Biology subjects

Harris, J.

Publications and source records attributed to Harris, J..

32 records · Page 2Linked to original sources

bioRxiv2023

Cold temperature induces a TRPM8-independent calcium release from the endoplasmic reticulum in human platelets

Platelets are sensitive to temperature changes and akin to sensory neurons, are activated by a decrease in temperature. However, the molecular mechanism of this temperature-sensing ability is unknown. Yet, platelet activation by temperature could contribute to numerous clinical sequelae, most importantly to reduced quality of ex vivo-stored platelets for transfusion. In this interdisciplinary study, we present evidence for the expression of the temperature-sensitive ion channel transient receptor potential cation channel subfamily member 8 (TRPM8) in human platelets and precursor cells. We found the TRPM8 mRNA and protein in MEG-01 cells and platelets. Inhibition of TRPM8 prevented temperature-induced platelet activation and shape change. However, chemical agonists of TRPM8 did not seem to have an acute effect on platelets. When exposing platelets to below-normal body temperature, we detected a cytosolic calcium increase which was independent of TRPM8 but was completely dependent on the calcium release from the endoplasmic reticulum. Because of the high interindividual variability of TRPM8 expression, a population-based approach should be the focus of future studies. Our study suggests that the cold response of platelets is complex and TRPM8 appears to play a role in early temperature-induced activation of platelets, while other mechanisms likely contribute to later stages of temperature-mediated platelet response.

cell biology↗

bioRxiv2023

Multiple Pleistocene refugia for Arctic White Heather (Cassiope tetragona) supported by population genomics analyses of contemporary and Little-Ice-Age samples

AimArctic plants survived the Pleistocene glaciations in unglaciated refugia, but the number of these refugia is often unclear. We use high-resolution genomic data from present-day and Little-Ice-Age populations of Arctic White Heather (Cassiope tetragona) to re-evaluate the biogeography of this species and determine whether it had multiple independent refugia or a single refugium in Beringia. LocationCircumpolar Arctic and Coastal British Columbia (BC) alpine TaxonCassiope tetragona L., subspecies saximontana and tetragona, outgroup C. mertensiana (Ericaceae) MethodsWe built genotyping-by-sequencing (GBS) libraries using Cassiope tetragona tissue from 36 Arctic locations, including two [~]250-500-year-old populations collected under glacial ice on Ellesmere Island, Canada. We assembled a de novo GBS reference and called variants in dDocent. Population structure, genetic diversity, and demography were inferred from PCA, ADMIXTURE, fastsimcoal2, SplitsTree, and several population genomics statistics. ResultsPopulation structure analyses identified 4-5 clusters that align with geographic locations. Nucleotide diversity was highest in Beringia and decreased eastwards across Canada. Demographic coalescent analysis of the site-frequency-spectrum dated the following splits from Alaska: BC subspecies saximontana (6 mya), Russia (1.5 mya), Europe (>300-600 kya), Greenland (100 kya). Northern Canada populations appear to be from the current interglacial (7-9 kya). Genetic variants from Alaska appeared more frequently in present-day than historic plants on Ellesmere Island. ConclusionsDemographic analyses show BC, Alaska, Russia, Europe, and Greenland all had separate refugia during the last major glaciations. Northern Canadian populations appear to be founded during the current interglacial with genetic contributions from Alaska, Europe, and Greenland. On Ellesmere Island, there is evidence for continued, recent gene flow with foreign variants introduced in the last 250-500 years. These results suggest that a re-analysis of other Arctic species with shallow population structure using higher resolution genomic markers and demographic analyses may help reveal deeper structure and other circumpolar glacial refugia.

genomics↗

bioRxiv2021

Antiviral activity of Pacific oyster (Crassostrea gigas) hemolymph against a human coronavirus.

