Biology subjects

Clarke, J.

Publications and source records attributed to Clarke, J..

At least 19 recordsLinked to original sources

bioRxiv2026

Extracellular matrix defects destabilise apical cytoarchitecture and mechanical properties during early Down syndrome neurodevelopment

Down syndrome (DS) is associated with altered brain development, especially in the cerebellum. However, how trisomy 21 (TS21) is linked to cerebellar defects remains poorly understood. Cerebellar organoids reveal that TS21 leads to extracellular matrix (ECM) alterations, perturbing downstream pathways linked to reduction of apical RAB11+ endosomes. These changes lead to impaired apical maintenance and altered progenitor composition. Significantly, ECM-enriched culture rescues apical defects in TS21 organoids, supporting a functional role for ECM in preserving apical organisation and progenitor niche integrity. In the developing human DS cerebellum and 2D neural cultures, where exogenous ECM is present, severe epithelial disorganisation is attenuated. Nevertheless, ECM organisation and mechanical properties remain altered in 2D cultures. Our findings identify TS21-driven ECM abnormalities as a mechanism impairing apical integrity, progenitor composition and mechanical proprieties in neural stem cell models of DS. Altogether, we established apical maintenance instability as an early developmental defect contributing for DS neuropathology.

Developmental Biology↗

bioRxiv2026

Measurement and comparison of acoustic space use in vocalizations of humans and close primate relatives

The human larynx, compared to those of closely related primates, lies deeper in the throat and lacks vocal membranes and air sacs. These shifts are usually analyzed regarding their acoustic effects on vowel-like vocalizations, since the evolution of speech was long thought to require an expansion of vocal range driven by vocal tract modifications. However, vowels are just one type of phoneme, and speech is just one class of human utterance. To understand the evolutionary underpinnings of known shifts in human vocal morphology, a broader bioacoustic comparison is needed. Specifically, the range of sounds used in human speech must be compared to that employed in other human vocalizations and in the repertoires of extant close primate relatives. Here, we measure the acoustic-feature space occupied by human speech, non-linguistic, and musical vocalizations along with the calls of chimpanzees, bonobos, and chacma baboons. We use Mel-frequency cepstral coefficients to create an acoustic space depicting the spectro-temporal features of over 750,000 brief vocal segments sourced from published databases and other verified sources. Speech and song occupied significantly less volume in this acoustic space than human non-linguistic vocalizations. In addition, the acoustic-feature volumes of speech and song were not statistically distinct from those of non-human primates. These results suggest that speech was not enabled by an expansion of human vocal acoustic space. Anatomical shifts unique to humans may have led to an elaboration of non-linguistic utterances, but learned vocalizations use a surprisingly small fraction of this space. Our understanding of human vocal evolution will be further informed by additional systematic comparisons of the function and homology of non-speech vocalizations, along with the collection and incorporation of more complete non-human primate vocal datasets, especially from Gorilla and Orangutan.

animal behavior and cognition↗

bioRxiv2026

OpenSplice: the impact of half a million mutations on the alternative splicing of 600 human exons

Alternative splicing of mRNA precursors is an important step in gene regulation and a major mechanism by which genetic variants cause human disease. However, changes in splicing have only been quantified for a tiny fraction of possible variants in the human genome, limiting our ability to interpret clinical variants, evaluate and develop machine learning models, and understand the splicing regulatory code. Here, to address this data gap, we present OpenSplice, a well-calibrated experimental dataset that quantifies the impact of >590,000 variants on the splicing of >600 human alternatively spliced exons. OpenSplice increases the number of exons with site saturation mutagenesis data [~]28-fold, quantifying the impact of all possible exonic and proximal intronic single nucleotide (nt) substitutions, as well as all 1, 3, 6 and 21nt deletions. Hundreds of thousands of variants affect splicing and we use the data to evaluate machine learning models, to interpret clinical variants, and to map splicing regulatory architectures. Exons and introns exhibit a broad spectrum of regulatory landscapes, including configurations dominated by enhancers or silencers, checkerboard-like patterns with interleaved enhancers and silencers, and sparse architectures with minimal regulatory content. Silencers are particularly important for determining inclusion levels. OpenSplice provides a complete atlas of variants that impact splicing, a rich testing and training dataset for machine learning models, and a comprehensive map of regulatory elements for mechanistic studies.

