Abstract Background At a global scale, the SARS-CoV-2 virus did not remain in its initial genotype for a long period of time, with the first global reports of variants of concern (VOCs) in late 2020. ...Subsequently, genome sequencing has become an indispensable tool for characterizing the ongoing pandemic, particularly for typing SARS-CoV-2 samples obtained from patients or environmental surveillance. For such SARS-CoV-2 typing, various in vitro and in silico workflows exist, yet to date, no systematic cross-platform validation has been reported. Results In this work, we present the first comprehensive cross-platform evaluation and validation of in silico SARS-CoV-2 typing workflows. The evaluation relies on a dataset of 54 patient-derived samples sequenced with several different in vitro approaches on all relevant state-of-the-art sequencing platforms. Moreover, we present UnCoVar, a robust, production-grade reproducible SARS-CoV-2 typing workflow that outperforms all other tested approaches in terms of precision and recall. Conclusions In many ways, the SARS-CoV-2 pandemic has accelerated the development of techniques and analytical approaches. We believe that this can serve as a blueprint for dealing with future pandemics. Accordingly, UnCoVar is easily generalizable towards other viral pathogens and future pandemics. The fully automated workflow assembles virus genomes from patient samples, identifies existing lineages, and provides high-resolution insights into individual mutations. UnCoVar includes extensive quality control and automatically generates interactive visual reports. UnCoVar is implemented as a Snakemake workflow. The open-source code is available under a BSD 2-clause license at github.com/IKIM-Essen/uncovar .
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Zusammenfassung
Die humanmedizinische Anwendung von Barbituraten als Schlafmittel gilt heutzutage als obsolet. Während Pentobarbital in Deutschland lediglich in der Veterinärmedizin angewendet wird, ...nutzt man es aufgrund der bei Überdosierungen schnell eintretenden Atemdepression z. B. in der Schweiz bei Freitodbegleitungen oder in den USA bei der Vollstreckung von Todesstrafen. In Deutschland ist der Zugriff auf Pentobarbital für unheilbar Kranke mit Wunsch zur selbstbestimmten Einnahme mit großen Hürden verbunden. Der vorgestellte Fall zeigt, dass der „letzte Weg“ bei stark ausgeprägter Todessehnsucht dennoch unter Zuhilfenahme von Pentobarbital – auch in Deutschland – gegangen werden kann.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Styrene–maleic acid (SMA) copolymers are a promising alternative to detergents for the solubilization of membrane proteins. Here we employ Escherichia coli membranes containing KcsA as a model ...protein to investigate the influence of different environmental conditions on SMA solubilization efficiency. We show that SMA concentration, temperature, incubation time, ionic strength, presence of divalent cations and pH all influence the amount of protein that is extracted by SMA. The observed effects are consistent with observations from lipid-only model membrane systems, with the exception of the effect of pH. Increasing pH from 7 to 9 was found to result in an increase of the solubilization yield of E. coli membranes, whereas in lipid-only model systems it decreased over the same pH range, based on optical density (OD) measurements. Similar opposite pH-dependent effects were observed in OD experiments comparing solubilization of native yeast membranes and yeast lipid-only membranes. We propose a model in which pH-dependent electrostatic interactions affect binding of the polymers to extramembraneous parts of membrane proteins, which in turn affects the availability of polymer for membrane solubilization. This model is supported by the observations that a similar pH-dependence as for SMA is observed for the anionic detergent SDS, but not for the nonionic detergent DDM and that the pH-dependence can be largely overcome by increasing the SMA concentration. The results are useful as guidelines to derive optimal conditions for solubilization of biological membranes by SMA.
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•The amount of protein that is extracted from E.coli membranes by SMA strongly depends on a number of environmental factors.•Effects of these factors are similar for E.coli membranes and lipid-only model membranes, with the exception of pH.•Solubilization of E.coli membranes is more efficient at higher pH.•pH-dependent effects can be largely overcome by increasing polymer concentration.•Turbidimetry is useful as a tool to monitor membrane solubilization in biological membranes.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract
Aims
Early detection of atrial fibrillation (AF) is essential for stroke prevention. Emerging technologies such as smartphone cameras using photoplethysmography (PPG) and mobile, ...internet-enabled electrocardiography (iECG) are effective for AF screening. This study compared a PPG-based algorithm against a cardiologist’s iECG diagnosis to distinguish between AF and sinus rhythm (SR).
