Biology subjects

Wilson, A.

Publications and source records attributed to Wilson, A..

At least 55 records · Page 3Linked to original sources

bioRxiv2022

Atypical speech production of multisyllabic words by children with developmental dyslexia

The prevalent core phonological deficit model of dyslexia proposes that the reading and spelling difficulties characterizing affected children stem from prior developmental difficulties in processing speech sound structure, for example perceiving and identifying syllable stress patterns, syllables, rhymes and phonemes. Yet spoken word production appears normal. This suggests an unexpected disconnect between speech input and speech output processes. Here we investigated the output side of this disconnect from a speech rhythm perspective by measuring the speech amplitude envelope (AE) of multisyllabic spoken phrases. The speech AE contains crucial information regarding stress patterns, speech rate, tonal contrasts and intonational information. We created a novel computerized speech copying task in which participants copied aloud familiar spoken targets like "Aladdin". Seventy-five children with and without dyslexia were tested, some of whom were also receiving an oral intervention designed to enhance multi-syllabic processing. Similarity of the childs productions to the target AE was computed using correlation and mutual information metrics. Similarity of pitch contour, another acoustic cue to speech rhythm, was used for control analyses. Children with dyslexia were significantly worse at producing the multi-syllabic targets as indexed by both similarity metrics for computing the AE. However, children with dyslexia were not different from control children in producing pitch contours. Accordingly, the spoken production of multisyllabic phrases by children with dyslexia is atypical regarding the AE. Children with dyslexia may not appear to listeners to exhibit speech production difficulties because their pitch contours are intact. Research HighlightsO_LISpeech production of syllable stress patterns is atypical in children with dyslexia. C_LIO_LIChildren with dyslexia are significantly worse at producing the amplitude envelope of multi-syllabic targets compared to both age-matched and reading-level-matched control children. C_LIO_LINo group differences were found for pitch contour production between children with dyslexia and age-matched control children. C_LIO_LIIt may be difficult to detect speech output problems in dyslexia as pitch contours are relatively accurate. C_LI

neuroscience↗

bioRxiv2022

A human mesenchymal spheroid prototype to replace moderate severity animal procedures in leukaemia drug testing

Patient derived xenograft (PDX) models are regarded as gold standard preclinical models in leukaemia research, especially in testing new drug combinations where typically 45-50 animals are used per assay. 9000 animal experiments are performed annually in leukaemia research with these expensive procedures being described as moderate severity, meaning they cause significant pain, suffering and visible distress to animals state. Furthermore, not all clinical leukaemia samples engraft and when they do data turnaround time can be between 6-12 months. Heavy dependence on animal models is because clinical leukaemia samples do not proliferate in vitro. Alternative cell line models though popular for drug testing are not biomimetic - they are not dependent on the microenvironment for survival, growth and treatment response and being derived from relapse samples they do not capture the molecular complexity observed at disease presentation. Here we have developed an in vitro platform to rapidly establish co-cultures of patient-derived leukaemia cells with 3D bone marrow mesenchyme spheroids, BM-MSC-spheroids. We optimise protocols for developing MSC-spheroid leukaemia co-culture using clinical samples and deliver drug response data within a week. Using three patient samples representing distinct cytogenetics we show that patient-derived-leukaemia cells show enhanced proliferation when co-cultured with MSC-spheroids. In addition, MSC-spheroids provided improved protection against treatment. This makes our spheroids suitable to model treatment resistance - a major hurdle in current day cancer management Given this 3Rs approach is 12 months faster (in delivering clinical data), is a human cell-based biomimetic model and 45-50 animals/drug-response assay cheaper the anticipated target end-users would include academia and pharmaceutical industry. This animal replacement prototype would facilitate clinically translatable research to be performed with greater ethical, social and financial sustainability. Research HighlightsO_ST_ABSScientific BenefitC_ST_ABSA 3D spheroid-based approach for ex vivo co-culture of clinical leukaemia samples for further investigation into cancer biology such as blast-niche interactions, blast proliferation and treatment resistance 3Rs BenefitTo replace moderate severity animal procedures in leukaemia research and drug testing Practical Benefit3Rs approach that yields drug response data quickly and is more ethically, socially and financially sustainable than its in vivo counterparts Current applicationsExploration of leukaemia biology such as blasts proliferation, blast-niche interactions, niche-impacted treatment resistance and obtain drug response data Potential applicationsExtend the approach to include other haematological cancers as well as bone cancers.