genomics↗

bioRxiv2026

Iridescence in pterosaur pycnofibers and the evolution of integumentary coloration

The bodies of pterosaurs, the first flying vertebrates, are covered with integumentary filaments (pycnofibres) thought to be homologous to feathers in dinosaurs, but their coloration remains unknown. Here, we report a layered internal arrangement of melanosomes containing a photonic nanostructure within the monofilaments in a previously undescribed specimen of tapejarid pterosaur Sinopterus dongi from the Early Cretaceous Jehol Biota. Optical simulations showed that this structure reflects green to magenta iridescent coloration, confirming the presence of melanosome-based iridescent coloration previously thought to be unique to birds. This finding deepens our understanding of structure/color gamut relationships in amniotes, while supporting further shared characteristics associated with derived genetic and regulatory shifts in archosaurs.

paleontology↗

bioRxiv2026

Identification and Masking of Artefactual and Misleading Within-Host Variants in Deep-Sequencing SARS-CoV-2 Data

Deep sequencing data are increasingly used to study within-host viral diversity and to inform evolutionary inference. For SARS-CoV-2, analyses based on intra-host single-nucleotide variants (iSNVs) have been widely applied to quantify within-host diversity and infer transmission dynamics. However, these applications critically depend on the reliable identification of low-frequency variants, which remain vulnerable to systematic and technical artefacts. In this study, we show that recurrent artefactual iSNVs are common in large-scale SARS-CoV-2 sequencing data and can persist even under conservative minor allele frequency (MAF) thresholds. Using data from the UKs Office for National Statistics COVID-19 Infection Survey, we demonstrate that such artefacts are predominantly sequencing centre-rather than protocol-specific. Each centre exhibits a modest, distinct set of recurrent artefactual variants showing little overlap with sites routinely masked at the consensus level. To address this, we developed a systematic, dataset-aware framework that uses recurrence within sequencing datasets to identify small, noise-adapted sets of artefactual iSNVs to mask. Applying this framework reduces spurious sharing of low-frequency variants between samples and qualitatively alters downstream inferences, including estimates of within-host diversity and transmission bottleneck sizes. Together, these findings highlight the importance of explicit, dataset-aware artefact control for robust inference from within-host variation, particularly as genomic studies increasingly seek to exploit sub-consensus diversity in rapidly evolving pathogens such as SARS-CoV-2.

genomics↗

bioRxiv2026

Nanobodies against the S2 region of the spike protein potently neutralize SARS-CoV-2 viruses and show resistance to virus escape

Entry of coronaviruses into cells is mediated by the viral spike (S) glycoproteins each consisting of S1 receptor binding and S2 membrane fusion subunits. The sequence of the S2 region is very highly conserved amongst variants of SARS-CoV-2 and compared to the S1 unit shares significant sequence identity amongst different beta-coronavirus lineages. By targeting the S2 of SARS-CoV-2 we have identified two selective and potent neutralizing nanobodies (BA.1-C2 and BA.1-D3) that bind to two different quaternary epitopes in the S2 formed by the Heptad Repeat 2 (HR2) trimer at the base of the spike protein. The HR2 sequence is identical in SARS-CoV and SARS-CoV-2 but differs in other beta-coronaviruses explaining the lack of binding to the spike proteins of MERS-CoV or HuCoV-OC43. No viral escape was observed following serial passaging of SARS-CoV-2 (JN.1) with a combination of BA.1-C2 and BA.1-D3 and the most potent of these nanobodies reduced viral load in the hamster model of COVID-19, following intranasal administration. Overall, the results show the value of nanobody technology for identifying novel neutralising epitopes in the S2 region of beta-coronaviruses with potential for the development of new selective anti-viral agents.