Coronaviruses can cause severe respiratory infections in humans. In this study we assessed the antiviral activity of Pacific oyster (Crassostrea gigas) hemolymph against a human coronavirus, HCoV-229E. An eight-fold reduction in infectivity of HCoV-229E on Huh-7 cells was observed in the presence of 10% C. gigas hemolymph. Antiviral activity of C. gigas hemolymph positively correlated with its concentration and appears to be active during an intracellular stage of HCoV-229E infection.

microbiology↗

bioRxiv2021

Precision neoantigen discovery using large-scale immunopeptidomes and composite modeling of MHC peptide presentation

Major histocompatibility complex (MHC)-bound peptides that originate from tumor-specific genetic alterations, known as neoantigens, are an important class of anti-cancer therapeutic targets. Accurately predicting peptide presentation by MHC complexes is a key aspect of discovering therapeutically relevant neoantigens. Technological improvements in mass-spectrometry-based immunopeptidomics and advanced modeling techniques have vastly improved MHC presentation prediction over the past two decades. However, improvement in the sensitivity and specificity of prediction algorithms is needed for clinical applications such as the development of personalized cancer vaccines, the discovery of biomarkers for response to checkpoint blockade and the quantification of autoimmune risk in gene therapies. Toward this end, we generated allele-specific immunopeptidomics data using 25 mono-allelic cell lines and created Systematic HLA Epitope Ranking Pan Algorithm (SHERPA), a pan-allelic MHC-peptide algorithm for predicting MHC-peptide binding and presentation. In contrast to previously published large-scale mono-allelic data, we used an HLA-null K562 parental cell line and a stable transfection of HLA alleles to better emulate native presentation. Our dataset includes five previously unprofiled alleles that expand MHC binding pocket diversity in the training data and extend allelic coverage in underprofiled populations. To improve generalizability, SHERPA systematically integrates 128 mono-allelic and 384 multi-allelic samples with publicly available immunoproteomics data and binding assay data. Using this dataset, we developed two features that empirically estimate the propensities of genes and specific regions within gene bodies to engender immunopeptides to represent antigen processing. Using a composite model constructed with gradient boosting decision trees, multiallelic deconvolution and 2.15 million peptides encompassing 167 alleles, we achieved a 1.44 fold improvement of positive predictive value compared to existing tools when evaluated on independent mono-allelic datasets and a 1.15 fold improvement when evaluating on tumor samples. With a high degree of accuracy, SHERPA has the potential to enable precision neoantigen discovery for future clinical applications.

bioinformatics↗

bioRxiv2020

Diverse Changes in Microglia Morphology and Axonal Pathology Over One Year after Mild Traumatic Brain Injury in Pigs

Over 2.8 million people experience mild traumatic brain injury (TBI) in the United States each year, which may lead to long-term neurological dysfunction. The mechanical forces that occur due to TBI propagate through the brain to produce diffuse axonal injury (DAI) and trigger secondary neuroinflammatory cascades. The cascades may persist from acute to chronic time points after injury, altering the homeostasis of the brain. However, the relationship between the hallmark axonal pathology of diffuse TBI and potential changes in glial cell activation or morphology have not been established in a clinically relevant large animal model at chronic time points. In this study, we assessed tissue from pigs subjected to rapid head rotation in the coronal plane to generate mild TBI. Neuropathological assessments for axonal pathology, microglial morphological changes, and astrocyte reactivity were conducted in specimens out to 1 year post injury. We detected an increase in overall amyloid precursor protein pathology, as well as periventricular white matter and fimbria/fornix pathology after a single mild TBI. We did not detect changes in corpus callosum integrity or astrocyte reactivity. However, detailed microglial skeletal analysis revealed changes in morphology, most notably increases in the number of microglial branches, junctions, and endpoints. These subtle changes were most evident in periventricular white matter and certain hippocampal subfields, and were observed out to 1 year post injury in some cases. These ongoing morphological alterations suggest persistent change in neuroimmune homeostasis. Additional studies are needed to characterize the underlying molecular and neurophysiological alterations, as well as potential contributions to neurological deficits.

pathology↗

bioRxiv2020

Visual QC Protocol for FreeSurfer Cortical Parcellations from Anatomical MRI

Quality control of morphometric neuroimaging data is essential to improve reproducibility. Owing to the complexity of neuroimaging data and subsequently the interpretation of their results, visual inspection by trained raters is the most reliable way to perform quality control. Here, we present a protocol for visual quality control of the anatomical accuracy of FreeSurfer parcellations, based on an easy-to-use open source tool called VisualQC. We comprehensively evaluate its utility in terms of error detection rate and inter-rater reliability on two large multi-site datasets, and discuss site differences in error patterns. This evaluation shows that VisualQC is a practically viable protocol for community adoption.

