Biology subjects

Wood, R.

Publications and source records attributed to Wood, R..

12 recordsLinked to original sources

bioRxiv2026

Deciphering Coccolith Formation: Advanced Microscopy Insights from the Biomineralisation of Gephyrocapsa huxleyi

Coccolithophores are unicellular marine phytoplankton that produce complex and intricately shaped mineralised scales called coccoliths. Coccoliths are produced in an intracellular vesicle where crystal nucleation occurs, from which several individual calcite units develop with anisotropic crystallographic facets, prompting studies into the cellular mechanisms which control crystal growth within the cell. Here, we characterise those morphological developments in 3D that occur during the formation of coccoliths by the species Gephyrocapsa huxleyi using cryo-ptychographic X-ray computed tomography. This technique is ideally suited to study coccolith mineral development, as intracellular structures can be imaged intact in their native state without needing to disrupt cells. Combined with additional imaging of developing coccoliths using cryo-transmission electron microscopy and scanning electron microscopy, we report the developmental stages involved in coccolith growth across the complete mineralisation period, while also showing that the constrained space created by individual crystal units growing in close confinement affects the final crystal morphology and overall mineral structure. These findings provide clarification on the mineralisation pathways that coccolithophores and other biomineralising organisms use to control the formation of highly functionalised crystalline structures, particularly relevant in the design of materials with tunable properties.

biophysics↗

bioRxiv2025

Population-scale cellular GUIDE-seq-2 and biochemical CHANGE-seq-R profiles reveal human genetic variation frequently affects Cas9 off-target activity

Genome editing enzymes can introduce targeted changes to DNA in living cells1-4, transforming biological research and enabling the first approved gene editing therapy for sickle cell disease5. However, their activity can be altered by genetic variation at on- or off-target sites6-8, potentially impacting both their precision and therapeutic safety. Due to a lack of scalable methods to measure genome-wide editing activity in cells from large populations and diverse target libraries, the frequency and extent of these variant effects on editing remain unknown. Here, we present the first systematic, population-scale study of how genetic variation affects the cellular genome-wide activity of CRISPR-Cas9, enabled by a novel, sensitive, and unbiased cellular assay, GUIDE-seq-2, with improved scalability and accuracy compared to the original broadly adopted method9. Analyzing Cas9 genome-wide activity at 1,115 on- and off-target sites across six guide RNAs in cells from 95 individuals spanning four genetically diverse populations, we found that genetic variants frequently overlap off-target sites, with 14% significantly altering Cas9 editing activity. To understand the effect of mismatches in more diverse sequence contexts, we developed a novel method, combinatorial high-throughput analysis of nuclease cleavage effects (CHANCE-seq), the first massively parallel biochemical approach that can quantify Cas9 activity across millions of mismatched target sites. We leveraged this large-scale CHANCE-seq dataset to train a context-aware deep neural network model, CHANCE-net, to accurately predict and interpret the effects of single-nucleotide variants on off-targets with up to six mismatches. Our deep combinatorial profiling of Cas9 off-target activity revealed frequent, context-dependent synergistic effects of mismatches. Taken together, our findings illuminate an approach to accounting for genetic variation when designing genome-editing strategies for research and therapeutics.

genomics↗

bioRxiv2024

Dynamic change of calcium-rich compartments during coccolithophore biomineralization

Coccolithophores are abundant marine phytoplankton that produce biomineralized calcite scales, called coccoliths, which sequester substantial amounts of carbon and play a significant role in biogeochemical cycles. However, mechanisms underlying the storage and transport of ions essential for calcification remain unresolved. We used ptychographic X-ray computed tomography under cryogenic conditions to visualize intracellular calcium-rich structures involved in the storage of calcium ions in the coccolithophore species Chrysotila carterae. During calcification, we observed a range of structures, from small electron-dense bodies within larger compartments, to denser and distributed globular compartments, before returning to small bodies once scale formation is complete. Nanobeam-scanning X-ray fluorescence measurements further revealed these electron-dense bodies are rich in P and Ca (molar ratio of [~]4:1). We infer from the dynamic nature of structures that these bodies are part of required cellular calcium ion transport pathways, a fundamental process critical for understanding the response of coccolithophores to climate change.

