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Williams, C. M.

Publications and source records attributed to Williams, C. M..

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A developmental atlas of the mouse brain by single-cell mass cytometry

Development of the mammalian brain requires precisely controlled differentiation of neurons, glia, and nonneural cells. To investigate protein-level changes in these diverse cell types and their progenitors, we performed single-cell mass cytometry on whole brain (E11.5/E12.5) and microdissected telencephalon, diencephalon, mesencephalon, and rhombencephalon (E13.5-P4) collected at daily timepoints from C57/BL6 mice. Measuring 24,290,787 cells from 112 sample replicates with a 40-antibody panel, we quantified 85 molecularly distinct cell populations across embryonic and postnatal development, including microglia putatively phagocytosing neurites, neural cells, and myelin. Differentiation trajectory analysis also identified two separate pathways for producing oligodendrocyte precursor cells. Comparison with previous studies revealed considerable discrepancies between protein and mRNA abundances in the developing brain, demonstrating the value of protein-level measurements for identifying functional cell states. Overall, our findings demonstrate the utility of mass cytometry as a high-throughput, scalable platform for single-cell profiling of brain tissue.

neuroscience↗

Increased homozygosity due to endogamy results in fitness consequences in a human population

Recessive alleles have been shown to directly affect both human Mendelian disease phenotypes and complex traits like height. Pedigree studies also suggest that consanguinity results in increased childhood mortality and adverse health phenotypes, presumably through penetrance of recessive mutations. Here, we test whether the accumulation of homozygous, recessive alleles decreases reproductive success in a human population. We address this question among the Namibian Himba, an endogamous agro-pastoralist population, who until very recently practiced natural fertility. Using a sample of 681 individuals, we show that Himba exhibit elevated levels of "inbreeding", calculated as the fraction of the genome in runs of homozygosity (FROH). Many individuals contain multiple long segments of ROH in their genomes, indicating that their parents had high kinship coefficients. However, we did not find evidence that this is explained by first-cousin consanguinity, despite a reported social preference for cross-cousin marriages. Rather, we show that elevated haplotype sharing in the Himba is due to a bottleneck, likely in the past 60 generations. We test whether increased recessive mutation load results in observed fitness consequences by assessing the effects of FROH on completed fertility in a cohort of post-reproductive women (n=69). We find that higher FROH is significantly associated with lower fertility among women who have had at least one child (p<0.006). Our data suggest a multi-locus genetic effect on fitness driven by the expression of deleterious recessive alleles, especially those in long ROH. However, these effects are not the result of consanguinity but rather elevated background identity by descent.

genetics↗

A developmental atlas of somatosensory diversification and maturation in the dorsal root ganglia by single-cell mass cytometry

Precisely controlled development of the somatosensory system is essential for detecting pain, itch, temperature, mechanical touch, and body position. To investigate the protein-level changes that occur during somatosensory development, we performed single-cell mass cytometry on dorsal root ganglia from C57/BL6 mice, with litter replicates collected daily from E11.5 to P4. Measuring nearly 3 million cells, we quantified 30 molecularly distinct somatosensory glial and 41 distinct neuronal states across all time points. Analysis of differentiation trajectories revealed rare cells that coexpress two or more Trk receptors and overexpress stem cell markers, suggesting that these neurotrophic factor receptors play a role in cell fate specification. Comparison to previous RNA-based studies identified substantial differences between many protein/mRNA pairs, demonstrating the importance of protein-level measurements to identify functional cell states. Overall, this study demonstrates that mass cytometry is a high-throughput, scalable platform to rapidly phenotype somatosensory tissues.

neuroscience↗

Receptor-like kinases FER and SRK mediate Lotus japonicus freezing tolerance and climate adaptation

Many plant species have succeeded in colonizing a wide range of diverse climates through local adaptation, but the underlying molecular genetics remain obscure. We previously found that winter survival was a direct target of selection during colonization of Japan by the perennial legume Lotus japonicus and identified associated candidate genes. Here, we show that two of these, the FERONIA-receptor like kinase (LjFER) and a novel S-receptor-like kinase (LjSRK) are required for non-acclimated freezing tolerance and show haplotype-dependent cold-responsive expression. Our work demonstrates that recruiting a conserved growth regulator, FER, and a novel receptor-like kinase, SRK, into the set of cold-responsive genes has contributed to freezing tolerance and local climate adaptation in L. japonicus, offering new functional genetic insight into perennial herb evolution.

