Biology subjects

James, D.

Publications and source records attributed to James, D..

15 recordsLinked to original sources

bioRxiv2026

Covalent bond formation caught in a LOV photoreceptor

Light-oxygen-voltage (LOV) domains are blue-light photoreceptors of plants, algae and fungi, and among the most widely used tools in optogenetics. They switch on by forming a covalent thioether bond between a conserved cysteine and their flavin chromophore, in a reaction that needs a proton to cross from the cysteine to the flavin through a pocket containing essentially no water. Its mechanism has been debated for two decades1, and because the chemistry is over within a microsecond its elementary steps have stayed hidden. Here we combine 10 time-resolved serial femtosecond crystallography snapshots and infrared spectroscopy with QM/MM calculations to resolve the entire sequence of events at 1.4 [A] resolution: from excited-state distortion of the flavin ring (10-100 ps), through hydration of a surface channel (10 ns) and a single ordered water reaching the active site as the reactive cysteine shifts between its conformations (100-500 ns), to the thioether bond itself, caught half-formed at 1 {micro}s (half the molecules reacted, half still poised) and complete at 10-100 {micro}s. That water bridges the cysteine and the flavin and shuttles the proton, lowering the barrier from [~]35 to [~]15 kcal/mol and accelerating the reaction by roughly fourteen orders of magnitude (without it, the half-life would be [~]237,000 years), then departs before the bond forms. Proteins can therefore hydrate a dehydrated active site transiently and on demand to overcome otherwise prohibitive reaction barriers, a catalytic strategy that reaches well beyond photoreceptors.

biophysics↗

bioRxiv2026

Rac1 deficiency reduces mitochondrial respiratory capacity, impairs fatty acid metabolism and causes muscle wasting

BackgroundThe age-related progressive decline in skeletal muscle function is characterised by declining mitochondrial quality control and perturbed fatty acid metabolism, contributing to frailty and increased mortality. The actin cytoskeleton, a key structural component of skeletal muscle, has recently been implicated in mitochondrial anchoring and dynamics. However, the role of actin-regulating proteins, including the Rho GTPase Rac1, in mitochondrial function and age-associated metabolic and functional muscle deterioration remains undefined. MethodsSkeletal muscle from mice with inducible muscle-specific deletion of Rac1 (Rac1 imKO) underwent unbiased mass spectrometry-based proteomic profiling. Mitochondrial morphology was assessed by transmission electron microscopy, and physiological parameters, including muscle mass and contraction-stimulated palmitate oxidation in isolated soleus muscle, were evaluated. Mitochondrial respiratory function was determined by high-resolution respirometry in permeabilised gastrocnemius skeletal muscle fibre bundles. Biochemically, muscular triacylglycerol (TG) content, mRNA (qPCR) and protein (immunoblotting) content were determined. In vastus lateralis muscle biopsies from healthy, untrained young (20-30 years) and old, sarcopenic (83-94 years) men, Rac1 and mitochondrial respiratory protein abundances were measured. A complementary human genetic association analysis was performed using the FinnGen dataset. ResultsRac1 deficiency triggered muscle wasting in middle-aged mice (Gastrocnemius: -10%; Quadriceps: -7%). Preceding muscle wasting, gene set enrichment analysis identified enrichment in fatty acid metabolism and oxidative phosphorylation pathways, consistent with increased mitochondrial volume density in Rac1 imKO muscle (subsarcolemmal: +467%; intermyofibrillar: +166%). Despite mitochondrial expansion at this stage, Rac1 deficiency attenuated the increase in palmitate oxidation in response to muscle contraction (-62%). At the muscle-wasting stage, Rac1 imKO muscle exhibited reduced mitochondrial respiratory capacity (-25-32%). Additionally, the mitochondrial dysfunction was associated with an accumulation of muscle TG (+78%, p = 0.096) and upregulation of fatty acid transporter, CD36 protein content (+25%), indicative of altered fatty acid handling. In humans, Rac1 muscle protein content was increased in old, sarcopenic subjects compared to young (+41%), and negatively correlated with quadriceps cross-sectional area (CSA) (r = -0.475) and type II fibre CSA (r = -0.466). In old, sarcopenic muscle, Rac1 protein content correlated negatively with protein content of multiple mitochondrial respiratory complexes (CI: r = -0.690, CIV: r = -0.938, CV: r = -0.704). GWAS further identified associations between Rac1 SNP variants and lipid metabolic and muscle-wasting diseases. ConclusionsMuscle Rac1 deficiency reduces mitochondrial respiratory capacity and metabolic flexibility through impaired fatty acid metabolism, leading to muscle wasting and highlighting a potential therapeutic target in age-related functional decline.

