SARS-CoV-2 outbreak in a tri-national urban area is dominated by a B.1 lineage variant linked to a mass gathering event

The first case of SARS-CoV-2 in Basel, Switzerland was detected on February 26^th 2020. We present a phylogenetic study to explore viral introduction and evolution during the exponential early phase of the local COVID-19 outbreak from February 26^th until March 23^rd. We sequenced SARS-CoV-2 naso-oropharyngeal swabs from 746 positive tests that were performed at the University Hospital Basel during the study period. We successfully generated 468 high quality genomes from unique patients and called variants with our COVID-19 Pipeline (COVGAP), and analysed viral genetic diversity using PANGOLIN taxonomic lineages. To identify introduction and dissemination events we incorporated global SARS-CoV-2 genomes and inferred a time-calibrated phylogeny. Epidemiological data from patient questionnaires was used to facilitate the interpretation of phylogenetic observations. The early outbreak in Basel was dominated by lineage B.1 (83·6%), detected first on March 2^nd, although the first sample identified belonged to B.1.1. Within B.1, 68·2% of our samples fall within a clade defined by the SNP C15324T (‘Basel cluster’), including 157 identical sequences at the root of the ‘Basel cluster’, some of which we can specifically trace to regional spreading events. We infer the origin of B.1-C15324T to mid-February in our tri-national region. The other genomes map broadly over the global phylogenetic tree, showing several introduction events from and/or dissemination to other regions of the world via travellers. Family transmissions can also be traced in our data. A single lineage variant dominated the outbreak in the Basel area while other lineages, such as the first (B.1.1), did not propagate. A mass gathering event was the predominant initial source of cases, with travel returners and family transmissions to a lesser extent. We highlight the importance of adding specific questions to epidemiological questionnaires, to obtain data on attendance of large gatherings and their locations, as well as travel history, to effectively identify routes of transmissions in up-coming outbreaks. This phylogenetic analysis in concert with epidemiological and contact tracing data, allows connection and interpretation of events, and can inform public health interventions.Trial Registration: ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT04351503","term_id":"NCT04351503"}}NCT04351503.     

Characterization of the minimum domain required for targeting budding yeast myosin II to the site of cell division

Background  

All eukaryotes with the exception of plants use an actomyosin ring to generate a constriction force at the site of cell division (cleavage furrow) during mitosis and meiosis. The structure and filament forming abilities located in the C-terminal or tail region of one of the main components, myosin II, are important for localising the molecule to the contractile ring (CR) during cytokinesis. However, it remains poorly understood how myosin II is recruited to the site of cell division and how this recruitment relates to myosin filament assembly. Significant conservation between species of the components involved in cytokinesis, including those of the CR, allows the use of easily genetically manipulated organisms, such as budding yeast (Saccharomyces cerevisiae), in the study of cytokinesis. Budding yeast has a single myosin II protein, named Myo1. Unlike most other class II myosins, the tail of Myo1 has an irregular coiled coil. In this report we use molecular genetics, biochemistry and live cell imaging to characterize the minimum localisation domain (MLD) of budding yeast Myo1.     

Obesity‐related hypertension: Pathogenesis,cardiovascular risk,and treatment—A position paper of the The Obesity Society and the American Society of Hypertension

In light of the worldwide epidemic of obesity, and in recognition of hypertension as a major factor in the cardiovascular morbidity and mortality associated with obesity, The Obesity Society and The American Society of Hypertension agreed to jointly sponsor a position paper on obesity‐related hypertension to be published jointly in the journals of each society. The purpose is to inform the members of both societies, as well as practicing clinicians, with a timely review of the association between obesity and high blood pressure, the risk that this association entails, and the options for rational, evidenced‐based treatment. The position paper is divided into six sections plus a summary as follows: pathophysiology, epidemiology and cardiovascular risk, the metabolic syndrome, lifestyle management in prevention and treatment, pharmacologic treatment of hypertension in the obese, and the medical and surgical treatment of obesity in obese hypertensive patients. Obesity (2012)     

Parabiosis in Mice: A Detailed Protocol

Parabiosis is a surgical union of two organisms allowing sharing of the blood circulation. Attaching the skin of two animals promotes formation of microvasculature at the site of inflammation. Parabiotic partners share their circulating antigens and thus are free of adverse immune reaction. First described by Paul Bert in 1864¹, the parabiosis surgery was refined by Bunster and Meyer in 1933 to improve animal survival². In the current protocol, two mice are surgically joined following a modification of the Bunster and Meyer technique. Animals are connected through the elbow and knee joints followed by attachment of the skin allowing firm support that prevents strain on the sutured skin. Herein, we describe in detail the parabiotic joining of a ubiquitous GFP expressing mouse to a wild type (WT) mouse. Two weeks after the procedure, the pair is separated and GFP positive cells can be detected by flow cytometric analysis in the blood circulation of the WT mouse. The blood chimerism allows one to examine the contribution of the circulating cells from one animal in the other.     

Exogenous Administration of a Recombinant Variant of TWEAK Impairs Healing after Myocardial Infarction by Aggravation of Inflammation

Background

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor fibroblast growth factor-inducible 14 (Fn14) are upregulated after myocardial infarction (MI) in both humans and mice. They modulate inflammation and the extracellular matrix, and could therefore be important for healing and remodeling after MI. However, the function of TWEAK after MI remains poorly defined.

