Biology subjects

Robinson, P.

Publications and source records attributed to Robinson, P..

18 recordsLinked to original sources

bioRxiv2026

Unmasking Supervillin: SVIL haploinsufficiency causes hypertrophic cardiomyopathy by impairing mechanotransduction and cellular energetics

BackgroundRare heterozygous loss-of-function (LoF) variants in SVIL, encoding the Z-disk and costameric protein supervillin, have recently been identified as a cause of hypertrophic cardiomyopathy (HCM). Although supervillin is implicated in actin-dependent mechanotransduction, the mechanisms linking SVIL deficiency to cardiomyopathy remain poorly understood. Homozygous LoF cause a novel skeletal Myofibrillar Myopathy-10 (MFM-10) while heterozygous LoF cause HCM without skeletal myopathy. In this study we use a human model system to disentangle the LoF pathomechanism of the scaffolding protein supervillin in cardiomyocytes and its clinical implications. MethodsUsing CRISPR/Cas-9 we engineered a representative pathogenic LoF variant Q255X into an isogenic induced pluripotent stem cell (iPSC) line creating the heterozygous SVILQ255X/+ and homozygous SVILQ255X/Q255X cell lines. These lines were differentiated into iPSC-derived cardiomyocytes (iPSC-CMs) and cellular phenotypes were assessed using bulk RNA-sequencing, LC-MS proteomics, electrophysiological and calcium handling analyses, contractility measurements, sarcomere organization analysis, Seahorse metabolic flux assay, and pharmacological intervention with mavacamten. ResultsThe Q255X variant resulted in SVIL haploinsufficiency at both RNA and protein levels with no evidence of a truncated protein. Compared with isogenic controls, SVILQ255X/+ iPSC-CMs demonstrated action potential shortening, calcium transient elongation, sarcomeric disorganization and hypertrophy, and impaired mitochondrial respiration. Multi-omic analyses of SVILQ255X/+ iPSC-CMs showed a profile of cellular stress and inflammation, hypertrophic and pro-fibrotic signalling, and a pseudohypoxic state driven by decreased respiration and a HIF-induced glycolytic shift. These abnormalities were not present in SVILQ255X/Q255X cardiomyocytes, consistent with a relatively limited cardiac phenotype reported in homozygous variant carriers. Mavacamten improved sarcomeric disorganization and hypertrophy in SVILQ255X/+ cells but did not rescue energetic compromise. ConclusionsPathogenic heterozygous SVIL LoF produces a distinct cellular phenotype characterized by impaired mechanotransduction, mitochondrial dysfunction, and maladaptive metabolic remodelling that promotes hypertrophic and pro-fibrotic signalling. These findings define a mechanistic basis for SVIL-associated cardiomyopathy and identify metabolic dysfunction as a potential therapeutic target beyond sarcomere-directed therapy. Clinical PerspectiveO_ST_ABSWhat Is New?C_ST_ABSO_LISVIL haploinsufficiency causes HCM through a mechanism distinct from canonical sarcomeric disease, characterized by impaired mechanotransduction, mitochondrial dysfunction, and pseudohypoxia-driven metabolic remodeling. C_LIO_LIHeterozygous SVIL loss of function produces a substantially more severe cardiomyocyte phenotype than homozygous loss of function, providing a mechanistic explanation for the predominance of cardiac disease in heterozygous variant carriers. C_LIO_LIMavacamten improves sarcomeric organization but does not restore impaired mitochondrial respiration, demonstrating that energetic dysfunction persists despite sarcomere-directed therapy. C_LI What Are the Clinical Implications?O_LIOur findings give functional evidence to support SVIL as a clinically relevant HCM disease gene and its inclusion in clinical genetic testing panels. C_LIO_LIThese findings establish SVIL-associated cardiomyopathy as a mechanistically distinct form of HCM and offer insight into the pathomechanism of Z-disk and costameric HCM C_LIO_LIThe persistence of mitochondrial dysfunction despite myosin inhibition suggests that drugs targeting mitochondrial bioenergetics may be a therapeutic strategy in patients with SVIL-associated cardiomyopathy. C_LI

cell biology↗

bioRxiv2025

Contemporary seasonal human coronaviruses display differences in cellular tropism compared to laboratory-adapted reference strains

