical, structural and cell biological approaches, we demonstrate a direct interaction between talin and CDK1 that occurs at sites of integrin-mediated adhesion. Mutagenesis demonstrated that CDK1 contains a functional talin-binding LD motif, and the binding site within talin was pinpointed to helical bundle R8 through the use of recombinant fragments. Talin also contains a consensus CDK1 phosphorylation motif centred on S1589; a site that was phosphorylated by CDK1 in vitro. A phosphomimetic mutant of this site within talin lowered the binding affinity of KANK and weakened the mechanical response of the region, potentially altering downstream mechanotransduction pathways. The direct binding of the master cell cycle regulator, CDK1, to the primary integrin effector, talin, therefore provides a primordial solution for coupling the cell proliferation and cell adhesion machineries, and thereby enables microenvironmental control of cell division in multicellular organisms. SummaryThe direct binding of the master cell cycle regulator, CDK1, to the primary integrin effector, talin, provides a primordial solution for coupling the cell proliferation and cell adhesion machineries, and thereby enables microenvironmental control of cell division.

cell biology↗

bioRxiv2020

FHL-1 interacts with human RPE cells through the α5β1 integrin and confers protection against oxidative stress

Retinal pigment epithelial (RPE) cells that underlie the neurosensory retina are essential for the maintenance of photoreceptor cells and hence vision. Interactions between the RPE and their basement membrane, i.e. the inner layer of Bruchs membrane, are essential for RPE cell health and function, but the signals induced by Bruchs membrane engagement, and their contributions to RPE cell fate determination remain poorly defined. Here, we studied the functional role of the soluble complement regulator and component of Bruchs membrane, Factor H-like protein 1 (FHL-1). Human primary RPE cells adhered to FHL-1 in a manner that was eliminated by either mutagenesis of the integrin-binding RGD motif in FHL-1 or by using competing antibodies directed against the 5 and {beta}1 integrin subunits. The results obtained from primary RPE cells were replicated using the hTERT-RPE cell line. RNAseq expression analysis of hTERT-RPE cells bound to FHL-1 showed an increased expression of the heat-shock protein genes HSPA6, CRYAB, HSPA1A and HSPA1B when compared to cells bound to fibronectin (FN) or laminin (LA). Pathway analysis implicated changes in EIF2 signalling, the unfolded protein response, and mineralocorticoid receptor signalling as putative pathways. Subsequent cell survival assays using H2O2 to induce oxidative stress-induced cell death showed hTERT-RPE cells had significantly greater protection when bound to FHL-1 or LA compared to plastic or FN. These data show a non-canonical role of FHL-1 in protecting RPE cells against oxidative stress and identifies a novel interaction that has implications for ocular diseases such as age-related macular degeneration.

cell biology↗

bioRxiv2020

Glycocalyx-mediated Cell Adhesion and Migration

Cell migration is a force-dependent adaptive process mediated by integrin-dependent adhesion as well as other yet poorly defined interactions to the extracellular matrix. Using enzymatic multi-targeted digestion of sugar moieties on the surface of mesenchymal cells and leukocytes after interference with integrin function, we demonstrate that the surface glycocalyx represents an independent adhesion system. The glycocalyx mediates cell attachment to ECM ligand in the 100-500 pN force range and amoeboid migration in 3D environments in vitro and in vivo. Glycan-based adhesions consist of actin-rich membrane deformations and appositions associated with bleb-like and other protrusions forming complex-shaped sub-micron contact sites to ECM fibrils. These data implicate the glycocalyx in mediating generic stickiness to support nanoscale interactions (nanogrips) between the cell surface and ECM, mechano-coupling, and migration.

cell biology↗

bioRxiv2019

Maladaptive striatal plasticity and abnormal reward-learning in cervical dystonia

In monogenetic generalized forms of dystonia, in vitro neurophysiological recordings have demonstrated direct evidence for abnormal plasticity at the level of the cortico-striatal synapse. It is unclear whether similar abnormalities contribute to the pathophysiology of cervical dystonia, the most common type of focal dystonia. We investigated whether abnormal cortico-striatal synaptic plasticity contributes to abnormal reward-learning behavior in patients with focal dystonia. Forty patients and forty controls performed a reward-gain and loss-avoidance reversal learning task. Participants behavior was fitted to a computational model of the basal ganglia incorporating detailed cortico-striatal synaptic learning rules. Model comparisons were performed to assess the ability of four hypothesised receptor specific abnormalities of cortico-striatal long term potentiation (LTP) and Long Term Depression (LTD): increased or decreased D1:LTP/LTD and increased or decreased D2: LTP/LTD to explain abnormal behavior in patients. Patients were selectively impaired in the post-reversal phase of the reward task. Individual learning rates in the reward reversal task correlated with the severity of the patients motor symptoms. A model of the striatum with decreased D2:LTP/ LTD best explained the patients behavior, suggesting excessive D2 cortico-striatal synaptic depotentiation could underpin biased reward learning in patients with cervical dystonia. Reversal learning impairment in cervical dystonia may be a behavioural correlate of D2 specific abnormalities in cortico-striatal synaptic plasticity. Reinforcement learning tasks with computational modeling could allow the identification of molecular targets for novel treatments based on their ability to restore normal reward-learning behavior in these patients.

