SpikeSeq: A rapid, cost efficient and simple method to identify SARS-CoV-2 variants of concern by Sanger sequencing part of the spike protein gene.

In 2020, the novel coronavirus, SARS-CoV-2, caused a pandemic, which is still raging at the time of writing this. Here, we present results from SpikeSeq, the first published Sanger sequencing-based method for the detection of Variants of Concern (VOC) and key mutations, using a 1 kb amplicon from the recognized ARTIC Network primers. The proposed setup relies entirely on materials and methods already in use in diagnostic RT-qPCR labs and on existing commercial infrastructure offering sequencing services. For data analysis, we provide an automated, open source, and browser-based mutation calling software (https://github.com/kblin/covid-spike-classification, https://ssi.biolib.com/covid-spike-classification). We validated the setup on 195 SARS-CoV-2 positive samples, and we were able to profile 85% of RT-qPCR positive samples, where the last 15% largely stemmed from samples with low viral count. We compared the SpikeSeq results to WGS results. SpikeSeq has been used as the primary variant identification tool on > 10.000 SARS-CoV-2 positive clinical samples during 2021. At approximately 4€ per sample in material cost, minimal hands-on time, little data handling, and a short turnaround time, the setup is simple enough to be implemented in any SARS-CoV-2 RT-qPCR diagnostic lab. Our protocol provides results that can be used to choose antibodies in a clinical setting and for the tracking and surveillance of all positive samples for new variants and known ones such as Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1) Delta (B.1.617.2), Omicron BA.1(B.1.1.529), BA.2, BA.4/5, BA.2.75.x, and many more, as of October 2022.

Postconditioning with curaglutide, a novel GLP-1 analog, protects against heart ischemia-reperfusion injury in an isolated rat heart.

AIM: GLP-1(7-36)amide (GLP-1) is an intestinal hormone with effects on glucose metabolism and feeding behavior, including insulinotropic, insulinomimetic, glucagonostatic and anorectic actions. In experimental settings, GLP-1 has also been shown to diminish infarct size following heart ischemia-reperfusion. GLP-1 analogs with extended half-lives are continuously being developed against type 2 diabetes mellitus. Of these, only exendin-4 (exenatide, registered as Byetta) has been shown to mimic the infarct size-limiting effect of GLP-1 in a clinically relevant application as a postconditioning agent. The aim of this work was to test, in a postconditioning mode, a novel, proteolysis-resistant GLP-1 analog N-Ac-GLP-1(7-34)amide, herein termed curaglutide, for its cardioprotective ability. METHOD: Global ischemia (35 min)-reperfusion (120 min) was applied in isolated, retrogradely perfused rat hearts. Peptides were present for 15 min at the onset of reperfusion. Cardiac function parameters (beats per minute, left ventricle developed and diastolic pressures, rate-pressure product) were measured. Infarct size was determined by 2,3,5-tripehyltetrazolium chloride staining and planimetry. RESULTS: Curaglutide did not affect any of the functional heart parameters when administered without preceding ischemia. Curaglutide 0.3 nM diminished significantly the postischemic hypercontracture, with no significant effect on the left ventricle developed pressure or rate-pressure product. Infarct size was reduced by curaglutide postconditioning from 24.8% (SEM 2.8, N=14) to 11.4% (SEM 3.2, N=8; P<0.05). These effects of curaglutide on postischemic hypercontracture and infarct size were similar in magnitude to corresponding effects of GLP-1 receptor agonist exendin-4. The cardioprotective effects of both agents were abolished in the presence of a GLP-1 receptor antagonist exendin(9-39). CONCLUSION: Curaglutide is a new, proteolysis-resistant GLP-1 analog with a beneficial effect on reperfusion-injury in an isolated rat heart. Curaglutide was here shown to act through GLP-1 receptors. Based on the present results, more extensive experimental studies in vivo, comparing dose-response characteristics and efficacy of curaglutide and exendin-4 appear warranted.