Inferring single-cell protein levels and cell cycle behavior in heterogeneous cell populations

Individual cells in a genetically identical population can show highly variable behavior. Single-cell measurements allow us to study this variability, but the available measurement techniques have limitations: live-cell microscopy is typically restricted to one or a few molecular markers, while techniques that simultaneously measure large numbers of molecular markers are destructive and cannot be used to follow cells over time. To help overcome these limitations, we present here scMeMo (single cell Mechanistic Modeler): a mechanistic modeling framework that can leverage diverse sets of measurements in order to infer unobserved variables in heterogeneous single cells. We used this framework to construct a model describing cell cycle progression in human cells, and show that it can predict the levels of several proteins in individual cells, based on live-cell microscopy measurements of only one marker and information learned from other experiments. The framework incorporates an uncertainty calibration step that makes the posterior distributions robust against partial model misspecification. Our modeling framework can be used to integrate information from separate experiments with diverse readouts, and to infer single cell variables that may be difficult to measure directly

Endovascular navigation with Fiber Optic RealShape technology

Objective: Fiber Optic RealShape (FORS) technology has recently been introduced as an adjunctive guidance technology that allows real-time three-dimensional visualization of dedicated endovascular devices while avoiding radiation exposure. It consists of equipment which sends pulses of light through hair-thin optical fibers that run within a dedicated hydrophilic wire and selective catheters. The purpose of the study was to report the observed benefits and limitations related to the first edition of FORS technology. Methods: Data were collected prospectively from the first 50 patients undergoing FORS-guided endovascular repair at a single center between February 2020 and February 2021 as part of the global multicenter FORS Learn registry. All consecutive, elective procedures with one or more navigation tasks attempted with FORS were included. Factors related to FORS navigation task success were assessed. The time required for the catheterization of each task as well as the amount of radiation exposure (fluoroscopy time, dose area product, and estimated skin dose) were collected. A per-task analysis was conducted. End points included the success rate in achieving a stable FORS-guided catheterization, catheterization time, and radiation dose during catheterization. Results: During the study period from February 2020 to February 2021, 50 patients were treated using FORS technology. Forty-five patients were treated for aortic aneurysm, 4 for iliac artery aneurysm, and 1 for splenic artery aneurysm. Overall, 201 navigation tasks were completed for these procedures and FORS was used in 186 tasks (92.5%). No FORS-related complication was recorded and a success rate of 60.2% (n = 116) was observed. Target vessel (TV) angle of 45° or greater, TV stenosis, and the renal arteries as navigation tasks (compared with celiac artery or superior mesenteric artery) were associated with a lower success rate. Catheterization of a TV through a branch more frequently required a standard catheter in combination with the FORS-enabled guidewire. Successful task catheterization using FORS guidance was associated with a shorter catheterization time 6 minutes (interquartile range, 3-11 minutes) versus 16 minutes (interquartile range, 10-24 minutes) (P < .001) and lower radiation exposure compared with unsuccessful catheterization (dose area product, 4.4 cGy/cm2 vs 12.5 cGy/cm2; P < .001). Conclusions: FORS technology was implemented successfully as a new guidance technology in a complex endovascular aortic repair program and was associated with an encouraging success rate and a high potential for radiation reduction

Direct-Acting Antiviral Treatment for Hepatitis C Genotypes Uncommon in High-Income Countries:A Dutch Nationwide Cohort Study

Background. The majority of hepatitis C virus (HCV) infections are found in low- and middle-income countries, which harbor many region-specific HCV subtypes. Nevertheless, direct-acting antiviral (DAA) trials have almost exclusively been conducted in high-income countries, where mainly epidemically spread HCV subtypes are present. Recently, several studies have demonstrated suboptimal DAA efficacy for certain nonepidemic subtypes, which could hamper global HCV elimination. Therefore, we aimed to evaluate DAA efficacy in patients treated for a nonepidemic HCV genotype infection in the Netherlands. Methods. We performed a nationwide retrospective study including patients treated with interferon-free DAAs for an HCV genotype other than 1a/1b/2a/2b/3a/4a/4d. The genotype was determined by NS5B region phylogenetic analysis. The primary end point was SVR-12. If stored samples were available, NS5A and NS5B sequences were obtained for resistance-associated substitutions (RAS) evaluation. Results. We included 160 patients, mainly infected with nonepidemic genotype 2 (41%) and 4 (31%) subtypes. Most patients were from Africa (45%) or South America (24%); 51 (32%) were cirrhotic. SVR-12 was achieved in 92% (140/152) of patients with available SVR-12 data. Only 73% (8/11) genotype 3-infected patients achieved SVR-12, the majority being genotype 3b patients with 63% (5/8) SVR. Regardless of SVR, all genotype 3b patients had 30K and 31M RAS. Conclusions. (T)he DAA efficacy we observed in most nonepidemic genotypes in the Netherlands seems reassuring. However, the low SVR-12 rate in subtype 3b infections is alarming, especially as it is common in several HCV-endemic countries. Alongside earlier results, our results indicate that a remaining challenge for global HCV elimination is confirming and monitoring DAA efficacy in nonepidemic genotypes

