Biology subjects

Hall, M. D.

Publications and source records attributed to Hall, M. D..

At least 37 records · Page 2Linked to original sources

bioRxiv2021

Characterization and tissue localization of zebrafish homologs of the human ABCB1 multidrug transporter

Given its similarities with mammalian systems, the zebrafish has emerged as a potential model to study the blood-brain barrier (BBB). Capillary endothelial cells at the human BBB express high levels of P-glycoprotein (P-gp, encoded by the ABCB1 gene) and ABCG2 (encoded by the ABCG2 gene). However, little information has been available about ATP-binding cassette transporters expressed at the zebrafish BBB. In this study, we focus on the characterization and tissue localization of two genes that are similar to human ABCB1, zebrafish abcb4 and abcb5. Cytotoxicity assays with stably-transfected cell lines revealed that zebrafish Abcb5 cannot efficiently transport the substrates doxorubicin and mitoxantrone compared to human P-gp and zebrafish Abcb4. Additionally, zebrafish Abcb5 did not transport the fluorescent probes BODIPY-ethylenediamine or LDS 751, while they were readily transported by Abcb4 and P-gp. A high-throughput screen conducted with 90 human P-gp substrates confirmed that zebrafish Abcb4 has overlapping substrate specificity with P-gp. Basal ATPase activity of zebrafish Abcb4 and Abcb5 was comparable to that of human P-gp. In the brain vasculature, RNAscope probes to detect abcb4 colocalized with staining by the P-gp antibody C219, while abcb5 was not detected. Zebrafish abcb4 also colocalized with claudin-5 expression in brain endothelial cells. Abcb4 and Abcb5 had different tissue localizations in multiple zebrafish tissues, consistent with different functions. The data suggest that zebrafish Abcb4 most closely phenocopies P-gp and that the zebrafish may be a viable model to study the role of the multidrug transporter P-gp at the BBB.

pharmacology and toxicology↗

bioRxiv2021

Sexually antagonistic evolution of mitochondrial and nuclear linkage

Across eukaryotes, genes encoding bioenergetic machinery are located in both mitochondrial and nuclear DNA, and incompatibilities between the two genomes can be devastating. Mitochondria are often inherited maternally, and theory predicts sex-specific fitness effects of mitochondrial mutational diversity. Yet how evolution acts on linkage patterns between mitochondrial and nuclear genomes is poorly understood. Using novel mito-nuclear population genetic models, we show that the interplay between nuclear and mitochondrial genes maintains mitochondrial haplotype diversity within populations, and it selects both for sex-independent segregation of mitochondrion-interacting genes and for paternal leakage. These effects of genetic linkage evolution can eliminate male-harming fitness effects of mtDNA mutational diversity. With maternal mitochondrial inheritance, females maintain a tight mitochondrial-nuclear match, but males accumulate mismatch mutations because of the weak statistical associations between the two genomic components. Sex-independent segregation of mitochondria-interacting loci improves the mito-nuclear match. In a sexually antagonistic evolutionary process, male nuclear alleles evolve to increase the rate of recombination, while females evolve to suppress it. Paternal leakage of mitochondria can evolve as an alternative mechanism to improve the mito-nuclear linkage. Our modelling framework provides an evolutionary explanation for the observed paucity of mitochondrion-interacting genes on mammalian sex chromosomes and for paternal leakage in protists, plants, fungi, and some animals.

evolutionary biology↗

bioRxiv2020

Minimal information for Chemosensitivity assays (MICHA): A next-generation pipeline to enable the FAIRification of drug screening experiments

Chemosensitivity assays are commonly used for preclinical drug discovery and clinical trial optimization. However, data from independent assays are often discordant, largely attributed to uncharacterized variation in the experimental materials and protocols. We report here the launching of MICHA (Minimal Information for Chemosensitivity Assays), accessed via https://micha-protocol.org. Distinguished from existing efforts that are often lacking support from data integration tools, MICHA can automatically extract publicly available information to facilitate the assay annotation including: 1) compounds, 2) samples, 3) reagents, and 4) data processing methods. For example, MICHA provides an integrative web server and database to obtain compound annotation including chemical structures, targets, and disease indications. In addition, the annotation of cell line samples, assay protocols and literature references can be greatly eased by retrieving manually curated catalogues. Once the annotation is complete, MICHA can export a report that conforms to the FAIR principle (Findable, Accessible, Interoperable and Reusable) of drug screening studies. To consolidate the utility of MICHA, we provide FAIRified protocols from five major cancer drug screening studies, as well as six recently conducted COVID-19 studies. With the MICHA webserver and database, we envisage a wider adoption of a community-driven effort to improve the open access of drug sensitivity assays.

