Biology subjects

Green, C.

Publications and source records attributed to Green, C..

At least 19 recordsLinked to original sources

bioRxiv2026

Cryo-EM Structure of a Triazole alpha-Conotoxin GI Mimetic Bound to the Muscle-Type Nicotinic Acetylcholine Receptor

Disulfide-rich peptides possess exceptional potency and selectivity but are often limited by the instability and synthetic challenges associated with native disulfide bonds. Here, we report the design, synthesis, pharmacological evaluation, and structural characterisation of triazole-based peptidomimetics of the -GI conotoxin, a selective antagonist of the muscle-type nicotinic acetylcholine receptor (nAChR). A series of 1,4- and 1,5-disubstituted triazole analogues were prepared entirely on resin using CuAAC and RuAAC chemistry to replace the native Cys3/13 disulfide bridge. Functional evaluation against human muscle nAChRs revealed that 1,5-triazole analogues retained low-nanomolar potency, with the lead mimetic exhibiting activity comparable to native -GI. Cryo-electron microscopy of the lead compound bound to the muscle-type nAChR provided the first structure of a disulfide-isostere peptidomimetic in complex with a membrane receptor. The structure demonstrates that the 1,5-triazole reproduces the native peptide fold with high fidelity while contributing receptor-facing interactions not available to the native disulfide bridge. Molecular dynamics simulations further revealed conserved hydration networks and similar conformational sampling between the native peptide and lead mimetic. Together, these findings establish triazoles as effective disulfide surrogates and provide a structural framework for the rational design of stabilised conotoxin therapeutics.

biochemistry↗

bioRxiv2026

Structures of muscle-type nicotinic acetylcholine receptor with α-conotoxins reveal determinants of receptor-subtype specificity

-Conotoxins (-CTX) are disulfide-rich peptide antagonists with exceptional potency and subtype selectivity across nicotinic acetylcholine receptors (nAChRs), making them promising leads for therapeutic development. The molecular basis of their specificity has remained unresolved for decades, limiting rational drug design. Here, we present the first high-resolution cryo-EM structures of full-length muscle-type nAChR bound to three distinct -CTXs, revealing their precise binding modes at both acetylcholine binding sites. Our integrated approach, combining structural biology, pharmacological profiling, and computational hydration mapping, uncovers conserved pharmacophore features, water-mediated interactions, and subunit-specific determinants that govern potency and selectivity. These findings provide a long-sought molecular framework for -conotoxin recognition and inhibition, addressing a critical gap required to realise the full potential of conotoxins as lead compounds in drug discovery.

molecular biology↗

bioRxiv2025

Evolution of the reproductive interactome: proteomic divergence and the origins of postmating-prezygotic reproductive incompatibilities

Reproductive proteins evolve rapidly, making them strong candidates for driving postmating-prezygotic (PMPZ) reproductive incompatibilities between populations. While most previous studies have focused on protein sequence divergence as a likely driver of PMPZ incompatibilities, reproductive proteomes may also diverge compositionally and/or quantitatively. Here, we combine quantitative proteomics, molecular evolutionary analyses, and protein-protein interaction (PPI) modeling to predict the molecular basis of reproductive incompatibilities between Drosophila mojavensis and D. arizonae. We demonstrate multidimensional divergence in reproductive proteomes including changes at the sequence, compositional, and quantitative levels. We further demonstrate that three divergent male seminal fluid proteins affect the size of the insemination reaction mass and/or fertilization success in D. arizonae. Despite high sequence divergence, predicted protein-protein interactions involving a conserved set of proteases and/or protease inhibitors were predicted to be maintained in heterospecific crosses. In contrast, predicted interspecies protein incompatibilities arose from proteome compositional divergence, suggesting that such changes may play a disproportionate role in PMPZ incompatibilities, at least for the subset of the interactome that we tested. Furthermore, extensive quantitative divergence, particularly for proteases and inhibitors, suggests pervasive stoichiometric mismatches in heterospecific matings. Altogether, our findings indicate that reproductive proteins are evolutionarily labile at multiple levels, and that compositional and quantitative divergence, rather than sequence changes alone, may be central to the early evolution of reproductive isolation.

