Biology subjects

Hill, C.

Publications and source records attributed to Hill, C..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

RNA editing is a molecular clock in unmodified human cells

Despite major advances in spatial RNA sequencing, the ability to extract temporal information in RNA sequencing experiments is still limited. Here, we describe Transcriptome Timestamping (T2), a system which harnesses naturally occurring A-to-I editing of RNA transcripts in unmodified human cells to infer transcriptional history. T2 provides age estimates for individual RNA transcripts, and serves as an endogenous molecular recorder, differentiating between complex transcriptional programs. We show that T2 can identify transient and transitional transcriptional programs in primary differentiating monocytes that are not apparent from gene expression analysis alone, including a regulatory module in the monocyte-to-macrophage transition that, to our knowledge, has not yet been described in humans. Finally, we show that T2 can also be applied to single cell data, allowing us to identify transcriptional programs in heterogeneous populations, such as asynchronously dividing cells. T2 is a scalable approach to temporal transcriptomics that can be applied to track the activity of thousands of genes in unmodified, primary human cells and tissues, with no genetic engineering.

genomics↗

bioRxiv2024

Adaptations in gut Bacteroidales facilitate stable co-existence with their lytic bacteriophages

BackgroundBacteriophages (phages) and bacteria within the gut microbiome persist in long-term stable coexistence. These interactions are driven by eco-evolutionary dynamics, where bacteria employ a variety of mechanisms to evade phage infection, while phages rely on counterstrategies to overcome these defences. Among the most abundant phages in the gut are the crAss-like phages that infect members of the Bacteroidales, in particular Bacteroides. In this study, we explored some of the mechanisms enabling the co-existence of four phage-Bacteroidales host pairs in vitro using a multi-omics approach (transcriptomics, proteomics and metabolomics). These included three Bacteroides species paired with three crAss-like phages (Bacteroides intestinalis and {phi}crAss001, Bacteroides xylanisolvens and {phi}crAss002, and an acapsular mutant of Bacteroides thetaiotaomicron with DAC15), and Parabacteroides distasonis paired with the siphovirus {phi}PDS1. ResultsWe show that phase variation of individual capsular polysaccharides (CPSs) is the primary mechanism promoting phage co-existence in Bacteroidales, but this is not the only strategy. Alternative resistance mechanisms, while potentially less efficient than CPS phase variation, can be activated to support bacterial survival by regulating gene expression and resulting in metabolic adaptations, particularly in amino acid degradation pathways. These mechanisms, also likely regulated by phase variation, enable bacterial populations to persist in the presence of phages, and vice versa. An acapsular variant of B. thetaiotaomicron demonstrated broader transcriptomic, proteomic, and metabolomic changes, supporting the involvement of additional resistance mechanisms beyond CPS variation. ConclusionsThis study advances our understanding of long-term phage-host interaction, offering insights into the long-term persistence of crAss-like phages and extending these observations to other phages, such as {phi}PDS1. Knowledge of the complexities of phage-bacteria interactions is essential for designing effective phage therapies and improving human health through targeted microbiome interventions.

microbiology↗

bioRxiv2024

Large scale capsid-mediated mobilisation of bacterial genomic DNA in the gut microbiome

Transducing bacteriophages and gene transfer agents (GTA) are constrained by their capsids structural properties in the length of host DNA they can package. Nanopore sequencing of intact capsid-packaged DNA molecules with full-sized reads can be used to establish the precise lengths and identity of individual packaged DNA molecules and their association with specific bacterial hosts. This approach was validated using a few well-characterised transducing systems, and then applied to study bacterial DNA encapsidation in the faecal microbiomes from three healthy human donors. Bacterial DNA encapsidation appears to be widespread in the microbiome with up to 5.4% of capsid-packaged DNA in the gut virome being of bacterial (non-prophage) origin. Generalised transduction and GTA activity are especially prevalent in the families Oscillospiraceae and Ruminococcaceae, whereas an example of lateral transduction was observed in genus Bacteroides. In addition to that, induction of prophages in a variety of highly prevalent gut bacteria was observed.

microbiology↗

bioRxiv2024

Assessment of a high-throughput mass spectrometry method to accelerate biomarker discovery in clinical cancer cohorts using volumetric absorptive microsampling (VAMS) devices.