Seasonal human coronaviruses (sHCoVs) cause 15-30% of common colds. The reference strains used for research were isolated decades ago and have been passaged extensively but contemporary sHCoVs have been challenging to study as they are notoriously difficult to grow in standard immortalized cell lines. Here we addressed these issues by utilizing primary human nasal epithelial cells (HNECs) and immortalized human bronchial epithelial cells (BCi) differentiated at an air-liquid interface, as well as human embryonic stem cell-derived alveolar type II (AT2) cells to recover contemporary sHCoVs from human nasopharyngeal specimens. From 21 specimens we recovered four 229E, three NL63 and eight OC43 viruses. All contemporary sHCoVs showed sequence differences from lab-adapted CoVs, particularly within the spike gene. Evidence of nucleotide changes in the receptor binding domains within 229E and detection of recombination for both 229E and OC43 isolates was also observed. Importantly, we developed methods for the amplification of high titre stocks of NL63 and 229E, that maintained sequence identity, and we established methods for the titration of contemporary sHCoV isolates. Comparison of lab-adapted and contemporary strains in immortalised cell lines and airway epithelial cells revealed differences in cell tropism, growth kinetics and cytokine production between lab-adapted and contemporary sHCoV strains. These data confirm that contemporary sHCoVs differ from lab-adapted reference strains and, using the methods established here, should be used for study of CoV biology and evaluation of medical countermeasures. ImportanceZoonotic coronaviruses have caused significant public health emergencies. The occurrence of a similar spillover event in the future is likely and efforts to further understand coronavirus biology should be a high priority. Several seasonal coronaviruses circulate within the human population. Efforts to study these viruses have been limited to reference strains isolated decades ago due to the difficulty in isolating clinical isolates. Here, we use human airway and alveolar epithelial cultures to recover contemporary isolates of NL63, 229E and OC43. We establish methods to make high titre stocks and titrate 229E and NL63 isolates. We show that contemporary isolates of NL63 and OC43 have a different tropism within the respiratory epithelium compared to lab-adapted strains. Although 229E clinical and lab-adapted strains similarly infect the respiratory epithelium, differences in host response and replication kinetics are observed. Using the methods developed here, future research should include contemporary isolates when studying coronavirus biology.

microbiology↗

bioRxiv2025

Two-photon activation, deactivation, and coherent control of melanopsin in live cells

Intrinsically photosensitive retinal ganglion cells are photoreceptors discovered in the last 20 years. These cells project to the suprachiasmatic nucleus of the brain to drive circadian rhythms, regulated by ambient light levels. The photopigment responsible for photoactivation in these cells, melanopsin, has been shown to exhibit many unique activation features among opsins. Notably, the photopigment can exist in three states dependent on the intensity and spectrum of ambient light, which affects its function. Despite increasing knowledge about these cells and melanopsin, tools that can manipulate their three states, and do so with single-cell precision, are limited. This reduces the extent to which circuit-level phenomena, and studying the implications of melanopsin tri-stability in living systems, can be pursued. In this report, we evoke and modulate calcium transients in live cells and intrinsically photosensitive retinal ganglion cells from isolated retinal tissues following two-photon excitation using near-infrared light pulses. We demonstrate that two-photon activation of melanopsin can successfully stimulate melanopsin-expressing cells with high spatio-temporal precision. Moreover, we demonstrate that the functional tri-stability of the photopigment can be interrogated by multiphoton excitation using spectral-temporal modulation of a broadband, ultrafast laser source.

bioengineering↗

bioRxiv2024

Building Inclusive Leadership: A Biosciences Approach

One-size-fits-all Equity, Diversity, and Inclusion (EDI) strategies and provisioning are no longer suitable for a growing and diversifying higher education landscape; as each research discipline faces its own unique challenges to inclusion. High impact research needs reliable, decisive, and holistic approaches to leadership, but managerial and leadership skills are often considered an add-on as opposed to a necessity in modern research environments. An estimated 70% of EDI initiatives in UK Higher Education Institutions fail to flourish (Marchiondo et al. 2023) - a combinatory effect of conservative approaches to change, cultural adaptation, and departmental stability. We developed an Inclusive Leadership Programme to empower bioscientists to work autonomously and inclusively. Using a co-creation approach, the Inclusive Leadership Programme collectively defined inclusive leadership traits to create a resource. The interactive resource shares inclusive approaches to communication, feedback, recognition, and development - prioritising the experiences of those typically excluded from leadership conversations. By embedding a self-reflective scoring system, we share changes in approaches to inclusivity. After using the resource, self-perception normalises in distribution, indicating the resource provides a baseline for best practice, and space to understand personal approaches to inclusive practice in biosciences. Ultimately, the Inclusive Leadership Programme and Resource offers a template for other biological research, learning, and teaching environments to build their own tailored approach to integrating inclusivity and empowering all members to work as autonomous and inclusive leaders, regardless of seniority.

