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Congratulations are in order for Postdoctoral Research Scientist Dr Samuel Malone who was been awarded the Marie Sklodowska Curie Individual Fellowship, funded by the European Commission.
Computational modelling of biological systems now and then: revisiting tools and visions from the beginning of the century
Since the turn of the millennium, computational modelling of biological systems has evolved remarkably and sees matured use spanning basic and clinical research. While the topic of the peri-millennial debate about the virtues and limitations of ‘reductionism and integrationism’ seems less controversial today, a new apparent dichotomy dominates discussions: mechanistic versus data-driven modelling. In light of this distinction, we provide an overview of recent achievements and new challenges with a focus on the cardiovascular system. Attention has shifted from generating a universal model of the human to either models of individual humans (digital twins) or entire cohorts of models representative of clinical populations to enable in silico clinical trials. Disease-specific parametrization, inter-individual and intra-individual variability, uncertainty quantification as well as interoperable, standardized and quality-controlled data are important issues today, which call for open tools, data and metadata standards, as well as strong community interactions. The quantitative, biophysical and highly controlled approach provided by in silico methods has become an integral part of physiological and medical research. In silico methods have the potential to accelerate future progress also in the fields of integrated multi-physics modelling, multi-scale models, virtual cohort studies and machine learning beyond what is feasible today. In fact, mechanistic and data-driven modelling can complement each other synergistically and fuel tomorrow’s artificial intelligence applications to further our understanding of physiology and disease mechanisms, to generate new hypotheses and assess their plausibility, and thus to contribute to the evolution of preventive, diagnostic and therapeutic approaches. This article is part of the theme issue ‘Science into the next millennium: 25 years on’.
ATR-hippo drives force signaling to nuclear F-actin and links mechanotransduction to neurological disorders.
The mechanical environment is sensed through cell-matrix contacts with the cytoskeleton, but how signals transit the nuclear envelope to affect cell fate decisions remains unknown. Nuclear actin coordinates chromatin motility during differentiation and genome maintenance, yet it remains unclear how nuclear actin responds to mechanical force. The DNA-damage kinase ataxia telangiectasia and Rad3-related protein (ATR) translocates to the nuclear envelope to protect the nucleus during cell motility or compression. Here, we show that ATR drives nuclear actin assembly via recruitment of Filamin-A to the inner nuclear membrane through binding of the hippo pathway scaffold and ATR substrate, RASSF1A. Moreover, we demonstrate how germline RASSF1 mutation disables nuclear mechanotransduction resulting in cerebral cortex thinning and associates with common psychological traits. Thus, defective mechanical-regulated pathways may contribute to complex neurological disorders.
Redefining respiratory sinus arrhythmia as respiratory heart rate variability: an international Expert Recommendation for terminological clarity.
The variation of heart rate in phase with breathing, known as 'respiratory sinus arrhythmia' (RSA), is a physiological phenomenon present in all air-breathing vertebrates. RSA arises from the interaction of several physiological mechanisms but is primarily mediated by rhythmic changes in cardiac parasympathetic (vagal) activity, increasing heart rate during inspiration and decreasing heart rate during expiration. RSA amplitude is an indicator of autonomic and cardiac health; RSA is diminished or absent in common pathological conditions such as chronic heart failure and hypertension. In this Expert Recommendation, we argue that the term 'RSA', although historically important, is semantically inaccurate and carries misleading pathological connotations, contributing to misunderstanding and misinterpretation of the origin and the physiological importance of the phenomenon. We propose replacing 'RSA' with the term 'respiratory heart rate variability' (RespHRV), which avoids pathological connotations and emphasizes the specific respiratory contribution to heart rate variability. We clarify that RespHRV encompasses respiratory-related heart rate variations in both the low-frequency and high-frequency bands traditionally defined in heart rate variability analysis, and that its amplitude should not be misconstrued as a measure of vagal tone. Adopting the proposed term 'RespHRV' is expected to unify understanding and stimulate further experimental and clinical research into the physiological mechanisms and functional importance of this phenomenon.
A multi-color flow cytometric method for characterizing murine reticulated platelets using SYTO 13.