cancer biology↗

bioRxiv2022

Decoding of Speech Information using EEG in Children with Dyslexia: Less Accurate Low-Frequency Representations of Speech, Not "Noisy" Representations

The amplitude envelope of speech carries crucial low-frequency acoustic information that assists linguistic decoding. The sensory-neural Temporal Sampling (TS) theory of developmental dyslexia proposes atypical encoding of speech envelope information <10 Hz, leading to atypical phonological representations. Here a backward linear TRF model and story listening were employed to estimate the speech information encoded in the electroencephalogram in the canonical delta, theta and alpha bands by 9-year-old children with and without dyslexia. TRF decoding accuracy provided an estimate of how faithfully the childrens brains encoded low-frequency envelope information. Between-group analyses showed that the children with dyslexia exhibited impaired reconstruction of speech information in the delta band. However, when the quality of speech encoding for each child was estimated using child-by-child decoding models, then the dyslexic children did not differ from controls. This suggests that children with dyslexia encode neither "noisy" nor "normal" representations of the speech signal, but different representations.

neuroscience↗

bioRxiv2022

DLK-dependent biphasic reactivation of herpes simplex virus latency established in the absence of antivirals

Understanding the molecular mechanism of herpes simplex virus type 1 (HSV-1) latent infection and reactivation in neurons requires the use of model systems. However, establishing a quiescent infection in cultured neurons is problematic as infectious virus released from any productively infected neuron can superinfect the cultures. Here, we describe a new reporter virus, HSV-1 Stayput-GFP, that is defective for cell-to-cell spread and can be used to model latency and reactivation at the single neuron level. Importantly, quiescent infection of neurons with Stayput-GFP can be established without the use of a viral DNA replication inhibitor. The establishment of a quiescent state requires a longer time frame than previous models of HSV latency using DNA replication inhibitors. This results in a decreased ability of the virus to reactivate, and the use of multiple reactivation triggers is required. Using this system, we demonstrate that an initial Phase I wave of lytic gene expression occurs independently of histone demethylase activity and viral DNA replication but is dependent on the neuronal cell stress protein, DLK. Progression into the later, Phase II wave of reactivation, characterized by detectable late viral protein synthesis and a requirement for histone demethylase activity, is also observed with the Stayput-GFP model system. These data demonstrate that the two waves of viral gene expression following HSV-1 reactivation are independent of secondary infection and occur when latent infections are established in the absence of a viral DNA replication inhibitor. ImportanceHerpes simplex virus-1 (HSV-1) enters a latent infection in neurons and periodically reactivates. Reactivation manifests as a variety of clinical symptoms. Studying latency and reactivation in vitro is invaluable, allowing the molecular mechanisms behind both processes to be targeted by therapeutics that reduce the clinical consequences. Here, we describe a novel in vitro model system using a cell-to-cell spread defective HSV-1, known as Stayput-GFP, which allows for the study of latency and reactivation at the single neuron level. We anticipate this new model system will be an incredibly valuable tool for studying the establishment and reactivation of HSV-1 latent infection in vitro. Using this model, we find that initial reactivation events are dependent on cellular stress kinase DLK, but independent of histone demethylase activity and viral DNA replication. Our data therefore demonstrate the essential role of DLK in mediating a wave of lytic gene expression unique to reactivation.

microbiology↗

bioRxiv2022

Oligomerization processes limit photoactivation and recovery of the Orange Carotenoid Protein.