cell biology↗

bioRxiv2024

Mitochondrial Dysfunction and Fatigue in Sjögren's Disease

ObjectivesSjogrens disease (SjD) is a common exocrine disorder typified by chronic inflammation and dryness, but also profound fatigue, suggesting a pathological basis in cellular bioenergetics. In healthy states, damaged or dysfunctional mitochondrial components are broken down and recycled by mitophagy, a specialized form of autophagy. In many autoimmune disorders, however, evidence suggests that dysfunctional mitophagy allows poorly functioning mitochondria to persist and contribute to a cellular milieu with elevated reactive oxygen species. We hypothesized that mitophagic processes are dysregulated in SjD and that dysfunctional mitochondria contribute to overall fatigue. We sought to link fatigue with mitochondrial dysfunction directly in SjD, heretofore unexamined, and further sought to assess the pathogenic extent and implications of dysregulated mitophagy in SjD. MethodsWe isolated pan T cells via negative selection from the peripheral blood mononuclear cells of 17 SjD and 8 age-matched healthy subjects, all of whom completed fatigue questionnaires prior to phlebotomy. Isolated T cells were analyzed for mitochondrial oxygen consumption rate (OCR) and glycolysis using Seahorse, and linear correlations with fatigue measures were assessed. A mitophagy transcriptional signature in SjD was identified by reanalysis of whole-blood microarray data from 190 SjD and 32 healthy subjects. Differential expression analyses were performed by case/control and subgroup analyses comparing SjD patients by mitophagy transcriptional cluster against healthy subjects followed by bioinformatic interpretation using gene set enrichment analysis. ResultsBasal OCR, ATP-linked respiration, maximal respiration, and reserve capacity were significantly lower in SjD compared to healthy subjects with no observed differences in non-mitochondrial respiration, basal glycolysis, or glycolytic stress. SjD lymphocytic mitochondria show structural alterations compared to healthy subjects. Fatigue scores related to pain/discomfort in SjD correlated with the altered OCR. Results from subgroup analyses by mitophagic SjD clusters revealed highly variable inter-cluster differentially expressed genes (DEGs) and expanded the number of SjD-associated gene targets by tenfold within the same dataset. ConclusionMitochondrial dysfunction, associated with fatigue, is a significant problem in SjD and warrants further investigation.

immunology↗

bioRxiv2023

Central and peripheral tau retention modulated by an anti-tau antibody.

Tau protein blood levels dependent on its distribution to peripheral organs and possible elimination from the body. Thus, the peripheral distribution of CSF-derived tau protein was explored, especially since there is a transition to blood-based biomarkers and the emerging idea that tau pathology may spread beyond brain. Near infrared fluorescence (NIRF) was mainly used to analyze tau (tau-NIRF) distribution after its intracisternal or intravenous injection. There was a striking uptake of blood- or CSF-derived tau-NIRF protein by the skeletal structures, liver, small intestine (duodenum), gall bladder, kidneys, urinary bladder, lymph nodes, heart, and spleen. In aging and in older APP/PS1 mice, tau uptake in regions, such as the brain, liver, and skeleton, was increased. In bone (femur) injected tau protein was associated with integrin-binding sialoprotein (IBSP), a major non-collagenous glycoprotein that is associated with mineralization. Tau-NIRF was cleared slowly from CSF via mainly across the cribriform plate, and cervical lymph nodes. In brain, some of the CSF injected tau protein was associated with NeuN-positive and PDGFRy-positive cells, which may explain its retention. The presence of tau in the bladders suggested excretion routes of tau. CSF anti-tau antibody increased CSF tau clearance, while blood anti-tau antibody decreased tau accumulation in the femur but not in liver, kidney, and spleen. Thus, the data show a body-wide distribution and retention of CSF-derived tau protein, which increased with aging and in older APP/PS1 mice. Further work is needed to elucidate the relevance of tau accumulation in each organ to tauopathy.

physiology↗

bioRxiv2023

Rat hepatitis E virus (HEV) cross-species infection and transmission in pigs

Rocahepevirus ratti, an emerging hepatitis E virus (HEV), has recently been found to be infectious to humans. Rats are a primary reservoir of the virus; thus, it is referred to as "rat HEV". Rats are often found on swine farms in close contact with pigs. Our goal was to determine whether swine may serve as a transmission host for rat HEV by characterizing an infectious cDNA clone of a zoonotic rat HEV, strain LCK-3110, in vitro and in vivo. RNA transcripts of LCK-3110 were constructed and assessed for their replicative capacity in cell culture and in gnotobiotic pigs. Fecal suspension from rat HEV-positive gnotobiotic pigs was inoculated into conventional pigs cohoused with naive pigs. Our results demonstrated that capped RNA transcripts of LCK-3110 rat HEV replicated in vitro and successfully infected conventional pigs that transmit the virus to cohoused animals. The infectious clone of rat HEV may afford an opportunity to study the genetic mechanisms of rat HEV cross-species infection and tissue tropism. Significance StatementNew zoonotic strains of Rocahepevirus ratti (rat HEV) have emerged infecting both immunocompetent and immunosuppressed people through unknown transmission sources. Pigs are a primary source of transmission for human HEV strains and could be serving a similar role for rat HEV transmission as rats are a common pest found on swine farms worldwide. Rats could be transmitting rat HEV to pigs which could then be transmitted to humans. Determining susceptibility of pigs to emerging zoonotic rat HEV strains can define potentially new transmission routes to inform public health policy and could provide pathology models for rat HEV disease.