evolutionary biology↗

bioRxiv2025

SAMP-Score: A morphology-based machine learning classification method for screening pro-senescence compounds in p16 positive cancer cells

BackgroundSenescence identification is rendered challenging due to a lack of universally available biomarkers. This represents a bottleneck in efforts to develop pro-senescence therapeutics - agents designed to induce the arrest of cellular proliferation associated with a senescence response in cancer cells for therapeutic gain. This is particularly true in contexts such as basal-like breast cancer (BLBC), which often express high levels of widely reported senescence hallmarks, which has led to the designation of these subtypes as senescence marker positive (Sen-Mark+). Unfortunately, these are often cancers with the most limited treatment options, where novel pro-senescence compounds would be of potential clinical utility. ResultsTo address these challenges, we have developed SAMP-Score, a machine learning classification tool for identifying senescence induction in Sen-Mark+ cancers. This technique builds upon our previous observation that senescent cells develop distinct senescence-associated morphological profiles (SAMPs), which can be assessed readily in traditionally challenging contexts for senescence identification, including high-throughput screens. ConclusionsThrough application of SAMP-Score, we have identified QM5928, a novel pro-senescence compound, that is able to induce senescence in a variety of Sen-Mark+ cancers and has potential utility as a tool molecule to explore the mechanisms and pathways through which senescence induction occurs in these cells.

cancer biology↗

bioRxiv2025

Dynamic structural connectivity changes in cortical and cortico-striatal strokes in mice

Stroke is a primary global health concern, leading to significant mortality and long-term disability. Beyond immediate neuronal damage, functional and structural connectivity is altered brain-wide with implications for functional deficits and recovery. It remains unclear, however, if the level of degeneration, i.e. reduced myelination and axonal damage, as well as compensatory plasticity, i.e., axonal sprouting and remyelination, depend on the lesion size and topology. This study compares for the first time two different stroke models in adult male mice, with the aim of uncovering the dynamics in white matter changes. Repetitive diffusion magnetic resonance imaging (dMRI) over four weeks post photothrombotic cortical (1.41{+/-}0.92% of brain volume), and middle cerebral artery occlusion (MCAO) cortico-striatal (11.53{+/-}2.8% of brain volume), strokes were used to map structural connectivity changes at the whole-brain level. We quantified inter- and intra-hemispheric seed strength changes over time, with seed strength reflecting how strongly each region is connected to the rest of the brain. Differences between groups and time points were assessed using a mixed model corrected for multiple comparisons. In conclusion, large cortico-striatal lesions led to increased structural connectivity in sensorimotor regions, whereas small cortical lesions induced asymmetric connectivity changes: an increase extending globally from the ischemic hemisphere and a decrease expanding globally from the healthy hemisphere. These findings highlight that stroke severity and lesion size significantly affect the temporal dynamics and spatial distribution of connectivity disruptions, emphasizing the need for targeted monitoring of neural changes post-stroke. Significance statementPathophysiological changes close to an ischemic lesion are well understood. But structurally connected regions also undergo stroke-related changes potentially contributing to functional deficits. Animal studies using in vivo diffusion MRI to quantify white matter changes have been limited to single connections, time points after stroke, and basic diffusion measures. We provide the first in-depth characterization of whole-brain fiber tracking to compare small cortical and large cortico-striatal strokes. We found a complex dynamic of decreasing and increasing structural connectivity in single sensorimotor-related brain regions within the brain network - even remote areas from the stroke lesion. This study provides a better understanding of the interactions of regions during stroke recovery, which may help in developing therapies targeting localized white matter repair.

neuroscience↗

bioRxiv2025

Glucosylceramide induced ectosomes propagate pathogenic α-synuclein in Parkinson's disease

Intercellular transmission of -synuclein contributes to Parkinsons disease pathology. Yet, the mechanisms of -synuclein spread are not fully understood. Here, we used live-cell microscopy to examine the impact of Parkinsons disease associated lipid alterations on -synuclein release. We discovered that increased glucosylceramides induce ectosome shedding from primary neurons, and from dopaminergic neurons derived from Parkinsons disease patient iPSCs harboring mutations in GBA1 (N370S, L444P and W378G) and LRRK2 (G2019S and R1441H) compared to their isogenic control. We show that elevated glucosylceramide similarly increases vesicle release and uptake by other neurons in living mouse brains using 2-photon microscopy. Finally, we show that ectosomes are loaded with pathogenic -synuclein and lead to the transmission of -synuclein pathology to neighbouring neurons. These data reveal ectosomes as the predominant route for -synuclein transmission that can only be appreciated by live-cell imaging technologies.