Reticulated platelets (RP) are immature platelets with heightened RNA content. An increased level of RP in the circulation is associated with various pathological conditions. In this study, we employed a novel flow cytometry approach for RP detection in mice, utilizing the nucleic acid dye SYTO 13 in conjunction with a platelet-specific marker (anti-mouse CD42b-DyLight 649). The efficacy of SYTO 13 for RP identification was confirmed by higher circulating RP levels in platelet-depleted mice during the recovery phase (35% ± 13%) compared to untreated mice (11% ± 1%, n = 9, p
FoxO1-zDHHC4-CD36 S-Acylation Axis Drives Metabolic Dysfunction in Diabetes.
BACKGROUND: The fatty acid (FA) transporter CD36 (FA translocase/cluster of differentiation 36) is the gatekeeper of cardiac FA metabolism. Preferential localization of CD36 to the sarcolemma is one of the initiating cellular responses in the development of muscle insulin resistance and the type 2 diabetic heart. Posttranslational S-acylation controls protein trafficking, and in this study, we hypothesized that increased CD36 S-acylation may underpin the preferential sarcolemmal localization of CD36, driving metabolic and contractile dysfunction in diabetes. METHODS AND RESULTS: Type 2 diabetes increased cardiac CD36 S-acylation, CD36 sarcolemmal localization, FA oxidation rates, and triglyceride storage in the diabetic heart. CD36 S-acylation was increased in diabetic rats, db/db mice, diabetic pigs, and insulin-resistant human iPSC-derived cardiomyocytes, demonstrating conservation between species. The enzyme responsible for S-acylating CD36, zDHHC4, was transcriptionally upregulated in the diabetic heart, and genetic silencing of zDHHC4 using siRNA or lentiviral shRNA decreased CD36 S-acylation. We identified that zDHHC4 expression is under the regulation of the transcription factor FoxO (forkhead box O) 1, as FoxO1 binds to the promotor of zDHHC4 and induces its transcription, as assessed using chromatin immunoprecipitation-seq, chromatin immunoprecipitation-quantitative PCR, luciferase assays, and siRNA silencing. Diabetic mice with cardiomyocyte-specific FoxO1 deletion had decreased cardiac zDHHC4 expression and decreased CD36 S-acylation, which was further confirmed using diabetic mice treated with the FoxO1 inhibitor AS1842856. Pharmacological inhibition of zDHHC enzymes in diabetic hearts decreased CD36 S-acylation, sarcolemmal CD36 content, FA oxidation rates, and triglyceride storage, culminating in improved cardiac function in diabetes. Conversely, inhibiting the deacylating enzymes in control hearts increased CD36 S-acylation, sarcolemmal CD36 content, and FA metabolic rates in control hearts, recapitulating the metabolic phenotype seen in diabetic hearts. CONCLUSIONS: Activation of the FoxO1-zDHHC4-CD36 S-acylation axis in diabetes drives metabolic and contractile dysfunction in type 2 diabetic heart.
Working memory filtering at encoding and maintenance in healthy ageing, Alzheimer's and Parkinson's disease.
The differential impact on working memory (WM) performance of distractors presented at encoding or during maintenance was investigated in Alzheimer's Disease (AD), Parkinson's Disease (PD) patients, elderly (EHC) and young healthy controls (YHC), (n = 28 per group). Participants reported the orientation of an arrow from a set of either two or three items, with a distractor present either at encoding or at maintenance. MRI data with hippocampal volumes was also acquired. Mean absolute error and mixture model metrics i.e., memory precision, target detection, misbinding (swapping the features of an object with another probed item) and guessing were computed. EHC and PD patients showed good filtering abilities both at encoding and maintenance. However, AD patients exhibited significant filtering deficits specifically when the distractor appeared during maintenance. In healthy ageing there was a prominent decline in WM memory precision, whilst in AD lower target detection and higher guessing were the main sources of error. Conversely, PD was associated only with higher guessing rates. Hippocampal volume was significantly correlated with filtering during maintenance - but not at encoding. These findings demonstrate how healthy ageing and neurodegenerative diseases exhibit distinct patterns of WM impairment, including when filtering irrelevant material either at encoding and maintenance.
Cholinergic degeneration in prodromal and early Parkinson's: a link to present and future disease states.
