Biology subjects

Young, G.

Publications and source records attributed to Young, G..

At least 19 recordsLinked to original sources

bioRxiv2026

Defective HIRA driven chromatin maintenance underlies age related decline in oocyte quality

The rapid fertility decline in women over 35 years of age has been attributed to decreasing oocyte quality. Aging oocytes accumulate DNA damage, chromosome segregation defects and altered epigenetic modifications. Critically, the molecular mechanisms underlying these changes remain poorly understood. Here we uncovered that in the mouse, the HIRA driven H3.3 histone replacement is attenuated during oocyte aging leading to the loss of chromatin homeostasis. The loss of HIRA function is caused by its SUMOylation resulting in the disruption of HIRA-UBN1-CABIN1 complex and its association with chromatin. We further show that preventing HIRA SUMOylation restores H3.3 incorporation and chromatin integrity in aged mouse oocytes leading to improved oocyte maturation and preimplantation development rates. Importantly, similar chromatin homeostasis defect is observed in aging human oocytes pointing towards a conserved process. Our findings provide novel insights into the molecular mechanisms underlying age-related oocyte quality decline and pave the way for clinical interventions.

developmental biology↗

bioRxiv2026

Identification and functional assessment of GPCRs across human adipogenesis

Clinical obesity, defined as the presence of excess adiposity in conjunction with the presence of at least one clinical presentation of disease, remains a significant social and economic burden. Pharmacological weight loss agents based on glucagon-like peptide-1 receptor (GLP-1R) targeting are effective but exhibit significant side-effects leading to cessation of treatment, weight regain and, importantly, re-development of co-morbidities. Adipose tissue, now established as a central mediator of energy balance, endocrine signalling and inflammation, plays a significant role in the protection against metabolic disease onset. Loss of adipose tissue expandability, insulin sensitivity and function is thought to be a pivotal event in the transition to clinical obesity. Targeting adipose tissue dysfunction prior to or following development of metabolic disease remains a key therapeutic strategy either in conjunction with incretin-based weight loss therapy, or as a stand-alone therapy. The recent confirmation of functional glucose-dependent insulinotropic polypeptide receptor (GIPR) in mature adipocytes has led to a significant shift in our mechanistic understanding of dual GLP-1R/GIPR agonists such as tirzepatide, with the addition of adipocyte-specific targeting thought to underpin its clinical superiority to GLP-1R agonism alone. However, the expression, regulation and mechanistic targets of GIPR, and indeed many G protein-coupled receptors (GPCRs), in human adipocytes remains unclear, with adipocyte development being particularly under-studied in this regard. Here we use unbiased transcriptomic analyses of human adipocyte development with high temporal resolution to identify the onset of human GPCR expression, uncovering a previously undocumented surge in expression following adipogenic induction and elegant gene waves throughout adipocyte development that may provide attractive pharmacological targets for adipose tissue dysfunction with or without weight loss. We functionally characterise GIPR, GLP-1 and CALCR/RAMP (Amylin) receptor activation at key differentiation timepoints, and identify a novel amylin response in early adipogenesis, presenting committed adipogenic precursors as a primary target of amylin signalling. HighlightsO_LIComprehensive transcriptomic analysis of human adipogenesis with improved temporal resolution and depth C_LIO_LIIdentification and classification of >150 GPCRs differentially regulated across adipocyte differentiation C_LIO_LIFunctional validation highlighting targeting of adipose stem cells and adipocytes using clinically approved receptor agonists C_LIO_LIGeneration of novel human adipocyte stem cell lines from healthy and obese individuals to drive early target validation and exploration of adipocyte biology with increased pre-clinical power. C_LI SummaryComprehensive temporal RNA sequencing across human adipocyte development with a focus on G-protein coupled receptor expression and activity.

cell biology↗

bioRxiv2025

PCNA encircling primer/template junctions is eliminated by exchange of RPA for Rad51: Implications for the interplay between human DNA damage tolerance pathways

