Rapid cloning-free mutagenesis of new SARS-CoV-2 variants using a novel reverse genetics platform.

Reverse genetic systems enable the engineering of RNA virus genomes and are instrumental in studying RNA virus biology. With the recent outbreak of the coronavirus disease 2019 pandemic, already established methods were challenged by the large genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Herein we present an elaborated strategy for the rapid and straightforward rescue of recombinant plus-stranded RNA viruses with high sequence fidelity using the example of SARS-CoV-2. The strategy called CLEVER (CLoning-free and Exchangeable system for Virus Engineering and Rescue) is based on the intracellular recombination of transfected overlapping DNA fragments allowing the direct mutagenesis within the initial PCR-amplification step. Furthermore, by introducing a linker fragment - harboring all heterologous sequences - viral RNA can directly serve as a template for manipulating and rescuing recombinant mutant virus, without any cloning step. Overall, this strategy will facilitate recombinant SARS-CoV-2 rescue and accelerate its manipulation. Using our protocol, newly emerging variants can quickly be engineered to further elucidate their biology. To demonstrate its potential as a reverse genetics platform for plus-stranded RNA viruses, the protocol has been successfully applied for the cloning-free rescue of recombinant Chikungunya and Dengue virus.

Rapid cloning-free mutagenesis of new SARS-CoV-2 variants using a novel reverse genetics platform.

Reverse genetic systems enable the engineering of RNA virus genomes and are instrumental in studying RNA virus biology. With the recent outbreak of the COVID-19 pandemic, already established methods were challenged by the large genome of SARS-CoV-2. Herein we present an elaborated strategy for the rapid and straightforward rescue of recombinant plus-stranded RNA viruses with high sequence fidelity, using the example of SARS-CoV-2. The strategy called CLEVER (CLoning-free and Exchangeable system for Virus Engineering and Rescue) is based on the intracellular recombination of transfected overlapping DNA fragments allowing the direct mutagenesis within the initial PCR-amplification step. Furthermore, by introducing a linker fragment - harboring all heterologous sequences - viral RNA can directly serve as a template for manipulating and rescuing recombinant mutant virus, without any cloning step. Overall, this strategy will facilitate recombinant SARS-CoV-2 rescue and accelerate its manipulation. Using our protocol, newly emerging variants can quickly be engineered to further elucidate their biology. To demonstrate its potential as a reverse genetics platform for plus-stranded RNA viruses, the protocol has been successfully applied for the cloning-free rescue of recombinant Chikungunya and Dengue virus.

ROR2 regulates self-renewal and maintenance of hair follicle stem cells.

Hair follicles undergo cycles of regeneration fueled by hair follicle stem cells (HFSCs). While ß-catenin-dependent canonical Wnt signaling has been extensively studied and implicated in HFSC activation and fate determination, very little is known about the function of ß-catenin-independent Wnt signaling in HFSCs. In this study, we investigate the functional role of ROR2, a Wnt receptor, in HFSCs. By analyzing Ror2-depleted HFSCs, we uncover that ROR2 is not only essential to regulate Wnt-activated signaling that is responsible for HFSC activation and self-renewal, but it is also required to maintain proper ATM/ATR-dependent DNA damage response, which is indispensable for the long-term maintenance of HFSCs. In analyzing HFSCs lacking ß-catenin, we identify a compensatory role of ROR2-PKC signaling in protecting ß-catenin-null HFSCs from the loss of stem cell pool. Collectively, our study unveils a previously unrecognized role of ROR2 in regulation of stem cell self-renewal and maintenance.

Wnt Signaling Pathways in Keratinocyte Carcinomas.