microbiology↗

bioRxiv2022

Keratose sponges in ancient carbonates - a problem of interpretation

Increasing current interest in sponge fossils includes numerous reports of diverse vermicular and peloidal structures interpreted as keratose sponges in Neoproterozoic to Mesozoic carbonates and in various open marine to peritidal and restricted settings. Reports of their occurrence are fundamental and far-reaching for understanding microfacies and diagenesis where they occur; and fossil biotic assemblages, as well as wider aspects of origins of animals, sponge evolution/ecology and the systemic recovery from mass extinctions. Keratose sponges: 1) have elaborate spongin skeletons but no spicules, thus lack mineral parts and therefore have poor preservation potential so that determining their presence in rocks requires interpretation; and 2) are presented in publications as interpreted fossil structures almost entirely in two-dimensional (thin section) studies, where structures claimed as sponges comprise diverse layered, network, particulate and amalgamated fabrics involving calcite sparite in a micritic groundmass. There is no verification of sponges in these cases and almost all of them can be otherwise explained; some are certainly not correctly identified. The diversity of structures seen in thin sections may be reinterpreted to include: a) meiofaunal activity; b) layered, possibly microbial (spongiostromate) accretion; c) sedimentary peloidal to clotted micrites; d) fluid escape and capture resulting in birdseye to vuggy porosities; and e) molds of siliceous sponge spicules. Without confirmation of keratose sponges in ancient carbonates, interpretations of their role in ancient carbonate systems, including facies directly after mass extinctions, are unsafe, and alternative explanations for such structures should be considered. This study calls for greater critical appraisal of evidence, to seek confirmation or not, of keratose sponge presence. (259/300 max, for Sedimentology)

paleontology↗

bioRxiv2021

Aerosolization of Mycobacterium tuberculosis by tidal breathing

RationaleInterrupting tuberculosis (TB) transmission requires an improved understanding of how - and when - the causative organism, Mycobacterium tuberculosis (Mtb), is aerosolized. Although Cough is commonly assumed to be the dominant source of Mtb aerosols, recent evidence of Cough-independent Mtb release implies the contribution of alternative mechanisms. ObjectiveTo compare the aerosolization of Mtb and particulate matter from GeneXpert-positive patients during three separate respiratory manoeuvres: Tidal Breathing (TiBr), Forced Vital Capacity (FVC), and Cough. MethodologyBioaerosol sampling and Mtb detection were combined with real-time assessments of CO2 production and particle counts from 39 confirmed TB patients. Measurements and Main ResultsTiBr and FVC produced comparable numbers of particles, with Cough producing >4-fold more. For all manoeuvres, the proportions of particles detected across size categories from 0.5 - 5 m were similar, with minor differences observed only in particles between 1.5 - 2 m (p = 0.014) and >5 m (p = 0.020). Viable Mtb bacilli were detected in 66%, 70%, and 65% of TiBr, FVC, and Cough samples, respectively. Notably, while Cough produced 3-fold more Mtb than TiBr, the relative infrequency of coughing compared to breathing implies that TiBr likely contributes >90% of the daily aerosolised Mtb across a range of Cough frequencies. ConclusionsOur results suggest that, while Cough increases particle aerosolization compared to TiBr, this is not associated with increased Mtb aerosolization. Instead, TiBr produces more Mtb per particle than Cough. Assuming the number of viable Mtb organisms detected provides a proxy measure of patient infectiousness, these observations imply a significant contribution of TiBr to TB transmission.

microbiology↗

bioRxiv2021

Half a century of stable antibiotic resistance in livestock-associated Staphylococcus aureus and its dynamic readaptation to humans

