Biology subjects

Foster, R.

Publications and source records attributed to Foster, R..

At least 19 recordsLinked to original sources

bioRxiv2026

Spatially-resolved multimodal profiling identifies functionally-heterogeneous cancer-associated fibroblasts associated with poor radiotherapy outcomes in muscle-invasive bladder cancer.

Background: Cancer-associated fibroblasts (CAFs) contribute to systemic therapy resistance in muscle-invasive bladder cancer (MIBC), but their functional heterogeneity and relevance to curative bladder-preserving radiotherapy, are poorly understood. Methods: Transcriptomic analysis was performed on 279 tumours from BC2001, a phase 3 radiotherapy clinical trial. To study CAF heterogeneity, we integrated bulk RNA-seq, single-cell spatial analysis of multiplex immunofluorescence images and quantification of extracellular matrix (ECM) features in 155 MIBC biopsies. The functional heterogeneity of distinct CAF populations was evaluated by single-nuclear RNA-seq. Spatial interactions between CAF populations and CD8+ T-cells was assessed and the relevance of lymphocytes to radiation responses was evaluated in a CAF-enriched murine bladder cancer model (BBN963). Results: BC2001 patients with CAF-enriched tumours had worse overall survival (HR=1.671, 95% CI 1.221-2.287, Log-rank p=0.0012). CAF abundance and antigen expression was highly heterogenous. Podoplanin (PDPN) was expressed on the majority of CAFs and was associated with inflammatory pathways. Enrichment of CAF gene signatures was associated with a significant increase in CAFs expressing fibroblast activation protein (FAP) (p=0.004) and dense ECM features (p=0.0004). Fifty-three percent of tumours exhibited stromal CD8+ T-cell exclusion with significant enrichment in FAP-dominant neighbourhoods (p<0.001). In vivo, lymphocytes were critical for radiation-induced tumour control, indicating that immune cold or excluded tumours may have limited radiotherapy responses. Conclusion: In MIBC, CAFs are associated with poor radiotherapy outcomes. Multiple mechanisms are deployed by functionally-heterogeneous CAFs, including promotion of chronic inflammation by PDPN+ CAFs and ECM remodelling by FAP+ CAFs which impact CD8+ T-cell distribution and radiation responses.

cancer biology↗

bioRxiv2026

Multimodal molecular profiling of the metabolic penumbra in hyperacute stroke

BackgroundThe ischemic penumbra, a metabolically compromised yet potentially salvageable region surrounding the ischemic core, is a prime target for acute stroke intervention. Yet an objective molecular definition of the penumbra, particularly during the earliest stages of ischemia, remains lacking. Methods and ResultsWe applied principal component analysis (PCA) followed by k-means clustering to high-resolution mass spectrometry imaging data covering multiple metabolic pathways to identify a metabolically defined penumbra in a mouse model of hyperacute stroke (45 min middle cerebral artery occlusion, MCAO). Targeted spatial metabolomic profiling by matrix-assisted laser desorption/ionization (MALDI) and desorption electrospray ionization (DESI) reveals a distinct penumbral metabolic profile, marked by relative preservation of high-energy phosphates, comparable lactate accumulation, and reduced succinate accumulation relative to the core. Spatial transcriptomics revealed selective induction of immediate-early genes, including Npas4, Fos and Junb, within the penumbra. Consistently, imaging mass cytometry shows enrichment of phospho-histone H3 (pHH3) within the penumbra, suggesting a chromatin-associated response potentially linked to immediate-early gene activation. ConclusionTogether, these findings provide a multimodal molecular atlas of the hyperacute metabolically defined penumbra and reveal molecular features that facilitates its identification and inform future therapeutic strategies.

neuroscience↗

bioRxiv2025

Dynamic proteome profiling uncovers age-related impairments in proteostasis and the protective effects of resistance exercise in human skeletal muscle