molecular biology↗

bioRxiv2025

MDFIC2 is a sensory neuron-specific PIEZO channel auxiliary subunit

PIEZO channels are critical for sensory mechanotransduction. While MyoD-family inhibitor proteins were identified as PIEZO1 auxiliary subunits, their broader regulatory roles, particularly in sensory cells, remained unclear. Here we demonstrate native MDFIC and MDFI regulate endogenous PIEZO channel currents in various non-sensory cell types. However, neither MDFIC nor MDFI are expressed in primary sensory neurons. In these cell types we identified an uncharacterised member of this family, Mdfic2/Gm765, that shares the ability to physically bind to PIEZO1 and PIEZO2. MDFIC2 is selectively expressed in subsets of mechanosensitive neurons, including dorsal root ganglia, trigeminal ganglia, and vagal sensory neurons. Like its paralogues, MDFIC2 alters PIEZO1/2 mechanosensitivity and inactivation kinetics, converting them into high-threshold slowly inactivating mechanoreceptors. Extensive cryo-EM reveals a conserved binding pocket for these auxiliary subunits in the pore modules of both PIEZO1 and PIEZO2 mediated by the post-translationally modified distal C-termini of MyoD-family inhibitor proteins. This provides a comprehensive structural and functional characterisation of MyoD-family inhibitor proteins as PIEZO1/2 channel regulators and offers new insights into sensory physiology and mechanical pain mechanisms.

biophysics↗

bioRxiv2025

The phylogenetic signal of extinction through the rise and fall of early vertebrates: field of bullets or clustered strike?

We investigate patterns of phylogenetic selectivity of extinction in early aquatic vertebrates from the Silurian to the Carboniferous, an interval punctuated by one of the Big Five mass extinctions and marked by many critical anatomical (e.g. jaws, limbs) and ecological (e.g. macrophagy, terrestrialization) vertebrate evolutionary innovations. Using a new >1300 taxon, formally inferred supertree, we show that phylogenetic extinction clustering in early vertebrates varied through time and between major ecomorphological divisions of the tree. At the end of the Silurian and into the Early Devonian, jawless fishes became marginalized components of vertebrate faunas and show more strongly clustered extinction than jawed fishes, which became ecologically dominant during this interval. Clustered extinction (contemporaneous extinction of close relatives) is typical of most stages of the Devonian, particularly during the Late Devonian extinctions. By contrast, the subsequent early Carboniferous interval of putative recovery is characterized by overdispersed extinction (contemporaneous extinction of distant relatives), consistent with widespread persistence of phylogenetically distinct lineages. This work shows how varying patterns of extinction selectivity pruned the vertebrate tree at the time when the first-order patterns of diversity apparent in modern aquatic vertebrate faunas were first established.

paleontology↗

bioRxiv2025

Discovery and biosynthesis of biffamycin A, a novel glycotetrapeptide antibiotic

The clinical deployment of antibiotics is undermined by antimicrobial resistance. Without new agents to treat antibiotic resistant bacterial infections, mortality rates are predicted to reach 10 million people per year by 2050. Most antibiotics are derived from natural products (NPs) produced by bacteria; however, this resource was abandoned by industry because of high rediscovery rates. We are amid a natural product renaissance fuelled by inexpensive access to genome sequencing and sophisticated bioinformatic tools, which have highlighted that most of the biosynthetic pathways for NPs are not expressed in the laboratory. Here, we engineered the expression of a silent biosynthetic gene cluster harboured by an environmental isolate of Streptomyces albidoflavus. By using a bioinformatics-guided approach, we isolated and structurally characterised a novel glycopeptide antibiotic (GPA) named biffamycin A, which is the smallest GPA known and harbours unprecedented 5-chloro-4-methoxy tryptophan and 3-hydroxy(-O_SCPLOWDC_SCPLOW-mannoysl)-O_SCPLOWDC_SCPLOW-lysine moieties. Biffamycin A possesses antimycobacterial and antistaphylococcal bioactivity, including methicillin-vancomycin-resistant Staphylococcus aureus.

