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De Vargas Roditi, Laura ; Jacobs, Andrea ; Rueschoff, Jan Hendrik ; Bankhead, Pete ; Chevrier, Stéphane ; Jackson, Hartland W. ; Hermanns, Thomas ; Fankhauser, Christian D. ; Poyet, Cedric ; Chun, Felix ; Rupp, Niels Jan Felix ; Tschäbunin, Alexandra ; Bodenmiller, Bernd ; Wild, Peter Johannes
Localized prostate cancer exhibits multiple genomic alterations and heterogeneity at the proteomic level. Single-cell technologies capture important cell-to-cell variability responsible for heterogeneity in biomarker expression that may be overlooked when molecular alterations are based on bulk tissue samples. This study aims to identify prognostic biomarkers and describe the heterogeneity of prostate cancer and the associated microenvironment by simultaneously quantifying 36 proteins using single-cell mass cytometry analysis of over 1.6 million cells from 58 men with localized prostate cancer. We perform this task, using a high-dimensional clustering pipeline named Franken to describe subpopulations of immune, stromal, and prostate cells, including changes occurring in tumor tissues and high-grade disease that provide insights into the coordinated progression of prostate cancer. Our results further indicate that men with localized disease already harbor rare subpopulations that typically occur in castration-resistant and metastatic disease.
Bruijn, Clara M. A. de ; Safder, Shaista ; Rolle, Udo ; Mosiello, Giovanni ; Marshall, David ; Christiansen, Albert B. ; Benninga, Marc A.
Objective: To develop a reliable and valid scoring tool, the Pediatric Bowel Management Scoring Tool (PBMST), to better guide management of constipation in pediatric patients.
Study design: The project comprised 2 stages, development of the questionnaire and construction of the bowel management score. Two questionnaires were created, one for children aged 8-18 years to self-report and one parent proxy-report for children aged 4-8 years. Questions regarding physical symptoms (n = 6), emotional aspects (n = 2), social activities/school (n = 1), and treatment (n = 1) were included. Patients (or parents of patients) with symptoms of constipation completed the questionnaire. The reproducibility of each question was computed using the Cohen weighted kappa coefficient (κ). A bowel management score was developed using logistic regression analysis, assessing the associations between the questions and impact on self-reported quality of life (QoL). Questions with adequate reproducibility and significantly associated with QoL were incorporated into the score.
Results: The questionnaire was completed by 385 patients. Six questions met the inclusion criteria and were incorporated into the score: stool shape (range, 0-3 points), anorectal pain (0-4 points), abdominal pain (0-3 points), frequency of fecal incontinence (0-3 points), assistance of caregivers (0-3 points), and interference with social activities (0-6 points). Differences in bowel management scores among patients reporting no, little, some, or major impact on QoL were statistically significant (P < .001).
Conclusions: The newly developed and validated PBMST is a reliable tool for evaluating bowel management strategies in children with constipation.
Hahn, Alexander ; Parey, Kristian ; Bublitz, Maike ; Mills, Deryck J. ; Zickermann, Volker ; Vonck, Janet ; Kühlbrandt, Werner ; Meier, Thomas Kurt
Highlights
• Cryo-EM structure of a yeast F1Fo-ATP synthase dimer
• Inhibitor-free X-ray structure of the F1 head and rotor complex
• Mechanism of ATP generation by rotary catalysis
• Structural basis of cristae formation in the inner mitochondrial membrane
Summary
We determined the structure of a complete, dimeric F1Fo-ATP synthase from yeast Yarrowia lipolytica mitochondria by a combination of cryo-EM and X-ray crystallography. The final structure resolves 58 of the 60 dimer subunits. Horizontal helices of subunit a in Fo wrap around the c-ring rotor, and a total of six vertical helices assigned to subunits a, b, f, i, and 8 span the membrane. Subunit 8 (A6L in human) is an evolutionary derivative of the bacterial b subunit. On the lumenal membrane surface, subunit f establishes direct contact between the two monomers. Comparison with a cryo-EM map of the F1Fo monomer identifies subunits e and g at the lateral dimer interface. They do not form dimer contacts but enable dimer formation by inducing.