Expert consensus recommendations on the cardiogenetic care for patients with thoracic aortic disease and their first-degree

Background: Thoracic aortic aneurysm (TAA) is a potentially life-threatening disorder with a strong genetic component. The number of genes implicated in TAA has increased exponentially over the last decade. Approximately 20% of patients with TAA have a positive family history. As most TAA remain asymptomatic for a long time, screening of at risk relatives is warranted to prevent complications. Existing international guidelines lack detailed instructions regarding genetic evaluation and family screening of TAA patients. We aimed to develop a consensus document to provide medical guidance for all health care professionals involved in the recognition, diagnosis and treatment of patients with thoracic aortic disease and their relatives. Methods: A multidisciplinary panel of experts including cardiologists, cardiothoracic surgeons, clinical geneticists and general practitioners, convened to review and discuss the current literature, guidelines and clinical practice on genetic testing and family screening in TAA. Results: There is a lack of high-quality evidence in the literature. This consensus statement, based on the available literature and expert opinions, summarizes our recommendations in order to standardize and optimize the cardiogenetic care for patients and families with thoracic aortic disease. In particular, we provide criteria to identify those patients most likely to have a genetic predisposition, and discuss the preferred modality and frequency of screening in their relatives. Conclusions: Age, family history, aortic size and syndromic features determine who is advised to have genetic testing as well as screening of first-degree relatives. There is a need for more prospective multicenter studies to optimize current recommendations

Expanding the genetic and phenotypic spectrum of ACTA2-related vasculopathies in a Dutch cohort

Purpose: Heterozygous pathogenic/likely pathogenic (P/LP) variants in the ACTA2 gene confer a high risk for thoracic aortic aneurysms and aortic dissections. This retrospective multicenter study elucidates the clinical outcome of ACTA2-related vasculopathies. Methods: Index patients and relatives with a P/LP variant in ACTA2 were included. Data were collected through retrospective review of medical records using a standardized questionnaire. Results: A total of 49 individuals from 28 families participated in our study. In total, 20 different ACTA2 variants were detected. Aortic events occurred in 65% of the cases (78.6% index patients and 47.6% relatives). Male sex and hypertension emerged as significantly associated with aortic events. Of 20 individuals, 5 had an aortic diameter of <45 mm (1.77 inches) at the time of the type A dissection. Mean age at first aortic event was 49.0 ± 12.4 years. Severe surgical complications for type A and type B dissection occurred in 25% and 16.7% of the cases and in-hospital mortality rates were 9.5% and 0%, respectively. Conclusion: P/LP ACTA2 variants are associated with an increased risk for an aortic event and age-related penetrance, which emphasizes the importance of early recognition of the disease. Caregivers should be aware of the risk for aortic dissections, even in individuals with aortic diameters within the normal range

Candidate gene resequencing in a large bicuspid aortic valve-associated thoracic aortic aneurysm cohort: SMAD6 as an important contributor