biochemistry↗

bioRxiv2025

Perceptual predictions track subjective, over objective, statistical structure

Over the past two decades, converging evidence from neuroscience and psychology has shown that predictions based on learnt statistical regularities exert a widespread influence on perception, action and cognition. Predictive processes in cognition and the brain are usually modelled as tracking objective event probabilities, deriving predictions and prediction errors from the statistical structure of the environment. However, our subjective models of our environments do not always align with these objective statistics. Currently we know little about how these subjective representations may determine the predictive functions. To separate subjective and objective contributions to prediction, we conducted three studies where cues (actions or tones) predicted visual outcomes (shapes or Gabors) with varying contingencies, and adult participants discriminated these outcomes. Uniquely to our paradigm, participants also reported their experiences of the statistical structure embedded in the task - the subjective probability (Experiment 1; N = 68), expectedness (Experiment 2; N = 35), or surprise (Experiment 3; N = 35) associated with the outcomes. When modelling subjective ratings alongside objective structure, the speed of perceptual decisions was best explained by independent, additive contributions of both. The decision itself was usually only explained by the subjective ratings, with little additional variance explained by objective statistical structure. These findings suggest that subjective experience may play a key, overlooked role in predictive processes, and open a host of interesting questions about the relative objective and subjective contributions to prediction, perception, and learning.

animal behavior and cognition↗

bioRxiv2025

AAV gene therapy for GBA-PD and Gaucher Disease

Mutations in GBA1, the gene encoding glucocerebrosidase (GCase), are the most common risk factor for Parkinsons Disease (PD). GBA-PD patients are a genetic subpopulation of PD carrying heterozygous mutations in GBA1. Additionally, bi-allelic mutations in GBA1 cause Gaucher Disease (GD), a lysosomal storage disorder. Loss of GCase activity, a lysosomal enzyme leads to the accumulation of lipid substrates, disrupting lipid homeostasis and promoting cellular toxicity. Here, we report an AAV-mediated GBA1 replacement strategy to treat GD and GBA-PD by a one-time infusion via intravenous (GD Type 1) or intra-CSF (GBA-PD) route of administration. We engineered human GCase to be readily secretable to facilitate broad cross-correction. We developed CBE (conduritol {beta}-epoxide) induced lipid accumulation models to assess efficacy in mice and non-human primates (NHPs) to assess efficacy of our engineered constructs. Based on data across species, across different routes of administration, we nominated AAV.GMU01 SS3-GBA1 as our lead candidate. SS3-GBA1 is robustly secreted, cross-corrected across tissues and promotes lipid clearance. By comparing human GCase levels in AAV-treated NHP brains to healthy human donor brains, we demonstrate that AAV.GMU01 SS3-GBA1 replenishes the GCase deficit seen in GBA-PD patients, thus, restoring GCase to near-physiological levels Importantly, AAV.GMU01 SS3-GBA1 is well-tolerated with no adverse findings. Collectively, we establish a therapeutic strategy for the treatment of Gaucher Disease and GBA-PD with a single gene therapy product. One Sentence SummaryA novel gene therapy strategy for GBA1-PD and Gaucher disease with an engineered payload that robustly cross-corrects enhancing therapeutic footprint

neuroscience↗

bioRxiv2025

Structural Identification of Major Molecular Determinants for Phosphotyrosine Recognition in Tyrosine Kinases Reveals Tumour Promoting and Suppressive Functions