Methods and results

Following ligation of the left coronary artery, mice were injected twice per week with a recombinant human serum albumin conjugated variant of TWEAK (HSA-Flag-TWEAK), mimicking the activity of soluble TWEAK. Treatment with HSA-Flag-TWEAK resulted in significantly increased mortality in comparison to the placebo group due to myocardial rupture. Infarct size, extracellular matrix remodeling, and apoptosis rates were not different after MI. However, HSA-Flag-TWEAK treatment increased infiltration of proinflammatory cells into the myocardium. Accordingly, depletion of neutrophils prevented cardiac ruptures without modulating all-cause mortality.

Conclusion

Treatment of mice with HSA-Flag-TWEAK induces myocardial healing defects after experimental MI. This is mediated by an exaggerated neutrophil infiltration into the myocardium.     

microRNA-210 is upregulated in hypoxic cardiomyocytes through Akt- and p53-dependent pathways and exerts cytoprotective effects

microRNA-210 (miR-210) is upregulated in hypoxia, but its function in cardiomyocytes and its regulation in response to hypoxia are not well characterized. The purpose of this study was to identify upstream regulators of miR-210, as well as to characterize miR-210's function in cardiomyocytes. We first showed miR-210 is upregulated through both hypoxia-inducible factor (HIF)-dependent and -independent pathways, since aryl hydrocarbon nuclear translocator (ARNT) knockout mouse embryonic fibroblasts (MEF), lacking intact HIF signaling, still displayed increased miR-210 levels in hypoxia. To determine the mechanism for HIF-independent regulation of miR-210, we focused on p53 and protein kinase B (Akt). Overexpression of p53 in wild-type MEFs induced miR-210, whereas p53 overexpression in ARNT knockout MEFs did not, suggesting p53 regulates miR-210 in a HIF-dependent mechanism. Akt inhibition reduced miR-210 induction by hypoxia, whereas Akt overexpression increased miR-210 levels in both wild-type and ARNT knockout MEFs, indicating Akt regulation of miR-210 is HIF-independent. We then studied the effects of miR-210 in cardiomyocytes. Overexpression of miR-210 reduced cell death in response to oxidative stress and reduced reactive oxygen species (ROS) production both at baseline and after treatment with antimycin A. Furthermore, downregulation of miR-210 increased ROS after hypoxia-reoxygenation. To determine a mechanism for the cytoprotective effects of miR-210, we focused on the predicted target, apoptosis-inducing factor, mitochondrion-associated 3 (AIFM3), known to induce cell death. Although miR-210 reduced AIFM3 levels, overexpression of AIFM3 in the presence of miR-210 overexpression did not reduce cellular viability either at baseline or after hydrogen peroxide treatment, suggesting AIFM3 does not mediate miR-210's cytoprotective effects. Furthermore, HIF-3α, a negative regulator of HIF signaling, is targeted by miR-210, but miR-210 does not modulate HIF activity. In conclusion, we demonstrate a novel role for p53 and Akt in regulating miR-210 and demonstrate that, in cardiomyocytes, miR-210 exerts cytoprotective effects, potentially by reducing mitochondrial ROS production.     

Comparison of renal effects of ibuprofen versus indomethacin during treatment of patent ductus arteriosus in contiguous historical cohorts

Background

The study aimed to investigate the pharmacokinetics of intravenous ciprofloxacin and the adequacy of 400 mg every 12 hours in critically ill Intensive Care Unit (ICU) patients on continuous veno-venous haemodiafiltration (CVVHDF) with particular reference to the effect of achieved flow rates on drug clearance.

Methods

This was an open prospective study conducted in the intensive care unit and research unit of a university teaching hospital. The study population was seven critically ill patients with sepsis requiring CVVHDF. Blood and ultrafiltrate samples were collected and assayed for ciprofloxacin by High Performance Liquid Chromatography (HPLC) to calculate the model independent pharmacokinetic parameters; total body clearance (TBC), half-life (t_1/2) and volume of distribution (Vd). CVVHDF was performed at prescribed dialysate rates of 1 or 2 L/hr and ultrafiltration rate of 2 L/hr. The blood flow rate was 200 ml/min, achieved using a Gambro blood pump and Hospal AN69HF haemofilter.

Results

Seventeen profiles were obtained. CVVHDF resulted in a median ciprofloxacin t_1/2 of 13.8 (range 5.15-39.4) hr, median TBC of 9.90 (range 3.10-13.2) L/hr, a median V_dss of 125 (range 79.5-554) L, a CVVHDF clearance of 2.47+/-0.29 L/hr and a clearance of creatinine (Cl_cr) of 2.66+/-0.25 L/hr. Thus CVVHDF, at an average flow rate of ~3.5 L/hr, was responsible for removing 26% of ciprofloxacin cleared. At the dose rate of 400 mg every 12 hr, the median estimated C_pmax/MIC and AUC_0-24/MIC ratios were 10.3 and 161 respectively (for a MIC of 0.5 mg/L) and exceed the proposed criteria of >10 for C_pmax/MIC and > 100 for AUC_0-24/MIC. There was a suggestion towards increased ciprofloxacin clearance by CVVHDF with increasing effluent flow rate.

Conclusions

Given the growing microbial resistance to ciprofloxacin our results suggest that a dose rate of 400 mg every 12 hr, may be necessary to achieve the desired pharmacokinetic - pharmacodynamic (PK-PD) goals in patients on CVVHDF, however an extended interval may be required if there is concomitant hepatic impairment. A correlation between ciprofloxacin clearance due to CVVHDF and creatinine clearance by the filter was observed (r² = 0.76), providing a useful clinical surrogate marker for ciprofloxacin clearance within the range studied.

Trial Registration

Current Controlled Trials ISRCTN52722850