Mobile genetic elements (MGEs) are agents of horizontal gene transfer in bacteria, but can also be vertically inherited by daughter cells. Establishing the dynamics that led to contemporary patterns of MGEs in bacterial genomes is central to predicting the emergence and evolution of novel and resistant pathogens. Methicillin-resistant Staphylococcus aureus (MRSA) clonal-complex (CC) 398 is the dominant MRSA in European livestock and a growing cause of human infections. Previous studies have identified three categories of MGEs whose presence or absence distinguishes livestock-associated CC398 from a closely related and less antibiotic-resistant human-associated population. Here we fully characterise the evolutionary dynamics of these MGEs using a collection of 1,180 CC398 genomes, sampled from livestock and humans, over 27 years. We find that the emergence of livestock-associated CC398 coincided with the acquisition of a Tn916 transposon carrying a tetracycline resistance gene, which has been stably inherited for 57 years. This was followed by the acquisition of a type V SCCmec that carries methicillin, tetracycline and heavy metal resistance genes, which has been maintained for 35 years, with occasional truncations and replacements with type IV SCCmec. In contrast, a class of prophages that carry a human immune evasion gene cluster and that are largely absent from livestock-associated CC398, have been repeatedly gained and lost in both human- and livestock-associated CC398. These contrasting dynamics mean that when livestock-associated MRSA is transmitted to humans, adaptation to the human host outpaces loss of antibiotic resistance. In addition, the stable inheritance of resistance-associated MGEs suggests that the impact of ongoing reductions in antibiotic and zinc oxide use in European farms on livestock-associated MRSA will be slow to be realised.

evolutionary biology↗

bioRxiv2019

Capture and visualization of live Mycobacterium tuberculosis bacilli from tuberculosis bioaerosols

The tuberculosis (TB) pandemic demands urgent interventions such as those designed to interrupt Mycobacterium tuberculosis (Mtb) transmission, a challenge exacerbated by our poor understanding of the events enabling successful transfer of infectious bacilli between hosts. To address this problem, we developed the Respiratory Aerosol Sampling Chamber (RASC), a personal clean-room equipped with high-efficiency filtration and sampling technologies that allow biosafe capture and isolation of particulate matter - including Mtb bacilli - released by patients during natural breathing and (non-induced) cough. Here, we demonstrate the use of DMN-trehalose labelling to detect and quantify live Mtb bacilli among complex bioaerosol samples arrayed in a bespoke nanowell device following capture in the RASC. A pilot study identified Mtb in more than 85 % of known TB patients, improving significantly on previous work which has relied on animal infection and cough sampling to estimate transmission events. Moreover, intra-patient comparisons of bioaerosol and sputum samples indicated that Mtb aerosols likely derive from a compartment other than sputum. These results support the utility of the RASC platform for research aimed at interrupting Mtb transmission, including the non-invasive detection of Mtb-infected individuals who are predicted to contribute to bacillary spread despite the absence of clinical symptoms.

microbiology↗

bioRxiv2019

The impact of delayed switch to second-line antiretroviral therapy on mortality, depending on failure time definition and CD4 count at failure

BackgroundLittle is known about the functional relationship of delaying second-line treatment initiation for HIV-positive patients and mortality, given a patients immune status. MethodsWe included 7255 patients starting antiretroviral therapy between 2004-2017, from 9 South African cohorts, with virological failure and complete baseline data. We estimated the impact of switch time on the hazard of death using inverse probability of treatment weighting (IPTW) of marginal structural models. The non-linear relationship between month of switch and the 5-year survival probability, stratified by CD4 count at failure, was estimated with targeted maximum likelihood estimation (TMLE). We adjusted for measured time-varying confounding by CD4 count, viral load and visit frequency. Results5-year mortality was estimated as 10.5% (2.2%; 18.8%) for immediate switch and as 26.6% (20.9%; 32.3%) for no switch (49.9% if CD4 count<100 cells/mm3). The hazard of death was estimated to be 0.40 (95%CI: 0.33-0.48) times lower if everyone had been switched immediately compared to never. The shorter the delay in switching, the lower the hazard of death, e.g. delaying 30-60 days reduced the hazard 0.52 (0.41-0.65) times, and 60-120 days 0.56 (0.47-0.66) times. ConclusionsEarly treatment switch is particularly important for patients with low CD4 counts at failure.

epidemiology↗

bioRxiv2018

Longitudinal studies at birth and age 7 reveal strong effects of genetic variation on ancestry-associated DNA methylation patterns in blood cells from ethnically admixed children

Epigenetic architecture is influenced by genetic and environmental factors, but little is known about their relative contributions or longitudinal dynamics. Here, we studied DNA methylation (DNAm) at over 750,000 CpG sites in mononuclear blood cells collected at birth and age 7 from 196 children of primarily self-reported Black and Hispanic ethnicities to study race-associated DNAm patterns. We developed a novel Bayesian method for high dimensional longitudinal data and showed that race-associated DNAm patterns at birth and age 7 are nearly identical. Additionally, we estimated that up to 51% of all self-reported race-associated CpGs had race-dependent DNAm levels that were mediated through local genotype and, quite surprisingly, found that genetic factors explained an overwhelming majority of the variation in DNAm levels at other, previously identified, environmentally-associated CpGs. These results not only indicate that race-associated DNAm patterns in blood are present at birth and are primarily genetically, and not environmentally, determined, but also that DNAm in blood cells overall is robust to many environmental exposures during the first 7 years of life.

genomics↗