Biology subjects

Stewart, R.

Publications and source records attributed to Stewart, R..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

A novel patient-derived cellular platform for validating microglia-targeted therapeutics for Alzheimer's disease

The scarcity of effective biomarkers and therapeutic strategies for predicting disease onset and progression in Alzheimers disease (AD) is a major challenge to improve much-needed therapeutic outcomes. Conventional drug discovery approaches have been unsuccessful in providing efficient interventions due to their one-size-fits-all nature. As an alternative, personalised drug development holds promise to pre-select responders and identify suitable drug efficacy indicators. In this study, we established a preclinical drug testing strategy by assessing the efficacy of anti-inflammatory drugs in 2D and 3D in vitro models of monocyte-derived microglia-like cells (MDMi) derived from AD and mild cognitive impairment (MCI) patients, and matched healthy individuals. We observed that the cytokine inflammatory profiles of MDMi in response to drugs clustered separately between cohorts, with the 3D model showing a more defined separation between healthy and patient donors than 2D. By ranking donor and cytokine responses to drugs, we identified that drug efficacy was limited in AD patients and involved cohort-specific responsive cytokines. Our findings suggest that MDMi models have the potential to predict disease progression, stratify responders and identify biomarkers for estimating the efficacy of microglia-targeted drugs. Together, our pipeline could serve as a valuable tool to enhance the clinical translational value of preclinical drug screens and ultimately improve drug outcomes for AD.

cell biology↗

bioRxiv2023

Serial KinderMiner (SKiM) Discovers and Annotates Biomedical Knowledge Using Co-Occurrence and Transformer Models

BackgroundThe PubMed database contains more than 34 million articles; consequently, it is becoming increasingly difficult for a biomedical researcher to keep up-to-date with different knowledge domains. Computationally efficient and interpretable tools are needed to help researchers find and understand associations between biomedical concepts. The goal of literature-based discovery (LBD) is to connect concepts in isolated literature domains that would normally go undiscovered. This usually takes the form of an A-B-C relationship, where A and C terms are linked through a B term intermediate. Here we describe Serial KinderMiner (SKiM), an LBD algorithm for finding statistically significant links between an A term and one or more C terms through some B term intermediate(s). The development of SKiM is motivated by the the observation that there are only a few LBD tools that provide a functional web interface, and that the available tools are limited in one or more of the following ways: 1) they identify a relationship but not the type of relationship, 2) they do not allow the user to provide their own lists of B or C terms, hindering flexibility, 3) they do not allow for querying thousands of C terms (which is crucial if, for instance, the user wants to query connections between a disease and the thousands of available drugs), or 4) they are specific for a particular biomedical domain (such as cancer). We provide an open-source tool and web interface that improves on all of these issues. ResultsWe demonstrate SKiMs ability to discover useful A-B-C linkages in three control experiments: classic LBD discoveries, drug repurposing, and finding associations related to cancer. Furthermore, we supplement SKiM with a knowledge graph built with transformer machine-learning models to aid in interpreting the relationships between terms found by SKiM. Finally, we provide a simple and intuitive open-source web interface (https://skim.morgridge.org) with comprehensive lists of drugs, diseases, phenotypes, and symptoms so that anyone can easily perform SKiM searches. ConclusionsSKiM is a simple algorithm that can perform LBD searches to discover relationships between arbitrary user-defined concepts. SKiM is generalized for any domain, can perform searches with many thousands of C term concepts, and moves beyond the simple identification of an existence of a relationship; many relationships are given relationship type labels from our knowledge graph.

bioinformatics↗

bioRxiv2023

Characterization of an Australian outbreak Japanese Encephalitis virus genotype 4 isolate in mice and human cortical brain organoids