The Orange Carotenoid Protein (OCP) is a photoactive protein involved in cyanobacterial photoprotection, by quenching of the excess of light harvested energy. The photoactivation mechanism remains elusive, in part due to absence of data pertaining to the timescales over which protein structural changes take place. It also remains unclear whether or not oligomerization of the dark-adapted and light-adapted OCP could play a role in the regulation of its energy quenching activity. Here, we probed photo-induced structural changes in OCP by a combination of static and time-resolved X-ray scattering and steady-state and transient optical spectroscopy in the visible range. Our results suggest that oligomerization partakes in regulation of the OCP photocycle, with different oligomers slowing down the overall thermal recovery of the dark-adapted state of OCP. They furthermore reveal that upon non-photoproductive excitation, a numbed-state forms, which remains in a non-photoexcitable structural state for at least [~]0.5 {micro}s after absorption of a first photon. Significance StatementThe orange carotenoid protein (OCP) is a photoactivatable protein involved in cyanobacterial photoprotection. Upon photoactivation, OCP becomes able to quench the excess of energy uptaken by the light-harvesting antennae, thereby evading damage to the cells. It remains unclear, however, what is the exact OCP photoactivation mechanism, and whether or not oligomerization partakes in the regulation of the OCP function. Here, we investigated these issues by combining static and time-resolved (TR) X-ray scattering and optical spectroscopy. Our results show that OCP oligomerizes in both the dark-adapted inactive and light-adapted active states, suggesting a functional role for oligomerization. TR scattering data furthermore reveal that the first large-scale conformational changes associated with OCP photoactivation take place on the {micro}s time scale.

biophysics↗

bioRxiv2022

Partial sex linkage and linkage disequilibrium on the guppy sex chromosome

The guppy Y chromosome has been considered a model system for the evolution of suppressed recombination between sex chromosomes, and it has been proposed that complete sex-linkage has evolved across about 3 Mb surrounding this fishs sex-determining locus, followed by recombination suppression across a further 7 Mb of the 23 Mb XY pair, forming younger "evolutionary strata". Sequences of the guppy genome show that Y is very similar to the X chromosome, making it important to understand which parts of the Y are completely non-recombining, and whether there is indeed a large completely non-recombining region. Here, we describe new evidence that supports a different interpretation of the data that suggested the presence of such a region. We analysed PoolSeq data in samples from multiple natural populations from Trinidad. This yields evidence for linkage disequilibrium (LD) between sequence variants and the sex-determining locus. Downstream populations have higher diversity than upstream ones (which display the expected signs of bottlenecks). The associations we observe conform to predictions for a genome region with infrequent recombination that carries one or more sexually antagonistic polymorphisms. They also suggest the region in which the sex-determining locus must be located. However, no consistently male-specific variants were found, supporting the suggestion that any completely sex-linked region may be very small.

evolutionary biology↗

bioRxiv2022

Structure-function-dynamics relationships in the peculiar Planktothrix PCC7805 OCP1: impact of his-tagging and carotenoid type.

The orange carotenoid protein (OCP) is a photoactive protein involved in cyanobacterial photoprotection. Here, we report on the functional, spectral and structural characteristics of the peculiar Planktothrix PCC7805 OCP (Plankto-OCP). We show that this OCP variant is characterized by higher photoactivation and recovery rates, and a stronger energy-quenching activity, compared to other OCP studied thus far. We characterize the effect of the functionalizing carotenoid and of his-tagging on these reactions, and identify the time scales on which these modifications affect photoactivation. The presence of a his-tag at the C-terminus has a large influence on photoactivation, thermal recovery and PBS-fluorescence quenching, and likewise for the nature of the carotenoid that additionally affects the yield and characteristics of excited states and the ns-s dynamics of photoactivated OCP. By solving the structures of Plankto-OCP in the ECN- and CAN-functionalized states, each in two closely-related crystal forms, we further unveil the molecular breathing motions that animate Plankto-OCP at the monomer and dimer levels. We finally discuss the structural changes that could explain the peculiar properties of Plankto-OCP. HighlightsO_LIComplete functional characterization of Synechocystis and Planktothrix OCP C_LIO_LIHitherto unknown structures of ECN- and CAN-functionalized Planktothrix OCP C_LIO_LIInsights into fs-s timescale photodynamics of ECN- and CAN-functionalized Synechocystis and Planktothrix OCP C_LI

biophysics↗

bioRxiv2021

A unifying perspective of the ultrafast photo-dynamics of Orange Carotenoid Protein from Synechocystis: peril of high-power excitation, existence of different S* states and influence of tagging