cell biology↗

bioRxiv2024

Requirement for Fc effector function is overcome by binding potency for broadly reactive anti-alphavirus antibodies

Alphaviruses are emerging public health threats. Broadly reactive anti-alphavirus monoclonal antibodies (mAbs) have been shown to be protective in mouse models of infection. However, the mechanism of Fc-dependent or Fc-independent heterologous protection remains ill-defined in vivo. Here, we used two vaccine-elicited, broadly reactive, anti-alphavirus mAbs, SKT05 and SKT20, to establish correlates of mAb-mediated protection during Venezuelan equine encephalitis virus (VEEV) challenge. SKT20 required Fc effector functions to prevent lethality. In contrast, SKT05-mediated survival was independent of Fc effector functions, which is likely linked to early viral control through potent egress inhibition. However, control of virus replication and spread with SKT05 was Fc-dependent; these findings extended to additional in vivo models with alternative VEEV subtypes and chikungunya virus. During therapeutic delivery of SKT05, Fc effector functions were only required at 3 days post-infection. The necessity of Fc effector functions for SKT20 was related to mAb binding avidity rather than epitope and could be overcome by increasing the dose of SKT20 relative to the functional avidity of SKT05. Collectively, this study identified antibody avidity as a correlate for in vivo efficacy and associated Fc-dependent mechanisms that can be leveraged for therapeutic development of monoclonal antibodies against alphaviruses. One sentence summaryFunctional avidity of broadly reactive anti-alphavirus antibodies dictates requirement for Fc-mediated protection.

microbiology↗

bioRxiv2024

Visualization of breast cancer using contrast-enhanced optical coherence elastography based on tissue heterogeneity

By mapping the mechanical properties of tissue, elastography can improve identification of breast cancer. On the macro-scale, ultrasound elastography and magnetic resonance elastography have emerged as effective clinical methods for the diagnosis of tumors. On the micro-scale, optical coherence elastography (OCE) shows promise for intraoperative tumor margin assessment during breast-conserving surgery. Whilst several OCE studies have demonstrated strong potential, the mechanical models used require the assumption of uniaxial stress throughout the sample. However, breast tissue is heterogeneous and contains compressible features (e.g., ducts and blood vessels) and collagen-rich fibrotic features (e.g., stroma). This heterogeneity can invalidate the assumption of uniaxial stress and reduce the accuracy of OCE, often making it challenging to interpret images. Here, we demonstrate a new variant of OCE based on mapping the Euler angle, i.e., the angle between the principal compression and the loading axis induced by tissue heterogeneity, which removes the assumption of uniaxial deformation. This is enabled by a hybrid three-dimensional (3-D) displacement estimation method that combines phase-sensitive detection and complex cross-correlation, providing access to the 3-D displacement and 3-D strain tensor on the micro-scale. We demonstrate this new OCE technique through experiments on phantoms and 10 fresh human breast specimens. Through close correspondence with histology, our results show that mapping the Euler angle provides additional contrast to both optical coherence tomography and a current OCE technique in identifying cancer. Mapping the Euler angle in breast tissue may provide a new biomarker for intraoperative tumor margin assessment.

bioengineering↗

bioRxiv2023

Dietary restriction of individual amino acids stimulates unique molecular responses in mouse liver

Dietary protein and essential amino acid (EAA) restriction promote favorable metabolic reprogramming, although the extent to which shared or EAA-specific mechanisms facilitate diet-associated phenotypes remains unclear. Here, we compared the physiological and molecular effects of dietary methionine, leucine, or isoleucine depletion (Met-D, Leu-D, and Ile-D) in C57BL/6J mice. Each diet elicited responses not phenocopied by mTORC1 inhibition, including reduced fat mass and hepatic amino acid catabolism. Ile-D yielded additional distinct responses, highlighted by histone H2A/H4 hypoacetylation and maintained hepatic acetyl-CoA levels despite downregulated FA {beta}-oxidation. Multi-Omics Factor Analysis of 14,139 data points objectively affirmed Ile-D phenotypes are distinct from Met-D or Leu-D and identified several metabolic and chromatin features as primary discriminators. Metabolic and epigenetic responses to Ile-D were recapitulated in vitro, suggesting underlying mechanisms represent fundamental cellular properties. Together, these results demonstrate EAAs can stimulate unique phenotypes and highlight distinct molecular mechanisms by which EAAs may inform metabolic health.