neuroscience↗

bioRxiv2020

Evaluation of ExoU inhibitors in a Pseudomonas aeruginosa scratch infection assay

Pseudomonas aeruginosa has recently been highlighted by the World Health Organisation (WHO) as a major threat with high priority for the development of new therapies. The type III secretion system of P. aeruginosa delivers the toxin ExoU into the cytosol of target host cells, where its plasma membrane directed phospholipase activity induces rapid cell lysis. Therefore, inhibition of the phospholipase activity of ExoU would be an important treatment strategy in P. aeruginosa infections. We evaluated a panel of ExoU small molecule inhibitors, previously identified from high throughput cellular based assays, and analysed their inhibition of ExoU phospholipase activity in vitro. A corneal epithelial (HCE-T) scratch and infection model using florescence microscopy, and cell viability assays, were used to test the efficacy of compounds to inhibit ExoU from P. aeruginosa. Compounds Pseudolipasin A, compound A and compound B were effective at mitigating ExoU mediated cytotoxicity after infection at concentrations as low as 0.5 M. Importantly, by using the antimicrobials moxifloxacin and tobramycin to control bacterial load, these assays were extended from 6 h to 24 h. P. aeruginosa remained cytotoxic to HCE-T cells with moxifloxacin, present at the minimal inhibitory concentration (MIC) for 24 h, but, when used in combination with either PSA, compound A or compound B, partial scratch healing was observed. These results provide evidence that ExoU inhibitors could be used in combination with certain antimicrobials as a novel means to treat clinical infections of ExoU producing P. aeruginosa.

biochemistry↗

bioRxiv2019

Genomic loci susceptible to systematic sequencing bias in clinical whole genomes

Accurate massively parallel sequencing (MPS) of genetic variants is key to many areas of science and medicine, such as cataloguing population genetic variation and diagnosing genetic diseases. Certain genomic positions can be prone to higher rates of systematic sequencing and alignment bias that limit accuracy, resulting in false positive variant calls. Current standard practices to differentiate between loci that can and cannot be sequenced with high confidence utilise consensus between different sequencing methods as a proxy for sequencing confidence. These practices have significant limitations and alternative methods are required to overcome these. We have developed a novel statistical method based on summarising sequenced reads from whole genome clinical samples and cataloguing them in "Incremental Databases" that maintain individual confidentiality. Allele statistics were catalogued for each genomic position that consistently showed systematic biases with the corresponding MPS sequencing pipeline. We found systematic biases present at [~]1-3% of the human autosomal genome across five patient cohorts. We identified which genomic regions were more or less prone to systematic biases, including large homopolymer flanks (odds ratio=23.29-33.69) and the NIST high confidence genomic regions (odds ratio=0.154-0.191). We confirmed our predictions on a gold-standard reference genome and showed that these systematic biases can lead to suspect variant calls within clinical panels. Our results recommend increased caution to address systematic biases in whole genome sequencing and alignment. This study provides the implementation of a simple statistical approach to enhance quality control of clinically sequenced samples by flagging variants at suspect loci for further analysis or exclusion.

bioinformatics↗

bioRxiv2019

Single-cell profiling guided combinatorial immunotherapy for fast-evolving CDK4/6 inhibitor resistant HER2-positive breast cancer