Schmidt, Florian ; Marx, Alexander ; Hebel, Marie ; Wegner, Martin ; Baumgarten, Nina ; Kaulich, Manuel ; Göke, Jonathan ; Vreeken, Jilles ; Schulz, Marcel Holger
Understanding the complexity of transcriptional regulation is a major goal of computational biology. Because experimental linkage of regulatory sites to genes is challenging, computational methods considering epigenomics data have been proposed to create tissue-specific regulatory maps. However, we showed that these approaches are not well suited to account for the variations of the regulatory landscape between cell-types. To overcome these drawbacks, we developed a new method called STITCHIT, that identifies and links putative regulatory sites to genes. Within STITCHIT, we consider the chromatin accessibility signal of all samples jointly to identify regions exhibiting a signal variation related to the expression of a distinct gene. STITCHIT outperforms previous approaches in various validation experiments and was used with a genome-wide CRISPR-Cas9 screen to prioritize novel doxorubicin-resistance genes and their associated non-coding regulatory regions. We believe that our work paves the way for a more refined understanding of transcriptional regulation at the gene-level.
Recktenwald, Steffen Michael ; Simionato, Greta ; Lopez, Marcelle Guedes de Medeiros ; Gamboni, Fabia ; Dzieciatkowska, Monika ; Meybohm, Patrick ; Zacharowski, Kai ; Knethen, Andreas von ; Wagner, Christian ; Kästner, Lars ; D’Alessandro, Angelo ; Quint, Stephan
Coronavirus disease 2019 (COVID-19) is caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and can affect multiple organs, among which is the circulatory system. Inflammation and mortality risk markers were previously detected in COVID-19 plasma and red blood cells (RBCs) metabolic and proteomic profiles. Additionally, biophysical properties, such as deformability, were found to be changed during the infection. Based on such data, we aim to better characterize RBC functions in COVID-19. We evaluate the flow properties of RBCs in severe COVID-19 patients admitted to the intensive care unit by using in vitro microfluidic techniques and automated methods, including artificial neural networks, for an unbiased RBC analysis. We find strong flow and RBC shape impairment in COVID-19 samples and demonstrate that such changes are reversible upon suspension of COVID-19 RBCs in healthy plasma. Vice versa, healthy RBCs immediately resemble COVID-19 RBCs when suspended in COVID-19 plasma. Proteomics and metabolomics analyses allow us to detect the effect of plasma exchanges on both plasma and RBCs and demonstrate a new role of RBCs in maintaining plasma equilibria at the expense of their flow properties. Our findings provide a framework for further investigations of clinical relevance for therapies against COVID-19 and possibly other infectious diseases.
Recktenwald, Steffen Michael ; Simionato, Greta ; Lopez, Marcelle Guedes de Medeiros ; Gamboni, Fabia ; Dzieciatkowska, Monika ; Meybohm, Patrick ; Zacharowski, Kai ; Knethen, Andreas von ; Wagner, Christian ; Kästner, Lars ; D’Alessandro, Angelo ; Quint, Stephan
Coronavirus disease 2019 (COVID-19) is caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and can affect multiple organs, among which is the circulatory system. Inflammation and mortality risk markers were previously detected in COVID-19 plasma and red blood cells (RBCs) metabolic and proteomic profiles. Additionally, biophysical properties, such as deformability, were found to be changed during the infection. Based on such data, we aim to better characterize RBC functions in COVID-19. We evaluate the flow properties of RBCs in severe COVID-19 patients admitted to the intensive care unit by using in vitro microfluidic techniques and automated methods, including artificial neural networks, for an unbiased RBC analysis. We find strong flow and RBC shape impairment in COVID-19 samples and demonstrate that such changes are reversible upon suspension of COVID-19 RBCs in healthy plasma. Vice versa, healthy RBCs immediately resemble COVID-19 RBCs when suspended in COVID-19 plasma. Proteomics and metabolomics analyses allow us to detect the effect of plasma exchanges on both plasma and RBCs and demonstrate a new role of RBCs in maintaining plasma equilibria at the expense of their flow properties. Our findings provide a framework for further investigations of clinical relevance for therapies against COVID-19 and possibly other infectious diseases.
In this special issue of Matrix Biology centered on proteoglycan biology we have assembled a blend of articles focused on the state-of-the-art of proteoglycanology. The field has greatly expanded in the past three decades and now encompasses all the areas of biology. This special issue is divided into five chapters describing hyaluronan metabolism, biosynthetic and catabolic pathways of proteoglycans and their roles in inflammation, cancer, repair and development. We hope that the new original work and the reviews from recognized leaders will stimulate investigations in this exciting and fertile field of research.
Kutyifa, Valentina ; Burch, Ashley E. ; Aßmus, Birgit ; Bonderman, Diana ; Bianco, Nicole R. ; Russo, Andrea M. ; Erath, Julia W.
Background: Despite known clinical benefits, guideline-recommended heart rate (HR) control is not achieved for a significant proportion of patients with HF with reduced ejection fraction. The wearable cardioverter-defibrillator (WCD) provides continuous HR monitoring and alerts that could aid medication titration.