neuroscience↗

bioRxiv2019

β1 integrin is a sensor of blood flow direction

The ability of endothelial cells (EC) to sense blood flow direction is a critical determinant of vascular health and disease. Unidirectional flow induces EC alignment and vascular homeostasis, whereas bidirectional flow has pathophysiological effects. EC express several mechanoreceptors that can respond to fluid flow (shear stress) but the mechanism for sensing the direction of shearing force is poorly understood. We observed using in vitro flow systems and magnetic tweezers that {beta}1 integrin is a key sensor of force direction because it is activated by unidirectional but not bidirectional shearing forces. Consistently, {beta}1 integrin was essential for Ca2+ signalling and cell alignment in response to unidirectional but not bidirectional shear stress. {beta}1 integrin activation by unidirectional force was amplified in EC that were pre-sheared in the same direction, indicating that alignment and {beta}1 integrin activity has a feedforward interaction which is a hallmark of system stability. En face staining and EC-specific genetic deletion studies of the murine aorta revealed that {beta}1 integrin is activated and is essential for EC alignment at sites of unidirectional flow but is not activated at sites of bidirectional flow. In summary, {beta}1 integrin sensing of unidirectional force is a key mechanism for decoding blood flow mechanics to promote vascular homeostasis.\n\nSUMMARY STATEMENTWe demonstrate using flow systems, magnetic tweezers and knockout mice that {beta}1 integrin is a sensor of shear stress direction in endothelial cells. {beta}1 integrin is activated by unidirectional hemodynamic shear force leading to calcium signalling and cell alignment, but it is not activated by bidirectional force.

cell biology↗

bioRxiv2018

Eps8 is a convergence point integrating EGFR and integrin trafficking and crosstalk

Crosstalk between adhesion and growth factor receptors plays a critical role in tissue morphogenesis and repair, and aberrations contribute substantially to neoplastic disease. However, the mechanisms by which adhesion and growth factor receptor signalling are integrated, spatially and temporally, are unclear.\n\nWe used adhesion complex enrichment coupled with quantitative proteomic analysis to identify rapid changes to adhesion complex composition and signalling following growth factor stimulation. Bioinformatic network and ontological analyses revealed a substantial decrease in the abundance of adhesion regulatory proteins and co-ordinators of endocytosis within 5 minutes of EGF stimulation. Together these data suggested a mechanism of EGF-induced receptor endocytosis and adhesion complex turnover.\n\nCombinatorial interrogation of the networks allowed a global and dynamic view of adhesion and growth factor receptor crosstalk to be assembled. By interrogating network topology we identified Eps8 as a putative node integrating 5{beta}1 integrin and EGFR functions. Importantly, EGF stimulation promoted internalisation of both 5{beta}1 and EGFR. However, perturbation of Eps8 increased constitutive internalisation of 5{beta}1 and EGFR; suggesting that Eps8 constrains 5{beta}1 and EGFR endocytosis in the absence of EGF stimulation. Consistent with this, Eps8 regulated Rab5 activity and was required for maintenance of adhesion complex organisation and for EGF-dependent adhesion complex disassembly. Thus, by co-ordinating 5{beta}1 and EGFR trafficking mechanisms, Eps8 is able to control adhesion receptor and growth factor receptor bioavailability and cellular contractility.\n\nWe propose that during tissue morphogenesis and repair, Eps8 functions to spatially and temporally constrain endocytosis, and engagement, of 5{beta}1 and EGFR in order to precisely co-ordinate adhesion disassembly, cytoskeletal dynamics and cell migration.

cell biology↗

bioRxiv2018

Insights into Parkinson’s disease from computational models of the basal ganglia

Movement disorders arise from the complex interplay of multiple changes to neural circuits. Successful treatments for these disorders could interact with these complex changes in myriad ways, and as a consequence their mechanisms of action and their amelioration of symptoms are incompletely understood. Using Parkinsons disease as a case-study, we review here how computational models are a crucial tool for taming this complexity, across causative mechanisms, consequent neural dynamics, and treatments. For mechanisms, we review models that capture the effects of losing dopamine on basal ganglia function; for dynamics, we discuss models that have transformed our understanding of how beta-band (15-30 Hz) oscillations arise in the parkinsonian basal ganglia. For treatments, we touch on the breadth of computational modelling work trying to understand the therapeutic actions of deep brain stimulation. Collectively, models from across all levels of description are providing a compelling account of the causes, symptoms, and treatments for Parkinsons disease.

neuroscience↗

bioRxiv2017

An ensemble code in medial prefrontal cortex links prior events to outcomes during learning

The prefrontal cortex is implicated in learning the rules of an environment through trial and error. But it is unclear how such learning is related to the prefrontal cor-texs role in short-term memory. Here we asked if the encoding of short-term memory in prefrontal cortex was used by rats learning decision rules in a Y-maze task. We found that a similar pattern of neural ensemble activity was selectively recalled after reinforcement for a correct decision. This reinforcement-selective recall only reliably occurred immediately before the abrupt behavioural transitions indicating successful learning of the current rule, and faded quickly thereafter. We could simultaneously decode multiple, retrospective task events from the ensemble activity, suggesting the recalled ensemble activity had multiplexed encoding of prior events. Our results suggest that successful trial-and-error learning is dependent on reinforcement tagging the relevant features of the environment to maintain in prefrontal cortex short-term memory.

neuroscience↗