A loss of proteostasis is a primary hallmark of ageing that has emerged from mechanistic studies in model organisms, but little is currently known about changes to proteostasis in the muscle of older humans. We used stable isotope labelling (deuterium oxide; D2O) in vivo, and peptide mass spectrometry of muscle samples to investigate differences in proteome dynamics between the muscle of younger (28 {+/-} 5 y; n=4) and older (69 {+/-} 3 y; n=4) men during either habitual activity or resistance exercise training. We quantified the abundance of 1787 proteins and the turnover rate of 1046 proteins in bi-lateral samples of vastus lateralis (n=32 samples total) taken before and after a 15-day program including 5 sessions of unilateral leg-press exercise (3 sets of 10 repetitions at 90% of 10 RM). Our protein abundance profiling revealed a stoichiometric imbalance within the proteostasis network in aged skeletal muscle, including subunits of eIF3, subunits of 40S and 60S ribosomal proteins. The rate of bulk, mixed-protein synthesis was not different between younger and older men, but most ribosomal proteins were less abundant in the muscle of older participants, suggesting ribosomes in older muscle may exhibit increased translational efficiency to maintain similar levels of protein turnover compared to ribosomes in younger muscle. Resistance exercise partially restored age-related disruptions to the proteostasis network. In older skeletal muscle, resistance exercise specifically increased the absolute turnover rate (ATR) of mixed mitochondrial proteins, with increased fractional turnover rate (FTR) of prohibitin 1 (PHB1) and profilin-1 (PROF1), and increased abundance of prohibitin 2 (PHB2). These adaptations may suggest resistance exercise promotes mitochondrial proteostasis by facilitating the synthesis and maintenance of key mitochondrial proteins. Thus, our Dynamic Proteome Profiling data provide an impetus for further exploration of the role of proteostasis in maintaining skeletal muscle quality and supports resistance exercise as a potential therapeutic strategy to promote healthy skeletal muscle ageing in humans. In BriefNishimura et al. used Dynamic Proteome Profiling to uncover whether the distorted proteomic landscape of ageing skeletal muscle is associated with altered turnover of specific proteins. Basal muscle from older men exhibits a divergence in protein abundance between subunits of eIF3 and subunits of 40S and 60S ribosomal proteins, whereas resistance exercise partially restored age-related disruptions in the muscle proteome. In older muscle, protein-specific turnover generally increases after resistance exercise, independent of changes in protein abundance, suggesting improved protein quality and renewal. Created in BioRender. Nishimura, Y. (2025) https://BioRender.com/p2a1aio HighlightsO_LIDynamic Proteome Profiling in human skeletal muscle ageing C_LIO_LIAgeing alters muscle proteostasis C_LIO_LIMixed-muscle protein synthesis does not differ between younger and older men C_LIO_LIResistance exercise increased mitochondrial protein turnover specifically in older muscle C_LIO_LIProtein-specific responses to resistance exercise differed between age groups C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/684531v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@11d0c8corg.highwire.dtl.DTLVardef@22f451org.highwire.dtl.DTLVardef@d62118org.highwire.dtl.DTLVardef@16eda9_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2025

Validated Antimalarial Drug Target Discovery using Genome-Scale Metabolic Modelling

Given the rapid resistance of Plasmodium falciparum to antimalarial drugs, there is a continual need for new treatments. A genome-scale metabolic (GSM) model was developed with integrated omic and constraint-based, experimental flux-balance data to predict genes essential for P. falciparum growth as drug targets. We selected the highly ranked P. falciparum UMP-CMP kinase (UCK) to test its necessity and the ability to inhibit growth with inhibitors. Conditional deletion mutants using the DiCre recombinase system, generated by CRISPR-Cas genome editing, exhibited defective asexual growth and stage-specific developmental arrest. Based on in silico and in vitro screening, inhibitors were identified that are selective for P. falciparum UCK and exhibit antiparasitic activity. This study, for the first time, shows assertions from a GSM model identifying novel, validated, "druggable" targets. These findings show a role for GSM models in antimalarial drug discovery and identify P. falciparum UCK as a novel, valid malaria drug target.

