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Harris, M.

Publications and source records attributed to Harris, M..

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Severity Index for Suspected Arbovirus (SISA): machine learning for accurate prediction of hospitalization in subjects suspected of arboviral infection

BackgroundDengue, chikungunya, and Zika are arboviruses of major global health concern. Decisions regarding the clinical management of suspected arboviral infection are challenging in resource-limited settings, particularly when deciding on patient hospitalization. The objective of this study was to determine if hospitalization of individuals with suspected arboviral infections could be predicted using subject intake data.\n\nMethodology/Principal FindingsTwo prediction models were developed using data from a surveillance study in Machala, a city in southern coastal Ecuador with a high burden of arboviral infections. Data was obtained from subjects who presented at sentinel medical centers with suspected arboviral infection (November 2013 to September 2017). The first prediction model--called the Severity Index for Suspected Arbovirus (SISA)-- used only demographic and symptom data. The second prediction model--called the Severity Index for Suspected Arbovirus with Laboratory (SISAL)--incorporated laboratory data. These models were selected by comparing the prediction ability of seven machine learning algorithms; the area under the receiver operating characteristic curve from the prediction of a test dataset was used to select the final algorithm for each model. After eliminating those with missing data, the SISA dataset had 534 subjects, and the SISAL dataset had 98 subjects. For SISA, the best prediction algorithm was the generalized boosting model, with an AUC of 0.91. For SISAL, the best prediction algorithm was the elastic net with an AUC of 0.94. A sensitivity analysis revealed that SISA and SISAL are not directly comparable to one another.\n\nConclusions/SignificanceBoth SISA and SISAL were able to predict arbovirus hospitalization with a high degree of accuracy in our dataset. These algorithms will need to be tested and validated on new data from future patients. Machine learning is a powerful prediction tool and provides an excellent option for new management tools and clinical assessment of arboviral infection.\n\nAuthor SummaryPatient triage is a critical decision for clinicians; patients with suspected arbovirus infection are difficult to diagnose as symptoms can be vague and molecular testing can be expensive or unavailable. Determining whether these patients should be hospitalized or not can be challenging, especially in resource-limited settings. Our study included data from 543 subjects with a diagnosis of suspected dengue, chikungunya, or Zika infection. Using a machine learning approach, we tested the ability of seven algorithms to predict hospitalization status based on the signs, symptoms, and laboratory data that would be available to a clinician at patient intake. Using only signs and symptoms, we were able to predict hospitalization with high accuracy (94%). Including laboratory data also resulted in highly accurate prediction of hospitalization (92%). This tool should be test in future studies with new subject data. Upon further development, we envision a simple mobile application to aid in the decision-making process for clinicians in areas with limited resources.

epidemiology↗

Integrated K+ channel and K+-Cl- cotransporter functions regulate fin proportionality in zebrafish

The coordination of growth during development establishes proportionality within and among the different anatomic structures of organisms. Innate memory of this proportionality is preserved, as shown in the ability of regenerating structures to return to their original size. Although the regulation of this coordination is incompletely understood, mutant analyses of zebrafish with long-finned phenotypes have uncovered important roles for bioelectric signaling in modulating growth and size of the fins and barbs. To date, long-finned mutants identified are caused by hypermorphic mutations, leaving unresolved whether such signaling is required for normal development. We isolated a new zebrafish mutant, schleier, with proportional overgrowth phenotypes caused by a missense mutation and loss of function in the K+-Cl- cotransporter Kcc4a. Genetic depletion of Kcc4a in wild-type fish leads to a dose-dependent loss of growth restriction in fins and barbs, supporting a requirement for Kcc4a in regulation of proportion. Epistasis experiments suggest that Kcc4a and the two-pore potassium channel Kcnk5b both contribute to a common bioelectrical signaling response in the fin. These data suggest that an integrated bioelectric signaling pathway is required for the coordination of size and proportion during development.\n\nGraphical Abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=145 SRC=\"FIGDIR/small/621243v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (26K):\norg.highwire.dtl.DTLVardef@2ba7b0org.highwire.dtl.DTLVardef@e3df5org.highwire.dtl.DTLVardef@105b878org.highwire.dtl.DTLVardef@920b54_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

