Effects of antiplatelet therapy on stroke risk by brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases: subgroup analyses of the RESTART randomised, open-label trial

Background Findings from the RESTART trial suggest that starting antiplatelet therapy might reduce the risk of recurrent symptomatic intracerebral haemorrhage compared with avoiding antiplatelet therapy. Brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases (such as cerebral microbleeds) are associated with greater risks of recurrent intracerebral haemorrhage. We did subgroup analyses of the RESTART trial to explore whether these brain imaging features modify the effects of antiplatelet therapy

The importance of examining movements within the US health care system: sequential logit modeling

Background: Utilization of specialty care may not be a discrete, isolated behavior but rather, a behavior of sequential movements within the health care system. Although patients may often visit their primary care physician and receive a referral before utilizing specialty care, prior studies have underestimated the importance of accounting for these sequential movements. Methods: The sample included 6,772 adults aged 18 years and older who participated in the 2001 Survey on Disparities in Quality of Care, sponsored by the Commonwealth Fund. A sequential logit model was used to account for movement in all stages of utilization: use of any health services (i.e., first stage), having a perceived need for specialty care (i.e., second stage), and utilization of specialty care (i.e., third stage). In the sequential logit model, all stages are nested within the previous stage. Results: Gender, race/ethnicity, education and poor health had significant explanatory effects with regard to use of any health services and having a perceived need for specialty care, however racial/ethnic, gender, and educational disparities were not present in utilization of specialty care. After controlling for use of any health services and having a perceived need for specialty care, inability to pay for specialty care via income (AOR = 1.334, CI = 1.10 to 1.62) or health insurance (unstable insurance: AOR = 0.26, CI = 0.14 to 0.48; no insurance: AOR = 0.12, CI = 0.07 to 0.20) were significant barriers to utilization of specialty care. Conclusions: Use of a sequential logit model to examine utilization of specialty care resulted in a detailed representation of utilization behaviors and patient characteristics that impact these behaviors at all stages within the health care system. After controlling for sequential movements within the health care system, the biggest barrier to utilizing specialty care is the inability to pay, while racial, gender, and educational disparities diminish to non-significance. Findings from this study represent how Americans use the health care system and more precisely reveals the disparities and inequalities in the U.S. health care system

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

Optimized De Novo Eriodictyol Biosynthesis in Streptomyces albidoflavus Using an Expansion of the Golden Standard Toolkit for Its Use in Actinomycetes

Eriodictyol is a hydroxylated flavonoid displaying multiple pharmaceutical activities, such as antitumoral, antiviral or neuroprotective. However, its industrial production is limited to extraction from plants due to its inherent limitations. Here, we present the generation of a Streptomyces albidoflavus bacterial factory edited at the genome level for an optimized de novo heterologous production of eriodictyol. For this purpose, an expansion of the Golden Standard toolkit (a Type IIS assembly method based on the Standard European Vector Architecture (SEVA)) has been created, encompassing a collection of synthetic biology modular vectors (adapted for their use in actinomycetes). These vectors have been designed for the assembly of transcriptional units and gene circuits in a plug-and-play manner, as well as for genome editing using CRISPR-Cas9-mediated genetic engineering. These vectors have been used for the optimization of the eriodictyol heterologous production levels in S. albidoflavus by enhancing the flavonoid-3′-hydroxylase (F3’H) activity (by means of a chimera design) and by replacing three native biosynthetic gene clusters in the bacterial chromosome with the plant genes matBC (involved in extracellular malonate uptake and its intracellular activation into malonyl-CoA), therefore allowing more malonyl-CoA to be devoted to the heterologous production of plant flavonoids in this bacterial factory. These experiments have allowed an increase in production of 1.8 times in the edited strain (where the three native biosynthetic gene clusters have been deleted) in comparison with the wild-type strain and a 13 times increase in eriodictyol overproduction in comparison with the non-chimaera version of the F3′H enzyme

Gene Ontology (GO) Fisher enrichment analysis.

A better mechanistic understanding of virus-host dependencies can help reveal vulnerabilities and identify opportunities for therapeutic intervention. Of particular interest are essential interactions that enable production of viral proteins, as those could target an early step in the virus lifecycle. Here, we use subcellular proteomics, ribosome profiling analyses and reporter assays to detect changes in protein synthesis dynamics during SARS-CoV-2 (CoV2) infection. We identify specific translation factors and molecular chaperones that are used by CoV2 to promote the synthesis and maturation of its own proteins. These can be targeted to inhibit infection, without major toxicity to the host. We also find that CoV2 non-structural protein 1 (Nsp1) cooperates with initiation factors EIF1 and 1A to selectively enhance translation of viral RNA. When EIF1/1A are depleted, more ribosomes initiate translation from a conserved upstream CUG start codon found in all genomic and subgenomic viral RNAs. This results in higher translation of an upstream open reading frame (uORF1) and lower translation of the main ORF, altering the stoichiometry of viral proteins and attenuating infection. Replacing the upstream CUG with AUG strongly inhibits translation of the main ORF independently of Nsp1, EIF1, or EIF1A. Taken together, our work describes multiple dependencies of CoV2 on host biosynthetic networks and proposes a model for dosage control of viral proteins through Nsp1-mediated control of translation start site selection.</div

Translation initiation on CoV2 gRNA is non-optimal.