The DNA genome is constantly exposed to agents, such as ultraviolet radiation (UVR), that can alter or eliminate its coding properties through covalent modifications of the template bases. Many of these damaging modifications (i.e., lesions) persist into S-phase of the cell cycle where they may stall the canonical DNA replication machinery. In humans, these stalling events are circumvented by one of at least three interconnected DNA damage tolerance (DDT) pathways; translesion DNA synthesis (TLS), Template Switching (TS), and Homology-dependent Recombination (HDR). Currently, the functional interplay between these pathways is unclear, leaving wide gaps in our fundamental understanding of human DDT. To gain insights, we focus on the activation mechanisms of the DDT pathways. PCNA sliding clamps encircling primer/template (P/T) junctions of stalled replication sites are central to the activation of both TLS and TS whereas exchange of RPA for Rad51 filaments on the single strand DNA (ssDNA) sequences of stalled replication sites is central to HDR activation. Utilizing direct, ensemble FRET approaches developed by our lab, we independently monitor and directly compare PCNA occupancy and RPA/Rad51 exchange at P/T junctions under a variety of conditions that mimic in vivo scenarios. Collectively, the results reveal that assembly of stable Rad51 filaments at P/T junctions via RPA/Rad51 exchange causes complete and irreversible unloading of the resident PCNA, both in the presence and absence of an abundant PCNA-binding protein complex. Further investigations decipher the mechanism of RPA/Rad51 exchange-dependent unloading of PCNA. Collectively, these studies provide critical insights into the interplay between human DDT pathways and direction for future studies.

biophysics↗

bioRxiv2025

High resolution multi-scale profiling of embryonic germ cell-like cells derivation reveals pluripotent state transitions in humans

Primordial germ cells (PGCs) are the embryonic precursors of the gametes. In mice and rats, PGCs can readily acquire pluripotency in vitro by forming embryonic germ cells (EGCs). To date, a comparable in vitro system has not been established in humans, despite the fact that human PGCs (hPGCs) readily undergo pluripotent conversion in the context of germ cell tumorigenesis. Here we report that hPGC-like cells (hPGCLCs) undergo conversion to human embryonic germ-like cells (hEGCLCs) upon exposure to the same inductive signals previously used to derive mouse EGCs. This defined, feeder-free culture system allows efficient derivation of human EGCLCs which can be expanded and maintained in standard human pluripotent stem cell medium. hEGCLCs are transcriptionally similar to human pluripotent stem cells (hPSCs) and can differentiate into all three germ layers, as well as giving rise to PGCLCs once more - demonstrating the interconvertibility of pluripotent states. This is also evident at the epigenetic level, as the initial DNA demethylation that occurs in hPGCLCs is largely reversed in hEGCLCs, restoring DNA methylation to the level observed in hPSCs. This new in vitro model captures the transition from the pluripotent stem cell state to a germ cell identity and back again, and therefore represents a highly tractable system to study pluripotent and epigenetic transitions, including those which occur during human germ cell tumorigenesis. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=195 HEIGHT=200 SRC="FIGDIR/small/632914v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@14572bcorg.highwire.dtl.DTLVardef@6fa7c1org.highwire.dtl.DTLVardef@6eaa99org.highwire.dtl.DTLVardef@1822307_HPS_FORMAT_FIGEXP M_FIG C_FIG In briefWe report the first fully defined system to efficiently convert hPGCLCs to a pluripotent stem cell (PSC) state. We tracked pluripotent state transitions by multi-omic analysis and provided a high-resolution map of the transcriptional and epigenomic transitions upon entry to and exit from the human germline. HighlightsO_LIEfficient derivation of hEGCLC in fully defined feeder-free conditions C_LIO_LISingle-cell transcriptomic profiling of transitions from the hPSC state to hPGCLCs and back. C_LIO_LILongitudinal DNA methylation profiling highlights the overall reversibility of epigenetic states C_LIO_LIMulti-omic gene regulatory network analysis identifies key regulators of pluripotent transitions C_LI

developmental biology↗

bioRxiv2024

CRISPRa-mediated disentanglement of the Dux-MERVL axis in the 2C-like state, totipotency and cell death