Identification of biomarkers of early-stage disease typically requires analysis of very large cohorts which can only be reasonably achieved using high-throughput methods. We have developed and optimised novel methods for whole blood analysis using volumetric absorptive microsampling devices to produce over 3000 protein identifications by LCMS on a Q Exactive HF-X Orbitrap. These methods were tested using a set of whole blood samples from lung cancer patients and matching healthy controls finding 455 differentially expressed proteins, using a mid-throughput method enabling analysis of 18 samples per day. To increase throughput for larger clinical cohorts, a 60-sample per day method was tested on a Sciex ZenoTOF 7600. The high-throughput method produced 1.5-fold fewer protein identifications and a higher overall % CV compared to the mid-throughput method. Despite the lower numbers, it produced a set of 36 disease-relevant and discriminatory differentially expressed proteins that, using a machine learning model, could differentiate between the disease and control samples with an area under the ROC curve (AUC) of 88.9% using random forest algorithms. These data support the use of high-throughput mass spectrometry methods to screen large cohorts for diagnostic biomarkers that can then be followed up with more targeted analyses.

biochemistry↗

bioRxiv2024

Investigating the use of novel blood processing methods to boost the identification of biomarkers of non-small cell lung cancer

1.Background and objectivesDiagnosis of non-small cell lung cancer (NSCLC) currently relies on imaging and in-clinic visits, however these methods are not effective at detecting early-stage disease. The investigation into blood-based biomarkers aims to simplify the diagnostic process and has the potential for identifying disease-associated changes before they can be seen using imaging techniques. Methods and designIn this study, plasma and frozen whole blood cell pellets from patients with NSCLC and healthy controls were processed using both classical as well as novel techniques to produce a unique set of 4 sample types from a single blood draw. Samples were analysed using 12 commercially available immunoassay kits in addition to liquid chromatography mass spectrometry using a Q Exactive HF-X Orbitrap to collectively screen 3974 proteins as potential biomarkers. Results and conclusionsAnalysis of all sample types produced a set of 522 differentially expressed proteins, with conventional blood analysis (proteomic analysis of plasma) accounting for only 7 of that total. Boosted regression tree analysis of the differentially expressed proteins produced a panel of 13 proteins that were able to discriminate between controls and NSCLC patients with an area under the ROC curve (AUC) of 0.864 for the set. Our rapid and reproducible blood preparation and analysis methods enable the production of high-quality data from small aliquots of complex samples that are typically seen as requiring significant fractionation prior to proteomic analysis.

cancer biology↗

bioRxiv2024

Vps4 substrate binding and coupled mechanisms of Vps4p substrate recruitment and release from autoinhibition

The ESCRT pathways AAA+ ATPase, Vps4p, remodels ESCRT-III complexes to drive membrane fission. Here, we use peptide binding assays to further the understanding of substrate specificity and the mechanism of autoinhibition. Our results reveal unexpected sequence preference to the substrate binding groove and an elegant mechanism of regulation that couples localization to substrate with release from autoinhibition.

biochemistry↗

bioRxiv2024

The structure of full-length AFPK supports the ACP linker in a role that regulates iterative polyketide and fatty acid assembly

We present the full structures of two animal fatty acid synthase (FAS)-like polyketide synthases (AFPKs), PKS1 and PKS2 from Elysia chlorotica. Unlike the related FAS enzymes that use malonate to produce reduced lipids, EcPKS1 and EcPKS2 accept methylmalonyl-CoA to produce oxidized polypropionate products. When incubated with inhibitory malonyl-CoA (MC), the resulting EcPKS2(MC) structure revealed MC bound to the acyltransferase active site and the phosphopantetheinylated acyl carrier protein (ACP-pPant) bound to the ketosynthase (KS) active site. Remarkably, the entire linker from one modifying region to the ACP was visible in full-length EcPKS2(MC), revealing an asymmetric mega-enzyme structure. Mutations disrupting the affinity between the ACP linker and modifying domains altered substrate selectivity and active site selection, despite an expectation that the interactions are transient and that the wild-type linker alternates between the observed ordered and disordered conformations during the competing catalytic cycles of the homodimeric protein. A second structure, EcPKS2(AC), was acylated both on the KS catalytic cysteine and on the ACP-pPant. The ACP was docked at the dehydratase site, revealing further interactions between ACP and these FAS-like enzymes. The results suggest an unexpected role for the ACP linker in the control of substrate and product selectivity across the AFPK/FAS clade in animals and highlight ACP interfaces and mega-enzyme dynamics over the course of the catalytic cycle.

