Spatial dissection of the Arabidopsis thaliana transcriptional response to downy mildew using fluorescence activated cell sorting

Changes in gene expression form a crucial part of the plant response to infection. In the last decade, whole-leaf expression profiling has played a valuable role in identifying genes and processes that contribute to the interactions between the model plant Arabidopsis thaliana and a diverse range of pathogens. However, with some pathogens such as downy mildew caused by the biotrophic oomycete pathogen Hyaloperonospora arabidopsidis (Hpa), whole-leaf profiling may fail to capture the complete Arabidopsis response encompassing responses of non-infected as well as infected cells within the leaf. Highly localized expression changes that occur in infected cells may be diluted by the comparative abundance of non-infected cells. Furthermore, local and systemic Hpa responses of a differing nature may become conflated. To address this we applied the technique of Fluorescence Activated Cell Sorting (FACS), typically used for analyzing plant abiotic responses, to the study of plant-pathogen interactions. We isolated haustoriated (Hpa-proximal) and non-haustoriated (Hpa-distal) cells from infected seedling samples using FACS, and measured global gene expression. When compared with an uninfected control, 278 transcripts were identified as significantly differentially expressed, the vast majority of which were differentially expressed specifically in Hpa-proximal cells. By comparing our data to previous, whole organ studies, we discovered many highly locally regulated genes that can be implicated as novel in the Hpa response, and that were uncovered for the first time using our sensitive FACS technique

Genetic mechanisms of critical illness in COVID-19.

Host-mediated lung inflammation is present1, and drives mortality2, in the critical illness caused by coronavirus disease 2019 (COVID-19). Host genetic variants associated with critical illness may identify mechanistic targets for therapeutic development3. Here we report the results of the GenOMICC (Genetics Of Mortality In Critical Care) genome-wide association study in 2,244 critically ill patients with COVID-19 from 208 UK intensive care units. We have identified and replicated the following new genome-wide significant associations: on chromosome 12q24.13 (rs10735079, P = 1.65 × 10-8) in a gene cluster that encodes antiviral restriction enzyme activators (OAS1, OAS2 and OAS3); on chromosome 19p13.2 (rs74956615, P = 2.3 × 10-8) near the gene that encodes tyrosine kinase 2 (TYK2); on chromosome 19p13.3 (rs2109069, P = 3.98 ×  10-12) within the gene that encodes dipeptidyl peptidase 9 (DPP9); and on chromosome 21q22.1 (rs2236757, P = 4.99 × 10-8) in the interferon receptor gene IFNAR2. We identified potential targets for repurposing of licensed medications: using Mendelian randomization, we found evidence that low expression of IFNAR2, or high expression of TYK2, are associated with life-threatening disease; and transcriptome-wide association in lung tissue revealed that high expression of the monocyte-macrophage chemotactic receptor CCR2 is associated with severe COVID-19. Our results identify robust genetic signals relating to key host antiviral defence mechanisms and mediators of inflammatory organ damage in COVID-19. Both mechanisms may be amenable to targeted treatment with existing drugs. However, large-scale randomized clinical trials will be essential before any change to clinical practice

Multiple novel prostate cancer susceptibility signals identified by fine-mapping of known risk loci among Europeans

Genome-wide association studies (GWAS) have identified numerous common prostate cancer (PrCa) susceptibility loci. We have fine-mapped 64 GWAS regions known at the conclusion of the iCOGS study using large-scale genotyping and imputation in 25 723 PrCa cases and 26 274 controls of European ancestry. We detected evidence for multiple independent signals at 16 regions, 12 of which contained additional newly identified significant associations. A single signal comprising a spectrum of correlated variation was observed at 39 regions; 35 of which are now described by a novel more significantly associated lead SNP, while the originally reported variant remained as the lead SNP only in 4 regions. We also confirmed two association signals in Europeans that had been previously reported only in East-Asian GWAS. Based on statistical evidence and linkage disequilibrium (LD) structure, we have curated and narrowed down the list of the most likely candidate causal variants for each region. Functional annotation using data from ENCODE filtered for PrCa cell lines and eQTL analysis demonstrated significant enrichment for overlap with bio-features within this set. By incorporating the novel risk variants identified here alongside the refined data for existing association signals, we estimate that these loci now explain ∼38.9% of the familial relative risk of PrCa, an 8.9% improvement over the previously reported GWAS tag SNPs. This suggests that a significant fraction of the heritability of PrCa may have been hidden during the discovery phase of GWAS, in particular due to the presence of multiple independent signals within the same regio

Infection-site-specific responses of Arabidopsis thaliana to the biotrophic oomycete Hyaloperonospora arabidopsidis.