Transposable elements (TEs) provide sequences that are powerful cis-regulatory drivers of gene expression programmes. This is particularly apparent during early development when many TEs become de-repressed. MERVL elements are highly yet transiently upregulated in mouse totipotent 2-cell (2C) embryos during major zygotic genome activation (ZGA), and in 2C-like cells in vitro. One of the most powerful activators of MERVL is the pioneer transcription factor, Dux. However, apparent differences lie in the requirement for Dux versus MERVL activation in embryos, for unclear reasons. Moreover, sustained Dux activation causes cell toxicity in multiple cell types, which may or may not be linked to MERVL activation. Using a CRISPR-activation, 2C-GFP reporter system, we have unpicked the relative role of Dux and MERVL in ZGA, totipotent-like characteristics and cell toxicity. We find that direct MERVL activation comprises only a portion of the Dux-dependent transcriptome, and which is sufficient for expanded fate potential, but not other totipotency features. Conversely, Dux-induced pathology is independent of MERVL activation and involves induction of the pro-apoptotic factor, Noxa. Our study highlights the complexity of the Dux-MERVL transcriptional network and uncovers a new player in Dux-driven pathology.

developmental biology↗

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

Systemic extracellular acidification is a hallmark of aging

Understanding the critical pathophysiological processes that promote age-related disease is needed to uncover effective targets for preventive medicine. Here, we investigate how extracellular pH changes with age and its impact on longevity, using fly and mouse models. We find that extracellular acidification occurs in flies during aging and correlates to mortality rate. With age, flies also become more susceptible to die from acidotic stress, which can be prevented by alkalotic treatment. Acidification is caused by insufficient acid elimination, linked to downregulation of genes in the fly excretory tract that control pH and ATP production, essential for active secretion initiation. In mice, we show that lymph-drained interstitial fluids acidify with age. Expression of genes, whose pathogenic loss-of-function variants cause tubular acidosis in humans, is decreased in the kidneys of aging mice. Overall, this study sheds light on dysregulated systemic acid-base balance as a conserved pathophysiological mechanism of aging.

physiology↗

bioRxiv2024

Alpha-synuclein aggregates trigger anti-viral immune pathways and RNA editing in human astrocytes

RNA editing is a post transcriptional mechanism that targets changes in RNA transcripts to modulate innate immune responses. We report the role of astrocyte specific, ADAR1 mediated RNA editing in neuroinflammation in Parkinsons disease. We generated hiPSC-derived astrocytes, neurons and co-cultures and exposed them to small soluble alpha-synuclein aggregates. Oligomeric alpha-synuclein triggered an inflammatory glial state associated with TLR activation, viral responses, and cytokine secretion. This reactive state resulted in loss of neurosupportive functions, and the induction of neuronal toxicity. Notably, interferon response pathways were activated leading to upregulation, and isoform switching of the RNA deaminase enzyme, ADAR1. ADAR1 mediates A-to-I RNA editing, and increases in RNA editing were observed in inflammatory pathways in cells, as well as in post-mortem human PD brain. Aberrant, or dysregulated, ADAR1 responses and RNA editing may lead to sustained inflammatory reactive states in astrocytes triggered by alpha-synuclein aggregation, and this may drive the neuroinflammatory cascade in Parkinsons.

neuroscience↗

bioRxiv2023

Myosin-5 varies its steps along the irregular F-actin track

Molecular motors employ chemical energy to generate unidirectional mechanical output against a track. By contrast to the majority of macroscopic machines, they need to navigate a chaotic cellular environment, potential disorder in the track and Brownian motion. Nevertheless, decades of nanometer-precise optical studies suggest that myosin-5a, one of the prototypical molecular motors, takes uniform steps spanning 13 subunits (36 nm) along its F-actin track. Here, we use high-resolution interferometric scattering (iSCAT) microscopy to reveal that myosin takes strides spanning 22 to 34 actin subunits, despite walking straight along the helical actin filament. We show that cumulative angular disorder in F-actin accounts for the observed proportion of each stride length, akin to crossing a river on variably-spaced stepping stones. Electron microscopy revealed the structure of the stepping molecule. Our results indicate that both motor and track are soft materials that can adapt to function in complex cellular conditions.