A substantial number of Orange Carotenoid Protein (OCP) studies have aimed to describe the evolution of singlet excited states leading to the formation of photo-activated form, OCPR. The most recent one suggests that three picosecond-lived excited states are formed after the sub-100 fs decay of the initial S2 state. The S* state which has the longest reported lifetime of a few to tens of picoseconds is considered to be the precursor of the first red photoproduct P1. Here, we report the ultrafast photo-dynamics of the OCP from Synechocystis PCC 6803, carried out using Visible-NIR femtosecond time-resolved absorption spectroscopy as a function of the excitation pulse power and wavelength. We found that a carotenoid radical cation can form even at relatively low excitation power, obscuring the determination of photo-activation yields for P1. Moreover, the comparison of green (540 nm) and blue (470 nm) excitations revealed the existence of an hitherto uncharacterized excited state, denoted as S[~], living a few tens of picoseconds and formed only upon 470 nm excitation. Since neither the P1 quantum yield nor the photo-activation speed over hundreds of seconds vary under green and blue continuous irradiation, this S[~] species is unlikely to be involved in the photo-activation mechanism leading to OCPR. We also addressed the effect of His-tagging at the N- or C-termini on excited state photo-physical properties. Differences in spectral signatures and lifetimes of the different excited states were observed, at variance with the usual assumption that His-tagging hardly influences protein dynamics and function. Altogether our results advocate for careful consideration of the excitation power and His-tag position when comparing the photo-activation of different OCP variants, and beg to revisit the notion that S* is the precursor of photoactivated OCPR. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=166 SRC="FIGDIR/small/474187v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1064473org.highwire.dtl.DTLVardef@77e3c7org.highwire.dtl.DTLVardef@10b1e0eorg.highwire.dtl.DTLVardef@e24977_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2021

The ColR/S two-component system is a conserved determinant of host association across Pseudomonas species

Members of the bacterial genus Pseudomonas form mutualistic, commensal and pathogenic associations with diverse hosts. The prevalence of host association across the genus suggests that symbiosis may be a conserved ancestral trait and that distinct symbiotic lifestyles may be more recently evolved. Here we show that the ColR/S two-component system, part of the Pseudomonas core genome, is functionally conserved between Pseudomonas aeruginosa and Pseudomonas fluorescens. Using plant rhizosphere colonization and virulence in a murine abscess model, we show that colR is required for commensalism with plants and virulence in animals. Comparative transcriptomics revealed that the ColR regulon has diverged between P. aeruginosa and P. fluorescens and deleting components of the ColR regulon revealed strain-specific, but not hostspecific, requirements for ColR-dependent genes. Collectively, our results suggest that ColR/S allows Pseudomonas to sense and respond to a host, but that the ColR-regulon has diverged between Pseudomonas strains with distinct lifestyles.

microbiology↗

bioRxiv2021

Neurons in the dorsomedial hypothalamus promote, prolong, and deepen torpor

Torpor is a naturally occurring, hypometabolic, hypothermic state engaged by a wide range of animals in response to imbalance between the supply and demand for nutrients. Recent work has identified some of the key neuronal populations involved in daily torpor induction in mice, in particular projections from the preoptic area of the hypothalamus (POA) to the dorsomedial hypothalamus (DMH). The DMH plays a role in thermoregulation, control of energy expenditure, and circadian rhythms, making it well positioned to contribute to the expression of torpor. We used activity dependent genetic TRAPing techniques to target DMH neurons that were active during natural torpor bouts in female mice. Chemogenetic reactivation of torpor-TRAPed DMH neurons in calorie-restricted mice promoted torpor, resulting in longer and deeper torpor bouts. Chemogenetic inhibition of torpor-TRAPed DMH neurons did not block torpor entry, suggesting a modulatory role for the DMH in the control of torpor. This work adds to the evidence that the POA and the DMH form part of a circuit within the mouse hypothalamus that controls entry into daily torpor. SignificanceDaily heterotherms such as mice employ torpor to cope with environments in which the supply of metabolic fuel is not sufficient for the maintenance of normothermia. Daily torpor involves reductions in body temperature, as well as active suppression of heart rate and metabolism. How the central nervous system controls this profound deviation from normal homeostasis is not known, but a projection from the preoptic area to the dorsomedial hypothalamus has recently been implicated. We demonstrate that the dorsomedial hypothalamus contains neurons that are active during torpor. Activity in these neurons promotes torpor entry and maintenance, but their activation alone does not appear to be sufficient for torpor entry.