Development of acquired resistance to targeted cancer therapy is one of the most significant clinical challenges. Acquiring resistance under drug selection pressure is a result of evolutionary adaptation to a complex and dynamic tumor microenvironment (TME). New therapy regimens combining CDK4/6 inhibitor are under active investigation in clinical trials to treat HER2+ breast cancer patients. In parallel with clinical trial settings, in this study, we sought to prospectively model the tumor evolution in response to a targeted therapy regimen in vivo and identify a clinically actionable strategy to combat potential acquired resistance. Notably, despite a promising initial response, acquired resistance emerged rapidly to the anti-Her2/Neu antibody plus CDK4/6 inhibitor Palbociclib combination treatment. By leveraging high-throughput single-cell analyses of the evolving tumors over the course of treatments, we revealed a distinct immunosuppressive immature myeloid cell (IMC) population infiltrated in the resistant TME. Guided by single-cell transcriptome analysis, we demonstrated a combinatorial immunotherapy of IMC-targeting tyrosine kinase inhibitor cabozantinib and immune checkpoint blockades enhanced anti-tumor immunity, and overcame the resistance. Further, sequential combinatorial immunotherapy enabled a sustained control of the rapidly evolving CDK4/6 inhibitor-resistant tumors. Our study demonstrates a translational framework for treating rapidly evolving tumors through preclinical modeling and single-cell analyses. Our findings provide a rationale for an immediate clinical proposition of combinatorial immunotherapy for HER2+ breast cancer as a strategy to mitigate the emergence of resistance.

cancer biology↗

bioRxiv2019

KLK4 inhibition by cyclic and acyclic peptides: structural and dynamical insights into standard-mechanism protease inhibitors

Sunflower Trypsin Inhibitor (SFTI-1) is a 14-amino acid serine protease inhibitor. The dual anti-parallel {beta}-sheet arrangement of SFTI-1 is stabilized by a N-terminal-C-terminal backbone cyclization and a further disulfide bridge to form a final bicyclic structure. This constrained structure is further rigidified by an extensive network of internal hydrogen bonds. Thus, the structure of SFTI-1 in solution resembles the protease-bound structure, reducing the entropic penalty upon protease binding. When cleaved at the scissile bond, it is thought that the rigidifying features of SFTI-1 maintain its structure, allowing the scissile bond to be reformed. The lack of structural plasticity for SFTI-1 is proposed to favour initial protease binding and continued occupancy in the protease active site, resulting in an equilibrium between cleaved and uncleaved inhibitor in the presence of protease. We have determined, at 1.15 [A] resolution, the x-ray crystal structures of complexes between human kallikrein-related peptidase 4 (KLK4) and SFTI-FCQR(Asn14), and between KLK4 and an acyclic form of the same inhibitor, SFTI-FCQR(Asn14)[1,14], with the latter displaying a cleaved scissile bond. Structural analysis and MD simulations together reveal the roles of altered contact sequence, intramolecular hydrogen bonding network and backbone cyclization, in altering the state of SFTIs scissile bond ligation at the protease active site. Taken together, the data presented reveal insights into the role of dynamics in the standard-mechanism inhibition, and suggest that modifications on the noncontact strand may be a useful, underexplored approach for generating further potent or selective SFTI-based inhibitors against members of the serine protease family.

biochemistry↗

bioRxiv2018

Pioneer and nonpioneer cooperation drives lineage specific chromatin opening

Pioneer transcription factors are coined as having the unique property of "opening closed chromatin sites" for implementation of cell fates. We previously showed that the pioneer Pax7 specifies melanotrope cells through deployment of an enhancer repertoire: this allows binding of Tpit, a nonpioneer factor that determines the related lineages of melanotropes and corticotropes. Here, we investigated the relation between these two factors in the pioneer mechanism. Cell-specific gene expression and chromatin landscapes were defined by scRNAseq and chromatin accessibility profiling. We found that in vivo deployment of the melanotrope enhancer repertoire and chromatin opening requires both Pax7 and Tpit. In cells, binding of heterochromatin targets by Pax7 is independent of Tpit but Pax7-dependent chromatin opening requires Tpit. The present work shows that pioneer core properties are limited to the ability to recognize heterochromatin targets and facilitate nonpioneer binding. Chromatin opening per se may be provided through cooperation with nonpioneer factors.

genomics↗

bioRxiv2018

Connectional architecture of a mouse hypothalamic circuit node controlling social behavior