scientific communication and education↗

bioRxiv2024

An ALPK3 truncation variant causing autosomal dominant hypertrophic cardiomyopathy is partially rescued by mavacamten

The ALPK3 gene encodes alpha-protein kinase 3, a cardiac pseudo-kinase of unknown function. Heterozygous truncating variants (ALPK3tv) can cause dominant adult-onset Hypertrophic Cardiomyopathy (HCM). Here we confirm an excess of ALPK3tv in sarcomere-gene negative HCM patients. Moreover, we generated a novel knock-in mouse model carrying an ALPK3tv (K201X). Homozygous animals displayed hypertrophy and systolic dysfunction. Heterozygous animals demonstrated no obvious baseline; however, they had an aggravated hypertrophic response upon chronic adrenergic challenge. Isolated cardiomyocytes from heterozygous and homozygous mice showed reduced basal sarcomere length with prolonged relaxation, whilst calcium transients showed increased diastolic calcium levels and prolonged re-uptake. We also observed reduced fractional shortening. Protein kinase A-mediated phosphorylation, including that of cardiac troponin I, was significantly decreased. In agreement with the cellular HCM phenotype, reduced ratios of myosin heads in the super-relaxed state were measured. Contractile and calcium handling defects were partly corrected by treatment with mavacamten, a novel myosin inhibitor. For the first time with a non-sarcomere HCM variant, we have demonstrated hallmark changes in cardiac contractility and calcium handling, some resembling sarcomere-positive HCM, while others are distinct and DCM-like. Mavacamten is able to partially rescue the cellular phenotype, hence could be beneficial to HCM patients with ALPK3tv.

genetics↗

bioRxiv2024

Personalized stimulation therapies for disorders of consciousness: A computational approach to inducing healthy-like brain activity based on neural field theory

Disorders of consciousness (DoC) pose significant challenges in neurology. Conventional neuromodulation therapies for DoC have exhibited limited success, with varying effectiveness among patients. In this study, we introduce a computational approach for constructing personalized stimulus signals capable of inducing healthy-like neural activity patterns in DoC patients. Leveraging a simplified brain model based on neural field theory, we fit this model to the power spectrum of a patient with DoC and derive a personalized stimulus time series to induce a healthy-like power spectrum. By applying this stimulus to brain regions typically targeted by stimulation therapies such as deep brain stimulation and repetitive transcranial magnetic stimulation, we demonstrate in silico the ability of our method to elicit EEG power spectra resembling those of healthy individuals. We speculate that in the course of a long-term treatment, when the brain produces healthy-like activity, it may trigger intrinsic plasticity mechanisms, potentially leading to sustained improvements in the patients condition. While further clinical adjustments and validation are needed, this novel approach offers promise in tailoring brain stimulation therapies for DoC patients. Moreover, it presents potential extensions to other conditions that could also benefit from brain stimulation therapies. Author summaryIn this research, we tackled a challenging issue in the field of neurology - disorders of consciousness (DoC). These conditions, which include states like coma and the minimally consciousness state, have been difficult to treat effectively. While traditional brain stimulation treatments have been tried, they do not always work well, and their effects can vary greatly from one person to another. Thus, we set out to find a new way to help these patients. Our approach involves using computer models of the brain to design personalized treatments. We designed a method to generate stimulation signals tailored to individual DoC patients. These signals aim to trigger healthier brain activity patterns, similar to those found in individuals without consciousness disorders. What is exciting is that if the brain begins to produce these healthy-like patterns, it could potentially lead to lasting improvements in the patients consciousness level. We show that this approach works in different simulations, and can be applied in multiple neuromodulation techniques like deep brain stimulation and transcranial magnetic stimulation. This research opens up new possibilities for more effective treatments for people dealing with these challenging disorders. Plus, it might work for other conditions treated with similar brain stimulation therapies.

