Biology subjects

Hill, C.

Publications and source records attributed to Hill, C..

At least 55 records · Page 3Linked to original sources

bioRxiv2021

Simons Collaborative Marine Atlas Project (Simons CMAP): an open-source portal to share, visualize and analyze ocean data

Simons Collaborative Marine Atlas Project (Simons CMAP) is an open-source data portal that interconnects large, complex, and diverse public data sets currently dispersed in different formats across different Oceanography discipline-specific databases. Simons CMAP is designed to streamline the retrieval of custom subsets of data, the generation of data visualizations, and the analyses of diverse data, thus expanding the power of these potentially underutilized data sets for cross-disciplinary studies of ocean processes. We describe a unified architecture that allows numerical model outputs, satellite products, and field observations to be readily shared, mined, and integrated regardless of data set size or resolution. A current focus of Simons CMAP is integration of physical, chemical, and biological data sets essential for characterizing the biogeography of key marine microbes across ocean basins and seasonal cycles. Using a practical example, we demonstrate how our unifying data harmonization plans significantly simplifies and allows for systematic data integration across all Simons CMAP data sets.

ecology↗

bioRxiv2021

REPEATED INJURY PROMOTES TRACHEOBRONCHIAL TISSUE STEM CELL ATTRITION

Chronic lung disease has been attributed to stem cell aging and/or exhaustion. To address this issue, we investigated the lifespan of tracheobronchial tissue stem cells (TSC) over time and in response to repeated injury. Chromatin and nucleotide labeling studies in mice indicated that: 1) injury activated a subset of the TSC pool and that this process conserved TSC over time; and 2) activated TSC were predisposed to further proliferation and this activated state lead to terminal differentiation. Analysis of human TSC and clonal isolates indicated that repeated TSC proliferation led to telomere shortening and analysis of TSC from Dyskeratosis Congenita donors indicated that mutations in telomere biology genes accelerated TSC depletion. RNAseq and functional studies indicated that human TSC terminated as a secretory committed cell. These data support a model in which a repeated epithelial injury depletes the TSC pool and initiates the abnormal repair associated with chronic lung disease.

cell biology↗

bioRxiv2020

Long-term persistence of crAss-like phage crAss001 is associated with phase variation in Bacteroides intestinalis

The crAss-like phages are ubiquitous and highly abundant members of the human gut virome that infect commensal bacteria of the order Bacteroidales. Although incapable of classical lysogeny, these viruses demonstrate unexplained long-term persistence in the human gut microbiome, dominating the virome in some individuals. Here we demonstrate that rapid phase variation of alternate capsular polysaccharides plays an important role in dynamic equilibrium between phage sensitivity and resistance in B. intestinalis cultures, allowing phage and bacteria to multiply in parallel. The data also suggests the role of concomitant phage persistence mechanisms associated with delayed lysis of infected cells, such as carrier state infection. From an ecological and evolutionary standpoint this type of phage-host interaction is consistent with the Piggyback-the-Winner model, which suggests a preference towards lysogenic or other "benign" forms of phage infection when the host is stably present at high abundance. TeaserCrAss-like phage persistence in Bacteroides is associated with capsule phase-variation and additional unexplored mechanisms.

microbiology↗

bioRxiv2020

Microbiome-based environmental monitoring of a dairy processing facility highlights the challenges associated with low microbial-load samples

Food processing environments can harbor microorganisms responsible for food spoilage or foodborne disease. Efficient and accurate identification of microorganisms throughout the food chain can allow the identification of sources of contamination and the timely implementation of control measures. Currently, microbial monitoring of the food chain relies heavily on culture-based techniques. These assays are determined on the microbes expected to be present in the environment, and thus do not cater for unexpected contaminants. Many culture-based assays are also unable to distinguish between undesirable taxa and closely related harmless species. Furthermore, even when multiple culture-based approaches are used in parallel, it is still not possible to comprehensively characterize the entire microbiology of a food-chain sample. High throughput DNA sequencing represents a potential means through which microbial monitoring of the food chain can be enhanced. While sequencing platforms, such as the Illumina MiSeq, NextSeq and NovaSeq, are most typically found in research or commercial sequencing laboratories, newer portable platforms, such as the Oxford Nanopore Technologies (ONT) MinION, offer the potential for rapid analysis of food chain microbiomes. In this study, having initially assessed the ability of rapid MinION-based sequencing to discriminate between different microbes within a simple mock metagenomic mixture of related food spoilage, spore-forming microorganisms. Subsequently, we proceeded to compare the performance of both ONT and Illumina sequencing for environmental monitoring of an active food processing facility. Overall, ONT MinION sequencing provided accurate classification to species level, which was comparable to Illumina-derived outputs. However, while the MinION-based approach provided a means of easy library preparations and portability, the high concentrations of DNA needed to run the rapid sequencing protocols was a limiting factor, requiring the random amplification of template DNA in order to generate sufficient material for analysis.