Continent-scale phenotype mapping using photographs from citizen scientists

Field investigations of phenotypic variation in free-living organisms are often limited in scope owing to time and funding constraints. By collaborating with online communities of amateur naturalists, investigators can greatly increase the amount and diversity of phenotypic data in their analyses while simultaneously engaging with a public audience. Here, we present a method for quantifying phenotypes of individual organisms in citizen scientists photographs. We then show that our protocol for measuring wing phenotypes from photographs yields accurate measurements in two species of Calopterygid damselflies. Next, we show that, while most observations of our target species were made by members of the large and established community of amateur naturalists at iNaturalist.org, our efforts to increase recruitment through various outreach initiatives were successful. Finally, we present results from two case studies: (1) an analysis of wing pigmentation in male smoky rubyspots (Hetaerina titia) showing previously undocumented geographical variation in a seasonal polyphenism, and (2) an analysis of variation in the relative size of the wing spots of male banded demoiselles (Calopteryx splendens) in Great Britain questioning previously documented evidence for character displacement. Our results demonstrate that our protocol can be used to create high quality phenotypic datasets using citizen scientists photographs, and, when combined with metadata (e.g., date and location), can greatly broaden the scope of studies of geographical and temporal variation in phenotypes. Our analyses of the recruitment and engagement process also demonstrate that collaborating with an online community of amateur naturalists can be a powerful way to conduct hypothesis-driven research aiming to elucidate the processes that impact trait evolution at landscape scales.

evolutionary biology↗

Hidden in plain sight: What remains to be discovered in the eukaryotic proteome?

The first decade of genome sequencing stimulated an explosion in the characterization of unknown proteins. More recently, the pace of functional discovery has slowed, leaving around 20% of the proteins even in well-studied model organisms without informative descriptions of their biological roles. Remarkably, many uncharacterized proteins are conserved from yeasts to human, suggesting that they contribute to fundamental biological processes. To fully understand biological systems in health and disease, we need to account for every part of the system. Unstudied proteins thus represent a collective blind spot that limits the progress of both basic and applied biosciences.\n\nWe use a simple yet powerful metric based on Gene Ontology (GO) biological process terms to define characterized and uncharacterized proteins for human, budding yeast, and fission yeast. We then identify a set of conserved but unstudied proteins in S. pombe, and classify them based on a combination of orthogonal attributes determined by large-scale experimental and comparative methods. Finally, we explore possible reasons why these proteins remain neglected, and propose courses of action to raise their profile and thereby reap the benefits of completing the catalog of proteins biological roles.

molecular biology↗

Epigenome-wide meta-analysis of blood DNA methylation and its association with subcortical volumes: findings from the ENIGMA Epigenetics Working Group

DNA methylation, which is modulated by both genetic factors and environmental exposures, may offer a unique opportunity to discover novel biomarkers of disease-related brain phenotypes, even when measured in other tissues than brain, such as blood. A few studies of small sample sizes have revealed associations between blood DNA methylation and neuropsychopathology, however, large-scale epigenome-wide association studies (EWAS) are needed to investigate the utility of DNA methylation profiling as a peripheral marker for the brain. Here, in an analysis of eleven international cohorts, totalling 3,337 individuals, we report epigenome-wide meta-analyses of blood DNA methylation with volumes of the hippocampus, thalamus and nucleus accumbens (NAcc) -three subcortical regions selected for their associations with disease and heritability and volumetric variability. Analyses of individual CpGs revealed genome-wide significant associations with hippocampal volume at two loci. No significant associations were found for analyses of thalamus and nucleus accumbens volumes. CpG sites associated with hippocampus volume were significantly enriched within cancer-related genes and within regulatory elements containing the transcriptionally repressive histone H3K27 tri-methylation mark that is vital for stem cell fate specification. Cluster-based analyses revealed additional differentially methylated regions (DMRs) associated with hippocampal volume. DNA methylation at these loci affected expression of proximal genes involved in in learning and memory, stem cell maintenance and differentiation, fatty acid metabolism and type-2 diabetes. These DNA methylation marks, their interaction with genetic variants and their impact on gene expression offer new insights into the relationship between epigenetic variation and brain structure and may provide the basis for biomarker discovery in neurodegeneration and neuropsychiatric conditions.