The neuropathological process in Parkinson's disease (PD) and Lewy body disorders has been shown to extend well beyond the degeneration of the dopaminergic system, affecting other neuromodulatory systems in the brain which play crucial roles in the clinical expression and progression of these disorders. Here, we investigate the role of the macrostructural integrity of the nucleus basalis of Meynert (NbM), the main source of cholinergic input to the cerebral cortex, in cognitive function, clinical manifestation, and disease progression in non-demented subjects with PD and individuals with isolated REM sleep behaviour disorder (iRBD). Using structural MRI data from 393 early PD patients, 128 iRBD patients, and 186 controls from two longitudinal cohorts, we found significantly lower NbM grey matter volume in both PD (β=-12.56, p=0.003) and iRBD (β=-16.41, p=0.004) compared to controls. In PD, higher NbM volume was associated with better higher-order cognitive function (β=0.10, p=0.045), decreased non-motor (β=-0.66, p=0.026) and motor (β=-1.44, p=0.023) symptom burden, and lower risk of future conversion to dementia (Hazard ratio (HR)<0.400, p<0.004). Higher NbM volume in iRBD was associated with decreased future risk of phenoconversion to PD or dementia with Lewy bodies (DLB) (HR<0.490, p<0.016). However, despite similar NbM volume deficits to those seen in PD, associations between NbM structural deficits and current disease burden or clinical state were less pronounced in iRBD. These findings identify NbM volume as a potential biomarker with dual utility: predicting cognitive decline and disease progression in early PD, while also serving as an early indicator of phenoconversion risk in prodromal disease. The presence of structural deficits before clear clinical correlates in iRBD suggests complex compensatory mechanisms may initially mask cholinergic dysfunction, with subsequent failure of these mechanisms potentially contributing to clinical conversion.
Automated quality control of T1-weighted brain MRI scans for clinical research: methods comparison and design of a quality prediction classifier
T1-weighted (T1w) MRI is widely used in clinical neuroimaging for studying brain structure and its changes, including those related to neurodegenerative diseases, and as anatomical reference for analysing other modalities. Ensuring high-quality T1w scans is vital as image quality affects reliability of outcome measures. However, visual inspection can be subjective and time-consuming, especially with large datasets. The effectiveness of automated quality control (QC) tools for clinical cohorts remains uncertain. In this study, we used T1w scans from elderly participants within ageing and clinical populations to test the accuracy of existing QC tools with respect to visual QC and to establish a new quality prediction framework for clinical research use. Four datasets acquired from multiple scanners and sites were used (N = 2438, 11 sites, 39 scanner manufacturer models, 3 field strengths – 1.5T, 3T, 2.9T, patients and controls, average age 71 ± 8 years). All structural T1w scans were processed with two standard automated QC pipelines (MRIQC and CAT12). The agreement of the accept-reject ratings was compared between the automated pipelines and with visual QC. We then designed a quality prediction framework that combines the QC measures from the existing automated tools and is trained on clinical research datasets. We tested the classifier performance using cross-validation on data from all sites together, also examining the performance across diagnostic groups. We then tested the generalisability of our approach when leaving one site out and explored how well our approach generalises to data from a different scanner manufacturer and/or field strength from those used for training, as well as on an unseen new dataset of healthy young participants with movement related artefacts. Our results show significant agreement between automated QC tools and visual QC (Kappa=0.30 with MRIQC predictions; Kappa=0.28 with CAT12’s rating) when considering the entire dataset, but the agreement was highly variable across datasets. Our proposed robust undersampling boost (RUS) classifier achieved 87.7% balanced accuracy on the test data combined from different sites (with 86.6% and 88.3% balanced accuracy on scans from patients and controls respectively). This classifier was also found to be generalisable on different combinations of training and test datasets (average balanced accuracy of leave-one-site-out = 78.2%; exploratory models on field strengths and manufacturers = 77.7%; movement related artefact dataset when including 1% scans in the training = 88.5%). While existing QC tools may not be robustly applicable to datasets comprised of older adults, they produce quality metrics that can be leveraged to train a more robust quality control classifiers for ageing and clinical cohorts.