microbiology↗

bioRxiv2024

A workflow for practical training in ecological genomics using Oxford Nanopore long-read sequencing

Long-read single molecule sequencing technologies continue to grow in popularity for genome assembly and provide an effective way to resolve large and complex genomic variants. However, uptake of these technologies for teaching and training is hampered by the complexity of high molecular weight DNA extraction protocols, the time required for library preparation and the costs for sequencing, as well as challenges with downstream data analyses. Here, we present a full long-read workflow optimised for teaching, that covers each stage from DNA extraction, to library preparation and sequencing, to data QC and genome assembly and characterisation, that can be completed in under two weeks. We use a specific case study of plant identification, where students identify an anonymous plant sample by sequencing and assembling the genome and comparing it to other samples and to reference databases. In testing, long-read genome skimming of nine wild-collected plant species extracted with a modified kit-based approach produced an average of 8Gb of Oxford Nanopore data, enabling the complete assembly of plastid genomes, and partial assembly of nuclear genomes. In the classroom, all students were able to complete the protocols, and to correctly identify their plant samples based on BOLD searches of barcoding loci extracted from the plastid genome, coupled with phylogenetic analyses of whole plastid genomes. We supply all the learning material and raw data allowing this to be adapted to a range of teaching settings.

genomics↗

bioRxiv2023

The Cyclophilin Inhibitor Rencofilstat Decreases HCV-induced Hepatocellular Carcinoma Independently of Its Antiviral Activity

There is an urgent need for the identification of new drugs that inhibit HCV-induced hepatocellular carcinoma (HCC). Our work demonstrates that cyclophilin inhibitors (CypI) represent such new drugs. We demonstrated that the non-immunosuppressive cyclosporine A (CsA) analog (CsAa) rencofilstat possesses dual therapeutic activities for the treatment of HCV infection and HCV-induced HCC. Specifically, we showed that HCV infection of humanized mice results in the progressive development of HCC. This was true for four genotypes tested (1 to 4). Remarkably, we demonstrated that rencofilstat inhibits the development of HCV-induced HCC in mice even when added 16 weeks post-infection when HCC is well established. Importantly, we showed that rencofilstat drastically reduces HCC progression independently of its anti-HCV activity. Indeed, the CypI rencofilstat inhibits HCC while other anti-HCV agents such as NS5A (NS5Ai) and NS5B (NS5Bi) fail to reduce HCC. In conclusion, this study shows for the first time that the CypI rencofilstat represents a potent therapeutic agent for the treatment of HCV-induced HCC.

microbiology↗

bioRxiv2023

The virus-encoded ion channel "viroporin" activity of the agnoprotein is required for BK Polyomavirus release from infected kidney cells

BK polyomavirus (BKPyV) is a common opportunistic pathogen and the causative agent of several diseases in transplant patients and the immunosuppressed. Despite its importance, aspects of the virus lifecycle such as how the virus exits an infected cells, remain poorly understood. The late region of the BKPyV genome encodes an auxillery protein called agnoprotein. We and others have shown that agnoprotein is an essential factor in virus release, and the loss of agnoprotein results in an accumulation of virus particles within the nucleus of an infected cell. The functions of agnoprotein necessary for this egress phenotype are not known. Here we demonstrate that agnoprotein shows properties associated with viroporins, a group of virus-encoded membrane spanning proteins that play key roles in virus infection and release. We demonstrate that agnoprotein oligomerises and perturbs membranes in cells. The development of a novel recombinant agnoprotein expression system permitted the identification of the first small molecules targeting agnoprotein. These compounds abrogated agnoprotein viroporin activity in vitro and reduced virus release, indicating that viroporin activity contributes to the phenotype observed in agnoprotein knockout viruses. The identification of channel activity should enhance the future understanding of the physiological function of agnoprotein and could represent an important target for antiviral intervention.

microbiology↗

bioRxiv2022

Hypoconnectivity between anterior insula and amygdala in neonates with familial history of autism