BackgroundHuman infections with Japanese encephalitis virus (JEV) are a leading cause of viral encephalitis. An unprecedented outbreak of JEV genotype 4 was recently reported in Australia, with an isolate (JEVNSW/22) obtained from a stillborn piglet brain. MethodsHerein we compared the neuropathology of JEVNSW/22, JEVFU (genotype 2) and JEVNakayama (genotype 3) in adult C57BL/6J wild-type mice, mice deficient in interferon regulatory factor 7 (Irf7-/-), and mice deficient in type I interferon receptor (Ifnar-/-), as well as in human cortical brain organoids (hBOs). Using human serum post-Imojev vaccination, we performed neutralisation assays to determine JEVNSW/22 susceptibility to vaccine responses. FindingsIn C57BL/6J and Irf7-/- mice with lethal outcomes, brain infection and histopathological lesions recapitulated those seen in humans and primates. JEV was universally lethal in Ifnar-/- mice by day 3 with histological signs of brain hemorrhage, but produced no other detectable brain infection or lesions, with viral protein detected in blood vessels but not neurons. We thus describe a new Irf7-/- mouse model for JEVNSW/22, which had increased viremia compared to C57BL/6J mice, allowing for lethal neuroinvasive infection in one mouse. Overall, JEVNSW/22 was less neurovirulent than other JEV isolates in C57BL/6J and Irf7-/- mice, and was more sensitive to type I interferon. All JEV isolates showed robust cytopathic infection of human cortical brain organoids, albeit lower for JEVNSW/22. We also show that Imojev vaccination in humans induced neutralizing antibodies against JEVNSW/22, with the level of cross-neutralisation related to the conservation in envelope protein amino acid sequences for each isolate. InterpretationOur study establishes JEVNSW/22 mouse models of infection, allowing for possible lethal neuroinvasive infection that was rarer than for other JEV genotypes. JEV vaccination regimens may afford protection against this newly emerged JEV genotype 4 strain, although neutralizing antibody responses are sub-optimal. FundingQIMRB received a generous philanthropic donation from the Brazil Family Foundation awarded to D.J.R. to support Japanese Encephalitis virus research at QIMRB. A.S. holds an Investigator grant from the National Health and Medical Research Council (NHMRC) of Australia (APP1173880). We also acknowledge the intramural grant from QIMR Berghofer awarded to R.S. and D.J.R. for purchase of the CelVivo Clinostar incubator for producing human cortical brain organoids. The project "Japanese encephalitis vaccine via the intradermal route in children and adults (JEVID-2): A clinical trial comparing the immunogenicity and safety of Japanese encephalitis vaccine administered by subcutaneous and intradermal routes" being conducted by G.D., N.G., and N.W. was funded by the Sydney Childrens Hospitals Network and New South Wales Health. Research in context Evidence before the studyJEV from the historically rare genotype 4 recently emerged in Australia, causing an unprecedented outbreak, with 44 human cases and 7 fatalities. While a range of JEV mouse models have been reported, none of them infect adult mice with a genotype 4 isolate. The efficacy of current vaccines for this JEV genotype are also unclear. Added value of this studyWe establish well characterised adult and subcutaneously infected mouse models for JEV which recapitulate many aspects of human disease including lethal neuroinvasive infection and severe histopathological lesions. Prolonged viremia was significantly associated with lethal neuroinvasiveness in Irf7-/-mice. We demonstrate that a genotype 4 Australian isolate, JEVNSW/22, exhibited markedly diminished lethal neuroinvasion compared to other JEV genotypes. Using serum from Imojev vaccine recipients, neutralizing antibodies against JEVNSW/22 were present, albeit at sub-optimal titers. Implications of all the available evidenceThe establishment of well characterised adult mouse models of JEVNSW/22 with rare neuropenetrance after peripheral inoculation that recapitulate human disease is an important tool that can now be deployed in pre-clinical studies and to understand disease pathogenesis. Our study suggests that new vaccines should be developed against circulating JEV strains for optimal neutralizing antibody responses.

microbiology↗

bioRxiv2023

The impacts of contemporary logging after 250 years of deforestation and degradation on forest-dependent threatened species