neuroscience↗

bioRxiv2021

Multiplexed action-outcome representation by striatal striosome-matrix compartments detected with a novel cost-benefit foraging task

Learning about positive and negative outcomes of actions is crucial for survival and underpinned by conserved circuits including the striatum. How associations between actions and outcomes are formed is not fully understood, particularly when the outcomes have mixed positive and negative features. We developed a novel foraging ( bandit) task requiring mice to maximize rewards while minimizing punishments. By 2-photon Ca++ imaging, we monitored activity of 5831 identified anterodorsal striatal striosomal and matrix neurons. Surprisingly, we found that action-outcome associations for reward and punishment were combinatorially encoded rather than being integrated as overall outcome value. Single neurons could, for one action, encode outcomes of opposing valence. Striosome compartments consistently exhibited stronger representations of reinforcement outcomes than matrix, especially for high reward or punishment prediction errors. These findings demonstrate a remarkable multiplexing of action-outcome contingencies by single identified striatal neurons and suggest that striosomal neurons are differentially important in action-outcome learning.

neuroscience↗

bioRxiv2021

Prediction Of Plastic Degrading Microbes

Plastic pollution is one of the challenging problems in the environment. But a life without plastic we cannot imagine. This paper deals with the prediction of plastic degrading microbes using Machine Learning. Here we have used Decision Tree, Random Forest, Support vector Machine and K Nearest Neighbor algorithms in order to predict the plastic degrading microbes. Among the four classifiers, Random Forest model gave the best accuracy of 99.1%.

bioinformatics↗

bioRxiv2021

Human airway lineages derived from pluripotent stem cells reveal the epithelial responses to SARS-CoV-2 infection

There is an urgent need to understand how SARS-CoV-2 infects the airway epithelium and in a subset of individuals leads to severe illness or death. Induced pluripotent stem cells (iPSCs) provide a near limitless supply of human cells that can be differentiated into cell types of interest, including airway epithelium, for disease modeling. We present a human iPSC-derived airway epithelial platform, composed of the major airway epithelial cell types, that is permissive to SARS-CoV-2 infection. Subsets of iPSC-airway cells express the SARS-CoV-2 entry factors ACE2 and TMPRSS2. Multiciliated cells are the primary initial target of SARS-CoV-2 infection. Upon infection with SARS-CoV-2, iPSC-airway cells generate robust interferon and inflammatory responses and treatment with remdesivir or camostat methylate causes a decrease in viral propagation and entry, respectively. In conclusion, iPSC-derived airway cells provide a physiologically relevant in vitro model system to interrogate the pathogenesis of, and develop treatment strategies for, COVID-19 pneumonia. Highlights and eTOC blurbO_LISubsets of human iPSC-airway epithelial cells express SARS-Co-V entry factors ACE2 and TMPRSS2. C_LIO_LIiPSC-airway cells are permissive to SARS-CoV-2 infection via multiciliated cells. C_LIO_LISARS-CoV-2 infection of iPSC-airway leads to a robust interferon and inflammatory response. C_LIO_LIiPSC-airway is a physiologically relevant model to study SARS-CoV-2 infection. C_LI

cell biology↗

bioRxiv2021

Asymmetric peptidoglycan editing generates the curvature of predatory bacteria, optimizing invasion and replication within a spherical prey niche