Bicuspid aortic valve (BAV) is the most common congenital heart defect. Although many BAV patients remain asymptomatic, at least 20% develop thoracic aortic aneurysm (TAA). Historically, BAV-related TAA was considered as a hemodynamic consequence of the valve defect. Multiple lines of evidence currently suggest that genetic determinants contribute to the pathogenesis of both BAV and TAA in affected individuals. Despite high heritability, only very few genes have been linked to BAV or BAV/TAA, such as NOTCH1, SMAD6, and MAT2A. Moreover, they only explain a minority of patients. Other candidate genes have been suggested based on the presence of BAV in knockout mouse models (e.g., GATA5, NOS3) or in syndromic (e.g., TGFBR1/2, TGFB2/3) or non-syndromic (e.g., ACTA2) TAA forms. We hypothesized that rare genetic variants in these genes may be enriched in patients presenting with both BAV and TAA. We performed targeted resequencing of 22 candidate genes using Haloplex target enrichment in a strictly defined BAV/TAA cohort (n = 441; BAV in addition to an aortic root or ascendens diameter = 4.0 cm in adults, or a Z-score = 3 in children) and in a collection of healthy controls with normal echocardiographic evaluation (n = 183). After additional burden analysis against the Exome Aggregation Consortium database, the strongest candidate susceptibility gene was SMAD6 (p = 0.002), with 2.5% (n = 11) of BAV/TAA patients harboring causal variants, including two nonsense, one in-frame deletion and two frameshift mutations. All six missense mutations were located in the functionally important MH1 and MH2 domains. In conclusion, we report a significant contribution of SMAD6 mutations to the etiology of the BAV/TAA phenotype

High quality of SARS-CoV-2 molecular diagnostics in a diverse laboratory landscape through supported benchmark testing and External Quality Assessment

A two-step strategy combining assisted benchmark testing (entry controls) and External Quality Assessments (EQAs) with blinded simulated clinical specimens to enhance and maintain the quality of nucleic acid amplification testing was developed. This strategy was successfully applied to 71 diagnostic laboratories in The Netherlands when upscaling the national diagnostic capacity during the SARS-CoV-2 pandemic. The availability of benchmark testing in combination with advice for improvement substantially enhanced the quality of the laboratory testing procedures for SARS-CoV-2 detection. The three subsequent EQA rounds demonstrated high quality testing with regard to specificity (99.6% correctly identified) and sensitivity (93.3% correctly identified). Even with the implementation of novel assays, changing workflows using diverse equipment and a high degree of assay heterogeneity, the overall high quality was maintained using this two-step strategy. We show that in contrast to the limited value of Cq value for absolute proxies of viral load, these Cq values can, in combination with metadata on strategies and techniques, provide valuable information for laboratories to improve their procedures. In conclusion, our two-step strategy (preparation phase followed by a series of EQAs) is a rapid and flexible system capable of scaling, improving, and maintaining high quality diagnostics even in a rapidly evolving (e.g. pandemic) situation.</p

High quality of SARS-CoV-2 molecular diagnostics in a diverse laboratory landscape through supported benchmark testing and External Quality Assessment

A two-step strategy combining assisted benchmark testing (entry controls) and External Quality Assessments (EQAs) with blinded simulated clinical specimens to enhance and maintain the quality of nucleic acid amplification testing was developed. This strategy was successfully applied to 71 diagnostic laboratories in The Netherlands when upscaling the national diagnostic capacity during the SARS-CoV-2 pandemic. The availability of benchmark testing in combination with advice for improvement substantially enhanced the quality of the laboratory testing procedures for SARS-CoV-2 detection. The three subsequent EQA rounds demonstrated high quality testing with regard to specificity (99.6% correctly identified) and sensitivity (93.3% correctly identified). Even with the implementation of novel assays, changing workflows using diverse equipment and a high degree of assay heterogeneity, the overall high quality was maintained using this two-step strategy. We show that in contrast to the limited value of Cq value for absolute proxies of viral load, these Cq values can, in combination with metadata on strategies and techniques, provide valuable information for laboratories to improve their procedures. In conclusion, our two-step strategy (preparation phase followed by a series of EQAs) is a rapid and flexible system capable of scaling, improving, and maintaining high quality diagnostics even in a rapidly evolving (e.g. pandemic) situation.</p

AIDS-related mycoses: the way forward.

The contribution of fungal infections to the morbidity and mortality of HIV-infected individuals is largely unrecognized. A recent meeting highlighted several priorities that need to be urgently addressed, including improved epidemiological surveillance, increased availability of existing diagnostics and drugs, more training in the field of medical mycology, and better funding for research and provision of treatment, particularly in developing countries

Traumatic brain injury: integrated approaches to improve prevention, clinical care, and research

No abstract available