Despite the importance of safeguarding forests and woodlands for achieving global climate and biodiversity agendas, logging continues across most forested countries. Forestry advocates often claim logging has minimal impacts, but rarely consider the cumulative threat deforestation and degradation has had, and continue to have, on species. Using New South Wales (Australia) as a case study, we quantify the extent of deforestation and degradation from 1750 - current. Using these estimates of overall loss as a baseline, we then quantify the relative extent of contemporary (2000 - 2022) logging and the condition of the remaining native forest and woodland (quantified by measuring the similarity of a current ecosystem to a historical reference state with high ecological integrity). Using these data, we measure the impacts on distinct vegetation types and on 484 terrestrial forest-dependent now-threatened species. We show that more than half (29 million ha) of pre-1750 (pre-European colonization of Australia) native forest and woodland vegetation in NSW has been lost. Of the remaining 25 million ha, 9 million ha is degraded. We found contemporary degradation from logging affected 244 forest-dependent now-threatened species that had already been affected by this historical deforestation and degradation, but the impacts varied across species and vegetation types. We found that 70 now-threatened species that were impacted by historical deforestation and degradation and continue to be impacted by logging, now have [≤]50% of their pre-1750 extent remaining that is intact (with three species now having <20%). By quantifying the historical impacts of deforestation and degradation, our research sets the impact of contemporary degradation from logging in perspective and highlights shortfalls in current environmental assessments that fail to consider appropriate baselines when reporting on overall impact. Future land management decisions need to consider not only the extent of remaining habitat based on pre-1750 extents, but also its condition. Article impact statementThe impact of logging needs to be placed in perspective by considering past losses and degradation due to human land use decisions.

ecology↗

bioRxiv2022

Omicron BA.5 infects human brain organoids and is neuroinvasive and lethal in K18-hACE2 mice

The reduced pathogenicity of the omicron BA.1 sub-lineage compared to earlier variants is well described, although whether such attenuation is retained for later variants like BA.5 and XBB remains controversial. We show that BA.5 and XBB isolates were significantly more pathogenic in K18-hACE2 mice than a BA.1 isolate, showing increased neuroinvasiveness, resulting in fulminant brain infection and mortality, similar to that seen for original ancestral isolates. BA.5 also infected human cortical brain organoids to a greater extent than the BA.1 and original ancestral isolates. In the brains of mice, neurons were the main target of infection, and in human organoids neuronal progenitor cells and immature neurons were infected. Although fulminant brain infection is not a feature of COVID-19, evidence for brain infection and brain damage in some COVID-19 patients with severe disease is becoming compelling, with the results herein suggesting that evolving omicron variants may have increasing intrinsic neuropathogenic potential.

microbiology↗

bioRxiv2022

A report card methodology to showcase progress towards threatened species recovery

Among the conservation community, it is well known that Earths mass species extinction crisis is getting worse. Yet, an often neglected problem is the need for effectively communicating the species extinction crisis to diverse audiences in ways that catalyse immediate action. Here we generated a streamlined threatened species recovery report card methodology, which combined two input indicators including planning and funding, one output indicator capturing habitat protection, and one outcome indicator which highlights threatened species trajectories, to provide simple scores for all Australian threatened species. We show that just 41 (2.3%) of species achieved an A grade for the input indicator (i.e., recovery plans and federal funding), 240 (13.3%) achieved a C grade, and 1,521 (84.4%) achieved an F. Five hundred and twenty nine (29.3%) species achieved an A for the output indicator (i.e, habitat protection), 130 (7.2%) achieved a B, 158 (8.8%) achieved a C, 189 (10.5%) achieved a D, 212 (11.8%) achieved an E, and 584 (32.4%) achieved a F. While five (0.3%) species achieved an A for the output indicator (i.e., threat status improvement), every other species (99.7%) achieved an F. We provide a method to combine scores to test how individual jurisdictions are tracking and show that Australia is achieving an F for the input and outcome indicators, and a D for the output indicator. While the threatened species recovery report card highlighted a clear failure in many federal environmental legislation responsibilities, it provides a baseline from which different governments can track policy progress and outlines clear direction for immediate improvement including developing adequate recovery plans, funding the actions in the recovery plans, protecting habitat from further destruction, verifying recovery through monitoring and evaluation of species trajectories, and supporting transparency and collaboration on the execution on the plans through an improved data infrastructure. Without an immediate step change in how Australia communicates and faces its species crisis, we will leave a tragic legacy of extinction and fail our obligations to future generations of Australians, and the international community.