The skin functions as a barrier between the organism and the surrounding environment. Direct exposure to external stimuli and the accumulation of genetic mutations may lead to abnormal cell growth, irreversible tissue damage and potentially favor skin malignancy. Skin homeostasis is coordinated by an intricate signaling network, and its dysregulation has been implicated in the development of skin cancers. Wnt signaling is one such regulatory pathway orchestrating skin development, homeostasis, and stem cell activation. Aberrant regulation of Wnt signaling cascades not only gives rise to tumor initiation, progression and invasion, but also maintains cancer stem cells which contribute to tumor recurrence. In this review, we summarize recent studies highlighting functional evidence of Wnt-related oncology in keratinocyte carcinomas, as well as discussing preclinical and clinical approaches that target oncogenic Wnt signaling to treat cancers. Our review provides valuable insight into the significance of Wnt signaling for future interventions against keratinocyte carcinomas.

Multi-centre Randomised Controlled Trial of a Smartphone-based Event Recorder Alongside Standard Care Versus Standard Care for Patients Presenting to the Emergency Department with Palpitations and Pre-syncope: The IPED (Investigation of Palpitations in the ED) study.

BACKGROUND: Patients with palpitations and pre-syncope commonly present to Emergency Departments (EDs) but underlying rhythm diagnosis is often not possible during the initial presentation. This trial compares the symptomatic rhythm detection rate of a smartphone-based event recorder (AliveCor) alongside standard care versus standard care alone, for participants presenting to the ED with palpitations and pre-syncope with no obvious cause evident at initial consultation. METHODS: Multi-centre open label, randomised controlled trial. Participants ≥ 16 years old presenting to 10 UK hospital EDs were included. Participants were randomised to either (a) intervention group; standard care plus the use of a smartphone-based event recorder or (b) control group; standard care alone. Primary endpoint was symptomatic rhythm detection rate at 90 days. Trial registration number NCT02783898 (ClinicalTrials.gov). FINDINGS: Two hundred forty-three participants were recruited over an 18-month period. A symptomatic rhythm was detected at 90 days in 69 (n = 124; 55.6%; 95% CI 46.9-64.4%) participants in the intervention group versus 11 (n = 116; 9.5%; 95% CI 4.2-14.8) in the control group (RR 5.9, 95% CI 3.3-10.5; p < 0.0001). Mean time to symptomatic rhythm detection in the intervention group was 9.5 days (SD 16.1, range 0-83) versus 42.9 days (SD 16.0, range 12-66; p < 0.0001) in the control group. The commonest symptomatic rhythms detected were sinus rhythm, sinus tachycardia and ectopic beats. A symptomatic cardiac arrhythmia was detected at 90 days in 11 (n = 124; 8.9%; 95% CI 3.9-13.9%) participants in the intervention group versus 1 (n = 116; 0.9%; 95% CI 0.0-2.5%) in the control group (RR 10.3, 95% CI 1.3-78.5; p = 0.006). INTERPRETATION: Use of a smartphone-based event recorder increased the number of patients in whom an ECG was captured during symptoms over five-fold to more than 55% at 90 days. This safe, non-invasive and easy to use device should be considered part of on-going care to all patients presenting acutely with unexplained palpitations or pre-syncope. FUNDING: This study was funded by research awards from Chest, Heart and Stroke Scotland (CHSS) and British Heart Foundation (BHF) which included funding for purchasing the devices. MR was supported by an NHS Research Scotland Career Researcher Clinician award.

Multi-centre randomised controlled trial of a smart phone-based event recorder alongside standard care versus standard care for patients presenting to the Emergency Department with palpitations and pre-syncope - the IPED (Investigation of Palpitations in the ED) study: study protocol for a randomised controlled trial.

