Background
This study was performed to determine the clinical correlates and long‐term prognostic implications of microbleed burden and location in Chinese patients with ischemic stroke.
Methods and ...Results
We recruited 1003 predominantly Chinese patients with ischemic stroke who received magnetic resonance imaging at the University of Hong Kong. We determined the clinical correlates of microbleeds and the long‐term risks (3126 patient‐years of follow‐up) of recurrent ischemic stroke and intracerebral hemorrhage (ICH) by microbleed burden (0 versus 1, 2–4, and ≥5) and location, adjusting for age, sex, and vascular risk factors and stratified by antithrombotic use. Microbleeds were present in 450 of 1003 of the study population (119/450 had ≥5, 187/450 had mixed location). Having ≥5 microbleeds was independently associated with prior antiplatelet and anticoagulant use, whereas microbleeds of mixed location were independently associated with hypertension and prior anticoagulant use (all P<0.05). Microbleed burden was associated with an increased risk of ICH (microbleed burden versus no microbleeds: 1 microbleed: multivariate hazard ratio: 0.59 95% confidence interval, 0.07–5.05; 2–4 microbleeds: multivariate hazard ratio: 2.14 95% confidence interval, 0.50–9.12; ≥5 microbleeds: multivariate hazard ratio: 9.51 95% confidence interval, 3.25–27.81; Ptrend<0.0001), but the relationship of microbleed burden and risk of recurrent ischemic stroke was not significant (Ptrend=0.054). Similar findings were noted in the 862 of 1003 patients treated with antiplatelet agents only (ICH: Ptrend<0.0001; ischemic stroke Ptrend=0.096). Multivariate analysis revealed that, independent of vascular risk factors, antithrombotic use, and other neuroimaging markers of small vessel disease, having ≥5 microbleeds (multivariate hazard ratio: 6.08 95% confidence interval, 1.11–33.21; P=0.037) was identified as an independent predictor of subsequent ICH, but neither microbleed burden nor location was predictive of recurrent ischemic stroke risk.
Conclusions
In Chinese patients with ischemic stroke, a high burden of cerebral microbleeds was significantly associated with an increased risk of ICH; however, neither microbleed location nor burden was associated with recurrent ischemic stroke risk.
The aim of this nationwide cohort study was to evaluate whether the occurrence of isolated 3rd, 4th or 6th cranial nerve (CN) palsies is associated with a higher risk of ischemic stroke.
This study ...utilized data from Taiwan Longitudinal Health Insurance Database during 1995-2012. Subjects aged 20 years or older who had isolated CN 3/4/6 palsies diagnosed by a neurologist or ophthalmologist between January 2000 and December 2011 were included. A set of propensity score matched, randomly sampled patients who had never been diagnosed with CN 3/4/6 palsies were extracted to constitute the control group (cases and controls = 1:4). All subjects were followed until death, loss due to follow-up or completion of the study. Cox proportional hazard regression model stratified by matched pairs was used to estimate the hazards ratio (HR) of ischemic stroke.
A total of 657 patients with isolated CN 3/4/6 palsies (61.1% male, mean age 54.8 years) were identified. Compared with control group, the patients with isolated CN 3/4/6 palsies exhibited an increased risk of ischemic stroke (CN3: adjusted HR 3.69 (95% CI 2.20-6.19); CN4: 2.71 (95% CI 1.11-6.64); CN6: 2.15 (95% CI 1.31-3.52)). The association between CN 3/4/6 palsies and ischemic stroke was detected in both separate subgroup and sensitivity analyses.
The patients with CN 3/4/6 palsies exhibited an increased risk of developing ischemic stroke. Therefore, isolated ocular motor nerves palsies appear to represent an unrecognized risk factor for ischemic stroke, and these require further confirmation and exploration.
An iron(iii) complex bearing a cross-bridged cyclam ligand (4,11-dimethyl-1,4,8,11-tetraazabicyclo6.6.2hexadecane) is an efficient catalyst for the oxidation of both water and alcohols using sodium ...periodate as the oxidant. In catalytic water oxidation a maximum turnover number (TON) of 1030 is achieved, while in catalytic alcohol oxidation >95% conversions and yields can be obtained.
Host survival depends on the elimination of virus and mitigation of tissue damage. Herein, we report the modulation of D-mannose flux rewires the virus-triggered immunometabolic response cascade and ...reduces tissue damage. Safe and inexpensive D-mannose can compete with glucose for the same transporter and hexokinase. Such competitions suppress glycolysis, reduce mitochondrial reactive-oxygen-species and succinate-mediated hypoxia-inducible factor-1α, and thus reduce virus-induced proinflammatory cytokine production. The combinatorial treatment by D-mannose and antiviral monotherapy exhibits in vivo synergy despite delayed antiviral treatment in mouse model of virus infections. Phosphomannose isomerase (PMI) knockout cells are viable, whereas addition of D-mannose to the PMI knockout cells blocks cell proliferation, indicating that PMI activity determines the beneficial effect of D-mannose. PMI inhibition suppress a panel of virus replication via affecting host and viral surface protein glycosylation. However, D-mannose does not suppress PMI activity or virus fitness. Taken together, PMI-centered therapeutic strategy clears virus infection while D-mannose treatment reprograms glycolysis for control of collateral damage.