The vibrioid predatory bacterium Bdellovibrio bacteriovorus secretes prey wall-modifying enzymes to invade and replicate within the periplasm of Gram-negative prey bacteria. Studying self-modification of predator wall peptidoglycan during predation, we discover that Bd1075 generates self-wall curvature by exerting LD-carboxypeptidase activity in the vibrioid B. bacteriovorus strain HD100 as it grows inside spherical prey. Bd1075 localizes to the outer curved face of B. bacteriovorus, in contrast to most known shape-determinants. Asymmetric protein localization is determined by the novel function of a nuclear transport factor 2-like (NTF2) domain at the protein C-terminus. The solved structure of Bd1075 is monomeric, with key differences to other LD-carboxypeptidases. Rod-shaped {Delta}bd1075 mutants invade prey more slowly than curved wild-type predators, and stretch and deform the invaded prey cell from within. Vibrioid morphology increases the evolutionary fitness of wild predatory bacteria, facilitating efficient prey invasion and intracellular growth of curved predators inside a spherical prey niche.

microbiology↗

bioRxiv2021

A human iPSC derived bone marrow milieu identifies a novel target against niche-conferred leukaemia proliferation and treatment resistance

Leukaemia cells re-program their microenvironment to provide proliferation support and protection from standard chemotherapy, molecularly targeted therapies, and immunotherapy. Although much is becoming known about molecules that drive niche-dependent treatment resistance; means of targeting these in the clinics has remained a key obstacle. To address this challenge, we have developed human induced pluripotent stem cell engineered niches ex vivo to reveal insights into druggable cancer-niche dependencies. We show that mesenchymal (iMSC) and vascular niche-like (iANG) cells support ex vivo proliferation of patient-derived leukaemia cells, impact dormancy and mediate therapy resistance. iMSC protected both non-cycling and cycling blasts against dexamethasone treatment while iANG protected only dormant blasts. Leukaemia proliferation and protection from dexamethasone induced-apoptosis was dependent on direct cell-cell contact and mediated by CDH2. To explore the therapeutic potential of disrupting this cell-cell interaction, we tested the CDH2 antagonist ADH-1 (previously in phase I / II for solid tumours) in a very aggressive patient-derived xenograft leukaemia mouse model. ADH-1 showed high in vivo efficacy. ADH-1/ dexamethasone combination therapy was superior to dexamethasone alone with no ADH1 conferred additional toxicity. These findings provide a proof-of-concept starting point to develop novel, potentially safer therapeutics that target niche-mediated cancer cell dependencies in haematological malignancies. SummaryCDH2 mediated niche-dependent cancer proliferation and treatment resistance is clinically targetable via ADH-1, a low toxic agent that could be potentially repurposed for future clinical trials in acute leukaemia.

cancer biology↗

bioRxiv2021

Loss of TDP-43 function and rimmed vacuoles persist after T cell depletion in a xenograft model of sporadic inclusion body myositis

Sporadic inclusion body myositis (IBM) is the most common acquired muscle disease in adults over age 50, yet it remains unclear whether the disease is primarily driven by T cell-mediated autoimmunity. IBM muscle biopsies exhibit nuclear clearance and cytoplasmic aggregation of TDP-43 in muscle cells, a pathologic finding observed initially in neurodegenerative disease, and nuclear loss of TDP-43 in neurons causes aberrant RNA splicing. Here, we show that loss of TDP-43 splicing repression, as determined by inclusion of cryptic exons, occurs in skeletal muscle of IBM patients. Out of 119 muscle biopsies tested, RT-PCR-mediated detection of cryptic exon expression is 84% sensitive and 99% specific for diagnosing IBM, indicating utility as a functional and diagnostic biomarker. To determine the role of T cells in pathogenesis, we generated a novel xenograft model by transplanting human IBM muscle into the hindlimb of immunodeficient mice. Xenografts from IBM patients display robust regeneration of human myofibers and recapitulate both inflammatory and degenerative features of the disease. Myofibers in IBM xenografts are invaded by human, oligoclonal CD8+ T cells and exhibit MHC-I upregulation, rimmed vacuoles, mitochondrial pathology, p62-positive inclusions, and nuclear clearance and cytoplasmic aggregation of TDP-43, resulting in expression of cryptic exons. Depletion of human T cells within IBM xenografts by treating mice intraperitoneally with anti-CD3 (OKT3) suppresses MHC-I upregulation, but rimmed vacuoles and loss of TDP-43 function persist. These data suggest that myofiber degeneration occurs independent of T cells, and muscle cell-intrinsic mechanisms, such as loss of TDP-43 splicing repression, drive IBM pathogenesis. One Sentence SummaryDepletion of T cells in a xenograft model of sporadic inclusion body myositis suppresses inflammation but not TDP-43 pathology or muscle degeneration.