microbiology↗

bioRxiv2020

A toolkit to generate inducible and interconvertible Drosophila transgenes

The existence of three independent binary systems for conditional gene expression (Gal4/UAS; LexA/LexAop; QF/QUAST) has greatly expanded versatile genetic analyses in the fruit fly Drosophila melanogaster; however, the experimental application of these tools is limited by the need to generate multiple collections of non-interchangeable transgenic fly strains for each inducible gene expression system. To address this practical limitation, we developed a modular vector that contains the regulatory elements from all three binary systems, enabling Gal4-, LexA- or QF-dependent expression of transgenes. Our methods also incorporate DNA elements that facilitate independent site-specific recombination and elimination of regulatory UAS, LexAop or QUAST modules with spatial and temporal control, thus offering unprecedented possibilities and logistical advantages for in vivo genetic modulation and efficient interconversion of overexpression transgenic fly lines.

genetics↗

bioRxiv2020

The human oral phageome is highly diverse and rich in jumbo phages

Until recently, the contribution of bacteriophages to the composition and function of the human microbiome has been largely overlooked. Recent developments in discovering novel bacteriophages from human metagenomes have been mostly focused on the gut. Here we profile and compare the phageome of 633 human oral sites and 221 paired gut phageomes acquired from countries worldwide. Phageome profiles are specific to oral sites and the gut across individuals, with the largest separation between the oral cavity and the gut. The differences in phage genotypes is bolstered by distinct phage host compositions between these sites. The greatest diversity of phages is found on the tongue and in dental plaque. Notably, 37 unique circular jumbo phage genomes were identified in oral sites, particularly on the dorsum of the tongue, whilst none were found in the gut. Oral sites provide conducive environments, such as robust biofilms, that can harbour genetically diverse phages.Competing Interest StatementThe authors have declared no competing interest.View Full Text

genomics↗

bioRxiv2020

An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 and multiple endemic, epidemic and bat coronavirus

Coronaviruses (CoVs) traffic frequently between species resulting in novel disease outbreaks, most recently exemplified by the newly emerged SARS-CoV-2. Herein, we show that the ribonucleoside analog {beta}-D-N4-hydroxycytidine (NHC, EIDD-1931) has broad spectrum antiviral activity against SARS-CoV 2, MERS-CoV, SARS-CoV, and related zoonotic group 2b or 2c Bat-CoVs, as well as increased potency against a coronavirus bearing resistance mutations to another nucleoside analog inhibitor. In mice infected with SARS-CoV or MERS-CoV, both prophylactic and therapeutic administration of EIDD-2801, an orally bioavailable NHC-prodrug (b-D-N4-hydroxycytidine-5-isopropyl ester), improved pulmonary function, and reduced virus titer and body weight loss. Decreased MERS-CoV yields in vitro and in vivo were associated with increased transition mutation frequency in viral but not host cell RNA, supporting a mechanism of lethal mutagenesis. The potency of NHC/EIDD-2801 against multiple coronaviruses, its therapeutic efficacy, and oral bioavailability in vivo, all highlight its potential utility as an effective antiviral against SARS-CoV-2 and other future zoonotic coronaviruses.