biochemistry↗

bioRxiv2024

NAB2-STAT6 drives an EGR1-dependent neuroendocrine program in Solitary Fibrous Tumors

The pathogenesis of many rare tumor types is poorly understood, preventing the design of effective treatments. Solitary fibrous tumors (SFTs) are neoplasms of mesenchymal origin that affect 1/1,000,000 individuals every year and are clinically assimilated to soft tissue sarcomas. SFTs can arise throughout the body and are usually managed surgically. However, 30-40% of SFTs will relapse local-regionally or metastasize. There are no systemic therapies with durable activity for malignant SFTs to date. The molecular hallmark of SFTs is a gene fusion between the NAB2 and STAT6 loci on chromosome 12, resulting in a chimeric protein of poorly characterized function called NAB2-STAT6. We use primary samples and an inducible cell model to discover that NAB2-STAT6 operates as a transcriptional coactivator for a specific set of enhancers and promoters that are normally targeted by the EGR1 transcription factor. In physiological conditions, NAB2 is primarily localized to the cytoplasm and only a small nuclear fraction is available to operate as a co-activator of EGR1 targets. NAB2-STAT6 redirects NAB1, NAB2, and additional EGR1 to the nucleus and bolster the expression of neuronal EGR1 targets. The STAT6 moiety of the fusion protein is a major driver of its nuclear localization and further contributes to NAB2s co-activating abilities. In primary tumors, NAB2-STAT6 activates a neuroendocrine gene signature that sets it apart from most sarcomas. These discoveries provide new insight into the pathogenesis of SFTs and reveal new targets with therapeutic potential.

cancer biology↗

bioRxiv2024

Mice born preterm develop gait dystonia and reduced cortical parvalbumin immunoreactivity

Preterm birth leading to cerebral palsy (CP) is the most common cause of childhood dystonia, a movement disorder that is debilitating and often treatment refractory. Dystonia has been typically associated with dysfunction of striatal cholinergic interneurons, but clinical imaging data suggests that cortical injury may best predict dystonia following preterm birth. Furthermore, abnormal sensorimotor cortex inhibition has been found in many studies of non-CP dystonias. To assess the potential for a cortical etiology of dystonia following preterm birth, we developed a new model of preterm birth in mice. Noting that term delivery in mice on a C57BL/6J background is embryonic day 19.1 (E19.1), we induced preterm birth at the limits of pup viability at embryonic day (E) 18.3, equivalent to human 22 weeks gestation. Mice born preterm demonstrate display clinically validated metrics of dystonia during gait (leg adduction amplitude and variability) and also demonstrate reduced parvalbumin immunoreactivity in the sensorimotor cortex, suggesting dysfunction of cortical parvalbumin-positive inhibitory interneurons. Notably, reduced parvalbumin immunoreactivity or changes in parvalbumin-positive neuronal number were not observed in the striatum. These data support the association between cortical dysfunction and dystonia following preterm birth. We propose that our mouse model of preterm birth can be used to study this association and potentially also study other sequelae of extreme prematurity.

neuroscience↗

bioRxiv2023

Mother-Infant Gut Viruses and their Bacterial Hosts: Transmission Patterns and Dynamics during Pregnancy and Early Life