plant biology↗

Platelet generation from circulating megakaryocytes is triggered in the lung vasculature

Platelets, small hemostatic blood cells, are derived from megakaryocytes, although the generation process is not clear. Only small numbers of platelets have been produced in systems outside the body, where bone marrow and lung are proposed as sites of platelet generation. Here we show that perfusion of megakaryocytes ex vivo through the mouse lung vasculature generates very large numbers of platelets, up to 3,000 per megakaryocyte. Despite their large size, megakaryocytes were able repeatedly to passage through the lung vasculature, leading to enucleation and fragmentation to generate platelets intravascularly. Using the ex vivo lung and a novel in vitro microfluidic chamber we determined the contributions of oxygenation, ventilation and endothelial cell health to platelet generation, and showed a critical role for the actin regulator TPM4. One-Sentence SummaryMegakaryocytes form platelets intravascularly in the lung, dependent upon oxygenation, endothelium and megakaryocyte TPM4

physiology↗

Computer vision approach to characterize size and shape phenotypes of horticultural crops using high-throughput imagery

For many horticultural crops, variation in quality (e.g., shape and size) contribute significantly to the crops market value. Metrics characterizing less subjective harvest quantities (e.g., yield and total biomass) are routinely monitored. In contrast, metrics quantifying more subjective crop quality characteristics such as ideal size and shape remain difficult to characterize objectively at the production-scale due to the lack of modular technologies for high-throughput sensing and computation. Several horticultural crops are sent to packing facilities after having been harvested, where they are sorted into boxes and containers using high-throughput scanners. These scanners capture images of each fruit or vegetable being sorted and packed, but the images are typically used solely for sorting purposes and promptly discarded. With further analysis, these images could offer unparalleled insight on how crop quality metrics vary at the industrial production-scale and provide further insight into how these characteristics translate to overall market value. At present, methods for extracting and quantifying quality characteristics of crops using images generated by existing industrial infrastructure have not been developed. Furthermore, prior studies that investigated horticultural crop quality metrics, specifically of size and shape, used a limited number of samples, did not incorporate deformed or non-marketable samples, and did not use images captured from high-throughput systems. In this work, using sweetpotato (SP) as a use case, we introduce a computer vision algorithm for quantifying shape and size characteristics in a high-throughput manner. This approach generates 3D model of SPs from two 2D images captured by an industrial sorter 90 degrees apart and extracts 3D shape features in a few hundred milliseconds. We applied the 3D reconstruction and feature extraction method to thousands of image samples to demonstrate how variations in shape features across sweetptoato cultivars can be quantified. We created a sweetpotato shape dataset containing sweetpotato images, extracted shape features, and qualitative shape types (U.S. No. 1 or Cull). We used this dataset to develop a neural network-based shape classifier that was able to predict Cull vs. U.S. No. 1 sweetpotato with 84.59% accuracy. In addition, using univariate Chi-squared tests and random forest, we identified the most important features for determining qualitative shape (U.S. No. 1 or Cull) of the sweetpotatoes. Our study serves as the first step towards enabling big data analytics for sweetpotato agriculture. The methodological framework is readily transferable to other horticultural crops, particularly those that are sorted using commercial imaging equipment.

bioengineering↗

Combining multivariate genomic approaches to elucidate the comorbidity between ASD and ADHD