microbiology↗

bioRxiv2020

Rapid and scalable profiling of nascent RNA with fastGRO

Genome-wide profiling of nascent RNA has become a fundamental tool to study transcription regulation. Over the past decade, next-generation sequencing has fostered development of a handful of techniques (i.e. GRO-seq, PRO-seq, TT-seq and NET-seq) that map unprocessed transcripts originating from both the coding and the noncoding portion of the genome. Unlike steady-state RNA sequencing, nascent RNA profiling mirrors the real-time activity of RNA Polymerases and provides an accurate readout of transcriptome-wide variations that occur during short time frames (i.e. response to external stimuli or rapid metabolic changes). Some species of nuclear RNAs, albeit functional, have a short half-life and can only be accurately gauged by nascent RNA techniques (i.e. lincRNAs and eRNAs). Furthermore, these techniques capture uncapped post-cleavage RNA at termination sites or promoter-associated antisense RNAs, providing a unique insight into RNAPII dynamics and processivity. Here we present a run-on assay with 4s-UTP labelling, followed by reversible biotinylation and affinity purification via streptavidin. Our protocol allows streamlined sample preparation within less than 3 days. We named the technique fastGRO (fast Global Run-On). We show that fastGRO is highly reproducible and yields a more complete and extensive coverage of nascent RNA than comparable techniques. Importantly, we demonstrate that fastGRO is scalable and can be performed with as few as 0.5x10^6 cells.

genomics↗

bioRxiv2019

Noncoding microdeletion in mouse Hgf disrupts neural crest migration into the stria vascularis, reduces the endocochlear potential and suggests the neuropathology for human nonsyndromic deafness DFNB39

Hepatocyte growth factor (HGF) is a multifunctional protein that signals through the MET receptor. HGF stimulates cell proliferation, cell dispersion, neuronal survival and wound healing. In the inner ear, levels of HGF must be fine-tuned for normal hearing. In mouse, a deficiency of HGF expression limited to the auditory system, or over-expression of HGF, cause neurosensory deafness. In human, noncoding variants in HGF are associated with nonsyndromic deafness DFNB39. However, the mechanism by which these noncoding variants causes deafness was unknown. Here, we reveal the cause of this deafness using a mouse model engineered with a noncoding intronic 10bp deletion (del10) in Hgf, which is located in the 3UTR of a conserved short isoform (Hgf/NK0.5). Mice homozygous for del10 exhibit moderate-to-profound hearing loss at four weeks of age as measured by pure-tone auditory brainstem responses (ABRs). The wild type +80 millivolt endocochlear potential (EP) was significantly reduced in homozygous del10 mice compared to wild type littermates. In normal cochlea, EPs are dependent on ion homeostasis mediated by the stria vascularis (SV). Previous studies showed that developmental incorporation of neural crest cells into the SV depends on signaling from HGF/MET. We show by immunohistochemistry that in del10 homozygotes, neural crest cells fail to infiltrate the developing SV intermediate layer. Phenotyping and RNAseq analyses reveal no other significant abnormalities in other tissues. We conclude that, in the inner ear, the noncoding del10 mutation in Hgf leads to dysfunctional ion homeostasis in the SV and a loss of EP, recapitulating human DFNB39 deafness.\n\nSignificance StatementHereditary deafness is a common, clinically and genetically heterogeneous neurosensory disorder. Previously we reported that human deafness DFNB39 is associated with noncoding variants in the 3UTR of a short isoform of HGF encoding hepatocyte growth factor. For normal hearing, HGF levels must be fined-tuned as an excess or deficiency of HGF cause deafness in mouse. Using a Hgf mutant mouse with a small 10 base pair deletion recapitulating a human DFNB39 noncoding variant, we demonstrate that neural crest cells fail to migrate into the stria vascularis intermediate layer, resulting in a significantly reduced endocochlear potential, the driving force for sound transduction by inner ear hair cells. HGF-associated deafness is a neurocristopathy but, unlike many other neurocristopathies, it is not syndromic.

genetics↗

bioRxiv2019

Crumbs 2 mediates ventricular layer remodelling to form the adult spinal cord central canal