scientific communication and education↗

bioRxiv2022

An Alzheimer's disease patient-derived olfactory cell model identifies gene expression changes associated with cognition

An early symptom of Alzheimers disease (AD) is an impaired sense of smell, for which the molecular basis remains elusive. Here, we generated human olfactory neurosphere-derived (ONS) cells from people with AD and mild cognitive impairment (MCI), and performed global RNA sequencing to determine gene expression changes. ONS cells expressed markers of neuroglial differentiation, providing a unique cellular model to explore early AD-associated disease pathways. Our transcriptomics data from ONS cells revealed differentially expressed genes (DEGs) associated with cognitive processes in AD cells compared to MCI, or matched healthy controls (HC). A-Kinase Anchoring Protein 6 (AKAP6) was the most significantly altered gene in AD compared to both MCI and HC, and has been linked to cognitive function. The greatest change in gene expression of all DEGs occurred between AD and MCI. Gene pathway analysis revealed defects in multiple cellular processes with aging, intellectual deficiency and alternative splicing being the most significantly dysregulated in AD ONS cells. Our results demonstrate that ONS cells can provide a cellular model for AD that recapitulates disease-associated differences. We have revealed potential novel genes, including AKAP6 that may have a role in AD, particularly MCI to AD transition, and should be further examined.

neuroscience↗

bioRxiv2022

Computational design of peptides to target Nav1.7 channel with high potency and selectivity for the treatment of pain

The voltage-gated sodium NaV1.7 channel plays a key role as a mediator of action potential propagation in C-fiber nociceptors and is an established molecular target for pain therapy. ProTx-II is a potent and moderately selective peptide toxin from tarantula venom that inhibits human NaV1.7 activation. Here we used available structural and experimental data to guide Rosetta design of potent and selective ProTx-II-based peptide inhibitors of human NaV1.7 channels. Functional testing of designed peptides using electrophysiology identified the PTx2-3127 and PTx2-3258 peptides with IC50s of 7 nM and 4 nM for hNaV1.7 and more than 1,000-fold selectivity over human NaV1.1, NaV1.3, NaV1.4, NaV1.5, NaV1.8, and NaV1.9 channels. PTx2-3127 inhibits NaV1.7 currents in mouse and human sensory neurons and shows efficacy in rat models of chronic and thermal pain when administered intrathecally. Rationally-designed peptide inhibitors of human NaV1.7 channels have transformative potential to define a new class of biologics to treat pain.

biophysics↗

bioRxiv2022

SpatialCorr: Identifying Gene Sets with Spatially Varying Correlation Structure

Recent advances in spatially resolved transcriptomics technologies enable both the measurement of genome-wide gene expression profiles and their mapping to spatial locations within a tissue. A first step in spatial transcriptomics data analysis is identifying genes with expression that varies spatially, and robust statistical methods exist to address this challenge. While useful, these methods do not detect spatial changes in the coordinated expression within a group of genes. To this end, we present SpatialCorr, a method for identifying sets of genes with spatially varying correlation structure. Given a collection of gene sets pre-defined by a user, SpatialCorr tests for spatially induced differences in the correlation of each gene set within tissue regions, as well as between and among regions. An application to cutaneous squamous cell carcinoma demonstrates the power of the approach for revealing biological insights not identified using existing methods.

bioinformatics↗

bioRxiv2022

CRSP: Comparative RNA-seq pipeline for species lacking both of sequenced genomes and reference transcripts