Integrative role of CTPS cytoophidia in polyploid tissue growth and nutrient adaptation
Tissue growth and development are fundamental to organismal survival, requiring precise coordination of metabolic processes, nutrient availability, and signaling pathways. Cytidine triphosphate synthase (CTPS) is a rate-limiting enzyme in nucleotide biosynthesis and assembles filamentous cytoophidia, conserved across species. Despite increasing interest in cytoophidia, how CTPS filaments integrate metabolic and signaling cues to drive cell size and tissue growth remains incompletely understood. Using RNA interference and clustered regularly interspaces short palindromic repeats (CRISPR) / CRISPR-associate nuclease 9 gene editing, we generated CTPS-knockdown and point-mutated mutants to investigate the role of cytoophidia in cell growth. Specifically, we introduced the H355A mutation, which disrupts CTPS filament formation without affecting its enzymatic activity. Our findings revealed that CTPS depletion or filament disruption significantly impairs growth in polyploid organs, such as the fat body and salivary glands, underscoring the pivotal role of CTPS cytoophidia in cell growth regulation. Mutants lacking cytoophidia exhibited reduced DNA replication activity and smaller cell sizes compared to wild-type controls. Mechanistically, we found that nutrient-sensing pathways, particularly insulin-PI3K-Akt signaling pathway, regulate CTPS expression and cytoophidia formation in response to nutrient availability. Activation of the sterol regulatory element-binding protein partially rescued the growth defects caused by CTPS depletion. These findings provide new insights into the molecular mechanisms of the regulation of CTPS filaments, highlighting their role as critical mediators of tissue growth by integrating environmental demands, metabolism, and signaling pathways to regulate cell size and nutrient adaptation.
Distinct epicardial gene regulatory programs drive development and regeneration of the zebrafish heart.
Unlike the adult mammalian heart, which has limited regenerative capacity, the zebrafish heart fully regenerates following injury. Reactivation of cardiac developmental programs is considered key to successfully regenerating the heart, yet the regulation underlying the response to injury remains elusive. Here, we compared the transcriptome and epigenome of the developing and regenerating zebrafish epicardia. We identified epicardial enhancer elements with specific activity during development or during adult heart regeneration. By generating gene regulatory networks associated with epicardial development and regeneration, we inferred genetic programs driving each of these processes, which were largely distinct. Loss of Hif1ab, Nrf1, Tbx2b, and Zbtb7a, central regulators of the regenerating epicardial network, in injured hearts resulted in elevated epicardial cell numbers infiltrating the wound and excess fibrosis after cryoinjury. Our work identifies differences between the regulatory blueprint deployed during epicardial development and regeneration, underlining that heart regeneration goes beyond the reactivation of developmental programs.
Conserved N-terminal cysteine dioxygenases transduce responses to hypoxia in animals and plants.
Organisms must respond to hypoxia to preserve oxygen homeostasis. We identify a thiol oxidase, previously assigned as cysteamine (2-aminoethanethiol) dioxygenase (ADO), as a low oxygen affinity (high-K mO2) amino-terminal cysteine dioxygenase that transduces the oxygen-regulated stability of proteins by the N-degron pathway in human cells. ADO catalyzes the conversion of amino-terminal cysteine to cysteine sulfinic acid and is related to the plant cysteine oxidases that mediate responses to hypoxia by an identical posttranslational modification. We show in human cells that ADO regulates RGS4/5 (regulator of G protein signaling) N-degron substrates, modulates G protein-coupled calcium ion signals and mitogen-activated protein kinase activity, and that its activity extends to other N-cysteine proteins including the angiogenic cytokine interleukin-32. Identification of a conserved enzymatic oxygen sensor in multicellular eukaryotes opens routes to better understanding and therapeutic targeting of adaptive responses to hypoxia.
Hif-2α programs oxygen chemosensitivity in chromaffin cells.
The study of transcription factors that determine specialized neuronal functions has provided invaluable insights into the physiology of the nervous system. Peripheral chemoreceptors are neurone-like electrophysiologically excitable cells that link the oxygen concentration of arterial blood to the neuronal control of breathing. In the adult, this oxygen chemosensitivity is exemplified by type I cells of the carotid body, and recent work has revealed one isoform of the hypoxia-inducible transcription factor (HIF), HIF-2α, as having a nonredundant role in the development and function of that organ. Here, we show that activation of HIF-2α, including isolated overexpression of HIF-2α but not HIF-1α, is sufficient to induce oxygen chemosensitivity in adult adrenal medulla. This phenotypic change in the adrenal medulla was associated with retention of extra-adrenal paraganglioma-like tissues resembling the fetal organ of Zuckerkandl, which also manifests oxygen chemosensitivity. Acquisition of chemosensitivity was associated with changes in the adrenal medullary expression of gene classes that are ordinarily characteristic of the carotid body, including G protein regulators and atypical subunits of mitochondrial cytochrome oxidase. Overall, the findings suggest that, at least in certain tissues, HIF-2α acts as a phenotypic driver for cells that display oxygen chemosensitivity, thus linking 2 major oxygen-sensing systems.