genomics↗

Genetic Determinants of Cortical Structure (Thickness, Surface Area and Volumes) among Disease Free Adults in the CHARGE Consortium

Cortical thickness, surface area and volumes (MRI cortical measures) vary with age and cognitive function, and in neurological and psychiatric diseases. We examined heritability, genetic correlations and genome-wide associations of cortical measures across the whole cortex, and in 34 anatomically predefined regions. Our discovery sample comprised 22,822 individuals from 20 cohorts within the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) consortium and the United Kingdom Biobank. Significant associations were replicated in the Enhancing Neuroimaging Genetics through Meta-analysis (ENIGMA) consortium, and their biological implications explored using bioinformatic annotation and pathway analyses. We identified genetic heterogeneity between cortical measures and brain regions, and 161 genome-wide significant associations pointing to wnt/{beta}-catenin, TGF-{beta} and sonic hedgehog pathways. There was enrichment for genes involved in anthropometric traits, hindbrain development, vascular and neurodegenerative disease and psychiatric conditions. These data are a rich resource for studies of the biological mechanisms behind cortical development and aging.

genetics↗

A pivotal role of serine 225 phosphorylation in the function of hepatitis C virus NS5A revealed with the application of a phosphopeptide antiserum and super-resolution microscopy

NS5A is a multi-functional phosphoprotein that plays a key role in both viral replication and assembly. The identity of the kinases that phosphorylate NS5A, and the consequences for HCV biology, remain largely undefined. We previously identified serine 225 (S225) within low complexity sequence (LCS) I as a major phosphorylation site and used a phosphoablatant mutant (S225A) to define a role for S225 phosphorylation in the regulation of genome replication, interactions of NS5A with several host proteins and the sub-cellular localisation of NS5A. To investigate this further, we raised an antiserum to S225 phosphorylated NS5A (pS225). Western blot analysis revealed that pS225 was exclusively found in the hyper-phosphorylated NS5A species. Furthermore, using kinase inhibitors we demonstrated that S225 was phosphorylated by casein kinase 1 (CK1) and polo-like kinase 1 (PLK1). Using a panel of phosphoablatant mutants of other phosphorylation sites in LCSI we obtained the first direct evidence of bidirectional hierarchical phosphorylation initiated by phosphorylation at S225.\n\nUsing super-resolution microscopy (Airyscan and Expansion), we revealed a unique architecture of NS5A-positive clusters in HCV-infected cells - pS225 was concentrated on the surface of these clusters, close to lipid droplets. Pharmacological inhibition of S225 phosphorylation resulted in the condensation of NS5A-positive clusters into larger structures, recapitulating the S225A phenotype. Although S225 phosphorylation was not specifically affected by daclatasvir treatment, the latter also resulted in a similar condensation. These data are consistent with a key role for S225 phosphorylation in the regulation of NS5A function.\n\nImportanceNS5A has obligatory roles in the hepatitis C virus lifecycle, and is proposed to be regulated by phosphorylation. As NS5A is a target for highly effective direct-acting antivirals (DAAs) such as daclatasvir (DCV) it is vital to understand how phosphorylation occurs and regulates NS5A function. We previously identified serine 225 (S225) as a major phosphorylation site. Here we used an antiserum specific for NS5A phosphorylated at S225 (pS225-NS5A) to identify which kinases phosphorylate this residue. Using super-resolution microscopy we showed that pS225 was present in foci on the surface of larger NS5A-positive clusters likely representing genome replication complexes. This location would enable pS225-NS5A to interact with cellular proteins and regulate the function and distribution of these complexes. Both loss of pS225 and DCV treatment resulted in similar changes to the structure of these complexes, suggesting that DAA treatment might target a function of NS5A that is also regulated by phosphorylation.

microbiology↗

Multiple roles of the non-structural protein 3 (nsP3) alphavirus unique domain (AUD) during Chikungunya virus genome replication and transcription