bioinformatics↗

bioRxiv2020

A Workflow of Integrated Resources to Catalyze Network Pharmacology Driven COVID-19 Research

MotivationIn the event of an outbreak due to an emerging pathogen, time is of the essence to contain or to mitigate the spread of the disease. Drug repositioning is one of the strategies that has the potential to deliver therapeutics relatively quickly. The SARS-CoV-2 pandemic has shown that integrating critical data resources to drive drug-repositioning studies, involving host-host, hostpathogen and drug-target interactions, remains a time-consuming effort that translates to a delay in the development and delivery of a life-saving therapy. ResultsHere, we describe a workflow we designed for a semi-automated integration of rapidly emerging datasets that can be generally adopted in a broad network pharmacology research setting. The workflow was used to construct a COVID-19 focused multimodal network that integrates 487 host-pathogen, 74,805 host-host protein and 1,265 drug-target interactions. The resultant Neo4j graph database named "Neo4COVID19" is accessible via a web interface and via API calls based on the Bolt protocol. We believe that our Neo4COVID19 database will be a valuable asset to the research community and will catalyze the discovery of therapeutics to fight COVID-19. Availabilityhttps://neo4covid19.ncats.io

systems biology↗

bioRxiv2020

An Enzymatic TMPRSS2 Assay for Assessment of Clinical Candidates and Discovery of Inhibitors as Potential Treatment of COVID-19

SARS-CoV-2 is the viral pathogen causing the COVID19 global pandemic. Consequently, much research has gone into the development of pre-clinical assays for the discovery of new or repurposing of FDA-approved therapies. Preventing viral entry into a host cell would be an effective antiviral strategy. One mechanism for SARS-CoV-2 entry occurs when the spike protein on the surface of SARS-CoV-2 binds to an ACE2 receptor followed by cleavage at two cut sites ("priming") that causes a conformational change allowing for viral and host membrane fusion. TMPRSS2 has an extracellular protease domain capable of cleaving the spike protein to initiate membrane fusion. A validated inhibitor of TMPRSS2 protease activity would be a valuable tool for studying the impact TMPRSS2 has in viral entry and potentially be an effective antiviral therapeutic. To enable inhibitor discovery and profiling of FDA-approved therapeutics, we describe an assay for the biochemical screening of recombinant TMPRSS2 suitable for high throughput application. We demonstrate effectiveness to quantify inhibition down to subnanomolar concentrations by assessing the inhibition of camostat, nafamostat and gabexate, clinically approved agents in Japan. Also, we profiled a camostat metabolite, FOY-251, and bromhexine hydrochloride, an FDA-approved mucolytic cough suppressant. The rank order potency for the compounds tested are: nafamostat (IC50 = 0.27 nM), camostat (IC50 = 6.2 nM), FOY-251 (IC50 = 33.3 nM) and gabexate (IC50 = 130 nM). Bromhexine hydrochloride showed no inhibition of TMPRSS2. Further profiling of camostat, nafamostat and gabexate against a panel of recombinant proteases provides insight into selectivity and potency.

biochemistry↗

bioRxiv2020

Targeting ACE2-RBD interaction as a platform for COVID19 therapeutics: Development and drug repurposing screen of an AlphaLISA proximity assay

The COVID-19 pandemic, caused by SARS-CoV-2, is a pressing public health emergency garnering rapid response from scientists across the globe. Host cell invasion is initiated through direct binding of the viral spike protein to the host receptor angiotensin-converting enzyme 2 (ACE2). Disrupting the spike-ACE2 interaction is a potential therapeutic target for treating COVID-19. We have developed a proximity-based AlphaLISA assay to measure binding of SARS-CoV-2 spike protein Receptor Binding Domain (RBD) to ACE2. Utilizing this assay platform, a drug-repurposing screen against 3,384 small molecule drugs and pre-clinical compounds was performed, yielding 25 high-quality, small-molecule hits that can be evaluated in cell-based models. This established AlphaLISA RBD-ACE2 platform can facilitate evaluation of biologics or small molecules that can perturb this essential viral-host interaction to further the development of interventions to address the global health pandemic.

