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Motorists regarding COVID-19 Vaccine Customer base amidst Health-related Staff (HCWs) in Africa.
The CRISPR/Cas9 system allows scarless, marker-free genome editing. Current CRISPR/Cas9 systems for the fission yeast Schizosaccharomyces pombe rely on tedious and time-consuming cloning procedures to introduce a specific sgRNA target sequence into a Cas9-expressing plasmid. In addition, Cas9 endonuclease has been reported to be toxic to fission yeast when constitutively overexpressed from the strong adh1 promoter. To overcome these problems we have developed an improved system, SpEDIT, that uses a synthesised Cas9 sequence codon-optimised for S. pombe expressed from the medium strength adh15 promoter. The SpEDIT system exhibits a flexible modular design where the sgRNA is fused to the 3' end of the self-cleaving hepatitis delta virus (HDV) ribozyme, allowing expression of the sgRNA cassette to be driven by RNA polymerase III from a tRNA gene sequence. Lastly, the inclusion of sites for the BsaI type IIS restriction enzyme flanking a GFP placeholder enables one-step Golden Gate mediated replacement of GFP with synthesized sgRNAs for expression. The SpEDIT system allowed a 100% mutagenesis efficiency to be achieved when generating targeted point mutants in the ade6 + or ura4+ genes by transformation of cells from asynchronous cultures. SpEDIT also permitted insertion, tagging and deletion events to be obtained with minimal effort. Simultaneous editing of two independent non-homologous loci was also readily achieved. Importantly the SpEDIT system displayed reduced toxicity compared to currently available S. pombe editing systems. Thus, SpEDIT provides an effective and user-friendly CRISPR/Cas9 procedure that significantly improves the genome editing toolbox for fission yeast.Background With epidemiological transition, stroke has emerged as a public health priority in rural India. However, population-level information on secondary prevention of stroke from rural areas of India and other low- and middle-income countries remains exceedingly rare. Methods In a cross-sectional community-based survey, trained surveyors screened a well-defined population of 74,095 individuals living in 64 villages in Gadchiroli district of India for symptoms of stroke. A trained physician evaluated screen positive patients, diagnosed stroke, measured blood pressure and collected information on prior diagnosis of risk factors and current use of medications using a structured questionnaire. Selleckchem PTC-028 Results A total of 265 stroke survivors were identified. Prior diagnosis of hypertension was made in 57.4%, diabetes in 9.8%, hyperlipidaemia in 0.4%, ischaemic heart disease in 1.5%. and atrial fibrillation in 1.1%. Blood pressure was uncontrolled (>140/90) in 46% of stroke survivors. Among men 71.2% used tobacco and 30% used alcohol, while among women 38.2% used tobacco and none used alcohol. Only 40.8% of stroke survivors were receiving antihypertensive medications, while 10.6% were on antiplatelet agents and 4.9% were on statins. In a multivariate analysis, age less then 50 years (OR 0.2, 95% CI 0.1-0.5), male sex (OR 0.2, 95% CI 0.2-0.8) and lower economic status (no assets vs four assets; OR 0.3, 95% CI 0.1-0.9) were associated with lower odds of receiving medications for secondary prevention of stroke. Conclusions There were significant gaps in secondary prevention of stroke in rural Gadchiroli. Healthcare programmes for secondary prevention of stroke in rural areas will have to ensure that blood pressure is adequately controlled, alcohol and tobacco cessation is promoted and special attention is paid to those who are younger, men and economically weaker.Infection with SARS-CoV-2 is expected to result in substantial reorganization of host cell RNA metabolism. We identified 14 proteins that were predicted to interact with host RNAs or RNA binding proteins, based on published data for SARS-CoV and SARS-CoV-2. Here, we describe a series of affinity-tagged and codon-optimized expression constructs for each of these 14 proteins. Each viral gene was separately tagged at the N-terminus with Flag-His 8, the C-terminus with His 8-Flag, or left untagged. The resulting constructs were stably integrated into the HEK293 Flp-In T-REx genome. Each viral gene was expressed under the control of an inducible Tet-On promoter, allowing expression levels to be tuned to match physiological conditions during infection. Expression time courses were successfully generated for most of the fusion proteins and quantified by western blot. A few fusion proteins were poorly expressed, whereas others, including Nsp1, Nsp12, and N protein, were toxic unless care was taken to minimize background expression. All plasmids can be obtained from Addgene and cell lines are available. We anticipate that availability of these resources will facilitate a more detailed understanding of coronavirus molecular biology.Anticoagulant-associated traumatic intracranial hemorrhage (tICrH) is a devastating injury with high morbidity and mortality. For survivors, treating clinicians face the dilemma of restarting oral anticoagulation with scarce evidence to guide them. Thromboembolic risk is high from the bleeding event, patients' high baseline risks, that is, the pre-existing indication for anticoagulation, and the risk of immobility after the bleeding episode. This must be balanced with potentially devastating hematoma expansion or new hemorrhagic lesions. Retrospective evidence and expert opinion support restarting oral anticoagulants in most patients with tICrH, but timing is uncertain. Researchers have failed to make clear distinctions between tICrH and spontaneous intracranial hemorrhage (sICrH), which have differing natural histories. While both appear to benefit from restarting, sICrH has a higher rebleeding risk and similar or lower thrombotic risk. Clinical equipoise on restarting is also divergent. In sICrH, equipoise is centered on whether to restart. In tICrH, it is centered on when. Several prospective randomized clinical trials are ongoing or about to start to examine the risk-benefit of restarting. Most of them are restricted to patients with sICrH, with antiplatelet control groups. Most are also restricted to direct oral anticoagulants (DOACs), as they are associated with a lower overall risk of ICrH. There is some overlap with tICrH via subdural hematoma, and one trial is specific to restart timing with DOACs in only traumatic cases. This is a narrative review of the current evidence for restarting anticoagulation and restart timing after tICrH along with a summary of the ongoing and planned clinical trials.
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