biophysics↗

bioRxiv2023

Tiny Earth CURE improves student persistence in science

Course-based undergraduate research experiences (CUREs) enhance student retention in STEM, particularly among students who belong to historically excluded communities. Yet the mechanisms by which CUREs contribute to student integration and persistence are poorly understood. Utilizing the Tripartite Integration Model of Social Influence (TIMSI), this longitudinal study examines how Tiny Earth, an antibiotic-discovery CURE, impacts students scientific self-efficacy, scientific identity, endorsement of scientific community values, and intentions to persist in science. The study also explores how gains in TIMSI factors vary as a function of student demographics and course characteristics. Results of pre-and post-course measurements show that scientific self-efficacy and identity increased among students in Tiny Earth, and some student demographics and course characteristics moderated these gains. Gains in scientific self-efficacy, identity, and values correlated with gains in persistence intentions, whereas student demographics and course characteristics did not. Results of this study show that the Tiny Earth CURE enhanced students integration into the scientific community, which was linked to intentions of students of both historically underrepresented and majority groups to persist in STEM. We discuss how courses that provide opportunity to learn science skills in the context of a CURE can contribute toward enlarging and diversifying the STEM workforce.

scientific communication and education↗

bioRxiv2021

Dissemination and pathogenesis of toxigenic Clostridium perfringens strains linked to neonatal intensive care units and Necrotising Enterocolitis

BackgroundClostridium perfringens is an anaerobic toxin-producing bacterium that has long been associated with intestinal diseases, particularly in neonatal humans and animals. More recently, infant gut microbiome studies have suggested an important link between C. perfringens and the devastating preterm-associated disease Necrotising Enterocolitis (NEC), but in-depth studies on this pathogen (genomics and mechanistic) are lacking. Methods/MaterialsWe isolated and whole-genome sequenced 274 infant-associated C. perfringens isolates from 5 hospitals across the UK between 2011-2016 (including longitudinal samples from 31 individuals). We performed in-depth genomic analyses, phenotypically characterised pathogenic traits of 10 strains (including 4 C. perfringens from NEC patients) and established a novel oral-challenge C57BL/6 mouse infection model for microbe-host studies. ResultsPore-forming toxin encoding genes pfoA and cpb2 were enriched within hypervirulent lineages that exclusively consisted of C. perfringens-associated NEC (CPA-NEC) strains, in addition to overabundance of colonisation factors. Importantly, we identified a circulating C. perfringens variant, eventually linked to a fatal CPA-NEC case. The variant was detected consistently within 6 individuals in two sister hospitals across a 40-day window, demonstrating for the first time the intra- and inter-hospital dissemination of C. perfringens. CPA-NEC isolates were determined phenotypically to be more virulent (linked with overabundance of gene pfoA) than isolates obtained from non-NEC preterm babies. In addition, two pfoA-positive CPA-NEC C. perfringens strains were confirmed to induce clinical inflammatory tissue lesions in vivo. ConclusionsHypervirulent lineages are linked to CPA-NEC, potentially due to the production of pore-forming toxins, coupled with higher metabolic, transmission, and pathogenic capacities. These studies indicate C. perfringens is an important bacterial pathogen in preterm infants and highlights the requirement for further investigation into development of intervention and therapeutic strategies.