BackgroundSeveral lines of evidence point toward the presence of shared genetic factors underlying Autism Spectrum Disorder (ASD) and Attention Deficit Hyperactivity Disorder (ADHD). However, Genome-Wide Association Studies (GWAS) have yet to identify risk variants (i.e. Single-Nucleotide Polymorphisms, SNPs) shared by ADHD and ASD. MethodsTwo complementary multivariate analyses - genomic structural equation modelling (SEM) and colocalization analysis - were exploited to identify the shared SNPs for ASD and ADHD, using summary data from two independent GWAS of ASD (N=46,350) and ADHD individuals (N=55,374). ResultsGenomic SEM identified 7 novel SNPs shared between ASD and ADHD (pgenome-wide<5e-8), including three SNPs that were not identified in any of the original univariate GWAS of ASD and ADHD (rs227378, rs2391769 and rs325506). We also mapped 4 novel genes (MANBA, DPYD, INSM1, and PAX1) to SNPs shared by ASD and ADHD, as well as 4 genes that had already been mapped to SNPs identified in either ASD or ADHD GWAS (SORCS3, XRN2, PTBP2 and NKX2-4). All the shared genes between ADHD and ASD were more prominently expressed in the brain than the genes mapped to SNPs specific to ASD or ADHD. Colocalization analyses revealed that 44% percent of the SNPs associated with ASD (p<1e-6) colocalized with ADHD SNPs and 26% of the SNPs associated with ADHD (p<1e-6) colocalized with ASD SNPs. ConclusionsUsing multivariate genomic analyses, the present study reveals the shared genetic pathways that underlie ASD and ADHD. Further investigation of these pathways may help identify new targets for treatment of these disorders.

genetics↗

Identification of Human Immune Cell Subtypes Most Vulnerable to IL-1β-induced Inflammatory Signaling Using Mass Cytometry

IL-1{beta} has emerged as a key mediator of the cytokine storm linked to high morbidity and mortality from COVID-19 and blockade of the IL-1 receptor (IL-1R) with Anakinra has entered clinical trials in COVID-19 subjects. Yet, knowledge of the specific immune cell subsets targeted by IL-1{beta} and IL-1{beta}-induced signaling pathways in humans is limited. Utilizing mass cytometry (CyTOF) of human peripheral blood mononuclear cells, we identified effector memory CD4 T cells and CD4-CD8low/-CD161+ T cells as the circulating immune subtypes with the greatest expression of p-NF-{kappa}B in response to IL-1{beta} stimulation. Notably, CCR6 distinctly identified T cells most responsive to IL-1{beta}. Other subsets including CD11c myeloid dendritic cells (mDCs), classical monocytes (CM), two subsets of natural killer cells (CD16-CD56brightCD161- and CD16-CD56dimCD161+) and a population of lineage-(Lin-) cells expressing CD161 and CD25 also showed IL-1{beta}-induced expression of p-NF-kB. The IL-1R antagonist, Anakinra significantly inhibited IL-1{beta}-induced p-NF-kB in the CCR6+ T cells and CD11c mDCs with a trending inhibition in CD14 monocytes and Lin-CD161+CD25+ cells. IL-1{beta} also induced a rapid but much less robust increase in p-p38 expression as compared to p-NF-kB in the majority of these same immune cell subsets. Prolonged IL-1{beta} stimulation greatly increased p-STAT3 and to a much lesser extent p-STAT1 and p-STAT5 in T cell subsets, monocytes, DCs and the Lin-CD161+CD25+ cells suggesting IL-1{beta}-induced production of downstream STAT-activating cytokines, consistent with its role in cytokine storm. Interindividual heterogeneity and inhibition of this activation by Anakinra raises the intriguing possibility that assays to measure IL-1{beta}-induced p-NF-kB in CCR6+ T cell subtypes could identify those at higher risk of cytokine storm and those most likely to benefit from Anakinra therapy.

immunology↗

A rapid, accurate approach to inferring pedigrees in endogamous populations

Accurate reconstruction of pedigrees from genetic data remains a challenging problem. Pedigree inference algorithms are often trained only on European-descent families in urban locations. Many relationship categories can be difficult to distinguish (e.g. half-sibships versus avuncular) without external information. Furthermore, existing methods perform poorly in endogamous populations for which there may be reticulations within the pedigrees and elevated haplotype sharing. We present a simple, rapid algorithm which initially uses only high-confidence first-degree relationships to seed a machine learning step based on summary statistics of identity-by-descent (IBD) sharing. One of these statistics, our "haplotype score", is novel and can be used to: (1) distinguish half-sibling pairs from avuncular or grandparent-grandchildren pairs; and (2) assign individuals to ancestor versus descendant generation. We test our approach in a sample of 700 individuals from northern Namibia, sampled from an endogamous population called the Himba. Due to a culture of concurrent relationships in the Himba, there is a high proportion of half-sibships. We accurately identify first through fourth-degree relationships and distinguish between various second-degree relationships: half-sibships, avuncular pairs, and grandparent-grandchildren. We further validate our approach in a second diverse African-descent dataset, the Barbados Asthma Genetics Study (BAGS). Accurate reconstruction of pedigrees holds promise for tracing allele frequency trajectories, improved phasing and other population genomic questions.