neuroscience↗

bioRxiv2024

Blocking MIF secretion enhances CAR T-cell efficacy against neuroblastoma

While chimeric antigen receptor (CAR) T-cell therapies are showing highly promising first results in neuroblastoma, immunosuppressive tumor microenvironments (TME) limit T cell persistence and durable clinical efficacy. To improve CAR T-cell efficacy further, we applied a multi-omics approach including single-cell RNA sequencing and proteomics, which identified 13 targetable immunosuppressive factors in neuroblastoma. Of these, macrophage migration inhibitory factor (MIF) and midkine (MDK) were validated across multiple published RNA datasets. Moreover, they were secreted in high abundance by neuroblastoma tumoroids. Functional validation experiments revealed MIF as a potent inhibitor of CAR T-cells, in vitro and in vivo. Degradation of MIF by PROTAC technology significantly enhanced CAR T-cell activation targeting GPC2 and B7-H3, providing a potential intervention against MIF. By defining the immunosuppressive effects of neuroblastomas TME on CAR T-cell efficacy, particularly the pivotal role of MIF, we provide a therapeutic strategy for improving adoptive cell therapies for this pediatric malignancy.

cancer biology↗

bioRxiv2024

Ontology-based modeling, integration, and analysis of heterogeneous clinical, pathological, and molecular kidney data for precision medicine

Many resources are now generating, processing, storing, or providing kidney-related molecular, pathological, and clinical data. Reference ontologies offer an opportunity to support knowledge and data organization and integration. The Kidney Precision Medicine Project (KPMP) team contributed to the representation and addition of 329 kidney phenotype terms to the Human Phenotype Ontology (HPO) and identified many subcategories of acute kidney injury (AKI) or chronic kidney disease (CKD). The Kidney Tissue Atlas Ontology (KTAO) imports and integrates kidney-related terms from existing ontologies (e.g., HPO, CL, and Uberon) and represents 259 kidney-related biomarkers. We also developed a precision medicine metadata ontology (PMMO) to integrate 50 variables from KPMP and CellxGene resources and applied PMMO for integrative analysis. The gene expression profiles of kidney gene biomarkers were specifically analyzed in healthy controls or AKI/CKD disease states. This work demonstrates how ontology-based approaches support multi-domain data and knowledge organization and integration to advance precision medicine.

bioinformatics↗

bioRxiv2024

Proteostasis as a fundamental principle of Tau immunotherapy

The microtubule-associated protein Tau is a driver of neuronal dysfunction in Alzheimers disease and numerous other tauopathies. In this process, Tau initially undergoes subtle changes to its abundance, subcellular localisation and a vast array of post-translational modifications including phosphorylation, that progressively result in the proteins aggregation and dysregulation of multiple Tau-dependent cellular processes. Given the various loss- and gain-of-functions of Tau in disease and the brain-wide changes in the proteome that characterise tauopathies, we asked whether targeting Tau would restore the alterations in proteostasis observed in disease. To this end, we generated a novel pan-Tau antibody, RNJ1, that preferentially binds human Tau and neutralises proteopathic seeding activity in multiple cell lines and benchmarked it against a clinically tested pan-Tau antibody, HJ8.5 (murine version of tilavonemab). We next evaluated both antibodies, alone and in combination, in the K3 mouse model of tauopathy, showing reduced Tau pathology and improvements in neuronal function following 14 weekly treatments, without obtaining synergistic effects for the combination treatment. To gain insight into molecular mechanisms contributing to improvements in neuronal function, we employed quantitative proteomics and phosphoproteomics to first establish alterations in K3 mice relative to WT controls at the proteome level. This revealed 342 proteins with differential abundance in K3 mice, which are predominantly involved in metabolic and microtubule-associated processes, strengthening previously reported findings of defects in several functional domains in multiple tauopathy models. We next asked whether antibody-mediated Tau target engagement indirectly affects levels of deregulated proteins in the K3 model. Importantly, both immunotherapies, in particular RNJ1, induced abundance shifts in this protein subset towards a restoration to wild-type levels (proteostasis). A total of 257 of 342 ([~]75.1%) proteins altered in K3 were closer in abundance to WT levels after RNJ1 treatment. The same analysis indicated a similar response in K3 mice treated with HJ8.5, with approximately 72.5% of these altered proteins also showing changes in the same direction as wild-type. Furthermore, analysis of the phosphoproteome showed an even stronger restoration effect with RNJ1, with [~]82.1% of altered phosphopeptides in K3 showing a shift to WT levels, and 75.4% with HJ8.5. Gene set over-representation analysis (ORA) further confirmed that proteins undergoing restoration are involved in biological pathways affected in K3 mice. Together, our study suggests that a Tau immunotherapy-induced restoration of proteostasis links target engagement and treatment efficacy.