biochemistry↗

bioRxiv2023

Females translate male mRNA transferred during mating

Although RNA is found in the seminal fluid of diverse organisms, it is unknown whether this RNA is functional within females. Here, we develop an experimental proteomic method called VESPA (Variant Enabled SILAC Proteomic Analysis) to test the hypothesis that Drosophila male seminal fluid RNA is translated by females. We find strong evidence for 67 male-derived, female-translated proteins (mdFTPs) in female lower reproductive tracts at six hours postmating, many with predicted functions relevant to reproduction. Gene knockout experiments indicate that genes coding for mdFTPs play diverse roles in postmating interactions, with effects on fertilization efficiency, and the formation and persistence of the insemination reaction mass, a trait hypothesized to be involved in sexual conflict. These findings advance our understanding of reproduction by revealing a novel mechanism of postmating molecular interactions between the sexes that strengthens and extends male influences on reproductive outcomes in previously unrecognized ways. Given the diverse species known to carry RNA in seminal fluid, this discovery has broad significance for understanding molecular mechanisms of cooperation and conflict during reproduction.

evolutionary biology↗

bioRxiv2022

Pan-genome inversion index reveals evolutionary insights into the subpopulation structure of Asian rice (Oryza sativa)

Understanding and exploiting genetic diversity is a key factor for the productive and stable production of rice. Utilizing 16 high-quality genomes that represent the subpopulation structure of Asian rice (O. sativa), plus the genomes of two close relatives (O. rufipogon and O. punctata), we built a pan-genome inversion index of 1,054 non-redundant inversions that span an average of [~] 14% of the O. sativa cv. Nipponbare reference genome sequence. Using this index we estimated an inversion rate of 1,100 inversions per million years in Asian rice, which is 37 to 73 times higher than previously estimated for plants. Detailed analyses of these inversions showed evidence of their effects on gene regulation, recombination rate, linkage disequilibrium and agronomic trait performance. Our study uncovers the prevalence and scale of large inversions ([≥] 100 kb) across the pan-genome of Asian rice, and hints at their largely unexplored role in functional biology and crop performance.

genomics↗

bioRxiv2021

Symptomatic fever management in children A systematic review of national and international guidelines

IntroductionA comprehensive assessment of the recommendations made by clinical practice guidelines (CPGs) on symptomatic fever management in children has not been carried out. MethodsSearches were conducted on Pubmed, google scholar, pediatric society websites and guideline databases to locate CPGs from each country. Rather than assessing overall guideline quality, the level of evidence for each recommendation was evaluated according to criteria of the Oxford Centre for Evidence-Based Medicine (OCEBM). A GRADE assessment was undertaken to assess the body of evidence related to a single question: the threshold for initiating antipyresis. Results74 guidelines were retrieved. Recommendations for antipyretic threshold, type and dose; ambient temperature; dress/covering; activity; fluids; nutrition; proctoclysis; external applications; complementary/herbal recommendations; media; and age-related treatment differences all varied widely. OCEBM evidence levels for most recommendations were low (Level 3-4) or indeterminable. The GRADE assessment revealed a very low level of evidence for a threshold for antipyresis. ConclusionThere is no recommendation on which all guidelines agree, and many are inconsistent with the evidence - this is true even for recent guidelines. The threshold question is of fundamental importance and has not yet been answered. Guidelines for the most frequent intervention (antipyresis) remain problematic.

immunology↗

bioRxiv2020

C. elegans pharyngeal pumping provides a whole organism bio-assay to investigate anti-cholinesterase intoxication and antidotes