Type 1 Estrogen receptor-expressing neurons in the ventrolateral subdivision of the ventromedial hypothalamus (VMHvlEsr1) play a causal role in the control of social behaviors including aggression. Here we use six different viral-genetic tracing methods to map the connectional architecture of VMHvlEsr1 neurons. These data reveal a high level of input convergence and output divergence (\"fan-in/fan-out\") from and to over 30 distinct brain regions, with a high degree (~90%) of recurrence. Unlike GABAergic populations in other hypothalamic nuclei controlling feeding and parenting behavior, VMHvlEsr1 glutamatergic neurons collateralize to multiple targets. However, we identify two anatomically distinct subpopulations with anterior vs. posterior biases in their collateralization patterns. Surprisingly, these two subpopulations receive indistinguishable inputs. These studies suggest an overall system architecture in which an anatomically feed-forward sensory-to-motor processing stream is integrated with a dense, highly recurrent central processing circuit. This architecture differs from the \"brain-inspired\" feed-forward circuits used in certain types of artificial intelligence networks.\n\nSIGNIFICANCEHow the cellular heterogeneity of brain nuclei maps onto circuit connectivity, the relationship of this anatomical mapping to behavioral function, and whether there are general principles underlying this relationship, remains poorly understood. Here we systematically map the connectivity of estrogen receptor-1-expressing neurons in the ventromedial hypothalamus (VMHvlEsr1), which control aggression and other social behaviors. We find that a relatively sparse, anatomically feed-forward sensory-to-motor processing stream is integrated with a dense, highly recurrent central processing circuit. Further, the VMHvl contains at least two subpopulations of Esr1+ neurons with different cell body characteristics and locations, with distinct patterns of collateralization to downstream targets. Nevertheless, these projection-defined subpopulations receive similar inputs. This input-output organization appears distinct from those described in other hypothalamic nuclei.

neuroscience↗

bioRxiv2018

Quality circles for quality improvement in primary health care: their effectiveness, gaps of knowledge, origins and significance - a scoping review

BackgroundQuality circles, or similarly structured small groups in primary health care, such as peer review groups, consist of 6 to 12 professionals from the same background who meet regularly to improve their standard practice. This paper reports the results from a scoping search performed to clarify possible effectiveness, knowledge gaps, underlying concepts and significance.\n\nObjectivesTo gain insight into knowledge gaps and understanding of the effectiveness, origins and significance of quality circles.\n\nMethodsA search strategy was developed starting with quality circle in PubMed and the index terms from those articles revealed were then used as search terms to identify further papers. Repeating this process in collaboration with a librarian, search strings relating to quality circles were built, and databases searched up to December 2017. Any paper on structured quality circles or related small group work in primary health care was included when relevant to the objectives.\n\nResultsFrom 11973 citations, 82 background papers and 58 key papers were identified, in addition to 12 books and 10 websites. 19 studies, one paper summarizing three studies and one systematic review suggest that quality circles can be effective in behaviour change, though with varying effect sizes. Quality circles and their techniques are complex, as they are not standardized, and changes seem to depend on the topic and context, which requires further research into how and why they work in order to improve them. From their origins in industry, they are now used in primary health care in many countries for continuous medical education, continuous professional development and quality improvement.\n\nConclusionThe evidence on quality circles indicates that they can successfully change general practitioner behaviour. As they are a complex intervention, theory-driven research approaches are needed to understand and improve their effectiveness. This is of major importance because they play an important role in quality improvement in primary health care in many countries.

scientific communication and education↗

bioRxiv2018

Classification of electrophysiological and morphological types in mouse visual cortex

Understanding the diversity of cell types in the brain has been an enduring challenge and requires detailed characterization of individual neurons in multiple dimensions. To profile morpho-electric properties of mammalian neurons systematically, we established a single cell characterization pipeline using standardized patch clamp recordings in brain slices and biocytin-based neuronal reconstructions. We built a publicly-accessible online database, the Allen Cell Types Database, to display these data sets. Intrinsic physiological and morphological properties were measured from over 1,800 neurons from the adult laboratory mouse visual cortex. Quantitative features were used to classify neurons into distinct types using unsupervised methods. We establish a taxonomy of morphologically- and electrophysiologically-defined cell types for this region of cortex with 17 e-types and 35 m-types, as well as an initial correspondence with previously-defined transcriptomic cell types using the same transgenic mouse lines.

neuroscience↗