neuroscience↗

bioRxiv2020

Accuracy and precision of citizen scientist animal counts from drone imagery

Repeated counts of animal abundance can reveal changes in local ecosystem health and inform conservation strategies. Unmanned aircraft systems (UAS) such as drones are commonly used to photograph animals in remote locations; however, counting animals in images is a laborious task. Crowd-sourcing can reduce the time required to conduct these censuses considerably, but must first be validated against expert counts to measure sources of error. Our objectives were to assess the accuracy and precision of citizen science counts and make recommendations for future citizen science projects. We uploaded drone imagery from Ano Nuevo Island (California, USA) to a curated Zooniverse website that instructed citizen scientists to count seals and sea lions. Across 212 days, over 1,500 volunteers counted animals in 90,000 photographs. We quantified the error associated with several descriptive statistics to extract a single citizen science count per photograph from the 15 repeat counts and then compared the resulting citizen science counts to expert counts. Although proportional error was relatively low (9% for sea lions and 5% for seals during the breeding seasons) and improved with repeat sampling, the 12+ volunteers required to reduce error was prohibitively slow, taking on average 6 weeks to estimate animals from a single drone flight covering 25 acres, despite strong public outreach efforts. The single best algorithm was Median without the lowest two values, demonstrating that citizen scientists tended to under-estimate the number of animals present. Citizen scientists accurately counted adult seals, but accuracy was lower when sea lions were present during the summer and could be confused for seals. We underscore the importance of validation efforts and careful project design for researchers hoping to combine citizen science with drone imagery.

ecology↗

bioRxiv2020

Melanopsin Carboxy-terminus Phosphorylation Plasticity and Bulk Negative Charge, not Strict Site Specificity, Achieves Phototransduction Deactivation

Melanopsin is a visual pigment expressed in a small subset of ganglion cells in the mammalian retina known as intrinsically photosensitive retinal ganglion cells (ipRGCs) and is implicated in regulating non-image forming functions such as circadian photoentrainment and pupil constriction and contrast sensitivity in image formation. Mouse melanopsins Carboxy-terminus (C-terminus) possesses 38 serine and threonine residues, which can potentially serve as phosphorylation sites for a G-protein Receptor Kinase (GRK) and be involved in the deactivation of signal transduction. Previous studies suggest that S388, T389, S391, S392, S394, S395 on the proximal region of the C-terminus of mouse melanopsin are necessary for melanopsin deactivation. We expressed a series of mouse melanopsin C-terminal mutants in HEK293 cells and using calcium imaging, and we found that the necessary cluster of six serine and threonine residues, while being critical, are insufficient for proper melanopsin deactivation. Interestingly, the additional six serine and threonine residues adjacent to the required six sites, in either proximal or distal direction, are capable of restoring wild-type deactivation of melanopsin. These findings suggest an element of plasticity in the molecular basis of melanopsin phosphorylation and deactivation. In addition, C-terminal chimeric mutants and molecular modeling studies support the idea that the initial steps of deactivation and {beta}-arrestin binding are centered around these critical phosphorylation sites (S388-S395). This degree of functional versatility could help explain the diverse ipRGC light responses as well as non-image and image forming behaviors, even though all six sub types of ipRGCs express the same melanopsin gene OPN4.

neuroscience↗

bioRxiv2019

The mutational profile and clonal landscape of the inflammatory bowel disease affected colon