Changes in gene expression form a crucial part of the plant response to infection, and whole-leaf expression profiling has been valuable in our understanding of the interactions between the model plant Arabidopsis thaliana and a diverse range of pathogens. However, when studying the interaction between Arabidopsis and the biotrophic oomycete Hyaloperonospora arabidopsidis (Hpa), whole-leaf profiling may fail to capture the complete Arabidopsis response. Highly localised expression changes that occur in infected cells may be diluted by the comparative abundance of non-infected cells, or local and systemic responses of a differing nature may become convoluted. The aim of this project was to spatially dissect the transcriptional response to Hpa, allowing differentiation of local and more systemic responses. Fluorescence Activated Cell Sorting (FACS), utilising an infection-site-specific fluorescent marker ProDMR6::GFP, was performed in order to isolate Hpa-proximal and Hpa-distal cells from infected seedling samples, and global gene expression measured in these FACS-sorted samples using microarrays. When compared with an uninfected control, 278 transcripts were identified as differentially expressed, the vast majority of which were differentially expressed specifically in Hpa-proximal cells. By comparing our data to previous, whole organ studies, we discovered many locally responding genes that were detected for the first time using our sensitive FACS technique. A portion of locally-responding genes were selected for further study. The promoters of a subset of highly locally induced genes were selected to drive expression of Green Fluorescent Protein (GFP) as a marker of Hpa-contacting cells for further FACS experiments, and to further validate their localised induction. Although some evidence of localised induction was seen in these lines, further investigation is required. We also hypothesised that a number of locally-induced genes would have a functional influence on infection, and tested this through the use of genetic knockouts. Knockouts in 7 of these genes showed altered disease resistance or susceptibility, and the mechanism behind two of these genes was investigated through the use of microarrays. Overall, the use of FACS to study the Arabidopsis response to Hpa on a spatial scale has allowed identification of new genes with a putative role in the Arabidopsis-Hpa interaction, and contributes to a systems-level understanding of plant-pathogen interactions

Universal psychosocial screening and adverse pregnancy outcomes in an academic obstetric clinic

Objective: To test the hypothesis that women screened for psychosocial factors, including partner abuse, as recommended by the American College of Obstetricians and Gynecologists would have higher neonatal birth weight, longer gestational age at delivery, higher Apgar scores, and fewer maternal complications. Methods: We evaluated a universal psychosocial screening intervention using a retrospective cohort (n=881 prenatal care patients). Pregnancy outcomes among patients screened beginning in 2008 (n=464) were compared with outcomes among women receiving care before universal screening was implemented (n=417). Data were obtained from medical records between 2007 and 2009. Multivariable logistic regression and analysis of covariance were used to estimate the association between screening and pregnancy outcomes among singleton births adjusting for confounders (prior preterm births, insurance, and mode of delivery). Results: Screened women were less likely than women not universally screened to have low birth weight neonates (4.5% of screened, 10.3% of unscreened; adjusted odds ratio [OR] 0.41, 95% confidence interval [CI] 0.23-0.73), preterm births (9.9% of screened, 14.9% of unscreened; adjusted OR 0.62, 95% CI 0.41-0.96), and any maternal complication (30.0% of screened, 41.2% of unscreened; adjusted OR 0.67, 95% CI 0.50-0.88). Newborn Apgar scores were higher (P=.01) among screened relative to unscreened mothers. Conclusion: Our results provide evidence that universal screening was associated with improved pregnancy outcomes. © 2012 by The American College of Obstetricians and Gynecologists. Published by Lippincott Williams & Wilkins

TGFβ signaling in lung epithelium regulates bleomycin-induced alveolar injury and fibroblast recruitment

The response of alveolar epithelial cells (AECs) to lung injury plays a central role in the pathogenesis of pulmonary fibrosis, but the mechanisms by which AECs regulate fibrotic processes are not well defined. We aimed to elucidate how transforming growth factor-β (TGFβ) signaling in lung epithelium impacts lung fibrosis in the intratracheal bleomycin model. Mice with selective deficiency of TGFβ receptor 2 (TGFβR2) in lung epithelium were generated and crossed to cell fate reporter mice that express β-galactosidase (β-gal) in cells of lung epithelial lineage. Mice were given intratracheal bleomycin (0.08 U), and the following parameters were assessed: AEC death by terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling assay, inflammation by total and differential cell counts from bronchoalveolar lavage, fibrosis by scoring of trichrome-stained lung sections, and total lung collagen content. Mice with lung epithelial deficiency of TGFβR2 had improved AEC survival, despite greater lung inflammation, after bleomycin administration. At 3 wk after bleomycin administration, mice with epithelial TGFβR2 deficiency showed a significantly attenuated fibrotic response in the lungs, as determined by semiquantitatve scoring and total collagen content. The reduction in lung fibrosis in these mice was associated with a marked decrease in the lung fibroblast population, both total lung fibroblasts and epithelial-to-mesenchymal transition-derived (S100A4+/β-gal+) fibroblasts. Attenuation of TGFβ signaling in lung epithelium provides protection from bleomycin-induced fibrosis, indicating a critical role for the epithelium in transducing the profibrotic effects of this cytokine