biochemistry↗

bioRxiv2020

An OpenData portal to share COVID-19 drug repurposing data in real time

The National Center for Advancing Translational Sciences (NCATS) has developed an online open science platform - named the NCATS OpenData Portal (ODP) - for quickly and freely sharing complete NCATS translational datasets via an open-access, user-friendly interface. This paper describes the establishment of the ODP, initially deployed during the COVID-19 crisis, and provides a detailed analysis of COVID-19 drug repurposing screening datasets that served as the first large-scale use case for the platform. Over 10,000 compounds were tested across 17 quantitative high-throughput assays, covering a wide spectrum of the SARS-CoV-2 life cycle. In total, over 87,000 concentration-response curves and 426,000 data points were made publicly available on ODP in near real-time, enabling immediate access to complete datasets. The resource is flexible in accommodating various types and structures of data, and it has already expanded since its launch to host additional datasets for COVID-19, other viruses of pandemic potential, and beyond. The OpenData Portal has been designed as a scalable platform for real-time data sharing across drug discovery campaigns, regardless of disease area, with the overarching goal of accelerating discovery at NCATS, the NIH, and the greater scientific community.

microbiology↗

bioRxiv2020

Metabolic scaling has diversified among species, despite an evolutionary constraint within species

Metabolic rate scales disproportionally with body mass, such that the energetic cost of living is relatively lower in larger organisms. Theory emphasises the importance of fixed physical constraints on metabolic scaling, yet empirical data are lacking with which to assess how evolutionary processes (e.g. mutation, drift, selection) contribute to the observed variation in metabolic scaling across the tree of life. Using a large-scale quantitative genetic study of growth in cockroaches, we show that ontogenetic metabolic scaling is evolutionarily constrained due to an absence of additive genetic variation in juvenile metabolic rate and mass. Using a phylogenetic analysis, we also show that ontogenetic metabolic scaling is more similar among closely related species than among distant relatives, suggesting that the constraints on metabolic scaling are subject to change during lineage diversification. Our results are consistent with growing evidence that there is strong stabilising selection on combinations of mass and metabolic rate within species.

physiology↗

bioRxiv2020

SARS-CoV-2 genomics beyond the consensus sequence: evidence for circulating mixed viral populations

Extensive global sampling and whole genome sequencing of the pandemic virus SARS-CoV-2 have enabled researchers to characterise its spread, and to identify mutations that may increase transmission or enable the virus to escape therapies or vaccines. Two important components of viral spread are how frequently variants arise within individuals, and how likely they are to be transmitted. Here, we characterise the within-host diversity of SARS-CoV-2, and the extent to which genetic diversity is transmitted, by quantifying variant frequencies in 1390 clinical samples from the UK, many from individuals in known epidemiological clusters. We show that SARS-CoV-2 infections are characterised by low levels of within-host diversity across the entire viral genome, with evidence of strong evolutionary constraint in Spike, a key target of vaccines and antibody-based therapies. Although within-host variants can be observed in multiple individuals in the same phylogenetic or epidemiological cluster, highly infectious individuals with high viral load carry only a limited repertoire of viral diversity. Most viral variants are either lost, or occasionally fixed, at the point of transmission, consistent with a narrow transmission bottleneck. These results suggest potential vaccine-escape mutations are likely to be rare in infectious individuals. Nonetheless, we identified Spike variants present in multiple individuals that may affect receptor binding or neutralisation by antibodies. Since the fitness advantage of escape mutations in highly-vaccinated populations is likely to be substantial, resulting in rapid spread if and when they do emerge, these findings underline the need for continued vigilance and monitoring.