RNA sequencing (RNA-seq) has been a widely used high-throughput method to characterize transcriptomic dynamics spatiotemporally. However, typical RNA-seq data analysis pipelines depend on either a sequenced genome or corresponding reference transcripts or them both. This restriction makes the use of RNA-seq for species lacking both of sequenced genomes and corresponding reference transcripts challenging. Nile grass rat (Arvicanthis niloticus) is a diurnal rodent species with several unique characteristics making it as a useful model to study diet-induced type 2 diabetes and other physiological or behavioral processes due to its diurnal nature. However, there is neither a high-quality annotated Nile grass rat genome nor a reference transcript sets available so far, making it technically challenging to perform large-scale RNA-seq based transcriptomic studies. Although we are working on the first draft of Nile grass rat genome, a well annotated genome typically requires several rounds of manually reviewing curated transcripts and can take years to achieve. To solve this problem, we developed a Comparative RNA-Seq Pipeline (CRSP), integrating a comparative species strategy but not depending on a specific sequenced genome or species-matched reference transcripts. Benchmarking suggests the CRSP tool can achieve high accuracy to quantify gene expression levels. In this study, we generated the first ultra-deep (2.3 billion x 2 paired-end) Nile grass rat RNA-seq data from 59 biopsy samples representing 22 major organs, providing a unique resource and spatial gene expression reference for using Nile grass rat as a model to study human diseases. To facilitate a general use of CRSP, we also characterized the number of RNA-seq reads required for accurate estimation via simulation studies. CRSP and documents are available at: https://github.com/pjiang1105/CRSP. HighlightsO_LICRSP is a novel software tool which can quantify gene expression levels from RNA-seq data for species lacking both a sequenced genome and corresponding reference transcripts. C_LIO_LINile grass rat is a unique diurnal rodent species (day active but not night active) with several unique characteristics making it as a useful model to study diet-induced type 2 diabetes and other physiological or behavioral processes due to its diurnal nature. C_LIO_LIWe generated the first ultra-deep (2.3 billion x 2 paired-end reads) Nile grass rat RNA-seq data from 59 biopsy samples representing 22 major organs, providing a unique resource and spatial transcriptomic reference (e.g., tissue gene expression baseline) for using Nile grass rat as a model to study human diseases. C_LI

bioinformatics↗

bioRxiv2021

Automated identification of chicken distress vocalisations using deep learning models

The annual global production of chickens exceeds 25 billion birds, and they are often housed in very large groups, numbering thousands. Distress calling triggered by various sources of stress has been suggested as an "iceberg indicator" of chicken welfare. However, to date, the identification of distress calls largely relies on manual annotations, which is very labour-intensive and time-consuming. Thus, a novel light-VGG11 was developed to automatically identify chicken distress calls using recordings (3,363 distress calls and 1,973 natural barn sounds) collected on intensive chicken farms. The light-VGG11 was modified from VGG11 with a significantly smaller size in parameters (9.3 million vs 128 million) and 55.88% faster detection speed while displaying comparable performance, i.e., precision (94.58%), recall (94.89%), F1-score (94.73%), and accuracy (95.07%), therefore more useful for model deployment in practice. To further improve the light-VGG11s performance, we investigated the impacts of different data augmentation techniques (i.e., time masking, frequency masking, mixed spectrograms of the same class, and Gaussian noise) and found that they could improve distress calls detection by up to 1.52%. In terms of precision livestock farming, our research opens new opportunities for developing technologies used to monitor the output of distress calls in large, commercial chicken flocks.

animal behavior and cognition↗

bioRxiv2021

A haplotype-resolved genome assembly of the Nile rat facilitates exploration of the genetic basis of diabetes

The Nile rat (Avicanthis niloticus) is an important animal model for biomedical research, including the study of diurnal rhythms and type 2 diabetes. Here, we report a 2.5 Gb, chromosome-level reference genome assembly with fully resolved parental haplotypes, generated with the Vertebrate Genomes Project (VGP). The assembly is highly contiguous, with contig N50 of 11.1 Mb, scaffold N50 of 83 Mb, and 95.2% of the sequence assigned to chromosomes. We used a novel workflow to identify 3,613 segmental duplications and quantify duplicated genes. Comparative analyses revealed unique genomic features of the Nile rat, including those that affect genes associated with type 2 diabetes and metabolic dysfunctions. These include 14 genes that are heterozygous in the Nile rat or highly diverged from the house mouse. Our findings reflect the exceptional level of genomic detail present in this assembly, which will greatly expand the potential of the Nile rat as a model organism for genetic studies.

genomics↗

bioRxiv2021

3D models of Alzheimer's disease patient microglia recapitulate disease phenotype and show differential drug responses compared to 2D

