Characterization of fluorescein arsenical hairpin (FIAsH) as a probe for single-molecule fluorescence spectroscopy

Sherpa Romeo green journal. Open access article. Creative Commons Attribution 4.0 International License (CC BY 4.0) appliesIn recent years, new labelling strategies have been developed that involve the genetic insertion of small amino-acid sequences for specific attachment of small organic fluorophores. Here, we focus on the tetracysteine FCM motif (FLNCCPGCCMEP), which binds to fluorescein arsenical hairpin (FlAsH), and the ybbR motif (TVLDSLEFIASKLA) which binds fluorophores conjugated to Coenzyme A (CoA) via a phosphoryl transfer reaction. We designed a peptide containing both motifs for orthogonal labelling with FlAsH and Alexa647 (AF647). Molecular dynamics simulations showed that both motifs remain solvent-accessible for labelling reactions. Fluorescence spectra, correlation spectroscopy and anisotropy decay were used to characterize labelling and to obtain photophysical parameters of free and peptide-bound FlAsH. The data demonstrates that FlAsH is a viable probe for single-molecule studies. Single-molecule imaging confirmed dual labeling of the peptide with FlAsH and AF647. Multiparameter single-molecule Förster Resonance Energy Transfer (smFRET) measurements were performed on freely diffusing peptides in solution. The smFRET histogram showed different peaks corresponding to different backbone and dye orientations, in agreement with the molecular dynamics simulations. The tandem of fluorophores and the labelling strategy described here are a promising alternative to bulky fusion fluorescent proteins for smFRET and single-molecule tracking studies of membrane proteins.Ye

Prehospital transdermal glyceryl trinitrate in patients with ultra-acute presumed stroke (RIGHT-2): an ambulance-based, randomised, sham-controlled, blinded, phase 3 trial

Background High blood pressure is common in acute stroke and is a predictor of poor outcome; however, large trials of lowering blood pressure have given variable results, and the management of high blood pressure in ultra-acute stroke remains unclear. We investigated whether transdermal glyceryl trinitrate (GTN; also known as nitroglycerin), a nitric oxide donor, might improve outcome when administered very early after stroke onset. Methods We did a multicentre, paramedic-delivered, ambulance-based, prospective, randomised, sham-controlled, blinded-endpoint, phase 3 trial in adults with presumed stroke within 4 h of onset, face-arm-speech-time score of 2 or 3, and systolic blood pressure 120 mm Hg or higher. Participants were randomly assigned (1:1) to receive transdermal GTN (5 mg once daily for 4 days; the GTN group) or a similar sham dressing (the sham group) in UK based ambulances by paramedics, with treatment continued in hospital. Paramedics were unmasked to treatment, whereas participants were masked. The primary outcome was the 7-level modified Rankin Scale (mRS; a measure of functional outcome) at 90 days, assessed by central telephone follow-up with masking to treatment. Analysis was hierarchical, first in participants with a confirmed stroke or transient ischaemic attack (cohort 1), and then in all participants who were randomly assigned (intention to treat, cohort 2) according to the statistical analysis plan. This trial is registered with ISRCTN, number ISRCTN26986053. Findings Between Oct 22, 2015, and May 23, 2018, 516 paramedics from eight UK ambulance services recruited 1149 participants (n=568 in the GTN group, n=581 in the sham group). The median time to randomisation was 71 min (IQR 45–116). 597 (52%) patients had ischaemic stroke, 145 (13%) had intracerebral haemorrhage, 109 (9%) had transient ischaemic attack, and 297 (26%) had a non-stroke mimic at the final diagnosis of the index event. In the GTN group, participants’ systolic blood pressure was lowered by 5·8 mm Hg compared with the sham group (p<0·0001), and diastolic blood pressure was lowered by 2·6 mm Hg (p=0·0026) at hospital admission. We found no difference in mRS between the groups in participants with a final diagnosis of stroke or transient ischaemic stroke (cohort 1): 3 (IQR 2–5; n=420) in the GTN group versus 3 (2–5; n=408) in the sham group, adjusted common odds ratio for poor outcome 1·25 (95% CI 0·97–1·60; p=0·083); we also found no difference in mRS between all patients (cohort 2: 3 [2–5]; n=544, in the GTN group vs 3 [2–5]; n=558, in the sham group; 1·04 [0·84–1·29]; p=0·69). We found no difference in secondary outcomes, death (treatment-related deaths: 36 in the GTN group vs 23 in the sham group [p=0·091]), or serious adverse events (188 in the GTN group vs 170 in the sham group [p=0·16]) between treatment groups. Interpretation Prehospital treatment with transdermal GTN does not seem to improve functional outcome in patients with presumed stroke. It is feasible for UK paramedics to obtain consent and treat patients with stroke in the ultraacute prehospital setting. Funding British Heart Foundation