The genome-editing Cas9 protein uses multiple amino-acid residues to bind the target DNA. Considering only the residues in proximity to the target DNA as potential sites to optimise Cas9's activity, ...the number of combinatorial variants to screen through is too massive for a wet-lab experiment. Here we generate and cross-validate ten in silico and experimental datasets of multi-domain combinatorial mutagenesis libraries for Cas9 engineering, and demonstrate that a machine learning-coupled engineering approach reduces the experimental screening burden by as high as 95% while enriching top-performing variants by ∼7.5-fold in comparison to the null model. Using this approach and followed by structure-guided engineering, we identify the N888R/A889Q variant conferring increased editing activity on the protospacer adjacent motif-relaxed KKH variant of Cas9 nuclease from Staphylococcus aureus (KKH-SaCas9) and its derived base editor in human cells. Our work validates a readily applicable workflow to enable resource-efficient high-throughput engineering of genome editor's activity.
Abstract
The Cas9 nuclease from Staphylococcus aureus (SaCas9) holds great potential for use in gene therapy, and variants with increased fidelity have been engineered. However, we find that existing ...variants have not reached the greatest accuracy to discriminate base mismatches and exhibited much reduced activity when their mutations were grafted onto the KKH mutant of SaCas9 for editing an expanded set of DNA targets. We performed structure-guided combinatorial mutagenesis to re-engineer KKH-SaCas9 with enhanced accuracy. We uncover that introducing a Y239H mutation on KKH-SaCas9’s REC domain substantially reduces off-target edits while retaining high on-target activity when added to a set of mutations on REC and RuvC domains that lessen its interactions with the target DNA strand. The Y239H mutation is modelled to have removed an interaction from the REC domain with the guide RNA backbone in the guide RNA-DNA heteroduplex structure. We further confirmed the greatly improved genome-wide editing accuracy and single-base mismatch discrimination of our engineered variants, named KKH-SaCas9-SAV1 and SAV2, in human cells. In addition to generating broadly useful KKH-SaCas9 variants with unprecedented accuracy, our findings demonstrate the feasibility for multi-domain combinatorial mutagenesis on SaCas9’s DNA- and guide RNA- interacting residues to optimize its editing fidelity.
Selecting the most suitable existing base editors and engineering new variants for installing specific base conversions with maximal efficiency and minimal undesired edits are pivotal for precise ...genome editing applications. Here, we present a platform for creating and analyzing a library of engineered base editor variants to enable head-to-head evaluation of their editing performance at scale. Our comprehensive comparison provides quantitative measures on each variant’s editing efficiency, purity, motif preference, and bias in generating single and multiple base conversions, while uncovering undesired higher indel generation rate and noncanonical base conversion for some of the existing base editors. In addition to engineering the base editor protein, we further applied this platform to investigate a hitherto underexplored engineering route and created guide RNA scaffold variants that augment the editor’s base-editing activity. With the unknown performance and compatibility of the growing number of engineered parts including deaminase, CRISPR-Cas enzyme, and guide RNA scaffold variants for assembling the expanding collection of base editor systems, our platform addresses the unmet need for an unbiased, scalable method to benchmark their editing outcomes and accelerate the engineering of next-generation precise genome editors.
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•Platform established for unbiased head-to-head benchmarking of base editors’ activity•Simultaneously engineer and profile base editor variants’ editing outcome in human cells•Profiling uncovers undescribed editing outcome features of existing base editors•Screen identifies engineered guide RNA scaffolds that enhance base editor’s activity
There are hurdles to compare the precise editing outcome across a large panel of existing base editors and engineer more variants to achieve greater performance. We present a one-pot solution to engineer base editors in a high-throughput manner and evaluate their precise editing outcome head-to-head en masse.
Reaction of RuVI(N)(L1)(MeOH)+ (L1 = N,N′-bis(salicylidene)-o-cyclohexylenediamine dianion) with excess pyridine in CH3CN produces RuIII(L1)(py)2+ and N2. The proposed mechanism involves initial ...equilibrium formation of RuVI(N)(L1)(py)+, which undergoes rapid N···N coupling to produce (py)(L1)RuIII NN−RuIII(L1)(py)2+; this is followed by pyridine substituion to give the final product. This ligand-induced N···N coupling of RuVIN is utilized in the preparation of a series of new ruthenium(III) salen complexes, RuIII(L)(X)2 ± (L = salen ligand; X = H2O, 1-MeIm, py, Me2SO, PhNH2, t BuNH2, Cl− or CN−). The structures of RuIII(L1)(NH2Ph)2(PF6) (6), KRuIII(L1)(CN)2 (9), RuIII(L2)(NCCH3)2AuI(CN)2 (11) (L2 = N,N′-bis(salicylidene)-o-phenylenediamine dianion) and N n Bu4RuIII(L3)Cl2 (12) (L3 = N,N′-bis(salicylidene)ethylenediamine dianion) have been determined by X-ray crystallography.
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