Alzheimers disease (AD) is an incurable neurodegenerative disorder with a rapidly increasing prevalence worldwide. Current approaches targeting hallmark pathological features of AD have had no consistent clinical benefit. Neuroinflammation is a major contributor to neurodegeneration and hence, microglia, the brains resident immune cells, are an attractive target for potentially more effective therapeutic strategies. However, there is no current in vitro model system that faithfully recapitulates patient-specific microglial characteristics. To address this shortcoming, we developed novel 3D models of monocyte-derived microglia-like cells (MDMi) from AD patients. MDMi in 3D exhibited mature microglial features, including a highly branched morphology and enhanced bonafide microglial marker expression compared to 2D. Moreover, AD MDMi in 3D co-cultures with neuro-glial cells showed altered cell-to-cell interactions, growth factor and cytokine secretion profiles and responses to amyloid-{beta}. Drug screening assays in 3D AD MDMi revealed different cytokine responses compared to 2D. Our study demonstrates disease- and drug-specific responses in 3D MDMi models that are not apparent in 2D and presents a new 3D platform for more effective and personalised drug testing.

neuroscience↗

bioRxiv2020

ALS monocyte-derived microglia reveal cytoplasmic TDP-43 accumulation, DNA damage, and cell-specific impairment of phagocytosis associated with disease progression.

AimsAmyotrophic lateral sclerosis (ALS) is a multifactorial neurodegenerative disease characterised by the loss of upper and lower motor neurons. Neuroinflammation mediated by microglial activation is evident in post-mortem brain tissues, and in brain imaging of patients with ALS. However, the exact role of microglia in ALS remains to be elucidated partly due to the lack of an accurate microglial model system that is able to recapitulate the clinical pathology of ALS. Moreover, direct sampling of microglia from patients with ALS is not feasible, further limiting the study of microglial function in ALS. To address this shortcoming, we describe an approach that generates monocyte-derived microglia (MDMi) that are capable of expressing molecular markers, and functional characteristics similar to resident human brain microglia. Importantly, MDMi can be routinely and reproducibly generated from ALS patient blood, and reveal patient heterogeneity associated with age, sex and disease subgroup. MethodsMDMi were successfully established from all 30 ALS patients, including 15 patients with slow disease progression, 6 with intermediate progression, and 9 with rapid progression, together with 20 non-affected heathy controls (HC). ResultsOur ALS MDMi model recapitulated canonical pathological features of ALS including non-phosphorylated and phosphorylated-TDP-43-positive pathological inclusions. We further observed significantly impaired phagocytosis, altered cytokine expression and microglial morphology, as well as elevated DNA damage in ALS compared to HC MDMi. Abnormal phagocytosis was observed in all ALS cases, and was correlated to the progression of disease. Moreover, in-depth analysis of individual microglia revealed cell-specific variation in phagocytic function that was significantly altered, and exacerbated in rapid disease progression. ConclusionsOur approach enabled us to generate ALS patient microglia from peripheral blood samples using a rapid, robust, cost-effective, and reproducible protocol. We have shown that ALS monocyte-derived microglia have significantly altered functional behaviour compared to age-matched HCs, with a major deficit in phagocytic activity. This is also the first demonstration of abnormal TDP-43 localisation in microglia grown from ALS patients. Overall, this approach is highly applicable to monitor disease progression and can be applied as a functional readout in clinical trials for anti-neuroinflammatory agents. Additionally, this model system can be used as a basis for personalised therapeutic treatment for ALS, as well as other neurodegenerative diseases.

neuroscience↗

bioRxiv2020

SKiM - A generalized literature-based discovery system for uncovering novel biomedical knowledge from PubMed

Literature-based discovery (LBD) uncovers undiscovered public knowledge by linking terms A to C via a B intermediate. Existing LBD systems are limited to process certain A, B, and C terms, and many are not maintained. We present SKiM (Serial KinderMiner), a generalized LBD system for processing any combination of A, Bs, and Cs. We evaluate SKiM via the rediscovery of discoveries by Don Swanson, who pioneered LBD. Using only literature from the 19th century up to a year before Swansons discoveries, SKiM uncovers all five discoveries. We apply SKiM to repurposing drugs for 26 conditions of high prevalence. Manual analysis confirmed 65 discoveries useful for four diseases from Swansons discoveries from one to 31 years prior to their first validation by clinical trials. SKiM predicts many new potential drug candidates representing prime targets for wet lab validation. SKiM can be applied to any biomedical inquiry sufficiently mentioned in the literature.