Methods and results
In this prospective, two-centre, international, clinical validation study, we recruited in-house patients with presumed AF and matched controls in SR at two university hospitals in Switzerland and Germany. In each patient, a PPG recording on the index fingertip using a regular smartphone camera followed by iECG was obtained. Photoplethysmography recordings were analysed using an automated algorithm and compared with the blinded cardiologist’s iECG diagnosis. Of 672 patients recruited, 80 were excluded mainly due to insufficient PPG/iECG quality, leaving 592 patients (SR: n = 344, AF: n = 248). Based on 5 min of PPG heart rhythm analysis, the algorithm detected AF with a sensitivity of 91.5% (95% confidence interval 85.9–95.4) and specificity of 99.6% (97.8–100). By reducing analysis time to 1 min, sensitivity was reduced to 89.9% (85.5–93.4) and specificity to 99.1% (97.5–99.8). Correctly classified rate was 88.8% for 1-min PPG analysis and dropped to 60.9% when the threshold for the analysed file was set to 5 min of good signal quality.
Conclusion
This is the first prospective clinical two-centre study to demonstrate that detection of AF by using a smartphone camera alone is feasible, with high specificity and sensitivity. Photoplethysmography signal analysis appears to be suitable for extended AF screening.
Clinical trial registration
ClinicalTrials.gov, number NCT02949180, https://clinicaltrials.gov/ct2/show/NCT02949180.
The definition of the metabolic syndrome consists of five components. The underlying measurements are subject to intra-individual variability. This repeated measurements study investigated the impact ...of intra-individual measurement variability on the stability of the diagnosis of metabolic syndrome over 12 months.
Twenty-five employees of the University Medicine Greifswald aged 22–70 years were examined once a month over one year. Examinations included blood sampling and anthropometric and blood pressure measurements. Laboratory measurements included glucose, cholesterol (high-density lipoprotein HDL, and low-density lipoprotein LDL), and triglycerides. The metabolic syndrome was defined according to the International Diabetes Federation modified for non-fasting blood samples. Variations in continuous metabolic markers were assessed using coefficients of variation (CV) and intra-class correlation coefficients (ICC).
Overall eight participants (32%) were categorized at least once within 12 months as having a metabolic syndrome; in none of those metabolic syndrome was found consistently over the study follow-ups. The Cohen's Kappa for metabolic syndrome was 0.57. CV was highest for triglycerides (27.5%) followed by glucose (10.1%), LDL- (9.5%), and HDL-cholesterol (8.6%). ICC's were lowest for glucose (0.51), triglycerides (0.65), systolic (0.68), and diastolic blood pressure (0.69).
We showed that the measurement of biomarkers defining the metabolic syndrome is a time-varying condition with implications for the concept of the metabolic syndrome. To account for this uncertainty in prevalence studies we propose to identify uncertain cases according to the current definition of the metabolic syndrome. For analysing associations we recommend to apply probabilistic sensitivity analyses.
•Measurement of the biomarkers for defining the metabolic syndrome is a time-varying condition with implications for the concept of the metabolic syndrome.•Serum glucose and triglycerides are the most varying components of the metabolic syndrome.•To account for variations in components of the metabolic syndrome we propose to identify uncertain cases according to the current definition of the metabolic syndrome.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
To date, colchicine and prednisolone are two effective therapies for the treatment of acute gout but have never been compared directly in a randomized clinical trial. In addition, in previous trials ...of treating acute gout patients with concomitant comorbidities were often excluded due to contraindications to naproxen.
This pragmatic, prospective, double-blind, double-dummy, parallel-group, randomized, non-inferiority trial compares prednisolone with colchicine in terms of non-inferiority in patients with acute gout. Patients presenting to their general practitioner with acute gout can be included if the gout attack has occurred within the last 2 days. A total of 60 practices in the vicinity of three university medical centers (Greifswald, Göttingen, and Würzburg) participate in the study. The intervention group receives 30 mg prednisolone for 5 days, while the group of standard care receives low-dose colchicine (day 1: 1.5 mg; days 2-5: 1 mg). The first dose of treatment is provided at day 0 when patients present to the general practitioner due to an acute gout attack. From day 0 to day 6, patients will be asked to complete a study diary on daily basis regarding pain quantification. For safety reasons, potential side effects and the course of systolic blood pressure are also assessed.
N = 314 patients have to be recruited to compensate for 10% of dropout and to allow for showing non-inferiority of prednisolone compared to colchicine with a power of 90%. We use permuted block randomization with block sizes of 2, 4, and 6 to avoid imbalanced treatment arms in this multi-center study; patients are randomized in a 1:1 ratio. The absolute level of pain on day 3 (in the last 24 h) is the primary outcome and measured on a numerical rating scale (NRS: 0-10). Using a multiple linear regression model adjusted for age, sex, and pain at baseline, prednisolone is considered non-inferior if the effect estimate including the confidence intervals is lower than a margin of 1 unit on the NRS. Average response to treatment, joint swelling and tenderness, physical function of the joint, and patients' global assessment of treatment success are secondary outcomes.