BackgroundAltered resting state functional connectivity (FC) involving the anterior insula (aINS), a key node in the salience network, has been reported consistently in autism. MethodHere we examined, for the first time, FC between the aINS and the whole brain in a sample of full-term, postmenstrual age (PMA) matched neonates (mean 44.0 weeks, SD=1.5) who due to family history have high likelihood (HL) for developing autism (n=12) and in controls (n=41) without family history of autism (low likelihood, LL). Behaviors associated with autism were evaluated between 12 and 18 months (M=17.3 months, SD=2.5) in a subsample (25/53) of participants using the First Year Inventory (FYI). ResultsCompared to LL controls, HL neonates showed hypoconnectivity between left aINS and left amygdala. Lower connectivity between the two nodes was associated with higher FYI risk scores in the social domain (r(25) = -.561, p=.003) and this association remained robust when maternal mental health factors were considered. Considering that a subsample of LL participants (n=14/41) underwent brain imaging during the fetal period at PMA 31 and 34 weeks, in an exploratory analysis, we evaluated prospectively development of the LaINS-Lamy connectivity and found that the two areas strongly coactivate throughout the third trimester of pregnancy. ConclusionsThe study identifies left lateralized anterior insula - amygdala connectivity as a potential target of further investigation into neural circuitry that enhances likelihood of future onset of social behaviors associated with autism during neonatal and potentially prenatal periods.

neuroscience↗

bioRxiv2021

The genome sequence of the lesser marbled fritillary, Brenthis ino, and evidence for a segregating neo-Z chromosome

The lesser marbled fritillary, Brenthis ino (Rottemburg, 1775), is a species of Palearctic butterfly. Male B. ino individuals have been reported to have between 12 and 14 pairs of chromosomes, a much reduced chromosome number than is typical in butterflies. Here we present a chromosome-level genome assembly for B. ino, as well as gene and transposable element annotations. The assembly is 411.8 Mb in span with contig and scaffold N50s of 9.6 and 29.5 Mb respectively. We also show evidence that the male individual from which we generated HiC data was heterozygous for a neo-Z chromosome, consistent with inheriting 14 chromosomes from one parent and 13 from the other. This genome assembly will be a valuable resource for studying chromosome evolution in Lepidoptera, as well as for comparative and population genomics more generally.

evolutionary biology↗

bioRxiv2021

Dystrophin regulates peripheral circadian SRF signalling

Dystrophin is a sarcolemmal protein essential for muscle contraction and maintenance, absence of which leads to the devastating muscle wasting disease Duchenne muscular dystrophy (DMD)[1, 2]. Dystrophin has an actin-binding domain [3-5], which specifically binds and stabilises filamentous (F)-actin[6], an integral component of the RhoA-actin-serum response factor (SRF)-pathway[7]. The RhoA-actin-SRF-pathway plays an essential role in circadian signalling whereby the hypothalamic suprachiasmatic nucleus, transmits systemic cues to peripheral tissues, activating SRF and transcription of clock target genes[8, 9]. Given dystrophin binds F-actin and disturbed SRF-signalling disrupts clock entrainment, we hypothesised that dystrophin loss causes circadian deficits. Here we show for the first time alterations in the RhoA-actin-SRF-signalling-pathway, in both dystrophin-deficient myotubes and dystrophic mouse models. Specifically, we demonstrate reduced F/G-actin ratios and nuclear MRTF, dysregulation of core clock and downstream target-genes, and down-regulation of key circadian genes in muscle biopsies from DMD patients harbouring an array of mutations. Further, disrupted circadian locomotor behaviour was observed in dystrophic mice indicative of disrupted SCN signalling, and indeed dystrophin protein was absent in the SCN of dystrophic animals. Dystrophin is thus a critically important component of the RhoA-actin-SRF-pathway and a novel mediator of circadian signalling in peripheral tissues, loss of which leads to circadian dysregulation.

cell biology↗

bioRxiv2021

Inhibitors of the Small Membrane (M) Protein Viroporin Prevent Zika Virus Infection