Hypoxic and pharmacological activation of HIF inhibits SARS-CoV-2 infection of lung epithelial cells.
COVID-19, caused by the novel coronavirus SARS-CoV-2, is a global health issue with more than 2 million fatalities to date. Viral replication is shaped by the cellular microenvironment, and one important factor to consider is oxygen tension, in which hypoxia inducible factor (HIF) regulates transcriptional responses to hypoxia. SARS-CoV-2 primarily infects cells of the respiratory tract, entering via its spike glycoprotein binding to angiotensin-converting enzyme 2 (ACE2). We demonstrate that hypoxia and the HIF prolyl hydroxylase inhibitor Roxadustat reduce ACE2 expression and inhibit SARS-CoV-2 entry and replication in lung epithelial cells via an HIF-1α-dependent pathway. Hypoxia and Roxadustat inhibit SARS-CoV-2 RNA replication, showing that post-entry steps in the viral life cycle are oxygen sensitive. This study highlights the importance of HIF signaling in regulating multiple aspects of SARS-CoV-2 infection and raises the potential use of HIF prolyl hydroxylase inhibitors in the prevention or treatment of COVID-19.
N-terminal cysteine acetylation and oxidation patterns may define protein stability.
Oxygen homeostasis is maintained in plants and animals by O2-sensing enzymes initiating adaptive responses to low O2 (hypoxia). Recently, the O2-sensitive enzyme ADO was shown to initiate degradation of target proteins RGS4/5 and IL32 via the Cysteine/Arginine N-degron pathway. ADO functions by catalysing oxidation of N-terminal cysteine residues, but despite multiple proteins in the human proteome having an N-terminal cysteine, other endogenous ADO substrates have not yet been identified. This could be because alternative modifications of N-terminal cysteine residues, including acetylation, prevent ADO-catalysed oxidation. Here we investigate the relationship between ADO-catalysed oxidation and NatA-catalysed acetylation of a broad range of protein sequences with N-terminal cysteines. We present evidence that human NatA catalyses N-terminal cysteine acetylation in vitro and in vivo. We then show that sequences downstream of the N-terminal cysteine dictate whether this residue is oxidised or acetylated, with ADO preferring basic and aromatic amino acids and NatA preferring acidic or polar residues. In vitro, the two modifications appear to be mutually exclusive, suggesting that distinct pools of N-terminal cysteine proteins may be acetylated or oxidised. These results reveal the sequence determinants that contribute to N-terminal cysteine protein modifications, with implications for O2-dependent protein stability and the hypoxic response.
Comparative analysis of N-terminal cysteine dioxygenation and prolyl-hydroxylation as oxygen-sensing pathways in mammalian cells.
In animals, adaptation to changes in cellular oxygen levels is coordinated largely by 2-oxoglutarate-dependent prolyl-hydroxylase domain (PHD) dioxygenase family members, which regulate the stability of their hypoxia-inducible factor (HIF) substrates to promote expression of genes that adapt cells to hypoxia. Recently, 2-aminoethanethiol dioxygenase (ADO) was identified as a novel O2-sensing enzyme in animals. Through N-terminal cysteine dioxygenation and the N-degron pathway, ADO regulates the stability of a set of non-transcription factor substrates; the regulators of G-protein signaling 4, 5. and 16 and interleukin-32. Here, we set out to compare and contrast the in cellulo characteristics of ADO and PHD enzymes in an attempt to better understand their co-evolution in animals. We find that ADO operates to regulate the stability of its substrates rapidly and with similar O2-sensitivity to the PHD/HIF pathway. ADO appeared less sensitive to iron chelating agents or transition metal exposure than the PHD enzymes, possibly due to tighter catalytic-site Fe2+ coordination. Unlike the PHD/HIF pathway, the ADO/N-degron pathway was not subject to feedback by hypoxic induction of ADO, and induction of ADO substrates was well sustained in response to prolonged hypoxia. The data also reveal strong interactions between proteolytic regulation of targets by ADO and transcriptional induction of those targets, that shape integrated cellular responses to hypoxia. Collectively, our comparative analysis provides further insight into ADO/N-degron-mediated oxygen sensing and its integration into established mechanisms of oxygen homeostasis.