Chikungunya virus (CHIKV) is a re-emerging Alphavirus causing fever, joint pain, skin rash, arthralgia, and occasionally death. Antiviral therapies and/or effective vaccines are urgently required. CHIKV biology is poorly understood, in particular the functions of the non-structural protein 3 (nsP3). Here we present the results of a mutagenic analysis of the alphavirus unique domain (AUD) of nsP3. Informed by the structure of the Sindbis virus AUD and an alignment of amino acid sequences of multiple alphaviruses, a series of mutations in the AUD were generated in a CHIKV sub-genomic replicon. This analysis revealed an essential role for the AUD in CHIKV RNA replication, with mutants exhibiting species- and cell-type specific phenotypes. To test if the AUD played a role in other stages of the virus lifecycle, the mutant panel was also analysed in the context of infectious CHIKV. Results indicated that, in addition to a role in RNA replication, the AUD was also required for virus assembly. Further analysis revealed that one mutant (P247A/V248A) specifically blocked transcription of the subgenomic RNA leading to a dramatic reduction in synthesis of the structural proteins and concomitant reduction in virus production. This phenotype could be explained by both a reduction in the binding of the P247A/V248A mutant nsP3 to viral genomic RNA in vivo, and the reduced affinity of the mutant AUD for the subgenomic promoter RNA in vitro. We propose that the AUD is a pleiotropic protein domain, with multiple functions during CHIKV RNA synthesis.\n\nAuthor summaryChikungunya virus (CHIKV) is an emerging threat to world health. It is transmitted by Aedes species mosquitos, and has caused massive epidemics across the globe. The virus causes fever, rash, arthritis and can sometimes be fatal. The biology of CHIKV is poorly understood, to address this deficiency we aimed to identify functions of one of the viral proteins, nsP3. We focussed on the central part of this protein, termed the alphavirus unique domain (AUD) because it is unique to the genus of viruses to which CHIKV belongs - the Alphaviruses - and not present in other related viruses. By making changes (mutations) in the AUD and analysing the effects of these changes we show that it is involved in multiple stages of the virus lifecycle. These observations identify nsP3 and the AUD in particular as a potential target for antiviral therapy or rational vaccine design.

microbiology↗

Targeting hepatitis C virus p7 channel activity reveals prospect for bimodal antiviral prophylaxis

Since the 1960s, a single class of agent has been licensed targeting virus-encoded ion channels, or \"viroporins\", contrasting the success of channel blocking drugs in other areas of medicine. Although resistance arose to these prototypic adamantane inhibitors of the influenza A virus (IAV) M2 proton channel, a growing number of clinically and economically important viruses are now recognised to encode essential viroporins providing potential targets for modern drug discovery.\n\nWe describe the first rationally designed viroporin inhibitor with a comprehensive structure-activity relationship (SAR). This step-change in understanding not only revealed a second biological function for the p7 viroporin from hepatitis C virus (HCV) during virus entry, but also enabled the synthesis of a labelled tool compound that retained biological activity. Hence, p7 inhibitors (p7i) represent a unique class of HCV antiviral targeting both the spread and establishment of infection, as well as a precedent for future viroporin-targeted drug discovery.

microbiology↗

Persistent Chikungunya Virus Replication in Human Cells is Associated with Presence of Stable Cytoplasmic Granules Containing Non-structural Protein 3