bioinformatics↗

bioRxiv2020

Enhancing biological signals and detection rates in single-cell RNA-seq experiments with cDNA library equalization

Considerable effort has been devoted to refining experimental protocols having reduced levels of technical variability and artifacts in single-cell RNA-sequencing data (scRNA-seq). We here present evidence that equalizing the concentration of cDNA libraries prior to pooling, a step not consistently performed in single-cell experiments, improves gene detection rates, enhances biological signals, and reduces technical artifacts in scRNA-seq data. To evaluate the effect of equalization on various protocols, we developed Scaffold, a simulation framework that models each step of an scRNA-seq experiment. Numerical experiments demonstrate that equalization reduces variation in sequencing depth and gene-specific expression variability. We then performed a set of experiments in vitro with and without the equalization step and found that equalization increases the number of genes that are detected in every cell by 17-31%, improves discovery of biologically relevant genes, and reduces nuisance signals associated with cell cycle. Further support is provided in an analysis of publicly available data.

genomics↗

bioRxiv2020

CHARTS: A web application for characterizing and comparing tumor subpopulations in publicly available single-cell RNA-seq datasets

BackgroundSingle-cell RNA-seq (scRNA-seq) enables the profiling of genome-wide gene expression at the single-cell level and in so doing facilitates insight into and information about cellular heterogeneity within a tissue. Perhaps nowhere is this more important than in cancer, where tumor and tumor microenvironment heterogeneity directly impact development, maintenance, and progression of disease. While publicly available scRNA-seq cancer datasets offer unprecedented opportunity to better understand the mechanisms underlying tumor progression, metastasis, drug resistance, and immune evasion, much of the available information has been underutilized, in part, due to the lack of tools available for aggregating and analysing these data. ResultsWe present CHARacterizing Tumor Subpopulations (CHARTS), a computational pipeline and web application for analyzing, characterizing, and integrating publicly available scRNA-seq cancer datasets. CHARTS enables the exploration of individual gene expression, cell type, malignancy-status, differentially expressed genes, and gene set enrichment results in subpopulations of cells across multiple tumors and datasets. ConclusionCHARTS is an easy to use, comprehensive platform for exploring single-cell subpopulations within tumors across the ever-growing collection of public scRNA-seq cancer datasets. CHARTS is freely available at charts.morgridge.org.

bioinformatics↗

bioRxiv2020

Rapid changes in chromatin structure during dedifferentiation of primary hepatocytes in vitro

Primary hepatocytes are widely used in the pharmaceutical industry to screen drug candidates for hepatotoxicity, but isolated hepatocytes quickly dedifferentiate and lose their mature metabolic function in culture. Attempts have been made to better recapitulate the in vivo liver environment in culture, but the full spectrum of signals required to maintain hepatocyte function in vitro remains elusive. Here we studied the dedifferentiation process in detail through RNA-sequencing of hepatocytes cultured over eight days. We identified three distinct phases of dedifferentiation. An early phase, where mature hepatocyte genes are rapidly downregulated in a matter of hours. A middle phase, where fetal genes are activated, leading to hepatocytes with a fetal phenotype. A late phase, where initially rare contaminating non-parenchymal cells over-grow the culture as the hepatocytes gradually die. Using genetically tagged hepatocytes, we demonstrate that the cells reactivating fetal marker alpha-fetoprotein arise from cells previously expressing the mature hepatocyte marker albumin, and not from albumin negative precursor cells, proving that hepatocytes undergo true dedifferentiation. To better understand the signaling events that result in the rapid down-regulation of mature hepatocyte genes, we examined changes in chromatin accessibility of hepatocytes during the first 24h of culture using ATAC-seq. We find that drastic and rapid changes in chromatin accessibility occurs immediately upon start of culture. Using binding motif analysis of the areas of open chromatin sharing similar temporal profiles, we identify several candidate transcription factors potentially involved in the dedifferentiation of primary hepatocytes in culture.

cell biology↗