BACKGROUND: Palpitations and pre-syncope are together responsible for 300,000 annual Emergency Department (ED) attendances in the United Kingdom (UK). Diagnosis of the underlying rhythm is difficult as many patients are fully recovered on ED arrival; and examination and presenting electrocardiogram (ECG) are commonly normal. The only way to establish the underlying heart rhythm is to capture an ECG during symptoms. Recent technology advances have led to several novel ECG monitoring devices appearing on the market. This trial aims to compare the symptomatic rhythm detection rate at 90 days of one such smart phone-based event recorder (AliveCor Heart Monitor and AliveECG) with standard care for participants presenting to the ED with palpitations and pre-syncope and no obvious cause in the ED. METHODS/DESIGN: This is a multi-centre hospital ED / Acute Medical Unit (AMU) open label, randomised controlled trial. Participants will be recruited in 10 tertiary and district general hospitals in the UK. Participants aged ≥ 16 years presenting with an episode of palpitations or pre-syncope with no obvious cause and whose underlying ECG rhythm during these episodes remains undiagnosed after clinical assessment will be included. Participants will be randomised to either: (1) the intervention arm, standard care plus the use of a smart phone-based event recorder; or (2) the control arm, standard care. Primary endpoint will be symptomatic rhythm detection rate at 90 days. A number of secondary clinical, process and cost-effectiveness endpoints will be collected and analysed. Analysis will be on an intention-to-treat basis. DISCUSSION: The Investigation of Palpitations in the ED (IPED) study aims to recruit 242 participants across 10 hospital sites. It will be the first study to investigate the ability of a smart phone-based event recorder to detect symptomatic cardiac rhythms compared to standard care for ED patients with palpitations and pre-syncope with no obvious cause in the ED. This smart phone event recorder will allow ED patients who have presented with palpitations or pre-syncope to record their ECG tracing if they have a further episode and may increase the rate of underlying rhythm diagnosis. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02783898 . Registered on 26 May 2016.

Integrating Moving Platforms in a SLAM Agorithm for Pedestrian Navigation.

In 3D pedestrian indoor navigation applications, position estimation based on inertial measurement units (IMUss) fails when moving platforms (MPs), such as escalators and elevators, are not properly implemented. In this work, we integrate the MPs in an upper 3D-simultaneous localization and mapping (SLAM) algorithm which is cascaded to the pedestrian dead-reckoning (PDR) technique. The step and heading measurements resulting from the PDR are fed to the SLAM that additionally estimates a map of the environment during the walk in order to reduce the remaining drift. For integrating MPs, we present a new proposal function for the particle filter implementation of the SLAM to account for the presence of MPs. In addition, a new weighting function for features such as escalators and elevators is developed and the features are learned and stored in the learned map. With this, locations of MPs are favored when revisiting the MPs again. The results show that the mean height error is about 0.1 m and the mean position error is less than 1 m for walks with long distances along the floors, even when using multiple floor level changes with different numbers of floors in a multistory environment. For walks with short walking distances and many floor level changes, the mean height error can be higher (about 0.5 m). The final floor number is in all cases except one correctly estimated.

Diagnostic yield of an ambulatory patch monitor in patients with unexplained syncope after initial evaluation in the emergency department: the PATCH-ED study.

OBJECTIVES: Diagnosing underlying arrhythmia in ED syncope patients remains problematic. This study investigates diagnostic yield, event prevalence, patient satisfaction and compliance, and influence on resource utilisation of an ambulatory patch monitor in unexplained ED syncope patients. METHODS: Prospective pilot study conducted in a single tertiary ED in Scotland between 17 November 2015 and 16 June 2017 with a historical unmatched comparator group. Patients 16 years or over presenting within 6 hours of unexplained syncope were fitted in the ED with an ambulatory patch ECG recorder (Zio XT monitor), which continuously records a single-lead ECG for up to 14 days. Patients with an obvious underlying cause were excluded. An unmatched historical group of 603 syncope patients with no obvious diagnosis in ED, recruited to a prior cohort study (2007-2008), were used as a comparator. Primary endpoint was symptomatic significant arrhythmia at 90-day follow-up. RESULTS: During the prospective study period, 86 patients were recruited. 90-day diagnostic yield for symptomatic significant arrhythmia was 10.5% (95% CI 4.0 to 16.9; 9 of 86) versus 2.0% (95% CI 0.9 to 3.1; 12 of 603) in the comparator group. 24 patients (27.9%) had a significant arrhythmia (five serious); 26 patients (30.2%) had serious outcomes (major adverse cardiac event and/or death). Blinded patch report review suggested the patch would significantly reduce requirement for standard outpatient ambulatory ECG monitoring. 56 of 76 returned patches had a diagnostic finding within±45 s of a triggered/diary event (73.7% diagnostic utility; 95% CI 63.7 to 83.6); 34 of 56 (61%) for sinus rhythm or ectopic beats only. CONCLUSIONS: Routine, early ambulatory ECG monitoring in ED patients with unexplained syncope is probably warranted. A large-scale trial comparing this approach to standard care with cost-effectiveness and safety analysis is now required. TRIAL REGISTRATION: NCT02683174.