Flaviviruses, including Zika virus (ZIKV), are a significant global health concern, yet no licensed antivirals exist to treat disease. The small Membrane (M) protein plays well-defined roles during viral egress and remains within virion membranes following release and maturation. However, it is unclear whether M plays a functional role in this setting. Here, we show that M forms oligomeric membrane-permeabilising channels in vitro, with increased activity at acidic pH and sensitivity to the prototypic channel-blocker, rimantadine. Accordingly, rimantadine blocked an early stage of ZIKV cell culture infection. Structure-based channel models, comprising hexameric arrangements of two trans-membrane domain protomers were shown to comprise more stable assemblages than other oligomers using molecular dynamics (MD) simulations. Models contained a predicted lumenal rimantadine binding site, as well as a second druggable target region on the membrane-exposed periphery. In silico screening enriched for repurposed drugs/compounds predicted to bind to either one site or the other. Hits displayed superior potency in vitro and in cell culture compared with rimantadine, with efficacy demonstrably linked to virion-resident channels. Finally, rimantadine effectively blocked ZIKV viraemia in preclinical models, supporting that M constitutes a physiologically relevant target. This could be explored by repurposing rimantadine, or development of new M-targeted-therapies.

microbiology↗

bioRxiv2021

Txikispora philomaios n. sp., n. g., a Micro-Eukaryotic Pathogen of Amphipods, Reveals Parasitism and Hidden Diversity in Class Filasterea

This study provides a morphological, ultrastructural, and phylogenetic characterization of a novel micro-eukaryotic parasite (2.3-2.6 {micro}m) infecting genera Echinogammarus and Orchestia. Longitudinal studies across two years revealed that infection prevalence peaked in late April and May, reaching 64% in Echinogammarus sp. and 15% in Orchestia sp., but was seldom detected during the rest of the year. The parasite infected predominantly haemolymph, connective tissue, tegument, and gonad, although hepatopancreas and nervous tissue were affected in heavier infections, eliciting melanization and granuloma formation. Cell division occurred inside walled parasitic cysts, often within host haemocytes, resulting in haemolymph congestion. Small subunit (18S) rRNA gene phylogenies including related environmental sequences placed the novel parasite as a highly divergent lineage within Class Filasterea, which together with Choanoflagellatea represent the closest protistan relatives of Metazoa. We describe the new parasite as Txikispora philomaios n. sp. n. g., the first confirmed parasitic filasterean lineage, which otherwise comprises four free-living flagellates and a rarely observed endosymbiont of snails. Lineage-specific PCR probing of other hosts and surrounding environments only detected T. philomaios in the platyhelminth Procerodes sp. We expand the known diversity of Filasterea by targeted searches of metagenomic datasets, resulting in 13 previously unknown lineages from environmental samples.

pathology↗

bioRxiv2020

The SARS-CoV-2 spike protein disrupts the cooperative function of human cardiac pericytes - endothelial cells through CD147 receptor-mediated signalling: a potential non-infective mechanism of COVID-19 microvascular disease

Severe coronavirus disease 2019 (COVID-19) manifests as a life-threatening microvascular syndrome. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses the Spike (S) protein to engage with its receptors and infect host cells. To date, it is still not known whether heart vascular pericytes (PCs) are infected by SARS-CoV-2, and if the S protein alone provokes PC dysfunction. Here, we aimed to investigate the effects of the S protein on primary human cardiac PC signalling and function. Results show, for the first time, that cardiac PCs are not permissive to SARS-CoV-2 infection in vitro, whilst a recombinant S protein alone elicits functional alterations in PCs. This was documented as: (1) increased migration, (2) reduced ability to support endothelial cell (EC) network formation on Matrigel, (3) secretion of pro-inflammatory molecules typically involved in the cytokine storm, and (4) production of pro-apoptotic factors responsible for EC death. Next, adopting a blocking strategy against the S protein receptors angiotensin-converting enzyme 2 (ACE2) and CD147, we discovered that the S protein stimulates the phosphorylation/activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) through the CD147 receptor, but not ACE2, in PCs. The neutralisation of CD147, either using a blocking antibody or mRNA silencing, reduced ERK1/2 activation and rescued PC function in the presence of the S protein. In conclusion, our findings suggest that circulating S protein prompts vascular PC dysfunction, potentially contributing to establishing microvascular injury in organs distant from the site of infection. This mechanism may have clinical and therapeutic implications. Clinical perspectiveO_LISevere COVID-19 manifests as a microvascular syndrome, but whether SARS-CoV-2 infects and damages heart vascular pericytes (PCs) remains unknown. C_LIO_LIWe provide evidence that cardiac PCs are not infected by SARS-CoV-2. Importantly, we show that the recombinant S protein alone elicits cellular signalling through the CD147 receptor in cardiac PCs, thereby inducing cell dysfunction and microvascular disruption in vitro. C_LIO_LIThis study suggests that soluble S protein can potentially propagate damage to organs distant from sites of infection, promoting microvascular injury. Blocking the CD147 receptor in patients may help protect the vasculature not only from infection, but also from the collateral damage caused by the S protein. C_LI