In the spinal cord, the adult central canal forms through a poorly-understood process termed dorsal collapse that involves attrition and remodelling of the pseudostratified dorsal ventricular layer. Here we show, in mouse, that dorsal ventricular layer cells adjacent to midline Nestin(+) radial glia downregulate the apical polarity proteins Crumbs2 (CRB2) and aPKC and delaminate in a step-wise manner; concomitantly, Nestin(+) radial glial end-feet ratchet down, to repeat this process. Nestin(+) radial glia secrete a factor that promotes cell delamination. This activity is mimicked by a secreted variant of CRB2 (CRB2S), which is specifically expressed by dorsal midline Nestin(+) radial glia. In cultured cells, CRB2S associated with apical membranes and decreased cell cohesion. Analysis of Crb2F/F/Nestin-Cre+/- mice further confirmed an essential role for CRB2 in dorsal collapse. We propose a model in which CRB2S promotes the progressive attrition of the ventricular layer without loss of overall integrity. This novel mechanism may operate more widely to promote orderly progenitor delamination.

developmental biology↗

bioRxiv2019

Immune Profiling of Atherosclerotic Plaques Identifies Innate and Adaptive Dysregulations Associated with Ischemic Cerebrovascular Events

Atherosclerosis is driven by multifaceted contributions of the immune system within the circulation and at vascular focal sites. Yet the specific immune dysregulations within the atherosclerotic lesions that lead to clinical cerebro- and cardiovascular complications (i.e. ischemic stroke and myocardial infarction) are poorly understood. Here, using single-cell mass cytometry with Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) we found that atherosclerotic plaques were enriched in activated, differentiated, and exhausted subsets of T cells vs. blood. Next, using single-cell proteomic, transcriptomic, and cell-to-cell interaction analyses we found unique functional dysregulations of both T cells and macrophages in plaques of patients with clinically symptomatic (SYM; recent stroke of TIA) or asymptomatic (ASYM, no recent stroke) carotid artery disease. SYM plaques were enriched with a distinct CD4+ T cell subset, and T cells were activated, differentiated and presented subset specific exhaustion. SYM macrophages presented alternatively activated phenotypes including subsets associated with plaque vulnerability. In ASYM plaques, T cells and macrophages were activated and displayed a strong IL-1{beta} signaling across cell types, that was absent in SYM plaques. The identification of plaque-specific innate and adaptive immune dysregulations associated with cerebrovascular events provides the basis for the design of precisely tailored cardiovascular immunotherapies.

immunology↗

bioRxiv2019

Complete single neuron reconstruction reveals morphological diversity in molecularly defined claustral and cortical neuron types

Ever since the seminal findings of Ramon y Cajal, dendritic and axonal morphology has been recognized as a defining feature of neuronal types. Yet our knowledge concerning the diversity of neuronal morphologies, in particular distal axonal projection patterns, is extremely limited. To systematically obtain single neuron full morphology on a brain-wide scale, we established a platform with five major components: sparse labeling, whole-brain imaging, reconstruction, registration, and classification. We achieved sparse, robust and consistent fluorescent labeling of a wide range of neuronal types by combining transgenic or viral Cre delivery with novel transgenic reporter lines. We acquired high-resolution whole-brain fluorescent images from a large set of sparsely labeled brains using fluorescence micro-optical sectioning tomography (fMOST). We developed a set of software tools for efficient large-volume image data processing, registration to the Allen Mouse Brain Common Coordinate Framework (CCF), and computer-assisted morphological reconstruction. We reconstructed and analyzed the complete morphologies of 1,708 neurons from the striatum, thalamus, cortex and claustrum. Finally, we classified these cells into multiple morphological and projection types and identified a set of region-specific organizational rules of long-range axonal projections at the single cell level. Specifically, different neuron types from different regions follow highly distinct rules in convergent or divergent projection, feedforward or feedback axon termination patterns, and between-cell homogeneity or heterogeneity. Major molecularly defined classes or types of neurons have correspondingly distinct morphological and projection patterns, however, we also identify further remarkably extensive morphological and projection diversity at more fine-grained levels within the major types that cannot presently be accounted for by preexisting transcriptomic subtypes. These insights reinforce the importance of full morphological characterization of brain cell types and suggest a plethora of ways different cell types and individual neurons may contribute to the function of their respective circuits.