Protein tyrosine kinases activate signaling pathways by catalyzing the phosphorylation of tyrosine residues in their substrates. Mounting evidence suggests that, in addition to recognizing phosphorylated tyrosine (pTyr) residues through specific phosphobinding modules, many protein kinases selectively recognize pTyr directly adjacent to the tyrosine residue they phosphorylate and catalyze the formation of twin pTyr-pTyr sites. Here, we demonstrate the importance of this phosphopriming-driven twin pTyr signaling in promoting cell cycle progression through the cell cycle-inhibitory protein p27Kip1. We identify, structurally resolve, and tune two distinct molecular determinants driving the selective recognition of pTyr directly N- and C-terminal to the target phospho-acceptor tyrosine site. We further show structural and biochemical conservation in this recognition, and identify cancer-associated alterations to these determinants that are unable to recognize phosphoprimed substrates. Finally, using an in vivo mouse model of leukemia we show that Bcr-Abl mutants unable to recognize phosphoprimed substrates paradoxically result in enhanced tumor development and progression. These data indicate that Bcr-Abl, like other proto-oncogenes such as Ras or Myc, engages both pro- and anti-oncogenic programs - but in the case of Bcr-Abl, this is accomplished through a mechanism involving traditional and phosphoprimed substrate recognition.

biochemistry↗

bioRxiv2025

A new resource of clonal pluripotent human stem cell lines exhibiting inter- and intra-embryo consistency and variability

Human naive pluripotent stem cells can generate all somatic tissues and extra-embryonic components of the blastocyst. We derived multiple clonal naive pluripotent stem cell lines from individual embryos by physical separation of inner cell mass cells and subsequent individual expansion of each resulting dome-shaped colony, providing the foundation for a resource to investigate intra- and inter-embryo variation. Twenty lines were derived from ten embryos donated from nine couples. While differences between lines are observed, the overarching pluripotency circuitry is preserved in each. They can differentiate into extra-embryonic lineages and readily acquire post-implantation pluripotent identity when exposed to culture conditions driving in vitro capacitation, subsequently to generate derivatives of the three germ layers: ectoderm, mesoderm and endoderm. Some lines exhibit intra-chromosomal amplification and deletions and are therefore anticipated to provide a valuable, accessible system for modelling chromosomal mosaicism and its potential consequences using chimeric organoids, such as blastoids and gastruloids.

developmental biology↗

bioRxiv2024

Iridescent structural coloration in a crested Cretaceous enantiornithine bird from Jehol Biota

A combination of sectioning and microscopy techniques, along with the application of finite-difference-time-domain modeling on a fossil feather, results in the novel estimation of the range of iridescent colors from the fossilized melanosome type and organization preserved in the elongate head crest feathers of a new Cretaceous enantiornithine bird. The densely packed rod-like melanosomes are estimated to have yielded from red to deep blue iridescent coloration of the head feathers. The shape and density of these melanosomes also may have further increased the feathers structural strength. This occurrence on a likely male individual is highly suggestive of both a signaling function of the iridescent crest, and a potential behavioral role in adjusting the angle of light incidence to control the display of this iridescent structural coloration.

evolutionary biology↗

bioRxiv2024

Cell state and transcription factor modulation during extended ex vivo CD8+ T-cell expansion.

Adoptive cell therapy is becoming a cornerstone of tumour immunotherapy. It relies on the relatively long-term (> 2 week) ex vivo expansion of T cells either in the form of tumour-infiltrating cells, or bulk cells modified with the expression of heterologous signalling proteins, e.g., chimeric antigen receptors. However, relatively little is known about the developmental trajectories of T cells under these conditions at the system level, or whether the pathways governing these trajectories could be manipulated for clinical advantage. Using bulk RNA-seq analysis of T cells expanded and rested over a 17-day period, we produce a resource revealing how gene expression changes as cells transition through distinct cellular states over the course of activation and ex vivo expansion. By integrating this resource with published single-cell RNA-seq data, we identify a member of the AP1 transcription factor (TF) family, FOSL1, that primes CD8+ T-cells towards an effector/killing phenotype. Remarkably, FOSL1 over-expression during T-cell expansion produced super engager-like T-cells, evidenced by their gene-expression signatures and enhanced cancer-cell killing capacity. This establishes proof-of-principle for the rational engineering of T cells via TF modification during ex vivo expansion, offering a route to improving adoptive T-cell therapy.

immunology↗

bioRxiv2024

Ongoing genome doubling promotes evolvability and immune dysregulation in ovarian cancer