cell biology↗

bioRxiv2020

Drug and siRNA screens identify ROCK2 as a therapeutic target for ciliopathies.

Primary cilia are microtubule-based organelles that act as cellular antennae to mediate vertebrate development and growth factor signalling. Defects in primary cilia result in a group of inherited developmental conditions known as ciliopathies. Ciliopathies often present with cystic kidney disease, a major cause of early renal failure that requires renal replacement therapies. Currently, only one drug, Tolvaptan, is licensed to slow the decline of renal function for the ciliopathy polycystic kidney disease. Novel therapeutic interventions for these conditions remain a pressing clinical need. We screened clinical development compounds for positive effects on cilia formation and function and identified fasudil hydrochloride as the top hit. Fasudil is a generic, off-patent drug that is a potent but broadly selective Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor. In a parallel whole genome siRNA-based reverse genetics phenotypic screen of positive modulators of cilia formation, we identified ROCK2 as the target molecule. We demonstrate that ROCK2 is a key mediator of cilium formation and function through effects on actin cytoskeleton remodelling. Our results indicate that specific ROCK2 inhibitors such as belumosudil (KD-025) could be repurposed for pharmacological intervention in cystic kidney disease. We propose that ROCK2 inhibition represents a novel, disease-modifying therapeutic approach for heterogeneous ciliopathies.

cell biology↗

bioRxiv2020

COVID Moonshot: Open Science Discovery of SARS-CoV-2 Main Protease Inhibitors by Combining Crowdsourcing, High-Throughput Experiments, Computational Simulations, and Machine Learning

We report the results of the COVID Moonshot, a fully open-science, crowd sourced, structure-enabled drug discovery campaign targeting the SARS-CoV-2 main protease. We discovered a non-covalent, non-peptidic inhibitor scaffold with lead-like properties that is differentiated from current main protease inhibitors. Our approach leveraged crowdsourcing, machine learning, exascale molecular simulations, and high-throughput structural biology and chemistry. We generated a detailed map of the structural plasticity of the SARS-CoV-2 main protease, extensive structure-activity relationships for multiple chemotypes, and a wealth of biochemical activity data. All compound designs (>18,000 designs), crystallographic data (>840 ligand-bound X-ray structures), assay data (>10,000 measurements), and synthesized molecules (>2,400 compounds) for this campaign were shared rapidly and openly, creating a rich open and IP-free knowledgebase for future anti-coronavirus drug discovery.