Early development of the gut ecosystem is crucial for lifelong health. While infant gut bacterial communities have been studied extensively, the infant gut virome remains under-explored. We longitudinally assessed the composition of gut viruses and their bacterial hosts in 322 total metagenomes and 205 metaviromes from 30 mothers during and after pregnancy and from their 32 infants during their first year of life. While the maternal gut virome composition remained stable during late pregnancy and after birth, the infant gut virome was dynamic in the first year of life and contained a higher abundance of active temperate phages compared to the maternal gut viromes. The infant gut virome composition was also influenced by infant feeding mode and place of delivery. Lastly, we provide evidence of viral-bacterial strains co-transmission from mothers to infants, demonstrating that infants acquire some of their virome from their mothers gut. Highlights- Longitudinal characterisation of the gut microbiome and virome in 30 mothers during pregnancy, at birth and 3 months after birth and in 32 infants from birth across the first year of life. - The maternal gut bacteriome changes from the first to the second trimester and then remains stable through birth and the first 3 months after birth. - The maternal gut virome remains stable during late pregnancy, birth and the first 3 months after birth. - The infant gut virome is highly dynamic during the first year of life and is shaped by infant feeding mode and place of delivery. - The infant gut harbours more temperate bacteriophages than the maternal gut, but their relative abundance decreases with increasing infant age. - Gut viral strains and their bacterial host strains are co-transmitted from mothers to their infants. - Gut viral strains are transferred from mother to infant around birth directly or via transfer of their bacterial hosts followed by the induction of prophages.

microbiology↗

bioRxiv2023

Ultrastructure of the nebenkern during spermatogenesis in the praying mantid Hierodula membranacea

Spermatogenesis leads to the formation of functional sperm cells. Here we have applied high-pressure freezing in combination with transmission electron microscopy (TEM) to study the ultrastructure of sperm development in subadult males of the praying mantid Hierodula membranacea. We show the ultrastructure of different stages of sperm development in this species. In addition, we have applied serial-section electron tomography of the nebenkern to demonstrate in three dimensions (3D) that this organelle is composed of two interwoven segments that are connected by a zipper-like structure at opposing positions. Our approach will enable further ultrastructural analyses of the nebenkern also in other organisms.

cell biology↗

bioRxiv2023

ChromDL: A Next-Generation Regulatory DNA Classifier

MotivationPredicting the regulatory function of non-coding DNA using only the DNA sequence continues to be a major challenge in genomics. With the advent of improved optimization algorithms, faster GPU speeds, and more intricate machine learning libraries, hybrid convolutional and recurrent neural network architectures can be constructed and applied to extract crucial information from non-coding DNA. ResultsUsing a comparative analysis of the performance of thousands of Deep Learning (DL) architectures, we developed ChromDL, a neural network architecture combining bidirectional gated recurrent units (BiGRU), convolutional neural networks (CNNs), and bidirectional long short-term memory units (BiLSTM), which significantly improves upon a range of prediction metrics compared to its predecessors in transcription factor binding site (TFBS), histone modification (HM), and DNase-I hypersensitive site (DHS) detection. Combined with a secondary model, it can be utilized for accurate classification of gene regulatory elements. The model can also detect weak transcription factor (TF) binding with higher accuracy as compared to previously developed methods and has the potential to accurately delineate TF binding motif specificities. AvailabilityThe ChromDL source code can be found at https://github.com/chrishil1/ChromDL.

genomics↗

bioRxiv2022

Residual Complex I activity supports glutamate catabolism and mtSLP via canonical Krebs cycle activity during acute anoxia without OXPHOS

Anoxia halts oxidative phosphorylation (OXPHOS) causing an accumulation of reduced compounds in mitochondrial matrix which impedes dehydrogenases. By simultaneously measuring oxygen concentration, NADH autofluorescence, mitochondrial membrane potential and ubiquinone reduction extent in organello in real-time, we show that Complex I utilized endogenous quinones to oxidize NADH under acute anoxia. Untargeted or [U-13C]glutamate-targeted metabolomic analysis of matrix and effluxed metabolites extracted during anoxia in the presence or absence of site-specific inhibitors of the electron transfer system inferred that NAD+ regenerated by Complex I is reduced by the 2-oxoglutarate dehydrogenase complex yielding succinyl-CoA supporting mitochondrial substrate-level phosphorylation (mtSLP), releasing succinate. Yet, targeted metabolomic analysis using [U-13C]malate also revealed concomitant succinate dehydrogenase reversal during anoxia yielding succinate by reducing fumarate, albeit to a small extent. Our results highlight the importance of quinone availability to Complex I oxidizing NADH, thus maintaining glutamate catabolism and mtSLP in the absence of OXPHOS.

biochemistry↗

bioRxiv2022

An IBD-associated pathobiont synergises with NSAID to promote colitis which is blocked by NLRP3 inflammasome and Caspase-8 inhibitors