Transtemporal Investigation of Brain Parenchyma Elasticity Using 2-D Shear Wave Elastography: Definition of Age-Matched Normal Values.

The goal of our research was to assess the possibility of reliable investigation of brain tissue stiffness using ultrasonographic brain parenchyma elastography with an intact temporal bone. We enrolled 108 patients after exclusion of intracranial pathology or healthy volunteers. All patients were subdivided by age into groups: 20-40, 40-60 and >60 y. For statistical analysis, the χ2 test and t-test were used. The mean values, regardless of age and other parameters, were 3.34 kPa (SD = 0.59) on the left side and 3.33 kPa (SD = 0.58) on the right side. We found no correlation between the values, body mass index (r = 0.07, p = 0.48) and sex (t = -0.11, p = 0.91), but we observed a highly significant correlation between the values and age (r = 0.43, p <0.0001). We found ultrasonographic brain parenchyma elastography to be a valid, reproducible and investigator-independent method that reliably determines brain parenchyma stiffness. Normal values should serve as a reference for studies on various intracranial lesions.

Concise Review: Wnt Signaling Pathways in Skin Development and Epidermal Stem Cells.

Mammalian skin and its appendages constitute the integumentary system forming a barrier between the organism and its environment. During development, skin epidermal cells divide rapidly and stratify into a multilayered epithelium, as well as invaginate downward in the underlying mesenchyme to form hair follicles (HFs). In postnatal skin, the interfollicular epidermal (IFE) cells continuously proliferate and differentiate while HFs undergo cycles of regeneration. Epidermal regeneration is fueled by epidermal stem cells (SCs) located in the basal layer of the IFE and the outer layer of the bulge in the HF. Epidermal development and SC behavior are mainly regulated by various extrinsic cues, among which Wnt-dependent signaling pathways play crucial roles. This review not only summarizes the current knowledge of Wnt signaling pathways in the regulation of skin development and governance of SCs during tissue homeostasis, but also discusses the potential crosstalk of Wnt signaling with other pathways involved in these processes. Stem Cells 2018;36:22-35.

47-year-old female with an apical mass.

CLINICAL INTRODUCTION: A 47-year-old female with no medical history presented with a sudden collapse. Physical examination, chest X-ray and high-sensitivity cardiac troponin I were normal, however ECG demonstrated anterior T-wave inversion. CT pulmonary angiography was performed which ruled out pulmonary embolism but revealed a non-calcified, homogenous mass at the left ventricular (LV) apex. It was not clear whether this mass was intramyocardial or pericardial. Transthoracic echocardiography confirmed the apical mass but was unable to establish its aetiology. Subsequent cardiac MR (CMR) demonstrated a highly vascular intramyocardial mass on perfusion imaging (Figure 1A, online supplementary video A), with striking, homogenous late gadolinium enhancement (Figure 1B) consistent with a diagnosis of cardiac fibroma.1 The patient underwent successful surgical excision of the mass (see online supplementary image A) and made a good symptomatic recovery, quickly mobilising around the ward. On examination, the patient was afebrile but had a blood pressure of 90/40 mm Hg and raised venous pressure. Postoperative imaging with echocardiography (see online supplementary video B) and CMR (Figure 1C, D and online supplementary video C) revealed some unexpected findings. Study the provided images. QUESTION: What is the next most appropriate management step? Antibiotic therapy for pericardial abscessAnticoagulation for LV thrombusIntravenous fluids with close clinical and imaging follow-up of the intramyocardial haemorrhage and pericardial haematomaReturn to theatre for excision of residual tumourUrgent pericardiocentesis to drain pericardial collection.