(a) Pairwise comparisons of individual protein abundance in the heavy polysome fractions. Ribosomal proteins and translation elongation factors are depleted, whereas translation initiation factors are enriched. (b) Line plots of individual translation initiation factors quantified by MS in each fraction. Each line represents a single replicate. P, two-tailed Student’s t-test p-value. (c) Translation initiation factors are highly represented in the CoV2 RNA interactome. Shown are pairwise comparisons of individual host protein abundance, quantified by MS, that specifically interact with either genomic or subgenomic CoV2 RNA during infection. Inset, cumulative distribution plots of 40S and 60S ribosomal protein interaction with CoV2 RNA. P, Mann-Whitney p-value. (d) Heavy polysome fractions contain more 40S ribosomal proteins in infected cells. Shown are cumulative distribution plots of 40S and 60S ribosomal proteins in heavy polysome fractions from infected and uninfected cells, across three replicates. P, Mann-Whitney p-value. (e) rRNA absorbance profiles from Fig 1D, showing lower abundance of free 40S subunits during CoV2 infection. (f) Translation initiation from CoV2 5’ untranslated region (UTR) is less efficient than GAPDH 5’UTR. mRNA encoding for nano-luciferase (nLuc) flanked by 5’ and 3’UTRs of either CoV2 (orange) or GAPDH (white) was transcribed in vitro, capped/polyadenylated, and transfected into Vero cells. At 4 hours post-transfection, luminescence was measured in parallel with qPCR using oligonucleotides specific to nLuc. Shown are means±SD of 3 independent replicates.</p

CoV2 infection remodels host biosynthetic complexes.

(a) Pairwise comparisons of differences in individual protein abundance upon CoV2 infection of Vero cells, in each pooled fraction, as quantified by MS. Right, proportion of proteins showing statistically significant differences (FDRb) Gene Ontology terms enriched in heavy polysome fractions from infected versus control cells. (c) Line plots of individual proteins quantified by MS in each fraction. Each line represents a single replicate. P, two-tailed Student’s t-test p-value.</p

Inefficient translation initiation on CoV2 gRNA.

(a) Change in abundance of individual translation initiation factors upon CoV2 infection, in each fraction. (b) Line plots of individual translation factors quantified by MS in each fraction. Each line represents a single replicate. P, two-tailed Student’s t-test p-value. (c) Translation initiation factors are highly represented in the CoV2 RNA interactome during infection. Shown are pairwise comparisons of individual host protein abundance, quantified by MS, that specifically interact with either genomic or subgenomic CoV2 RNA. Inset, cumulative distribution plots of 40S and 60S ribosomal protein interaction with CoV2 RNA. (d) rRNA absorbance profiles showing lower abundance of free 40S subunits during CoV2 infection. Sum of four replicates. (TIF)</p

Toxicity and antiviral effects of proteostasis modulators.

(a-b) Vero cells were infected with CoV2 at MOI = 0.5. Single drugs (a) or drug combinations (b) were added at the start of infection, and titers were determined by plaque assays at 16 hours post-infection. Toxicity was determined using CellTiter-Glo at 24h of drug treatment, in the absence of CoV2 infection. Shown are means±SD of 3 independent replicates, normalized to DMSO controls. (TIF)</p

Nsp1 promotes accurate start codon usage through EIF1/1A.

(a) Diagram of elements in CoV2 gRNA 5’UTR involved in translation. SL1, stem-loop 1. uORF1 and uORF2 can both be translated from two different start codons. (b) Vero cells were transfected with siRNAs targeting initiation factors 1A, 1, and 4B, compared to non-targeting (NT) controls. At 48h the same transfection was repeated. At 48h after the second transfection, cells were infected with either CoV2 (orange, left) polio (PV), Zika (ZIKV) or dengue (DENV) viruses (black, right) at MOI = 0.5. Viral titers were determined by plaque assays. Shown are means±SD of 4 independent replicates. (c) Vero cells were transfected with siRNA as above, infected with CoV2 at MOI = 5, and subjected to ribosome profiling analysis at 16 hpi. Traces show ribosome footprints on CoV2 RNA. Insets, ribosome footprints on nucleotides 20–80 of the gRNA. Representative of 2 independent replicates. (d) Ribosome footprints spanning 13 nt upstream and 12 nt downstream of the indicated start codons. Each bar represents a single biological replicate. P, Wilcoxon ranked-sum p-value. (e) Cumulative fraction plots of ribosome footprints on Orf1a, Spike and Nucleocapsid (N) open reading frames. Combined analysis of two independent replicates. P, Wilcoxon ranked-sum p-value. A shift of the curve to the left reflects lower ribosome occupancy and therefore lower translation. (f) Vero cells transfected with siRNA and infected as in (b) were subjected to immunoblot analysis of whole cell lysates using antibodies against CoV2 Spike, Nucleoprotein and Nsp1. Shown are representative blots of 4 independent repeats. (g) Vero cells were transfected with siRNA as above, followed by combined plasmid transfections of GFP and Nsp1 at the indicated amounts. The same total amount of DNA was used for each transfection. At 24 hours, cells were transfected again with nLuc mRNA flanked by UTRs from either GAPDH (left panel), CoV2 gRNA (middle panel) or CoV2 gRNA with CUG(59) mutated to AUG (right panel). At 4 hours post-second transfection, cells were subjected to luminescence measurements. Shown are means±SD of 3 independent replicates.</p