Spatial distribution and activity of Na +/K +-ATPase in lipid bilayer membranes with phase boundaries

Abstract We have reconstituted functional Na +/K +-ATPase (NKA) into giant unilamellar vesicles (GUVs) of well-defined binary and ternary lipid composition including cholesterol. The activity of the membrane system can be turned on and off by ATP. The hydrolytic activity of \NKA\} is found to depend on membrane phase, and the water relaxation in the membrane on the presence of NKA. By collapsing and fixating the \{GUVs\} onto a solid support and using high-resolution atomic-force microscopy (AFM) imaging we determine the protein orientation and spatial distribution at the single-molecule level and find that \{NKA\} is preferentially located at l o/l d interfaces in two-phase \{GUVs\ and homogeneously distributed in single-phase GUVs. When turned active, the membrane is found to unbind from the support suggesting that the protein function leads to a softening of the membrane

Optimizing the Definitions of Stroke, TIA and Infarction for Research and Application in Clinical Practice

Background and purposeUntil now, stroke and transient ischemic attack (TIA) have been clinically based terms which describe the presence and duration of characteristic neurological deficits attributable to intrinsic disorders of particular arteries supplying the brain, retina, or (sometimes) the spinal cord. Further, infarction has been pathologically defined as death of neural tissue due to reduced blood supply. Recently, it has been proposed we shift to definitions of stroke and TIA determined by neuroimaging results alone and that neuroimaging findings be equated with infarction.MethodsWe examined the scientific validity and clinical implications of these proposals using the existing published literature and our own experience in research and clinical practice.ResultsWe found that the proposals to change to imaging-dominant definitions, as published, are ambiguous and inconsistent. Therefore, they cannot provide the standardization required in research or its application in clinical practice. Further, we found that the proposals are scientifically incorrect because neuroimaging findings do not always correlate with the clinical status or the presence of infarction. In addition, we found that attempts to use the proposals are disrupting research, are otherwise clinically unhelpful and do not solve the problems they were proposed to solve.ConclusionWe advise that the proposals must not be accepted. In particular, we explain why the clinical focus of the definitions of stroke and TIA should be retained with continued sub-classification of these syndromes depending neuroimaging results (with or without other information) and that infarction should remain a pathological term. We outline ways the established clinically based definitions of stroke and TIA, and use of them, may be improved to encourage better patient outcomes in the modern era

Intravenous alteplase for stroke with unknown time of onset guided by advanced imaging: systematic review and meta-analysis of individual patient data

Background: Patients who have had a stroke with unknown time of onset have been previously excluded from thrombolysis. We aimed to establish whether intravenous alteplase is safe and effective in such patients when salvageable tissue has been identified with imaging biomarkers. Methods: We did a systematic review and meta-analysis of individual patient data for trials published before Sept 21, 2020. Randomised trials of intravenous alteplase versus standard of care or placebo in adults with stroke with unknown time of onset with perfusion-diffusion MRI, perfusion CT, or MRI with diffusion weighted imaging-fluid attenuated inversion recovery (DWI-FLAIR) mismatch were eligible. The primary outcome was favourable functional outcome (score of 0–1 on the modified Rankin Scale [mRS]) at 90 days indicating no disability using an unconditional mixed-effect logistic-regression model fitted to estimate the treatment effect. Secondary outcomes were mRS shift towards a better functional outcome and independent outcome (mRS 0–2) at 90 days. Safety outcomes included death, severe disability or death (mRS score 4–6), and symptomatic intracranial haemorrhage. This study is registered with PROSPERO, CRD42020166903. Findings: Of 249 identified abstracts, four trials met our eligibility criteria for inclusion: WAKE-UP, EXTEND, THAWS, and ECASS-4. The four trials provided individual patient data for 843 individuals, of whom 429 (51%) were assigned to alteplase and 414 (49%) to placebo or standard care. A favourable outcome occurred in 199 (47%) of 420 patients with alteplase and in 160 (39%) of 409 patients among controls (adjusted odds ratio [OR] 1·49 [95% CI 1·10–2·03]; p=0·011), with low heterogeneity across studies (I2=27%). Alteplase was associated with a significant shift towards better functional outcome (adjusted common OR 1·38 [95% CI 1·05–1·80]; p=0·019), and a higher odds of independent outcome (adjusted OR 1·50 [1·06–2·12]; p=0·022). In the alteplase group, 90 (21%) patients were severely disabled or died (mRS score 4–6), compared with 102 (25%) patients in the control group (adjusted OR 0·76 [0·52–1·11]; p=0·15). 27 (6%) patients died in the alteplase group and 14 (3%) patients died among controls (adjusted OR 2·06 [1·03–4·09]; p=0·040). The prevalence of symptomatic intracranial haemorrhage was higher in the alteplase group than among controls (11 [3%] vs two [<1%], adjusted OR 5·58 [1·22–25·50]; p=0·024). Interpretation: In patients who have had a stroke with unknown time of onset with a DWI-FLAIR or perfusion mismatch, intravenous alteplase resulted in better functional outcome at 90 days than placebo or standard care. A net benefit was observed for all functional outcomes despite an increased risk of symptomatic intracranial haemorrhage. Although there were more deaths with alteplase than placebo, there were fewer cases of severe disability or death. Funding: None

Enzymatic maceration of bone:a gentler technique than boiling

This proof of concept study investigates the removal of soft tissue from human ribs with the use of two common methods: boiling with a laundry detergent and using enzymes. Six individuals were autopsied, and one rib from each individual was removed for testing. Each rib was cut into pieces and afterwards macerated by one of the two methods. DNA extraction was performed to see the effect of the macerations on DNA preservation. Furthermore, the bone pieces were examined in a stereomicroscope to assess for any bone damage. The results demonstrated that both methods removed all flesh/soft tissue from the bones. The DNA analysis showed that DNA was preserved on all the pieces of bones which were examined. Finally, the investigation suggests that enzyme maceration could be gentler on the bones, as the edges appeared less frayed. The enzyme maceration was also a quicker method; it took three hours compared with the traditional method which took about 24 hours. However, a more standardised study should be performed to confirm this