Conflicting evidence exists on the association between consumption of non-steroidal anti-inflammatory drugs (NSAIDs) and symptomatic worsening of inflammatory bowel disease (IBD). We hypothesise that the heterogeneous prevalence of pathobionts [e.g., adherent-invasive Escherichia coli (AIEC)], might explain this inconsistent NSAIDs/IBD correlation. Using IL10 -/- mice, we show aggravation of colitis in AIEC-colonised animals fed NSAID. This is accompanied by activation of the NLRP3 inflammasome, Caspase-8, apoptosis and pyroptosis; features not seen in mice exposed to AIEC or NSAID alone, revealing an AIEC/NSAID synergistic effect. Inhibition of NLRP3 or Caspase-8 activity ameliorated colitis, with reduction in NLRP3 inflammasome activation, cell death markers and activated T-cells and macrophages, improved histology and increased abundance of Clostridium cluster XIVa species. Our findings provide mechanistic insights into how NSAID and an opportunistic gut-pathobiont can synergise to worsen IBD symptoms. Thus, targeting the NLRP3 inflammasome and Caspase-8 could be a potential therapeutic strategy in patients with NSAID-worsened inflammation.

immunology↗

bioRxiv2022

T cell Egress via Lymphatic Vessels Limits the Intratumoral T cell Repertoire in Melanoma

Antigen-specific CD8+ T cell accumulation in tumors is a prerequisite for effective immunotherapy, and yet, the mechanisms of lymphocyte transit remain poorly defined. We find that tumor-associated lymphatic vessels control T cell exit from tumors via the chemokine CXCL12, and intratumoral antigen encounter tunes CXCR4 expression on effector CD8+ T cells. Only high affinity antigen downregulates CXCR4 and upregulates the CXCL12 decoy receptor, ACKR3, thereby reducing CXCL12 sensitivity and promoting T cell retention. A diverse repertoire of functional tumor-specific CD8+ T cells exit the tumor, thereby limiting tumor control. CXCR4 inhibition and loss of lymphatic-specific CXCL12 boosts T cell retention and enhances response to therapeutic immune checkpoint blockade. Strategies that limit T cell egress, therefore, provide a new tool to boost immunotherapy response. One-Sentence SummaryLymphatic vessel-mediated, antigen-dependent CD8+ T cell egress limits T cell accumulation in melanomas and impairs anti-tumor immunity.

immunology↗

bioRxiv2021

CXCL8/CXCR2 signaling mediates bone marrow fibrosis and represents a therapeutic target in myelofibrosis

Pro-inflammatory signaling is a hallmark feature of human cancer, including in myeloproliferative neoplasms (MPNs), most notably myelofibrosis (MF). Dysregulated inflammatory signaling contributes to fibrotic progression in MF; however, the individual cytokine mediators elicited by malignant MPN cells to promote collagen-producing fibrosis and disease evolution remain yet to be fully elucidated. Previously we identified a critical role for combined constitutive JAK/STAT and aberrant NF-{kappa}B pro-inflammatory signaling in myelofibrosis development. Using single-cell transcriptional and cytokine-secretion studies of primary MF patient cells and two separate murine models of myelofibrosis, we extend this previous work and delineate the role of CXCL8/CXCR2 signaling in MF pathogenesis and bone marrow fibrosis progression. MF patient hematopoietic stem/progenitor cells are enriched in a CXCL8/CXCR2 gene signature and display dose-dependent proliferation and fitness in response to exogenous CXCL8 ligand in vitro. Genetic deletion of Cxcr2 in the hMPLW515L adoptive transfer model abrogates fibrosis and extends overall survival, and pharmacologic inhibition of the CXCR1/2 pathway improves hematologic parameters, attenuates bone marrow fibrosis, and synergizes with JAK inhibitor therapy. Our mechanistic insights provide a rationale for therapeutic targeting of the CXCL8/CXCR2 pathway in MF patients at risk for continued fibrotic progression.

cancer biology↗

bioRxiv2021

Developmental regulation and functional prediction of microRNAs in an expanded Fasciola hepatica miRNome