genomics↗

bioRxiv2020

The SARS-CoV-2 cytopathic effect is blocked with autophagy modulators

SARS-CoV-2 is a new type of coronavirus capable of rapid transmission and causing severe clinical symptoms; much of which has unknown biological etiology. It has prompted researchers to rapidly mobilize their efforts towards identifying and developing anti-viral therapeutics and vaccines. Discovering and understanding the virus’ pathways of infection, host-protein interactions, and cytopathic effects will greatly aid in the design of new therapeutics to treat COVID-19. While it is known that chloroquine and hydroxychloroquine, extensively explored as clinical agents for COVID-19, have multiple cellular effects including inhibiting autophagy, there are also dose-limiting toxicities in patients that make clearly establishing their potential mechanisms-of-action problematic. Therefore, we evaluated a range of other autophagy modulators to identify an alternative autophagy-based drug repurposing opportunity. In this work, we found that 6 of these compounds blocked the cytopathic effect of SARS-CoV-2 in Vero-E6 cells with EC50 values ranging from 2.0 to 13 µM and selectivity indices ranging from 1.5 to >10-fold. Immunofluorescence staining for LC3B and LysoTracker dye staining assays in several cell lines indicated their potency and efficacy for inhibiting autophagy correlated with the measurements in the SARS-CoV-2 cytopathic effect assay. Our data suggest that autophagy pathways could be targeted to combat SARS-CoV-2 infections and become an important component of drug combination therapies to improve the treatment outcomes for COVID-19.One Sentence Summary Blocking SARS-CoV-2 cytopathic effects with selective autophagy inhibitors underlying the clinical benefits of chloroquine and hydroxychloroquine.Competing Interest StatementThe authors have declared no competing interest.View Full Text

pharmacology and toxicology↗

bioRxiv2020

Trade-off between virulence and dispersal drives parasite evolution at experimental invasion front

Eco-evolutionary processes may play an important role in the spatial spread of infectious disease. Current theory predicts more exploitative parasites to evolve in highly connected populations or at the front of spreading epidemics. However, many parasites rely on host dispersal to reach new populations. This may lead to conflict between local transmission and global spread, possibly counteracting selection for higher virulence. Here, we used the freshwater host Paramecium caudatum and its bacterial parasite Holospora undulata to investigate parasite evolution under an experimental range expansion scenario with natural host dispersal. We find that parasites evolving at experimental range fronts favoured higher dispersal rates of infected hosts than did parasites evolving in core populations. Front parasites further showed lower levels of virulence (host division and survival) and delayed development of infection, consistent with parameter estimates from an epidemiological model that we fitted on experimental time-series data. This combined evidence suggests an evolutionary trade-off between virulence and host-mediated dispersal, with a concomitant reduction in the investment into horizontal transmission. Our experiment illustrates how parasite evolution can be shaped by divergent selection encountered in different segments of an epidemic wave. Such an interplay between demography and spatial selection has important implications for the understanding and management of emerging diseases, and, more generally, for biological invasions and other non-equilibrium scenarios of spreading populations. SIGNIFICANCE STATEMENTWhat drives parasite evolution in spatially expanding epidemics? Many parasites require dispersal of infected hosts to reach new patches, and this may produce specific adaptations enhancing spatial spread. We performed experimental range expansions in an aquatic model system, with natural dispersal of infected hosts. Parasites from experimental range fronts were less virulent and interfered less with host dispersal, but also invested less in horizontal transmission than parasites from the range core. Thus, dispersal adaptation at the front may come at a cost of reduced horizontal transmission, a trade-off rarely considered in theoretical models on parasite virulence evolution. These results have important implications in the context of emerging diseases, and for parasite evolution during biological invasions or other spatial non-equilibrium scenarios.

evolutionary biology↗

bioRxiv2020

Travelling with a parasite: the evolution of resistance and dispersal syndrome during experimental range expansion

Rapid evolutionary change during range expansions can lead to diverging range core and front populations, with the emergence of dispersal syndromes (coupled responses in dispersal and life-history traits). Besides intraspecific effects, range expansions may be impacted by interspecific interactions such as parasitism. Yet, despite the potentially large impact of parasites imposing additional selective pressures on the host, their role on range expansions remains largely unexplored. Using microcosm populations of the ciliate Paramecium caudatum and its bacterial parasite Holospora undulata, we studied experimental range expansions under parasite presence or absence. We found that the interaction of range expansion and parasite treatments affected the evolution of host dispersal syndromes. Namely, front populations showed different associations of population growth parameters and swimming behaviours than core populations, indicating divergent evolution. Parasitism reshaped trait associations, with hosts evolved in the presence of the parasite exhibiting overall increased resistance and reduced dispersal. Nonetheless, when comparing infected range core and front populations, we found a positive association, suggesting joint evolution of resistance and dispersal at the front. We conclude that host-parasite interactions during range expansions can change evolutionary trajectories; this in turn may feed back on the ecological dynamics of the range expansion and parasite epidemics.