Molecular Biology↗

bioRxiv2025

Real-Time Human Interaction with Virtual Swarms in Shared Physical Space

Human-swarm interaction (HSI) explores how humans engage with distributed collective systems, aiming to incorporate human cognition into scalable and robust robotic swarms. While most HSI research focuses on remote teleoperation via engineered interfaces, real-world integration of swarms into everyday tasks requires natural, embodied interactions in shared physical spaces. To address the limitations of traditional teleoperation studies, and the high resource demands of using physical robot swarms for HSI research, we introduce CoBe XR, a spatial augmented reality system that projects virtual swarms into the physical environment of the human operator. CoBe XR enables real-time, fine-grained, natural interaction between humans and swarm-like agents through full-body movement without dedicated control interfaces or prior training. As a proof-of-concept, we present a behavioral study involving 40 participants who influenced swarm behavior solely through walking. Our results show that human participants were able to adapt to the collective dynamics of the swarm and control it through natural perception-motion control in a shared physical space. We argue that similar extended reality systems can not only reveal how humans perceive and adapt to collective dynamics, but they offer a general platform to understand human behavior or an intermediate solution to design embodied robot swarms.

animal behavior and cognition↗

bioRxiv2025

Dysregulation of the actin cytoskeleton and FMRP causes polar body protrusion defects in human fragile X premutation and aged oocytes

Women carrying the fragile X premutation (55-200 CGG repeat expansion, PM) are at risk for developing fragile X-associated primary ovarian insufficiency (FXPOI), which is preceded by fragile X-associated diminished ovarian reserve (FXDOR). So far, the cause of FXDOR/FXPOI could not be comprehensively examined due to the scarcity of human ovarian tissue and oocytes. From studies in model systems, it was proposed that molecular abnormalities within the ovaries or a diminished primordial follicle pool cause FXDOR/FXPOI. To elucidate the defects instigating FXDOR/FXPOI, we examined human oocytes obtained from PM carriers undergoing in vitro fertilization (IVF). We found that the number of MII oocytes was reduced suggesting that the maturation of the oocytes is constrained in PM carriers. Furthermore, immature PM oocytes contained abnormal inclusions, irregular ubiquitin levels and DNA breaks. Despite these defects PM oocytes passed the DNA damage checkpoints. However, in anaphase I PM oocytes failed to initiate the protrusion of the first polar body. In addition, these oocytes amassed bundle actin structures, lacked an actin cap and had elevated profilin1 level. Profilin1 limits the formation of branched actin structures which are necessary for actin cap formation and membrane protrusions. Surprisingly, our results suggest that in PM oocytes an increase in FMRP elevates the profilin1 translation, which leads to the cytoskeleton defects and deficiencies in formation of the first polar body. We also analyzed the decline of MII oocytes in aging human ovaries. Similar, we found that the profilin1 expression and formation of the actin cytoskeleton were dysregulated due to appearance of cytoplasmatic FMRP foci in aged human oocytes. Thus, these results reveal that defects during anaphase I hinder the maturation of human oocytes resulting in FXDOR/FXPOI in PM carriers and a reduction in mature oocytes in women with advanced maternal age.

cell biology↗

bioRxiv2024

Targeting of PTP4A3 overexpression sensitises HGSOC cells towards chemotherapeutic drugs.