Anuria Secondary to Bilateral Obstructing Ureteral Stones in the Absence of Renal Colic.

BACKGROUND: Obstructing ureteral stones are a rare cause of anuria, which is typically from prerenal or renal etiologies. Classically, obstructive stones cause moderate to severe renal colic. Urolithiasis is rarely considered during evaluation of painless anuria. CASE PRESENTATION: We present an unusual case of a 73-year-old Caucasian female who presented with anuria and was found to have large bilateral obstructing ureteral stones in the absence of renal colic. CONCLUSION: Given that patients with obstructive anuria can be asymptomatic, urolithiasis should be considered in all patients presenting with anuria.

Surgical Management of a Heterotopic Cesarean Scar Pregnancy With Preservation of an Intrauterine Pregnancy.

BACKGROUND: Spontaneously conceived heterotopic pregnancies are rare. A heterotopic cesarean scar pregnancy involves an intrauterine pregnancy and a second pregnancy in the cesarean scar. Treatment approaches include both medical and surgical management. We present a surgical approach to the treatment of a heterotopic cesarean scar pregnancy with preservation of an intrauterine gestation. CASE: A 29-year-old woman, gravida 4 para 1021, presented at 5 weeks of gestation with spotting. Ultrasonography revealed a heterotopic cesarean scar pregnancy. The patient underwent resection of the ectopic pregnancy through minilaparotomy in an attempt to conserve the intrauterine pregnancy. She subsequently delivered an early-term neonate. CONCLUSION: Laparotomy with resection of the ectopic pregnancy is an option for treatment of an early heterotopic cesarean scar pregnancy when the patient desires conservation of the intrauterine pregnancy. Excision was not associated with pregnancy complications.

Development of Covalent Ligand-Receptor Pairs to Study the Binding Properties of Nonpeptidic Neurotensin Receptor 1 Antagonists.

The neurotensin receptor NTS1 has been suggested to be of pharmaceutical relevance, as it was found to exert modulatory effects on dopaminergic signal transduction and to be involved in tumor progression. Rational drug design of NTS1 receptor ligands requires molecular insights into the binding behavior of a particular lead compound. Although crystal structures of NTS1 have revealed the molecular determinants of peptide-agonist interactions, the binding mode of small-molecule antagonists remains largely unknown. Employing a disulfide-based tethering approach, we developed covalently binding molecular probes. The ligands 1 and 2 are based on the pharmacophore of the nonpeptidic NTS1 antagonist SR142948A, allowing the formation of a disulfide bond to an engineered cysteine residue of NTS1. The position of the covalent bond between Cys127(2.65) and the ligand was used to predict the binding mode of the covalent antagonist 1 and its parent compound SR142948A by molecular dynamics simulations.

Factors Affecting Bleeding and Stent Thrombosis in Clinical Trials Comparing Bivalirudin With Heparin During Percutaneous Coronary Intervention.