microbiology↗

bioRxiv2021

Label-free tracking and massmeasurement of single proteins onlipid bilayers

We introduce dynamic mass photometry, a method for label-free imaging, tracking and mass measurement of membrane-associated proteins. Our method enables quantitative studies of their mobility, membrane affinity and interactions at the single molecule level. Application to the membrane remodelling GTPase dynamin1 reveals heterogeneous mixtures of oligomers suggesting that the fundamental building block for oligomerisation is a dimer, challenging current tetramer-centric models. Dynamic mass photometry has the ability to transform our approach to studying biomolecular mechanisms in and on lipid bilayers.

biophysics↗

bioRxiv2020

Endocast and bony labyrinth of a stem gnathostome shed light on the earliest diversification of jawed vertebrates

Our understanding of the earliest evolution of jawed vertebrates depends on a credible phylogenetic assessment of the jawed stem gnathostomes collectively known as placoderms. However, their relationships, and even whether placoderms represent a single radiation or a paraphyletic array, remain contentious. Here we describe the endocranial cavity and inner ear of Brindabellaspis stensioi, commonly recovered as a taxon of uncertain affinity branching near the base of placoderms. While some features of its braincase and endocast resemble those of jawless vertebrates, its inner ear displays a repertoire of crown gnathostome characters. Both parsimony and Bayesian analyses suggest that established hypotheses of placoderm relationships are unstable, with newly-revealed anatomy pointing to a potentially radical revision of early gnathostome evolution. Our results call into question the appropriateness of fusiform placoderms as models of primitive gnathostome anatomy and raise questions of homology relating to key cranial features. One Sentence SummaryThe skull of a 400-million-year old fossil fish suggests that hypotheses of early jawed vertebrate relationships might have to be turned on their head.

paleontology↗

bioRxiv2020

Tissue-specific and interferon-inducible expression of non-functional ACE2 through endogenous retrovirus co-option

Angiotensin-converting enzyme 2 (ACE2) is an entry receptor for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), as well as a regulator of several physiological processes. ACE2 has recently been proposed to be interferon-inducible, suggesting that SARS-CoV-2 may exploit this phenomenon to enhance viral spread and questioning the efficacy of interferon treatment in Coronavirus disease 2019 (COVID-19). Using a recent de novo transcript assembly that captured previously unannotated transcripts, we describe a novel isoform of ACE2, generated by co-option of an intronic long terminal repeat (LTR) retroelement promoter. The novel transcript, termed LTR16A1-ACE2, exhibits specific expression patterns across the aerodigestive and gastrointestinal tracts and, importantly, is highly responsive to interferon stimulation. In stark contrast, expression of canonical ACE2 is completely unresponsive to interferon stimulation. Moreover, the LTR16A1-ACE2 translation product is a truncated, unstable ACE2 form, lacking domains required for SARS-CoV-2 binding and therefore unlikely to contribute to or enhance viral infection.

genomics↗

bioRxiv2020

Highly accurate long-read HiFi sequencing data for five complex genomes

The PacBio(R) HiFi sequencing method yields highly accurate long-read sequencing datasets with read lengths averaging 10-25 kb and accuracies greater than 99.5%. These accurate long reads can be used to improve results for complex applications such as single nucleotide and structural variant detection, genome assembly, assembly of difficult polyploid or highly repetitive genomes, and assembly of metagenomes. Currently, there is a need for sample data sets to both evaluate the benefits of these long accurate reads as well as for development of bioinformatic tools including genome assemblers, variant callers, and haplotyping algorithms. We present deep coverage HiFi datasets for five complex samples including the two inbred model genomes Mus musculus and Zea mays, as well as two complex genomes, octoploid Fragaria x ananassa and the diploid anuran Rana muscosa. Additionally, we release sequence data from a mock metagenome community. The datasets reported here can be used without restriction to develop new algorithms and explore complex genome structure and evolution. Data were generated on the PacBio Sequel II System.

genomics↗

bioRxiv2020

Dual blockade of IL-6 and CTLA-4 regresses pancreatic tumors in a CD4+ T cell-dependent manner.