Inhibition of acetylcholinesterase by either organophosphates or carbamates causes anti-cholinesterase poisoning. This arises through a wide range of neurotoxic effects triggered by the overstimulation of the cholinergic receptors at synapses and neuromuscular junctions. Without intervention, this poisoning can lead to profound toxic effects, including death, and the incomplete efficacy of the current treatments, particularly for oxime-insensitive agents, provokes the need to find better antidotes. Here we show how the non-parasitic nematode Caenorhabditis elegans offers an excellent tool for investigating the acetylcholinesterase intoxication. The C. elegans neuromuscular junctions show a high degree of molecular and functional conservation with the cholinergic transmission that operates in the autonomic, central and neuromuscular synapses in mammals. In fact, the anti-cholinesterase intoxication of the worm’s body wall neuromuscular junction has been unprecedented in understanding molecular determinants of cholinergic function in nematodes and other organisms. We extend the use of the model organism’s feeding behaviour as a tool to investigate carbamate and organophosphate mode of action. We show that inhibition of the cholinergic-dependent rhythmic pumping of the pharyngeal muscle correlates with the inhibition of the acetylcholinesterase activity caused by aldicarb, paraoxons and DFP exposure. Further, this bio-assay allows one to address oxime dependent reversal of cholinesterase inhibition in the context of whole organism recovery. Interestingly, the recovery of the pharyngeal function after such anti-cholinesterase poisoning represents a sensitive and easily quantifiable phenotype that is indicative of the spontaneous recovery or irreversible modification of the worm acetylcholinesterase after inhibition. These observations highlight the pharynx of C. elegans as a new tractable approach to explore anti-cholinesterase intoxication and recovery with the potential to resolve critical genetic determinants of these neurotoxins’ mode of action.Competing Interest StatementThe authors have declared no competing interest.View Full Text

pharmacology and toxicology↗

bioRxiv2020

Autophagy in T cells from aged donors is maintained by spermidine, and correlates with function and vaccine responses

Older adults are at high risk for infectious diseases such as the recent COVID-19 and vaccination seems to be the only long-term solution to the pandemic. While most vaccines are less efficacious in older adults, little is known about the molecular mechanisms that underpin this. Autophagy, a major degradation pathway and one of the few processes known to prevent aging, is critical for the maintenance of immune memory in mice. Here, we show induction of autophagy is specifically induced in human vaccine-induced antigen-specific T cells in vivo. Reduced IFN{gamma} secretion by vaccine-induced T cells in older vaccinees correlates with low autophagy. We demonstrate in human cohorts that levels of the endogenous autophagy-inducing metabolite spermidine, fall with age and supplementing it in vitro recovers autophagy and T cell function. Finally, our data show that endogenous spermidine maintains autophagy via the translation factor eIF5A and transcription factor TFEB. With these findings we have uncovered novel targets and biomarkers for the development of anti-aging drugs for human T cells, providing evidence for the use of spermidine in improving vaccine immunogenicity in the aged human population.

physiology↗

bioRxiv2020

Single-cell RNA sequencing of human, macaque, and mouse testes uncovers conserved and divergent features of mammalian spermatogenesis

Spermatogenesis is a highly regulated process that produces sperm to transmit genetic information to the next generation. Although extensively studied in mice, our current understanding of primate spermatogenesis is limited to populations defined by state-specific markers defined from rodent data. As between-species differences have been reported in the process duration and cellular differentiation hierarchy, it remains unclear how molecular markers and cell states are conserved or have diverged from mice to man. To address this challenge, we employ single-cell RNA-sequencing to identify transcriptional signatures of major germ and somatic cell-types of the testes in human, macaque and mice. This approach reveals differences in expression throughout spermatogenesis, including the stem/progenitor pool of spermatogonia, classical markers of differentiation, potential regulators of meiosis, the kinetics of RNA turnover during spermatid differentiation, and germ cell-soma communication. These datasets provide a rich foundation for future targeted mechanistic studies of primate germ cell development and in vitro gametogenesis.

genetics↗

bioRxiv2019

ZCWPW1 is recruited to recombination hotspots by PRDM9, and is essential for meiotic double strand break repair