BACKGROUND: Patients treated with bivalirudin in randomized clinical trials of percutaneous coronary intervention generally have less bleeding but more acute stent thrombosis (ST) than do patients treated with heparin, but differences have varied among trials. METHODS AND RESULTS: We modeled the risk of major hemorrhage and ischemic outcomes 30 days after percutaneous coronary intervention by treatment assignment and the use of adjunctive therapies in 18 randomized clinical trials enrolling 41,871 patients. Overall bivalirudin caused less major bleeding (odds ratio [OR], 0.64; 95% confidence interval [CI], 0.53-0.76), more ST (OR, 1.58; 95% CI, 1.19-2.09), and no difference in mortality (OR, 0.93; 95% CI, 0.77-1.14) than heparin. A risk-benefit analysis identified 19 fewer bleeds and 5 more STs for every 1000 patients treated with bivalirudin in place of heparin. No significant bleeding advantage of bivalirudin over heparin could be identified in randomized clinical trials when transradial access (OR, 0.89; 95% CI, 0.57-1.41) and planned glycoprotein IIb/IIIa inhibitors were used with bivalirudin in the majority of patients (OR, 1.07; 95% CI, 0.87-1.31). The use of prasugrel or ticagrelor eliminated bleeding differences (OR, 0.80; 95% CI, 0.63-1.03) but did not reduce the risk of ST after bivalirudin (OR, 2.20; 95% CI, 1.48-3.27). CONCLUSIONS: Substituting bivalirudin for heparin conferred a tradeoff between bleeding and ST. Transradial access, adjunctive glycoprotein IIb/IIIa inhibitors, and potent P2Y12 inhibitors attenuated the bleeding advantage of bivalirudin over heparin but had no apparent effect on early ST. New approaches to reduce bleeding and ischemic complications during percutaneous coronary intervention warrant further investigation.

Incidence and Predictors of Left Ventricular Thrombus After Primary Percutaneous Coronary Intervention for Anterior ST-Segment Elevation Myocardial Infarction.

BACKGROUND: The role of warfarin in anterior ST-segment elevation myocardial infarction (STEMI) complicated by left ventricular (LV) dysfunction in patients treated with primary percutaneous coronary intervention (PCI) and dual antiplatelet therapy is unclear. Warfarin may prevent cardioembolic events but significantly increases bleeding in the setting of dual antiplatelet therapy. HYPOTHESIS: The incidence of LV thrombus in anterior STEMI patients treated with PCI is low, and clinical predictors might be valuable in determining patients at risk. METHODS: We performed a retrospective, single-center study of 687 consecutive patients with anterior STEMI treated with PCI from 2006 to 2013. Baseline variables were evaluated in 310 patients at high risk for LV thrombus based on echocardiographic criteria. Patients with definite, probable, and no LV thrombus were compared by ANOVA, χ(2), or t test where appropriate. Logistic regression analysis was performed. RESULTS: The incidence of LV thrombus was 15% (n = 47 probable/definite thrombus). Cardiac arrest was the only independent characteristic associated with increased risk of LV thrombus (odds ratio [OR]: 4.06, 95% confidence interval [CI]: 1.3-12.7). Trends were observed for a lower risk in cardiogenic shock (OR: 0.33, 95% CI: 0.10-1.05) and aspirin use at baseline (OR: 0.43, 95% CI: 0.17-1.1). Treatment variables associated with LV thrombus included unfractionated heparin use post-PCI (OR: 2.43, 95% CI: 1.16-5.1) and use of balloon angioplasty without stent. CONCLUSIONS: In contemporary practice with primary PCI, definite LV thrombus following anterior STEMI with LV dysfunction is challenging to predict. Further investigation is needed to determine if there is a subset of patients that should be treated with prophylactic warfarin.

Infrared transmissometer to measure the thickness of NbN thin films.

We present an optical setup that can be used to characterize the thicknesses of thin NbN films to screen samples for fabrication and to better model the performance of the resulting superconducting nanowire single photon detectors. The infrared transmissometer reported here is easy to use, gives results within minutes, and is nondestructive. Thus, the thickness measurement can be easily integrated into the workflow of deposition and characterization. Comparison to a similar visible-wavelength transmissometer is provided.