bioinformatics↗

Stem-cell-ubiquitous genes spatiotemporally coordinate division through regulation of stem-cell-specific gene networks

Stem cells are responsible for generating all of the differentiated cells, tissues, and organs in a multicellular organism and, thus, play a crucial role in cell renewal, regeneration, and organization. A number of stem cell type-specific genes have a known role in stem cell maintenance, identity, and/or division. Yet, how genes expressed across different stem cell types, referred here as stem-cell-ubiquitous genes, contribute to stem cell regulation is less understood. Here, we find that, in the Arabidopsis root, a stem-cell-ubiquitous gene, TESMIN-LIKE CXC2 (TCX2), controls stem cell division by regulating stem cell-type specific networks. Development of a mathematical model of TCX2 expression allowed us to show that TCX2 orchestrates the coordinated division of different stem cell types. Our results highlight that genes expressed across different stem cell types ensure cross-communication among cells, allowing them to divide and develop harmonically together.

systems biology↗

Epigenetic regulation of PAR4-related platelet activation: mechanistic links between environmental exposure and cardiovascular disease

Protease-activated receptor 4 (PAR4) is a potent thrombin receptor. Epigenetic control of the F2RL3 locus (which encodes for PAR4) via DNA methylation is associated with both smoking and cardiovascular disease. We examined the association between DNA hypomethylation at F2RL3 and risk of cardiovascular disease, focusing on acute myocardial infarction (AMI) (n=853 cases / 2,352 controls). We used in vitro cell models to dissect the role of DNA methylation in regulating expression of F2RL3. We investigated the interplay between F2RL3 DNA methylation and platelet function in human (n=41). Lastly, we used Mendelian randomization to unify observational and functional work by assessing evidence for causal relationships using data from UK Biobank (n=407,141) and CARDIoGRAMplusC4D (n=184,305). Observationally, one standard deviation (SD) decrease in DNA methylation at F2RL3 was associated with a 25% increase in the odds of AMI. In vitro, short-term exposure of cells to cigarette smoke reduced F2RL3 DNA methylation and increased gene expression. Transcriptional assays flagged a role for a CEBP recognition sequence in modulating the enhancer activity of F2RL3 exon 2. Lower DNA methylation at F2RL3 was associated with increased platelet reactivity in human. The estimated casual odds ratio of ischaemic heart disease was 1.03 (95% CI: 1.00, 1.07) per 1 SD decrease in F2RL3 DNA. In conclusion, we show that DNA methylation-dependent platelet activation is part of a complex system of features contributing to cardiovascular health. Tailoring therapeutic intervention to new knowledge of F2RL3/PAR4 function should be explored to ameliorate the detrimental effects of this risk factor on cardiovascular health.\n\nOne sentence summaryDNA methylation-dependent platelet activation is a likely causal contributor to cardiovascular health.

epidemiology↗

High-throughput determination of the antigen specificities of T cell receptors in single cells

We present tetramer-associated T-cell receptor sequencing (TetTCR-Seq), a method to link T cell receptor (TCR) sequences to their cognate antigens in single cells at high throughput. Binding is determined using a library of DNA-barcoded antigen tetramers that is rapidly generated by in vitro transcription and translation. We applied TetTCR-Seq to identify patterns in TCR cross-reactivity with cancer neo-antigens and to rapidly isolate neo-antigen-specific TCRs with no cross-reactivity to the wild-type antigen.

immunology↗

Twenty-four hour face mask sampling in pulmonary tuberculosis reveals three distinct patterns of bacterial aerosol production dissociated from conventional markers of transmission risk.