During meiosis, homologous chromosomes pair (synapse) and recombine, enabling balanced segregation and generating genetic diversity. In many vertebrates, recombination initiates with double-strand breaks (DSBs) within hotspots where PRDM9 binds, and deposits H3K4me3 and H3K36me3. However, no protein(s) recognising this unique combination of histone marks have yet been identified.\n\nWe identified Zcwpw1, which possesses H3K4me3 and H3K36me3 recognition domains, as highly co-expressed with Prdm9. Here, we show that ZCWPW1 has co-evolved with PRDM9 and, in human cells, is strongly and specifically recruited to PRDM9 binding sites, with higher affinity than sites possessing H3K4me3 alone. Surprisingly, ZCWPW1 also recognizes CpG dinucleotides, including within many Alu transposons.\n\nMale Zcwpw1 homozygous knockout mice show completely normal DSB positioning, but persistent DMC1 foci at many hotspots, particularly those more strongly bound by PRDM9, severe DSB repair and synapsis defects, and downstream sterility. Our findings suggest a model where ZCWPW1 recognition of PRDM9-bound sites on either the homologous, or broken, chromosome is critical for synapsis, and hence fertility.\n\nGraphical Abstract LegendIn humans and other species, recombination is initiated by double strand breaks at sites bound by PRDM9. Upon binding, PRDM9 deposits the histone marks H3K4me3 and H3K36me, but the functional importance of these marks has remained unknown. Here, we show that PRDM9 recruits ZCWPW1, a reader of both these marks, to its binding sites genome-wide. ZCWPW1 does not help position the breaks themselves, but is essential for their downstream repair and chromosome pairing, and ultimately meiotic success and fertility in mice.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=153 SRC=\"FIGDIR/small/821678v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (31K):\norg.highwire.dtl.DTLVardef@141deb9org.highwire.dtl.DTLVardef@18559deorg.highwire.dtl.DTLVardef@1cf380borg.highwire.dtl.DTLVardef@1689072_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2019

Mechanisms of Chromosomal Instability in High-grade Serous Ovarian Carcinoma.

Chromosomal instability (CIN), the continual gain and loss of chromosomes or parts of chromosomes, occurs in the majority of cancers and confers poor prognosis. Mechanisms driving CIN remain unknown in most cancer types due to a scarcity of functional studies. High-grade serous ovarian carcinoma (HGSC), the most common subtype of ovarian cancer, is the major cause of death due to gynaecological malignancy in the Western world with chemotherapy resistance developing in almost all patients. HGSC exhibits high rates of chromosome aberrations and knowledge of causative mechanisms is likely to represent an important step towards combating the poor prognosis of this disease. However, very little is known about the nature of chromosomal instability exhibited by this cancer type in particular due to a historical lack of appropriate cell line models. Here we perform the first in-depth functional characterisation of mechanisms driving CIN in HGSC by analysing eight cell lines that accurately recapitulate HGSC genetics as defined by recent studies. We show, using a range of established functional CIN assays combined with live cell imaging and single molecule DNA fibre analysis, that multiple mechanisms co-exist to drive CIN in HGSC. These include supernumerary centrosomes, elevated microtubule dynamics and DNA replication stress. By contrast, the spindle assembly checkpoint was intact. These findings are relevant for developing therapeutic approaches to manipulating CIN in ovarian cancer, and suggests that such approaches may need to be multimodal to combat multiple co-existing CIN drivers.

cancer biology↗

bioRxiv2019

Tracing Autism Traits in Large Multiplex Families to Identify Endophenotypes of the Broader Autism Phenotype

Families comprising many individuals with Autism Spectrum Disorder (ASD) may carry a dominant predisposing mutation. Our aim was to use rigorous phenotyping of the Broader Autism Phenotype (BAP) in large multiplex ASD families to identify endophenotypes of the BAP for future genetic studies. We evaluated ASD/BAP features using standardised tests and a semi-structured interview to assess social, intellectual, executive and adaptive functioning in 109 individuals, including two large multiplex families (Family A: 30; Family B: 34) and an independent sample of small families (n=45). Our protocol identified four psychological endophenotypes of the BAP that were evident in both samples, and showed high sensitivity (97%) and specificity (82%) for individuals classified with the BAP. The patterns of inheritance of these endophenotypes varied in the two large families, supporting their utility for identifying genes in autism.

neuroscience↗