neuroscience↗

bioRxiv2019

The human gut virome is highly diverse, stable and individual-specific

The human gut contains a vast array of viruses, mostly bacteriophages. The majority remain uncharacterised and their roles in shaping the gut microbiome and in impacting on human health remain poorly understood. Here we performed a longitudinal focused metagenomic study of faecal bacteriophage populations in healthy adults. Our results reveal high temporal stability and individual specificity of bacteriophage consortia which correlates with the bacterial microbiome. We report the existence of a stable, numerically predominant individual-specific persistent personal virome. Clustering of bacteriophage genomes and de novo taxonomic annotation identified several groups of crAss-like and Microviridae bacteriophages as the most stable colonizers of the human gut. CRISPR-based host prediction highlighted connections between these stable viral communities and highly predominant gut bacterial taxa such as Bacteroides, Prevotella and Faecalibacterium. This study provides insights into the structure of the human gut virome and serves as an important baseline for hypothesis-driven research.

microbiology↗

bioRxiv2019

Autochthonous faecal virome transplantation (FVT) reshapes the murine microbiome after antibiotic perturbation

BackgroundIt has become increasingly apparent that establishing and maintaining a complex and diverse gut microbiome is fundamental to human health. There are growing efforts to identify methods that can modulate and influence the microbiome, especially in individuals who due to disease or circumstance have experienced a disruption in their native microbiome. Faecal microbial transplantation (FMT) is one method that restores diversity to the microbiome of an individual by introducing microbes from a healthy donor. FMT introduces a complete microbiome into the recipient, including the bacteriome, archaeome, mycome and virome. In this study we investigated whether transplanting an autochthonous faecal virome consisting primarily of bacteriophages could impact a bacteriome disrupted by antibiotic treatment (Faecal Virome Transplantation; FVT).\n\nResultsFollowing disruption of the bacteriome by penicillin and streptomycin, test mice (n=8) received a bacteria free, faecal transplant, while Control mice (n=8) received a heated and nuclease treated control. The bacteriomes (as determined via 16S rRNA sequencing) of mice that received an FVT, in which bacteriophages predominate, separated from those of the Control mice as determined by principle co-ordinate analysis (PCoA), and contained differentially abundant taxa that reshaped the bacteriome profile such that it more closely resembled that of the pre-treatment mice. Similarly, metagenomic sequencing of the virome confirmed that the bacteriophages present in the gut of treatment and Control mice differed over time in both abundance and diversity, with transplanted phages seen to colonise the FVT mice.\n\nConclusionsAn autochthonous virome transplant impacts on the bacteriome and virome of mice following antibiotic treatment. The virome, consisting mainly of bacteriophages, reshapes the bacteriome such that it more closely resembles the pre-antibiotic state. To date, faecal transplants have largely focussed on transferring living microbes, but given that bacteriophage are inert biological entities incapable of colonising in the absence of a sensitive host they could form a viable alternative that may have fewer safety implications and that could be delivered as a robust formulation.

microbiology↗

bioRxiv2019

Mülleriann mimicry in bumble bees is a transient continuum

Mullerian mimicry theory states that frequency dependent selection should favour geographic convergence of harmful species onto a shared colour pattern. As such, mimetic patterns are commonly circumscribed into discrete mimicry complexes each containing a predominant phenotype. Outside a few examples in butterflies, the location of transition zones between mimicry complexes and the factors driving them has rarely been examined. To infer the patterns and processes of Mullerian mimicry, we integrate large-scale data on the geographic distribution of colour patterns of all social bumble bees across the contiguous United States and use these to quantify colour pattern mimicry using an innovative machine learning approach based on computer vision and image recognition. Our data suggests that bumble bees exhibit a manifold of similar, but imperfect colour patterns, that continuously transition across the United States, supporting the idea that mimicry is not discrete. We propose that bumble bees are mimicking a perceptual colour pattern average that is evolutionarily transient. We examine three comimicking polymorphic species, Bombus flavifrons, B. melanopygus, and B. bifarius, where active selection is driving colour pattern frequencies and determine that their colour pattern transition zones differ in location and breadth within a broad region of poor mimicry. Furthermore, we explore factors driving these differences such as mimicry selection dynamics and climate.