Summary paragraphInflammatory bowel disease (IBD) is a chronic inflammatory disease associated with increased risk of gastrointestinal cancers. Here, we whole-genome sequenced 447 colonic crypts from 46 IBD patients, and compared these to 412 crypts from 41 non-IBD controls. The average mutation rate of affected colonic epithelial cells is 2.4-fold that of healthy colon and this increase is mostly driven by acceleration of mutational processes ubiquitously observed in normal colon. In contrast to the normal colon, where clonal expansions outside the confines of the crypt are rare, we observed widespread millimeter-scale clonal expansions. We discovered non-synonymous mutations in ARID1A, FBXW7, PIGR and ZC3H12A, and genes in the interleukin 17 and Toll-like receptor pathways, under positive selection in IBD. These results suggest distinct selection mechanisms in the colitis-affected colon and that somatic mutations potentially play a causal role in IBD pathogenesis.

genetics↗

bioRxiv2019

Effects of physiological parameter evolution on the dynamics of tonic-clonic seizures

The temporal and spectral characteristics of tonic-clonic seizures are investigated using a neural field model of the corticothalamic system in the presence of a temporally varying connection strength between the cerebral cortex and thalamus. Increasing connection strength drives the system into [~] 10 Hz seizure oscillations once a threshold is passed and a subcritical Hopf bifurcation occurs. In this study, the spectral and temporal characteristics of tonic-clonic seizures are explored as functions of the relevant properties of physiological connection strengths, such as maximum strength, time above threshold, and the ramp rate at which the strength increases or decreases. Analysis shows that the seizure onset time decreases with the maximum connection strength and time above threshold, but increases with the ramp rate. Seizure duration and offset time increase with maximum connection strength, time above threshold, and rate of change. Spectral analysis reveals that the power of nonlinear harmonics and the duration of the oscillations increase as the maximum connection strength and the time above threshold increase. A secondary limit cycle at [~] 18 Hz, termed a saddle-cycle, is also seen during seizure onset and becomes more prominent and robust with increasing ramp rate. If the time above the threshold is too small, the system does not reach the 10 Hz limit cycle, and only exhibits 18 Hz saddle-cycle oscillations. It is also seen that the times to reach the saturated large amplitude limit-cycle seizure oscillation from both the instability threshold and from the end of the saddle-cycle oscillations are inversely proportional to the square root of the ramp rate.\n\nAuthor SummaryEpilepsy, which is characterized by recurrent seizures, affects around 1% of the world population at some point in their lives. Tonic-clonic seizures are the most commonly encountered primary generalized seizures and it is widely considered that they can be induced by an increase in the connection strength between the cerebral cortex and the thalamus. In this paper, we analyze the detailed dynamics of tonic-clonic seizures along with their dependence on the parameters of the changing connection strength. We study the relationship of the seizure onset, offset, oscillation strength, and oscillation frequency to the duration, amplitude, and rate of change of the connection strength. A detailed understanding of the dynamics and their dependence on the physiological parameters of the brain may explain the variability of seizure dynamics among patients. It may also help to constitute successful seizure prediction.

neuroscience↗

bioRxiv2019

Protein secondary structure determines the temporal relationship between folding and disulfide formation

How and when disulfides form in proteins during their folding is a fundamental question in cell biology. Two models describe the relationship between disulfide formation and folding, the folded precursor model, in which formation of nascent structure occurs prior to the disulfides and the quasi-stochastic model where disulfides form prior to complete domain folding. Here we investigate oxidative folding within a cellular milieu of three structurally diverse substrates in order to understand the folding mechanisms required to achieve correct cysteine coupling. We use a eukaryotic translation system in which we can manipulate the redox conditions and produce stalled translation intermediates representative of different stages of translocation. We identify different disulfide bonded isomers by non-reducing SDS-PAGE. Using this approach, we determined whether each substrate followed a folding driven or disulfide driven mechanism. Our results demonstrate that the folding model is substrate-dependent with disulfides forming prior to complete domain folding in a cysteine-rich domain lacking secondary structure, whereas disulfide formation was absent in proteins with defined structural elements. In addition, we demonstrate the presence and rearrangement of non-native disulfides specifically in substrates following the quasi-stochastic model. These findings demonstrate why non-native disulfides are prevented from forming in proteins with well-defined secondary structure. Significance statementA third of human proteins contain structural elements called disulfide bonds that are often crucial for stability and function. Disulfides form between cysteines in the specialised environment of the endoplasmic reticulum (ER), during the complex process of protein folding. Many proteins contain multiple cysteines that can potentially form correct or incorrect cysteine pairings. To investigate how correct disulfide pairs are formed in a biological context, we developed an experimental approach to assess disulfide formation and rearrangement as proteins enter the ER. We found that a disulfide-dense protein domain with atypical secondary structure undergoes disulfide orchestrated folding as it enters the ER and is prone to incorrect disulfide formation. In contrast, proteins with defined secondary structure form folding dependent, native disulfides. These findings show how different mechanisms of disulfide formation can be rationalised from structural features of the folding domains.