Of all the gynecologic malignancies, Ovarian cancer (OC) has the highest mortality rate, partly attributable to its propensity for chemotherapy resistance. The most common sub-type of OC is serous, of which High-Grade Serous Ovarian Cancer (HGSOC) is the most lethal sub-type. Elevated expression of Protein Tyrosine Phosphatase 4 A3 (PTP4A3) is implicated in tumour cell invasion and metastasis, by upregulating the PI3K/Akt/mTORC1 axis. Previously we reported PTP4A3 increased the survival of non-serous OC cells in vitro by activating the autophagy pathway. The present study focused on understanding the impact of PTP4A3 on cell growth, proliferation, and autophagy in HGSOC cells. In particular, we sought to understand whether targeting PTP4A3 in cancer cells that overexpress this phosphatase would sensitise HGSOC cells to existing chemotherapeutic drugs. We report that shRNA-mediated gene silencing of PTP4A3 resulted in the upregulation of compensatory mechanisms that may render PTP4A3 targeting redundant as a monotherapy. However, pan-PTP4A inhibition with JMS-053 overcame this. Finally, silencing of PTP4A3 expression sensitized HGSOC cells to clinically relevant chemotherapeutic drugs. Since mAb therapies targeting PTP4A3 are already in clinical trials, therapeutic targeting of PTP4A3 may have significant value in improving outcomes for those patients with HGSOC, in the clinical setting.

cancer biology↗

bioRxiv2024

Xanthomonas oryzae pv. oryzae type-III effector TAL9b targets a broadly conserved disease susceptibility locus to promote pathogenesis in rice

Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial blight of rice, translocates multiple Transcription Activator-Like Effectors (TALEs) into rice cells. The TALEs localize to the host cell nucleus, where they bind to the DNA in a sequence-specific manner and enhance gene expression to promote disease susceptibility. Xoo strain PXO99A encodes nineteen TALEs, but the host targets of all these TALEs have not been defined. A meta-analysis of rice transcriptome profiles revealed a gene annotated as flavonol synthase/flavanone-3 hydroxylase (henceforth OsS5H/FNS-03g) to be highly induced upon Xoo infection. Further analyses revealed that this gene is induced by PXO99A using TAL9b, a broadly conserved TALE of Xoo. Disruption of tal9b rendered PXO99A less virulent. OsS5H/FNS-03g functionally complemented its Arabidopsis homologue AtDMR6, a well-studied disease susceptibility locus. Biochemical analyses suggested that OsS5H/FNS-03g is a bifunctional protein with Salicylic Acid 5 Hydroxylase (S5H) and Flavone Synthase-I (FNS-I) activities. Further, an exogenous application of apigenin on rice leaves, the flavone that is enzymatically produced by OsS5H/FNS-03g, promoted virulence of PXO99A tal9b-. Overall, our study suggests that OsS5H/FNS-03g is a bifunctional enzyme and its product apigenin is potentially involved in promoting Xoo virulence.

plant biology↗

bioRxiv2023

Capturing the blue-light activated state of the Phot-LOV1 domain from Chlamydomonas reinhardtii using time-resolved serial synchrotron crystallography