The trial will provide evidence from a direct comparison of colchicine and prednisolone regarding their efficacy of pain reduction in acute gout patients of primary care and to indicate possible safety signals.
ClinicalTrials.gov Identifier: NCT05698680 first posted on January 26, 2023 (retrospectively registered).
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Abstract
Background
Gout is the most common form of rheumatic disease in which monosodium urate crystals are deposited in the joints followed by acute inflammatory reactions. There are various ...approved drugs that can be prescribed for pain relief during an acute gout attack. However, to date, no direct comparison of efficacy of colchicine and prednisolone for the treatment of acute gout attacks has been investigated. Furthermore, the majority of previous research studies were not only conducted in tertiary centres but also excluded patients with common comorbidities due to contraindications to naproxen.
Methods
This pragmatic, prospective, double-blind, double-dummy, parallel-group, randomized, non-inferiority trial investigates whether prednisolone (intervention) is non-inferior to treatment with colchicine (active control) in patients with acute gout. Adult patients presenting with acute gout to their general practitioners in 60 practices across 3 university sites (Greifswald, Göttingen, and Würzburg) are eligible to participate in the study. Participants in the intervention group receive 30 mg prednisolone for 5 days. Those in the control group receive low-dose colchicine (day 1: 1.5 mg; days 2–5: 1 mg). The primary outcome is the absolute level of the most severe pain on day 3 (in the last 24 h) measured with an 11-item numerical rating scale. Day 0 is the day patients take their study medication for the first time. They are then asked to fill out a study diary the same time each day for pain quantification. Pain scores are used for comparison between the two medications. Secondary outcomes are average response to treatment, swelling, tenderness and physical function of the joint, patients’ global assessment of treatment success, use of additional pain medication and non-pharmacological pain therapies. For safety reasons, potential side effects and course of systolic blood pressure are assessed.
Discussion
This trial will provide evidence on the effectiveness of pain reduction and side effects of colchicine and prednisolone in acute gout in primary care.
Trial registration
ClinicalTrials.gov Identifier: NCT05698680 first posted on January 26, 2023 (retrospectively registered). URL of trial registry record:
https://clinicaltrials.gov/study/NCT05698680
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Abstract
Background
Chimeric antigen receptor (CAR) T cell therapy has been successfully translated to clinical practice for the treatment of B cell malignancies. The suppressive microenvironment of ...many malignancies is a bottleneck preventing treatment success of CAR T cells in a broader range of tumours. Among others, the immunosuppressive metabolite adenosine is present in high concentrations within many tumours and dampens anti-tumour function of immune cells and consequently therapeutic response.
Methods
Here, we present the impact of the selective adenosine A2
A
and A2
B
receptor antagonist AB928/etrumadenant on CAR T cell cytokine secretion, proliferation, and cytotoxicity. Using phosphorylation-specific flow cytometry, we evaluated the capability of AB928 to shield CAR T cells from adenosine-mediated signalling. The effect of orally administered AB928 on CAR T cells was assessed in a syngeneic mouse model of colon carcinoma.
Results
We found that immunosuppressive signalling in CAR T cells in response to adenosine was fully blocked by the small molecule inhibitor. AB928 treatment enhanced CAR T cell cytokine secretion and proliferation, granted efficient cytolysis of tumour cells in vitro and augmented CAR T cell activation in vivo.
Conclusions
Together our results suggest that combination therapy with AB928 represents a promising approach to improve adoptive cell therapy.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Chimeric antigen receptor T cells (CAR-T cells) have emerged as a powerful treatment option for individuals with B cell malignancies but have yet to achieve success in treating acute myeloid leukemia ...(AML) due to a lack of safe targets. Here we leveraged an atlas of publicly available RNA-sequencing data of over 500,000 single cells from 15 individuals with AML and tissue from 9 healthy individuals for prediction of target antigens that are expressed on malignant cells but lacking on healthy cells, including T cells. Aided by this high-resolution, single-cell expression approach, we computationally identify colony-stimulating factor 1 receptor and cluster of differentiation 86 as targets for CAR-T cell therapy in AML. Functional validation of these established CAR-T cells shows robust in vitro and in vivo efficacy in cell line- and human-derived AML models with minimal off-target toxicity toward relevant healthy human tissues. This provides a strong rationale for further clinical development.
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GEOZS, IJS, IMTLJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK, ZAGLJ
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