Rationale: Although tuberculosis (TB) is transmitted by Mycobacterium tuberculosis (Mtb) in aerosols, little is known of the dynamic characteristics of spontaneous output of bacilli in this form. We have developed and implemented a mask aerosol sampling system (MASS) for longitudinal capture and study of spontaneous aerosol.\n\nObjective: To determine patterns of Mtb output in aerosols, captured using the MASS over 24 hours and their association with existing criteria used to assess transmission risk in patients with pulmonary TB.\n\nMethods: Twenty-four hospitalised patients with newly diagnosed pulmonary TB recruited in Pretoria, South Africa, wore FFP1 masks for one hour out of every three for 24 hours. Aerosol was captured in a gelatine filter processed for Mtb quantitation by PCR. Serial sputum was collected and objective cough frequency monitoring performed over the same period.\n\nMeasurements and Main Results: Mtb was detected in 86.5% of 192 mask samples and 20.7% of 38 assessable sputum samples obtained from the cohort. Mtb was detected by MASS in all but two patients. Three dynamic patterns of expression were identifiable in Mtb aerosol producers: i. variable high; ii. consistent; and iii. variable low. No diurnal variation was apparent and there was no correlation between mask Mtb and either sputum Mtb levels or cough frequency. Sputum smear status, culture time to positivity and chest radiographic characteristics also failed to associate with MASS bacillary output.\n\nConclusions: Conventional markers of tuberculosis case infectivity do not predict bacillary aerosols detected by the MASS. The MASS provides a novel, non-invasive tool for tuberculosis diagnosis and control.

microbiology↗

Economic evaluation of a healthy lifestyle intervention for chronic low back pain: a randomised controlled trial

We performed an economic evaluation of a healthy lifestyle intervention targeting weight loss, physical activity and diet for patients with chronic low back pain, who are overweight or obese. Eligible patients with chronic low back pain (n=160) were randomised to an intervention or usual care control group. The intervention included brief advice, a clinical consultation and referral to a 6-month telephone-based healthy lifestyle coaching service. The primary outcome was quality-adjusted life years (QALYs). Secondary outcomes were pain intensity, disability, weight, and body mass index. Costs included intervention costs, healthcare utilisation costs and work absenteeism costs. An economic analysis was performed from the societal perspective. Mean total costs were lower in the intervention group than the control group (-$614; 95%CI: -3133 to 255). The intervention group had significantly lower healthcare costs (-$292; 95%CI: -872 to -33), medication costs (-$30; 95%CI: -65 to -4) and absenteeism costs (-$1000; 95%CI: -3573 to -210). For all outcomes, the intervention was on average less expensive and more effective than usual care, and the probability of the intervention being cost-effective compared to usual care was relatively high (i.e. 0.81) at a willingness-to-pay of $0/unit of effect. However, the probability of cost-effectiveness was not as favourable among sensitivity analyses. The healthy lifestyle intervention seems to be cost-effective from the societal perspective. However, variability in the sensitivity analyses indicates caution is needed when interpreting these findings.

clinical trials↗

Causal mechanisms of a healthy lifestyle intervention for patients with musculoskeletal pain who are overweight or obese

We assessed the causal mechanisms of a healthy lifestyle intervention for patients with chronic low back pain and knee osteoarthritis (OA), who are overweight or obese. We conducted causal mediation analyses of aggregated data from two RCTs; which included 160 patients with chronic low back pain, and 120 patients with knee OA. Participants were randomised via one central randomisation schedule, to the intervention, or usual care. The intervention consisted of brief advice and referral to a 6-month telephone-based healthy lifestyle coaching service. Participants in the back pain trial were also offered a single physiotherapy consultation. The hypothesised primary mediator was self-reported weight, and alternative mediators were diet, physical activity, and pain beliefs. Outcomes were pain intensity, disability, and quality of life (QoL). Data were analysed using causal mediation analyses with sensitivity analyses for sequential ignorability. All mediation models were specified a priori. The intervention had no effect on pain intensity, disability or physical QoL. The intervention significantly improved mental QoL, however, the intervention effect was not channelled via the selected mediators. The intervention did not reduce weight, or the alternative mediators (diet, physical activity, pain beliefs), and these mediators were not associated with the outcomes (with one exception; poor diet was associated with lower mental QoL). The sensitivity analyses showed that our estimates were stable across all possible levels of residual confounding. Our findings show that the intervention did not cause a meaningful change in the hypothesised mediators, and these mediators were not associated with patient outcomes.