pathology↗

bioRxiv2021

High-throughput screen using fluorescence lifetime detects compounds that modulate myosin-binding protein C interactions with actin

Cardiac myosin-binding protein C (cMyBP-C) interacts with actin and myosin to modulate cardiac contractility. These interactions are regulated by cMyBP-C phosphorylation. Heart failure patients often have decreased cMyBP-C phosphorylation and phosphorylation in model systems appears to be cardioprotective for heart failure. Therefore, cMyBP-C is a potential target for heart failure drugs that mimic phosphorylation and/or perturb its interactions with actin/myosin. We have used a novel fluorescence lifetime-based assay to identify small-molecule inhibitors of actin-cMyBP-C binding. Actin was labeled with a fluorescent dye (Alexa Fluor 568, AF568) near its cMyBP-C binding sites. When combined with cMyBP-C N-terminal fragment, C0-C2, the fluorescence lifetime of AF568-actin decreases. Using this reduction in lifetime as a readout of actin binding, a high-throughput screen of a 1280-compound library identified 3 reproducible Hit compounds that reduced C0-C2 binding to actin in the micromolar range. Binding of phosphorylated C0-C2 was also blocked by these compounds. That they specifically block binding was confirmed by a novel actin-C0-C2 time-resolved FRET (TR-FRET) binding assay. Isothermal titration calorimetry (ITC) and transient phosphorescence anisotropy (TPA) confirmed that the Hit compounds bind to cMyBP-C but not to actin. TPA results were also consistent with these compounds inhibiting C0-C2 binding to actin. We conclude that the actin-cMyBP-C lifetime assay permits detection of pharmacologically active compounds that affect cMyBP-Cs actin binding function. TPA, TR-FRET, and ITC can then be used to understand the mechanism by which the compounds alter cMyBP-C interactions with actin.

biophysics↗

bioRxiv2021

3D genomic capture of regulatory immuno-genetic profiles in COVID-19 patients for prognosis of severe COVID disease outcome

Human infection with the SARS-CoV-2 virus leads to coronavirus disease (COVID-19). A striking characteristic of COVID-19 infection in humans is the highly variable host response and the diverse clinical outcomes, ranging from clinically asymptomatic to severe immune reactions leading to hospitalization and death. Here we used a 3D genomic approach to analyse blood samples at the time of COVID diagnosis, from a global cohort of 80 COVID-19 patients, with different degrees of clinical disease outcomes. Using 3D whole genome EpiSwitch(R) arrays to generate over 1 million data points per patient, we identified a distinct and measurable set of differences in genomic organization at immune-related loci that demonstrated prognostic power at baseline to stratify patients with mild forms of illness and those with severe forms that required hospitalization and intensive care unit (ICU) support. Further analysis revealed both well established and new COVID-related dysregulated pathways and loci, including innate and adaptive immunity; ACE2; olfactory, G{beta}{psi}, Ca2+ and nitric oxide (NO) signalling; prostaglandin E2 (PGE2), the acute inflammatory cytokine CCL3, and the T-cell derived chemotactic cytokine CCL5. We identified potential therapeutic agents for mitigation of severe disease outcome, with several already being tested independently, including mTOR inhibitors (rapamycin and tacrolimus) and general immunosuppressants (dexamethasone and hydrocortisone). Machine learning algorithms based on established EpiSwitch(R) methodology further identified a subset of 3D genomic changes that could be used as prognostic molecular biomarker leads for the development of a COVID-19 disease severity test.

biochemistry↗