evolutionary biology↗

bioRxiv2018

Choice of assembly software has a critical impact on virome characterisation

BackgroundThe viral component of microbial communities play a vital role in driving bacterial diversity, facilitating nutrient turnover and shaping community composition. Despite their importance, the vast majority of viral sequences are poorly annotated and share little or no homology to reference databases. As a result, investigation of the viral metagenome (virome) relies heavily on de novo assembly of short sequencing reads to recover compositional and functional information. Metagenomic assembly is particularly challenging for virome data, often resulting in fragmented assemblies and poor recovery of viral community members. Despite the essential role of assembly in virome analysis and difficulties posed by these data, current assembly comparisons have been limited to subsections of virome studies or bacterial datasets.\n\nDesignThis study presents the most comprehensive virome assembly comparison to date, featuring 16 metagenomic assembly approaches which have featured in human virome studies. Assemblers were assessed using four independent virome datasets, namely; simulated reads, two mock communities, viromes spiked with a known phage and human gut viromes.\n\nResultsAssembly performance varied significantly across all test datasets, with SPAdes (meta) performing consistently well. Performance of MIRA and VICUNA varied, highlighting the importance of using a range of datasets when comparing assembly programs. It was also found that while some assemblers addressed the challenges of virome data better than others, all assemblers had limitations. Low read coverage and genomic repeats resulted in assemblies with poor genome recovery, high degrees of fragmentation and low accuracy contigs across all assemblers. These limitations must be considered when setting thresholds for downstream analysis and when drawing conclusions from virome data.

bioinformatics↗

bioRxiv2018

ΦCrAss001, a member of the most abundant bacteriophage family in the human gut, infects Bacteroides

{Phi}CrAss001, isolated from human faecal material, is the first member of the extensive crAssphage family to be grown in pure culture. The bacteriophage infects the human gut symbiont Bacteroides intestinalis, confirming in silico predictions of the likely host. Genome analysis demonstrated that the phage DNA is 102 kb in size, has an unusual genome organisation and does not possess any obvious genes for lysogeny. In addition, electron microscopy confirms that {varphi}crAss001 has a podovirus-like morphology. Despite the absence of lysogeny genes, {varphi}crAss001 replicates in a way that does not disrupt proliferation of the host bacterium and is able to maintain itself in continuous host culture.

microbiology↗

bioRxiv2018

Biology and taxonomy of crAss-like bacteriophages, the most abundant virus in the human gut

CrAssphage is yet to be cultured even though it represents the most abundant virus in the gut microbiota of humans. Recently, sequence based classification was performed on distantly related crAss-like phages from multiple environments, leading to the proposal of a familial level taxonomic group [Yutin N, et al. (2018) Discovery of an expansive bacteriophage family that includes the most abundant viruses from the human gut. Nat Microbiol 3(1):38-46]. Here, we assembled the metagenomic sequencing reads from 702 human faecal virome/phageome samples and obtained 98 complete circular crAss-like phage genomes and 145 contigs [≥]70kb. In silico comparative genomics and taxonomic analysis was performed, resulting in a classification scheme of crAss-like phages from human faecal microbiomes into 4 candidate subfamilies composed of 10 candidate genera. Moreover, laboratory analysis was performed on faecal samples from an individual harbouring 7 distinct crAss-like phages. We achieved propagation of crAss-like phages in ex vivo human faecal fermentations and visualised Podoviridae virions by electron microscopy. Furthermore, detection of a crAss-like phage capsid protein could be linked to metagenomic sequencing data confirming crAss-like phage structural annotations.\n\nSignificanceCrAssphage is the most abundant biological entity in the human gut, but it remains uncultured in the laboratory and its host(s) is unknown. CrAssphage was not identified in metagenomic studies for many years as its sequence is so different from anything present in databases. To this day, it can only be detected from sequences assembled from metagenomics or viromic datasets (crAss - cross Assembly). In this study, we identified 243 new crAss-like phages from human faecal metagenomic studies. Taxonomic analysis of these crAss-like phages highlighted their extensive diversity within the human microbiome. We also present the first propagation of crAssphage in faecal fermentations and provide the first electron micrographs of this extraordinary bacteriophage.

microbiology↗