clinical trials↗

Cost-effectiveness of telephone-based weight loss support for patients with knee osteoarthritis: a pragmatic randomised controlled trial

BackgroundTelephone-based support offers a promising option to provide widely accessible and cost-effective weight loss care to the people with knee osteoarthritis who are overweight. While telephone-based interventions targeting weight loss are used routinely in the general populations, the cost-effectiveness of referring patients with knee osteoarthritis to these is unknown. The aim of this study was to assess the cost-effectiveness of referral to a telephone-based weight management and healthy lifestyle service for patients with knee osteoarthritis, who are overweight or obese, compared to usual care. MethodsWe randomised 120 patients with knee osteoarthritis to an intervention or usual care control group in a 1:1 ratio. Participants in the intervention group received a referral to an existing non-disease specific 6-month telephone-based weight management and healthy lifestyle service. The primary outcome of the study was quality-adjusted life years (QALYs). Secondary outcomes included pain intensity, disability, weight, and body mass index (BMI). Costs included intervention costs, healthcare utilisation costs (healthcare services and medication use) and absenteeism costs due to knee pain. The primary cost-effectiveness analysis was performed from the societal perspective. ResultsMean cost differences between groups (intervention minus control) were, $454 (95%CI: -2735 to 4206) for healthcare costs, $-36, (95%CI: -73 to 2) for medication costs, and $-13 (95%CI: -225 to 235) for absenteeism costs. The total mean difference in societal costs was $1022 (95%CI: -2201 to 4771). For all outcomes, the probability of the intervention being cost-effective compared with usual care was less than 0.33 at all willingness-to-pay values. ConclusionFrom a societal perspective, telephone-based weight loss support, provided using an existing non-disease specific 6-month weight management and healthy lifestyle service was not cost-effective in comparison with usual care for overweight and obese patients with knee osteoarthritis for QALYs, pain intensity, disability, weight, and BMI.

clinical trials↗

Back pain, mental health and substance use are associated in adolescents

BackgroundDuring adolescence, prevalence of pain and health risk factors such as smoking, alcohol use, and poor mental health rise sharply. While these risk factors and mental health are accepted public health concerns, the same is not true for pain. The aim of this study was to describe the relationship between back pain and health risk factors in adolescents.\n\nMethodsCross-sectional data from the Healthy Schools Healthy Futures study, and the Australian Child Wellbeing Project was used. The mean age of participants was 14-15 years. Children were stratified according to the frequency they experienced back pain over the past 6 months. Within each strata, the proportion of children that reported drinking alcohol or smoking in the past month and the proportion that experienced feelings of anxiety or depression was reported. Test-for-trend analyses assessed whether increasing frequency of pain was associated with health risk factors.\n\nResultsData from approximately 2,500 and 3,900 children in the two studies was analysed. Larger proportions of children smoked or drank alcohol within each strata of increasing pain frequency. The trend with report of anxiety and depression was less clear, although there was a marked difference between the children that reported pain rarely or never, and those that experienced back pain more frequently.\n\nConclusionTwo large, independent samples show Australian adolescents that experience back pain more frequently are also more likely to smoke, drink alcohol and report feelings of anxiety and depression. Pain appears to be part of the picture of general health risk in adolescents.\n\nWhat is already known on this subject?The prevalence of back pain rises steeply during the adolescent years, and is responsible for considerable personal impact in a substantial minority. During this time, indicators of adverse health risk such as smoking, alcohol use, anxiety and depression also increase in prevalence. Pain and lifestyle-related health risk factors can have ongoing consequences that stretch into adulthood.\n\nWhat this study adds?This study shows a close relationship between increasing pain frequency, and tendency to engage in health risk behaviours and experience indicators of poor mental health in adolescents. This study shows that pain may be an important consideration in understanding the general health, and health risk in adolescents.

epidemiology↗