biochemistry↗

bioRxiv2018

The landscape of somatic mutation in normal colorectal epithelial cells

The colorectal adenoma-carcinoma sequence has provided a paradigmatic framework for understanding the successive somatic genetic changes and consequent clonal expansions leading to cancer. As for most cancer types, however, understanding of the earliest phases of colorectal neoplastic change, which may occur in morphologically normal tissue, is comparatively limited because of the difficulty of detecting somatic mutations in normal cells. Each colorectal crypt is a small clone of cells derived from a single recently-existing stem cell. Here, we whole genome sequenced hundreds of normal crypts from 42 individuals. Signatures of multiple mutational processes were revealed, some ubiquitous and continuous, others only found in some individuals, in some crypts or during some phases of the cell lineage from zygote to adult cell. Likely driver mutations were present in [~]1% of normal colorectal crypts in middle-aged individuals, indicating that adenomas and carcinomas are rare outcomes of a pervasive process of neoplastic change across morphologically normal colorectal epithelium.

cancer biology↗

bioRxiv2018

Direct measurement of myofilament calcium in living cardiomyocytes

Visualising when and where calcium appears and disappears in cardiomyocytes is a major goal of cardiovascular research. Surprisingly we find that the chemical dyes widely used for this purpose disrupt cell contractility, due at least in part due to direct inhibition of the acto-myosin ATPase required to generate force. In order to improve calcium detection methods, we have developed a genetically encoded indicator that sits within the myofilament to directly visualise the changes occurring at the sarcomere. This tool improves on established chemical dyes and untargeted genetically encoded indicators for analysing small molecule modulators of myofilament-based calcium signalling. Importantly this is achieved without any measurable change in contractile function.

cell biology↗

bioRxiv2017

NuMA is a negative regulator of 53BP1 in DNA double-strand break repair

Accumulation of 53BP1 at DNA breaks determines DNA repair pathway choice and promotes checkpoint activation. Here, we show regulation of 53BP1 beyond repair foci. 53BP1 movements are constrained in the nucleoplasm and increase in response to DNA damage. 53BP1 interacts with the structural protein NuMA, which controls 53BP1 diffusion. This interaction, and colocalization between the two proteins in vitro and in breast tissues, is reduced after DNA damage. In cell lines and breast carcinoma, NuMA prevents 53BP1 accumulation at DNA breaks and high NuMA expression predicts better patient outcomes. Manipulating NuMA expression alters PARP inhibitor sensitivity of BRCA1-null cells, end-joining activity, and immunoglobulin class switching that rely on 53BP1. We propose a new mechanism that involves the sequestration of 53BP1 by NuMA in the absence of DNA damage. Such mechanism may have evolved to disable repair functions and may be a decisive factor for tumor responses to genotoxic treatments.

cell biology↗

bioRxiv2016

The Monarch Initiative: Insights across species reveal human disease mechanisms

The principles of genetics apply across the whole tree of life: on a cellular level, we share mechanisms with species from which we diverged millions or even billions of years ago. We can exploit this common ancestry at the level of sequences, but also in terms of observable outcomes (phenotypes), to learn more about health and disease for humans and all other species. Applying the range of available knowledge to solve challenging disease problems requires unified data relating genomics, phenotypes, and disease; it also requires computational tools that leverage these multimodal data to inform interpretations by geneticists and to suggest experiments. However, the distribution and heterogeneity of databases is a major impediment: databases tend to focus either on a single data type across species, or on single species across data types. Although each database provides rich, high-quality information, no single one provides unified data that is comprehensive across species, biological scales, and data types. Without a big-picture view of the data, many questions in genetics are difficult or impossible to answer. The Monarch Initiative (https://monarchinitiative.org) is an international consortium dedicated to providing computational tools that leverage a computational representation of phenotypic data for genotype-phenotype analysis, genomic diagnostics, and precision medicine on the basis of a large-scale platform of multimodal data that is deeply integrated across species and covering broad areas of disease.

evolutionary biology↗