1The liver fluke, Fasciola hepatica, is a global burden on the wellbeing and productivity of farmed ruminants, and a zoonotic threat to human health. Despite the clear need for accelerated discovery of new drug and vaccine treatments for this pathogen, we still have a relatively limited understanding of liver fluke biology and host interactions. Noncoding RNAs, including micro (mi)RNAs, are key to transcriptional regulation in all eukaryotes, such that an understanding of miRNA biology can shed light on organismal function at a systems level. Four previous publications have reported up to 89 mature miRNA sequences from F. hepatica, but our data show that this does not represent a full account of this species miRNome. We have expanded on previous studies by sequencing, for the first time, miRNAs from multiple life stages (adult, newly excysted juvenile (NEJ), metacercariae and adult-derived extracellular vesicles (EVs)). These experiments detected an additional 61 high-confidence miRNAs, most of which have not been described in any other species, expanding the F. hepatica miRNome to 150 mature sequences. We used quantitative (q)PCR assays to provide the first developmental profile of miRNA expression across metacercariae, NEJ, adult and adult-derived Evs. The majority of miRNAs were expressed most highly in metacercariae, with at least six distinct expression clusters apparent across life stages. Intracellular miRNAs were functionally analysed to identify target mRNAs with inversely correlated expression in F. hepatica tissue transcriptomes, highlighting regulatory interactions with key virulence transcripts including cathepsin proteases, and neuromuscular genes that control parasite growth, development and motility. We also linked 28 adult-derived EV miRNAs with downregulation of 397 host genes in F. hepatica-infected transcriptomes from ruminant lymph node, peripheral blood mononuclear cell (PBMC) and liver tissue transcriptomes. These included genes involved in signal transduction, immune and metabolic pathways, adding to the evidence for miRNA-based immunosuppression during fasciolosis. These data expand our understanding of the F. hepatica miRNome, provide the first data on developmental miRNA regulation in this species, and provide a set of testable hypotheses for functional genomics interrogations of liver fluke miRNA biology. CONTRIBUTION TO THE FIELDPrevious studies identified 89 micro (mi)RNAs (non-coding RNAs responsible for regulating gene expression) in the trematode parasite, Fasciola hepatica, and attached functional annotations to the miRNAs that are secreted by liver fluke in vitro. This study expands the known miRNA complement of F. hepatica by 40%, adding an additional 61 miRNAs, many of which appear Fasciola-specific. We used this expanded dataset to perform the first analysis of developmental miRNA expression across intra-mammalian parasites, showing clear developmentally regulated expression profiles across infectious metacercariae, juvenile and adult parasites, and extracellular vesicles secreted by adult parasites. We performed rigorous functional annotation of cellular miRNAs, correlating their expression with mRNA transcriptomes to identify potential roles for specific miRNAs in regulating expression of important proteases and nerve/muscle transcripts, all of which are important for parasite virulence. We also functionally analysed miRNAs secreted by adult parasites in terms of interactions with host transcripts; for the first time this analysis was supported by host transcriptome data, linking secreted miRNAs with mRNAs that are downregulated by fluke infection in sheep and cattle. This work contributes new data, insights and analyses to the field, providing a rich source of hypotheses for future experimental validation.

molecular biology↗

bioRxiv2021

Alpha-synuclein alters the faecal viromes of rats in a gut-initiated model of Parkinson's disease

Parkinsons disease (PD) is a chronic neurological disorder associated with the misfolding of alpha-synuclein (-syn) into Lewy body aggregates within nerve cells that contribute to their neurodegeneration. Recent evidence suggests -syn aggregation may begin in the gut and travel to the brain along the vagus nerve, with microbes a potential trigger initiating the misfolding of -syn. However, changes in the gut virome in response to -syn alterations have not been investigated. In this study, we show longitudinal changes in the faecal virome of rats administered either monomeric or preformed fibrils (PFF) of -syn directly into their enteric nervous system. Differential changes in rat viromes were observed when comparing monomeric and PFF -syn. The virome {beta}-diversity changes after -syn treatment were compounded by the addition of LPS as an adjunct. Changes in the diversity of rat faecal viromes were observed after one month and did not resolve within the studys five month observational period. Overall, these results suggest that microbiome alterations associated with PD may, partially, be reactive to host -syn associated changes.

microbiology↗