Whole-genome doubling (WGD) is a critical driver of tumor development and is linked to drug resistance and metastasis in solid malignancies. Here, we demonstrate that WGD is an ongoing mutational process in tumor evolution. Using single-cell whole-genome sequencing, we measured and modeled how WGD events are distributed across cellular populations within tumors and associated WGD dynamics with properties of genome diversification and phenotypic consequences of innate immunity. We studied WGD evolution in 65 high-grade serous ovarian cancer (HGSOC) tissue samples from 40 patients, yielding 29,481 tumor cell genomes. We found near-ubiquitous evidence of WGD as an ongoing mutational process promoting cell-cell diversity, high rates of chromosomal missegregation, and consequent micronucleation. Using a novel mutation-based WGD timing method, doubleTime, we delineated specific modes by which WGD can drive tumor evolution: (i) unitary evolutionary origin followed by significant diversification, (ii) independent WGD events on a pre-existing background of copy number diversity, and (iii) evolutionarily late clonal expansions of WGD populations. Additionally, through integrated single-cell RNA sequencing and high-resolution immunofluorescence microscopy, we found that inflammatory signaling and cGAS-STING pathway activation result from ongoing chromosomal instability and are restricted to tumors that remain predominantly diploid. This contrasted with predominantly WGD tumors, which exhibited significant quiescent and immunosuppressive phenotypic states. Together, these findings establish WGD as an evolutionarily active mutational process that promotes evolvability and dysregulated immunity in late stage ovarian cancer.

cancer biology↗

bioRxiv2024

An analysis of contractile and protrusive cell behaviours at the superficial surface of the zebrafish neural plate

The motive forces underlying convergence and internalisation of the teleost neural plate remain unknown. To help understand this crucial morphogenetic movement we have analysed collective and individual cell behaviours at the superficial surface of the neural plate. Convergence to the midline does not rely on mediolateral cell intercalation, it is characterised by oscillatory contractile behaviours, a punctate distribution of Cdh2 and medially polarised actin-rich protrusions at the surface of the neural plate. Additionally, we characterise and discuss the intimate relationship and dynamic cell surfaces between the motile neural plate and the stationary overlying non-neural enveloping layer.

developmental biology↗

bioRxiv2023

Prebiotic proanthocyanidins inhibit bile reflux-induced esophageal adenocarcinoma through reshaping the gut microbiome and esophageal metabolome

The gut and local esophageal microbiome progressively shift from healthy commensal bacteria to inflammatory-linked pathogenic bacteria in patients with gastroesophageal reflux disease, Barretts esophagus and esophageal adenocarcinoma (EAC). However, mechanisms by which microbial communities and metabolites contribute to reflux-driven EAC remain incompletely understood and challenging to target. Herein, we utilized a rat reflux-induced EAC model to investigate targeting the gut microbiome-esophageal metabolome axis with cranberry proanthocyanidins (C-PAC) to inhibit EAC progression. Sprague Dawley rats, with or without reflux-induction received water or C-PAC ad libitum (700 {micro}g/rat/day) for 25 or 40 weeks. C-PAC exerted prebiotic activity abrogating reflux-induced dysbiosis, and mitigating bile acid metabolism and transport, culminating in significant inhibition of EAC through TLR/NF-{kappa}B/P53 signaling cascades. At the species level, C-PAC mitigated reflux-induced pathogenic bacteria (Clostridium perfringens, Escherichia coli, and Proteus mirabilis). C-PAC specifically reversed reflux-induced bacterial, inflammatory and immune-implicated proteins and genes including Ccl4, Cd14, Crp, Cxcl1, Il6, Il1{beta}, Lbp, Lcn2, Myd88, Nfkb1, Tlr2 and Tlr4 aligning with changes in human EAC progression, as confirmed through public databases. C-PAC is a safe promising dietary constituent that may be utilized alone or potentially as an adjuvant to current therapies to prevent EAC progression through ameliorating reflux-induced dysbiosis, inflammation and cellular damage.

cancer biology↗