evolutionary biology↗

bioRxiv2019

Keeping it clean: the cell culture quality control experience at the National Center for Advancing Translational Sciences

Quality control monitoring of cell lines utilized in biomedical research is of critical importance, and is critical for reproducibility of data. Two key pitfalls in tissue culture are 1) cell line authenticity, and 2) mycoplasma contamination. As a collaborative research institute, the National Center for Advancing Translational Sciences receives cell lines from a range of commercial and academic sources, that are adapted for high-throughput screening. Here, we describe the implementation of a routine NCATS-wide mycoplasma testing and short-tandem repeat (STR) testing for cell lines. Initial testing identified a >10% mycoplasma contamination rate, but the implementation of clearly defined protocols that included immediate destruction of contaminated cell lines wherever possible has led to a much-reduced mycoplasma contamination rate, and data for >2,000 cell line samples tested over 3 years, and case studies are provided. STR testing of 170 cell lines with established STR profiles revealed only 5 mis-identified cell lines received from external labs. The data collected over the three years since implementation of this systematic testing demonstrates the importance of continual vigilance for rapid identification of problem cell lines, for ensuring reproducible data in translational science research.

cell biology↗

bioRxiv2019

Identification of activators of human fumarate hydratase by quantitative high-throughput screening

Fumarate hydratase (FH) is a metabolic enzyme that is part of the Krebs-cycle, and reversibly catalyzes the hydration of fumarate to malate. Mutations of the FH gene have been associated with fumarate hydratase deficiency (FHD), hereditary leiomyomatosis, renal cell cancer (HLRCC), and other diseases. Currently there are no high-quality small molecule probes for studying human fumarate hydratase. To address this, we developed a quantitative high throughput screening (qHTS) FH assay and screened a total of 57,037 compounds from in-house libraries in dose-response. While no inhibitors of FH were confirmed, a series of phenyl-pyrrolo-pyrimidine-diones were identified as activators of human fumarate hydratase. These compounds were not substrates of fumarate hydratase, were inactive in a malate dehydrogenase counter screen, and showed no detectable reduction-oxidation activity. The binding of two compounds from the series to human fumarate hydratase was confirmed by microscale thermophoresis. The low hit rate in this screening campaign confirmed that FH is a tough target to modulate, and the small molecule activators of human fumarate hydratase reported here may serve as a starting point for further optimization and development into cellular probes of human FH and potential drug candidates.

biochemistry↗

bioRxiv2019

A High-Throughput Screen of a Library of Therapeutics Identifies Substrates of P-glycoprotein

The ATP-binding cassette transporter P-glycoprotein (P-gp) is known to limit brain penetration of many chemotherapy drugs. Although Food and Drug Administration guidelines require that potential interactions of investigational drugs with P-gp be explored, often this information does not enter into the literature. As such, we developed a high-throughput screen (HTS) to identify substrates of P-gp from a series of chemical libraries, testing a total of 10,804 compounds, most of which have known mechanisms of action. We used the CellTiter-Glo viability assay to test library compounds against parental KB-3-1 human cervical adenocarcinoma cells and the colchicine-selected sub-line KB-8-5-11 that over-expresses P-gp. KB-8-5-11 cells were also tested in the presence of a P-gp inhibitor (tariquidar) to assess reversability of transporter-mediated resistance. Of the tested compounds, a total of 90 P-gp substrates were identified including 55 newly identified P-gp substrates. Substrates were confirmed using an orthogonal killing assay against HEK-293 cells transfected with P-gp. We confirmed that AT7159 (cyclin-dependent kinase inhibitor); AT9283, (Janus kinase 2/3 inhibitor); ispinesib (kinesin spindle protein inhibitor); gedatolisib (PKI-587, phosphoinositide 3-kinase/mammalian target of rampamycin inhibitor); GSK-690693 (AKT inhibitor); and KW-2478 (heat shock protein 90 inhibitor) were substrates, and direct ATPase stimulation was assessed. ABCG2 was also found to confer high levels of resistance to AT9283, GSK-690693 and gedatolisib, while ispinesib, AT7519 and KW-2478 were weaker substrates. Combinations of P-gp substrates and inhibitors were assessed to demonstrate on-target synergistic cell killing. This data will be of use in determining understanding how chemotherapeutic agents will cross the blood-brain barrier.