Light-Oxygen-Voltage (LOV) domains are small photosensory flavoprotein modules that allow converting external stimuli (sunlight) into intracellular signals responsible for various cell behavior (e.g., phototropism and chloroplast relocation). This ability relies on the light-induced formation of a covalent thioether adduct between a flavin chromophore and a reactive cysteine from the protein environment, which triggers a cascade of structural changes that results in the activation of a serine/threonine (Ser/Thr) kinase. Recent developments in time-resolved crystallography may allow the observation of the activation cascade of the LOV domain in real-time, which has been elusive. In this study, we report a robust protocol for the production and stable delivery of microcrystals of the LOV domain of phototropin Phot-1 from Chlamydomonas reinhardtii (CrPhotLOV1) with a high-viscosity injector for time-resolved serial synchrotron crystallography (TR-SSX). The detailed process covers all aspects, from sample optimization to the actual data collection process, which may serve as a guide for soluble protein preparation for TR-SSX. In addition, we show that the obtained crystals preserve the photoreactivity using infrared spectroscopy. Furthermore, the results of the TR-SSX experiment provide high-resolution insights into structural alterations of CrPhotLOV1 from {Delta}t = 2.5 ms up to {Delta}t = 95 ms post-photoactivation, including resolving the geometry of the thioether adduct and the C-terminal region implicated in the signal transduction process.

biochemistry↗

bioRxiv2023

Joint sequence & chromatin neural networks characterize the differential abilities of Forkhead transcription factors to engage inaccessible chromatin

The DNA-binding activities of transcription factors (TFs) are influenced by both intrinsic sequence preferences and extrinsic interactions with cell-specific chromatin landscapes and other regulatory proteins. Disentangling the roles of these binding determinants remains challenging. For example, the FoxA subfamily of Forkhead domain (Fox) TFs are known pioneer factors that can bind to relatively inaccessible sites during development. Yet FoxA TF binding also varies across cell types, pointing to a combination of intrinsic and extrinsic forces guiding their binding. While other Forkhead domain TFs are often assumed to have pioneering abilities, how sequence and chromatin features influence the binding of related Fox TFs has not been systematically characterized. Here, we present a principled approach to compare the relative contributions of intrinsic DNA sequence preference and cell-specific chromatin environments to a TFs DNA-binding activities. We apply our approach to investigate how a selection of Fox TFs (FoxA1, FoxC1, FoxG1, FoxL2, and FoxP3) vary in their binding specificity. We over-express the selected Fox TFs in mouse embryonic stem cells, which offer a platform to contrast each TFs binding activity within the same preexisting chromatin background. By applying a convolutional neural network to interpret the Fox TF binding patterns, we evaluate how sequence and preexisting chromatin features jointly contribute to induced TF binding. We demonstrate that Fox TFs bind different DNA targets, and drive differential gene expression patterns, even when induced in identical chromatin settings. Despite the association between Forkhead domains and pioneering activities, the selected Fox TFs display a wide range of affinities for preexiting chromatin states. Using sequence and chromatin feature attribution techniques to interpret the neural network predictions, we show that differential sequence preferences combined with differential abilities to engage relatively inaccessible chromatin together explain Fox TF binding patterns at individual sites and genome-wide.

genomics↗

bioRxiv2022

ULTRAFAST STRUCTURAL CHANGES DIRECT THE FIRST MOLECULAR EVENTS OF VISION

Vision is initiated by the rhodopsin family of light-sensitive G protein-coupled receptors (GPCRs). A photon is absorbed by the 11-cis retinal chromophore of rhodopsin which isomerises within 200 femtoseconds to the all-trans conformation, thereby initiating the cellular signal transduction processes that ultimately lead to vision. However, the intramolecular mechanism by which the photoactivated retinal induces the activation events inside rhodopsin remains elusive. In this work, we use ultrafast time-resolved crystallography at room temperature to determine how an isomerised twisted all-trans retinal stores the photon energy required to initiate protein conformational changes associated with the formation of the G protein-binding signalling state. The distorted retinal at 1 ps time-delay of photoactivation has pulled away from half of its numerous interactions with its binding pocket, and the excess of the photon energy is released through an anisotropic protein breathing motion in the direction of the extracellular space. Strikingly, the very early structural motions in the protein side chains of rhodopsin appear in regions involved in later stages of the conserved Class A GPCR activation mechanism. Our work sheds light on the earliest stages of vision in vertebrates and points to fundamental aspects of the molecular mechanisms of agonist-mediated GPCR activation.