Chikungunya virus (CHIKV), a mosquito-borne human pathogen, causes a disabling disease characterized by severe joint pain that can persist for weeks, months or even years in patients. The non-structural protein 3 (nsP3) plays essential roles during acute infection, but little is known about the function of nsP3 during chronic disease. Here, we used sub-diffraction multi-color microscopy for a spatial and temporal analysis of CHIKV nsP3 within human cells that persistently replicate viral RNA. Round cytoplasmic granules of various sizes (i) contained nsP3 and G3BP Stress Granule Assembly factor; (ii) were next to double-stranded RNA foci, and nsP1-positive structures; and (iii) made contact with markers of the cytoskeleton and cellular structures, such as early endosomes and nucleopores. Analysis of protein turnover and mobility by live-cell microscopy revealed that granules could persist for hours to days, can accumulate newly synthesized protein, and move at differently through the cytoplasm. Granules also had a static internal architecture and were stable in cell lysates. Whereas cells with active replication and stable nsP3-granules did not respond to oxidative stress, refractory cells that had cleared the non-cytotoxic replicon could. In summary, nsP3 can form uniquely stable granular structures that persist long-term within the host cell. This continued presence of viral and cellular protein-complexes has implications for the study of the pathogenic consequences of lingering CHIKV infection and the development of strategies to mitigate the burden of chronic musculoskeletal disease brought about by a medically important arthropod-borne virus (arbovirus).\n\nImportanceChikungunya virus (CHIKV) is a re-emerging alphavirus transmitted by mosquitos and causes widespread transient sickness but also chronic disease affecting muscles and joints. Although no approved vaccines or antivirals are available, a better understanding of the viral life cycle and the role of individual viral proteins can aid in identifying new therapeutic targets. Advances in microscopy and persistent CHIKV model systems now allow researchers to study viral proteins within controlled laboratory environments. Here we established human cells that stably replicate viral RNA and express a tagged version of non-structural protein 3. The ability to track this viral protein within the host cell and during persistent replication can benefit fundamental research efforts to better understand long-term consequences of the persistence of viral protein complexes and thereby provide the foundation for new therapeutic targets to control CHIKV infection and treat chronic disease symptoms.

microbiology↗

Bioelectric-calcineurin signaling module regulates allometric growth and size of the zebrafish fin

The establishment of relative size of organs and structures is paramount for attaining final form and function of an organism. Importantly, variation in the proportions of structures frequently underlies adaptive change in morphology in evolution and maybe a common mechanism underlying selection. However, the mechanism by which growth is integrated within tissues during development to achieve proper proportionality is poorly understood. We have shown that signaling by potassium channels mediates coordinated size regulation in zebrafish fins. Recently, calcineurin inhibitors were shown to elicit changes in zebrafish fin allometry as well. Here, we identify the potassium channel kcnk5b as a key player in integrating calcineurins growth effects, in part through regulation of the cytoplasmic C-terminus of the channel. We propose that the interaction between Kcnk5b and calcineurin acts as a signaling node to regulate allometric growth. Importantly, we find that this regulation is epistatic to inherent mechanisms instructing overall size as inhibition of calcineurin is able to bypass genetic instruction of size as seen in sof and wild-type fins, however, it is not sufficient to re-specify positional memory of size of the fin. These findings integrate classic signaling mediators such as calcineurin with ion channel function in the regulation of size and proportion during growth.

developmental biology↗

Ghrelin-Reactive Autoantibodies are elevated in Children with Prader-Willi Syndrome

Prader-Willi Syndrome (PWS) is a complex genetic disorder characterized by developmental and growth abnormalities, insatiable appetite, and excessive eating (hyperphagia). The underlying cause of hyperphagia in PWS is currently unknown, however, elevated levels of the peptide hormone ghrelin is believed to contribute. Recently, ghrelin-reactive autoantibodies (isotype IgG) were identified in non-genetic obesity. These autoantibodies act as ghrelin carrier proteins and potentiate its orexigenic effects. Here, we describe the identification of ghrelin-reactive autoantibodies in a cohort of 16 children with PWS. In comparison to unaffected siblings, autoantibody levels are significantly increased in PWS children. We further show that autoantibody levels are unaffected by food intake, unlike plasma ghrelin which declines postprandially in both groups. Critically, we also demonstrate that the autoantibodies bind the major circulating ghrelin isoforms, unacylated ghrelin, which does not stimulate appetite, and the orexigen acylated ghrelin. In excess, unacylated ghrelin may compete with acylated ghrelin for autoantibody binding. Taken together, this is the first report on ghrelin-reactive antibodies in a pediatric population, and the first to demonstrate that the antibodies do not discriminate between orexigenic and non-orexigenic ghrelin isoforms. Our work suggests that ghrelin autoantibodies can be targeted using non-orexigenic forms of ghrelin, thereby providing a novel therapeutic target for PWS and for obesity in general.

physiology↗