pharmacology and toxicology↗

bioRxiv2019

Spatial control of oxygen delivery to 3D cultures alters cancer cell growth and gene expression

Commonly used monolayer cancer cell cultures fail to provide a physiologically relevant environment in terms of oxygen delivery. Here, we describe a three-dimensional bioreactor system where cancer cells are grown in Matrigel in modified 6-well plates. Oxygen is delivered to the cultures through a polydimethylsiloxane (PDMS) membrane at the bottom of the wells, with microfabricated PDMS pillars to control oxygen delivery. The plates receive 3% oxygen from below and 0% oxygen at the top surface of the media, providing a gradient of 3% to 0% oxygen. We compared growth and transcriptional profiles for cancer cells grown in Matrigel in the bioreactor, 3D cultures grown in 21% oxygen, and cells grown in a standard hypoxia chamber at 3% oxygen. Additionally, we compared gene expression of conventional 2D monolayer culture and 3D Matrigel culture in 21% oxygen. We conclude that controlled oxygen delivery may provide a more physiologically relevant 3D system.

bioengineering↗

bioRxiv2018

Cytotoxic Profiling of Annotated and Diverse Chemical Libraries Using Quantitative High-Throughput Screening

Cell-based phenotypic screening is a commonly used approach to discover biological pathways, novel drug targets, chemical probes and high-quality hit-to-lead molecules. Many hits identified from high-throughput screening campaigns are ruled out through a series of follow-up potency, selectivity/specificity, and cytotoxicity assays. Prioritization of molecules with little or no cytotoxicity for downstream evaluation can influence the future direction of projects, so cytotoxicity profiling of screening libraries at an early stage is essential for increasing the likelihood of candidate success. In this study, we assessed cell-based cytotoxicity of nearly 10,000 compounds in NCATS annotated libraries, and over 100,000 compounds in a diversity library, against four normal cell lines (HEK 293, NIH 3T3, CRL-7250 and HaCat) and one cancer cell line (KB 3-1, a HeLa subline). This large-scale library profiling was analyzed for overall screening outcomes, hit rates, pan-activity and selectivity. For the annotated library, we also examined the primary targets and mechanistic pathways regularly associated with cell death. To our knowledge, this is the first study to use high-throughput screening to profile a large screening collection (>100,000 compounds) for cytotoxicity in both normal and cancer cell lines. The results generated here constitutes a valuable resource for the scientific community and provides insight on the extent of cytotoxic compounds in screening libraries, identifying and avoiding compounds with cytotoxicity during high-throughput screening campaigns.

cell biology↗

bioRxiv2018

Resource-dependent dispersal and the irrelevance of extra-patch information for Daphnia carinata

Dispersal is fundamental to population dynamics and it is increasingly apparent that, despite most models treating dispersal as a constant, many organisms make dispersal decisions based upon information gathered from the environment. Ideally, organisms would make fully informed decisions, with knowledge of both intra-patch conditions (conditions in their current location) and extra-patch conditions (conditions in alternative locations). Acquiring information is energetically costly however, and extra-patch information will typically be costlier to obtain than intra-patch information. As a consequence, theory suggests that organisms will often make partially informed dispersal decisions, utilising intra-patch information only. We test this proposition in an experimental two-patch system using populations of the aquatic crustacean, Daphnia carinata. We manipulated conditions (food availability) in the populations home patch, and in its alternative patch. We found that D. carinata made use of intra-patch information (resource limitation in the home patch induced a ten-fold increase in dispersal probability) but made no use of extra-patch information (resource limitation in the alternative patch did not affect dispersal probability). Our work highlights the very large influence that information can have on dispersal probability, but also that dispersal decisions will often be made in only a partially informed manner. The magnitude of the response we observed also adds to the growing chorus that condition-dependence may be a significant driver of variation in dispersal.

ecology↗