biochemistry↗

bioRxiv2022

Lipids Maintain Genomic Stability and Developmental Potency of Murine Pluripotent Stem Cells

Lipids play vital roles in cellular homeostasis and regulate pluripotency of human stem cells. However, the impact of lipids on murine pluripotent stem cells is unclear. While Mek1/2 and Gsk3{beta} inhibition ("2i") supports the maintenance of murine embryonic stem cells (ESCs) in a homogenous naive state, prolonged culture in 2i results in aneuploidy and DNA hypomethylation that impairs developmental potential. Additionally, 2i fails to support derivation and culture of fully potent female ESCs. Here we find that mouse ESCs cultured in 2i/LIF supplemented with lipid-rich albumin (AlbuMAX) undergo pluripotency transition yet maintain genomic stability and full potency over long-term culture. Mechanistically, lipids in AlbuMAX impact intracellular metabolism including nucleotide biosynthesis, lipid biogenesis, and TCA cycle intermediates, with enhanced expression of ZCAN4 and DNMT3s that prevent telomere shortening and DNA hypomethylation. In concert with 2i, lipids induce a formative-like pluripotent state through direct stimulation of Mek-mediated Erk2 phosphorylation, which also alleviates X chromosome loss in female ESCs. Importantly, both male and female "all-ESC" mice can be generated from de novo derived ESCs using AlbuMAX-based media. Our findings underscore the importance of lipids to pluripotency and link nutrient cues to genome integrity in early development.

developmental biology↗

bioRxiv2022

Molecular snapshots of drug release from tubulin over eleven orders of magnitude in time

The binding and release of ligands from their protein targets is central to fundamental biological processes as well as to drug discovery. Photopharmacology introduces chemical triggers that allow the changing of ligand affinities and thus biological activity by light. Insight into the molecular mechanisms of photopharmacology is largely missing because the relevant transitions during the light-triggered reaction cannot be resolved by conventional structural biology. Using time-resolved serial crystallography at a synchrotron and X-ray free-electron laser, we captured the release of the anti-cancer compound azo-combretastatin A4 and the resulting conformational changes in tubulin. Nine structural snapshots from 1 ns to 100 ms complemented by simulations show how cis-to-trans isomerization of the azobenzene bond leads to a switch in ligand affinity, opening of an exit channel, and collapse of the binding pocket upon ligand release. The resulting global backbone rearrangements are related to the action mechanism of microtubule-destabilizing drugs.

molecular biology↗

bioRxiv2021

Coordinated overexpression of OsSUT1, OsSWEET11 and OsSWEET14 in rice impairs carbohydrate metabolism that has implications in plant growth, yield and susceptibility to Xanthomonas oryzae pv oryzae (Xoo)

Enhancing carbohydrate export to sink tissues is considered as a feasible approach for improving photosynthetic efficiency and crop yield. In Oryza sativa Sucrose Transporter OsSUT1 located in companion cells and Sugars Will Eventually be Exported Transporters (SWEETs); OsSWEET11 and OsSWEET14 present in phloem parenchyma mesophyll cell plasma membranes are involved in long distance sucrose transport. OsSWEET11 and OsSWEET14 also play important role in host-pathogen interaction of rice plants and Xanthomonas oryzae pv oryzae (Xoo) that causes bacterial leaf blight. Three genes, OsSUT1, OsSWEET11, and OsSWEET14 were overexpressed under the control of their native promoters in rice to modulate long distance sugar transport and disease resistance. The transgenics displayed several phenotypic aberrations such as reduced plant height and seed weight due to altered sucrose transport and metabolism. Lower sucrose transport rate in transgenics than the WT resulted in reduced sucrose, fructose and glucose and increased starch accumulation in their leaves at the end of dark period. Transcriptional analysis revealed a reduction in the expression of genes involved in sucrose synthesis pathway in transgenics. Normal growth and development of transgenic seedlings were restored in growth media supplemented with 3% sucrose demonstrating in planta sucrose limitation. Remarkably, transgenic lines had diminished susceptibility to Xoo than the WTs due to low sugar content in the leaves demonstrating that rice plants maintain an optimum level of SWEETs for proper plant growth and development, and upregulation of these SWEETs in rice mimicks Xoo attack impelling plants to reduce sugar content in the apoplasm to inhibit pathogen growth.