biochemistry↗

bioRxiv2020

Transient Commensal Clonal Interactions Can Drive Tumor Metastasis

To interrogate functional heterogeneity and crosstalk between tumor cells, we generated clonal populations from a patient-derived ovarian clear cell carcinoma model which forms malignant ascites and solid peritoneal tumors upon intraperitoneal transplantation in mice. The clonal populations were engineered with secreted Gaussia luciferase to monitor tumor growth dynamics and tagged with a unique DNA barcode to track their fate in multiclonal mixtures during tumor progression. Only one clone, CL31, grew robustly, generating exclusively malignant ascites. However, multiclonal mixtures formed large solid peritoneal metastases, populated almost entirely by CL31, suggesting that transient cooperative interclonal interactions were sufficient to promote metastasis of CL31. CL31 uniquely harbored ERBB2 amplification, and its acquired metastatic trait was dependent on transient exposure to amphiregulin, which was exclusively secreted by non-tumorigenic clones. Amphiregulin enhanced CL31 mesothelial clearance, a prerequisite for metastasis. These findings demonstrate that transient, ostensibly innocuous tumor subpopulations can promote metastases via "hit- and-run" commensal interactions.

cancer biology↗

bioRxiv2019

Site-directed M2 proton channel inhibitors enable synergistic combination therapy for rimantadine-resistant pandemic influenza

Pandemic influenza A virus (IAV) remains a significant threat to global health. Preparedness relies primarily upon a single class of neuraminidase (NA) targeted antivirals, against which resistance is steadily growing. The M2 proton channel is an alternative clinically proven antiviral target, yet a near-ubiquitous S31N polymorphism in M2 evokes resistance to licensed adamantane drugs. Hence, inhibitors capable of targeting N31 containing M2 (M2-N31) are highly desirable.\n\nRational in silico design and in vitro screens delineated compounds favouring either lumenal or peripheral M2 binding, yielding effective M2-N31 inhibitors in both cases. Hits included adamantanes as well as novel compounds, with some showing low micromolar potency versus pandemic \"swine\" H1N1 influenza (Eng195) in culture. Interestingly, a published adamantane-based M2-N31 inhibitor rapidly selected a resistant V27A polymorphism (M2-A27/N31), whereas this was not the case for non-adamantane compounds. Nevertheless, combinations of adamantanes and novel compounds achieved synergistic antiviral effects, and the latter synergised with the neuraminidase inhibitor (NAi), Zanamivir. Thus, site-directed drug combinations show potential to rejuvenate M2 as an antiviral target whilst reducing the risk of drug resistance.

microbiology↗

bioRxiv2019

Long metabarcoding of the eukaryotic rDNA operon to phylogenetically and taxonomically resolve environmental diversity

High-throughput environmental DNA metabarcoding has revolutionized the analysis of microbial diversity, but this approach is generally restricted to amplicon sizes below 500 base pairs. These short regions contain limited phylogenetic signal, which makes it impractical to use environmental DNA in full phylogenetic inferences. However, new long-read sequencing technologies such as the Pacific Biosciences platform may provide sufficiently large sequence lengths to overcome the poor phylogenetic resolution of short amplicons. To test this idea, we amplified soil DNA and used PacBio Circular Consensus Sequencing (CCS) to obtain a ~4500 bp region of the eukaryotic rDNA operon spanning most of the small (18S) and large subunit (28S) ribosomal RNA genes. The CCS reads were first treated with a novel curation workflow that generated 650 high-quality OTUs containing the physically linked 18S and 28S regions of the long amplicons. In order to assign taxonomy to these OTUs, we developed a phylogeny-aware approach based on the 18S region that showed greater accuracy and sensitivity than similarity-based and phylogenetic placement-based methods using shorter reads. The taxonomically-annotated OTUs were then combined with available 18S and 28S reference sequences to infer a well-resolved phylogeny spanning all major groups of eukaryotes, allowing to accurately derive the evolutionary origin of environmental diversity. A total of 1019 sequences were included, of which a majority (58%) corresponded to the new long environmental CCS reads. Comparisons to the 18S-only region of our amplicons revealed that the combined 18S-28S genes globally increased the phylogenetic resolution, recovering specific groupings otherwise missing. The long-reads also allowed to directly investigate the relationships among environmental sequences themselves, which represents a key advantage over the placement of short reads on a reference phylogeny. Altogether, our results show that long amplicons can be treated in a full phylogenetic framework to provide greater taxonomic resolution and a robust evolutionary perspective to environmental DNA.

microbiology↗