Pancreatic ductal adenocarcinoma (PDAC) is exceptionally resistant to immune checkpoint inhibition (ICI). We previously reported that elevated systemic interleukin-6 (IL-6) and increased numbers of T cells positive for circulating cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) correlate with worse overall survival in patients with PDAC. We postulated that combined blockade of IL-6 and CTLA-4 would significantly enhance anti-tumor immune responses to PDAC. Dual blockade of IL-6 and CTLA-4 in immune competent mice bearing subcutaneously injected pancreatic tumors significantly inhibited tumor growth, accompanied by overwhelming T cell infiltration. Therapeutic efficacy was confirmed in an orthotopic murine model of pancreatic cancer and T cell depletion studies unveiled a unique dependence on CD4+ T cells for anti-tumor activity of dual IL-6 and CTLA-4 blockade. In vitro studies utilizing T cells from a TRP-1 transgenic mouse as an antigen-specific model system demonstrate this combination therapy elicits increased IFN-{gamma} production by activated CD4+ T cells. Additionally, IFN-{gamma} stimulation of pancreatic tumor cells in vitro profoundly increased tumor cell production of CXCR3 specific chemokines (CXCL10 and CXCL9). Further studies blocking CXCR3 in the presence of combined IL-6 and CTLA-4 blockade prevented orthotopic tumor regression, demonstrating a dependence on the CXCR3 axis for anti-tumor efficacy. We also found combination therapy increased intratumoral CD4+ T cells and elicited systemic changes in T-helper subsets. These data represent the first report of IL-6 and CTLA-4 blockade as a means to regress pancreatic tumors with defined operative mechanisms of efficacy. Given these results, this therapeutic combination has potential for immediate clinical translation. One Sentence SummaryBlockade of interleukin-6 in pancreatic cancer enhances CTLA-4 immune checkpoint inhibition to regress tumors in a CD4+ T cell and CXCR3-dependent manner.

cancer biology↗

bioRxiv2019

Quantifying the heterogeneity of macromolecular machines by mass photometry

Sample purity is central to in vitro studies of protein function and regulation, as well as to the efficiency and success of structural studies requiring crystallization or computational alignment of individual molecules. Here, we show that mass photometry (MP) accurately reports on sample heterogeneity using minimal volumes with molecular resolution within minutes. We benchmark our approach by negative stain electron microscopy (nsEM), including workflows involving chemical crosslinking and multi-step purification of a multi-subunit ubiquitin ligase. When applied to proteasome stability, we detect and quantify assemblies invisible to nsEM. Our results illustrate the unique advantages of MP for rapid sample characterization, prioritization and optimization.

biophysics↗

bioRxiv2019

High-sensitivity monitoring of ctDNA by patient-specific sequencing panels and integration of variant reads

Circulating tumor-derived DNA (ctDNA) can be used to monitor cancer dynamics noninvasively. Patients with small tumors have few copies of ctDNA in plasma, resulting in limited sensitivity to detect low-volume or residual disease. We show that sampling limitations can be overcome and sensitivity for ctDNA detection can be improved by massively parallel sequencing when hundreds to thousands of mutations are identified by tumor genotyping. We describe the INtegration of VAriant Reads (INVAR) analysis pipeline, which combines patient-specific mutation lists with both custom error-suppression methods and signal enrichment based on biological features of ctDNA. In this framework, the sensitivity can be estimated independently for each sample based on the number of informative reads, which is the product of the number of mutations analyzed and the average depth of unique sequencing reads. We applied INVAR to deep sequencing data generated by custom hybrid-capture panels, and showed that when ~106 informative reads were obtained INVAR allowed detection of tumor-derived DNA fractions to parts per million (ppm). In serial samples from patients with advanced melanoma on treatment, we detected ctDNA when imaging confirmed tumor volume of ~1cm3. In patients with resected early-stage melanoma, ctDNA was detected in 40% of patients who later relapsed, with higher rates of detection when more informative reads were obtained. We further demonstrated that INVAR can be generalized and allows improved detection of ctDNA from whole-exome and low-depth whole-genome sequencing data.

cancer biology↗