plant biology↗

bioRxiv2020

hRUV: Hierarchical approach to removal of unwanted variation for large-scale metabolomics data

Liquid chromatography-mass spectrometry based metabolomics studies are increasingly applied to large population cohorts, running for several weeks to months, even extending to years of data acquisition. This inevitably introduces unwanted intra- and inter-batch variations over time that can overshadow true biological signals and thus hinder potential biological discoveries. To date, normalization approaches have struggled to mitigate the variability introduced by technical factors whilst preserving biological variance, especially for protracted acquisitions. Here, we designed an experiment with an arrangement to embed biological sample replicates to measure the variance within and between batches for over 1,000 human plasma samples run over 44 days. We integrate these replicates in a novel workflow to remove unwanted variation in a hierarchical structure (hRUV) by progressively merging the adjustments in neighbouring batches. We demonstrate significant improvement of hRUV over existing methods in maintaining biological signals whilst removing unwanted variation for large scale metabolomics studies.

bioinformatics↗

bioRxiv2020

Screening of PCRP transcription factor antibodies in biochemical assays

Antibodies offer a powerful means to interrogate specific proteins in a complex milieu. However, antibody availability and reliability are problematic and epitope tagging can be impractical in many cases. In an effort to improve this situation, the Protein Capture Reagents Program (PCRP) generated over a thousand renewable monoclonal antibodies (mAbs) against human-presumptive chromatin proteins. However, these reagents have not been widely field-tested. We therefore performed a screen to test their ability to enrich genomic regions via chromatin immunoprecipitation (ChIP) and a variety of orthogonal assays. 887 unique antibodies against 681 unique human transcription factors (TFs), were assayed by ultra-high resolution ChIP-exo/seq, primarily in a single pass in one cell type (K562). Deep systematic analyses of the resulting [~]1,200 ChIP-exo datasets can be found at www.PCRPvalidation.org. Subsets of PCRP mAbs were further tested in ChIP-seq, CUT&RUN, STORM super-resolution microscopy, immunoblots, and protein binding microarray (PBM) experiments. About 5% of the tested antibodies displayed target (i.e., cognate antigen) enrichment across at least one assay and are strong candidates for additional validation. An additional 34% produced ChIP-exo data that was distinct from background and thus warrant further testing. The remaining 61% were not substantially different from background, and likely require consideration of a much broader survey of cell types and/or assay optimizations. We demonstrate and discuss the metrics and challenges to antibody validation in chromatin-based assays.

biochemistry↗

bioRxiv2019

Proton uptake mechanism in bacteriorhodopsin captured by serial synchrotron crystallography

Conformational dynamics are essential for proteins to function. Here we describe how we adapted time-resolved serial crystallography developed at X-ray lasers to visualize protein motions using synchrotrons. We recorded the structural changes upon proton pumping in bacteriorhodopsin over 200 ms in time. The snapshot from the first 5 ms after photoactivation shows structural changes associated with proton release at comparable quality to previous X-ray laser experiments. From 10-15 ms onwards we observe large additional structural rearrangements up to 9 [A] on the cytoplasmic side. Rotation of Leu93 and Phe219 opens a hydrophobic barrier leading to the formation of a water chain connecting the intracellular Asp96 with the retinal Schiff base. The formation of this proton wire recharges the membrane pump with a proton for the next cycle.

biophysics↗