Objective: This study sought to evaluate sex differences in achieving guideline-recommended HR control during a period of WCD use.
Methods: Data from patients fitted with a WCD from 2015 to 2018 were obtained from the manufacturer’s database (ZOLL). The proportion of patients with adequate nighttime resting HR control at the beginning of use (BOU) and at the end of use (EOU) were compared by sex. Adequate HR control was defined as having a nighttime median HR <70 beats/min.
Results: A total of 21,440 women and a comparative sample of 17,328 men (median 90 [IQR 59–116] days of WCD wear) were included in the final dataset. Among patients who did not receive a shock, over half had insufficient HR control at BOU (59% of women, 53% of men). Although the proportion of patients with resting HR ≥70 beats/min improved by EOU, 43% of women and 36% of men did not achieve guideline-recommended HR control.
Conclusion: A significant proportion of women and men did not achieve adequate HR control during a period of medical therapy optimization. Compared with men, a greater proportion of women receiving WCD shocks had insufficiently controlled HR in the week preceding ventricular tachyarrhythmia/ventricular fibrillation and 43% of nonshocked women, compared with 36% of men, did not reach adequate HR control during the study period. The WCD can be utilized as a remote monitoring tool to record HR and inform adequate uptitration of beta-blockers, with particular focus on reducing the treatment gap in women.
Goldenberg, Ido ; Erath, Julia W. ; Russo, Andrea M. ; Burch, Ashley E. ; Aßmus, Birgit ; Bonderman, Diana ; McNitt, Scott ; Kutyifa, Valentina
Background: Data on the arrhythmic burden of women at risk for sudden cardiac death are limited, especially in patients using the wearable cardioverter-defibrillator (WCD).
Objective: We aimed to characterize WCD compliance, atrial and ventricular arrhythmic burden, and WCD outcomes by sex in patients enrolled in the Prospective Registry of Patients Using the Wearable Cardioverter Defibrillator (WEARIT-II U.S. Registry).
Methods: In the WEARIT-II Registry, we stratified 2000 patients by sex into women (n = 598) and men (n = 1402). WCD wear time, ventricular and atrial arrhythmic events during WCD use, and implantable cardioverter-defibrillator (ICD) implantation rates at the end of WCD use were evaluated.
Results: The mean WCD wear time was similar in women and men (94 days vs 90 days; P = .145), with longer daily use in women (21.4 h/d vs 20.7 h/d; P = .001). Burden of ventricular tachycardia or ventricular fibrillation was higher in women, with 30 events per 100 patient-years compared with 18 events per 100 patient-years in men (P = .017), with similar findings for treated and non-treated ventricular tachycardia/ventricular fibrillation. Recurrent atrial arrhythmias/sustained ventricular tachycardia was also more frequent in women than in men (167 events per 100 patient-years vs 73 events per 100 patient-years; P = .042). However, ICD implantation rate at the end of WCD use was similar in both women and men (41% vs 39%; P = .448).
Conclusion: In the WEARIT-II Registry, we have shown a higher burden of ventricular and atrial arrhythmic events in women than in men. ICD implantation rates at the end of WCD use were similar. Our findings warrant monitoring women at risk for sudden cardiac death who have a high burden of atrial and ventricular arrhythmias while using the WCD.
Martin, Virginie ; Groenendyk, Jody ; Steiner, Simone S. ; Guo, Lei ; Dabrowska, Monika ; Robert Parker, J.M. ; Müller-Esterl, Werner ; Opas, Michal ; Michalak, Marek
Calreticulin is a Ca2+ -binding chaperone that resides in the lumen of the endoplasmic reticulum and is involved in the regulation of intracellular Ca2+ homeostasis and in the folding of newly synthesized glycoproteins. In this study, we have used site-specific mutagenesis to map amino acid residues that are critical in calreticulin function. We have focused on two cysteine residues (Cys(88) and Cys(120)), which form a disulfide bridge in the N-terminal domain of calreticulin, on a tryptophan residue located in the carbohydrate binding site (Trp(302)), and on certain residues located at the tip of the "hairpin-like" P-domain of the protein (Glu(238), Glu(239), Asp(241), Glu(243), and Trp(244)). Calreticulin mutants were expressed in crt(-/-) fibroblasts, and bradykinin-dependent Ca2+ release was measured as a marker of calreticulin function. Bradykinin-dependent Ca2+ release from the endoplasmic reticulum was rescued by wild-type calreticulin and by the Glu(238), Glu(239), Asp(241), and Glu(243) mutants. The Cys(88) and Cys(120) mutants rescued the calreticulin-deficient phenotype only partially ( approximately 40%), and the Trp(244) and Trp(302) mutants did not rescue it at all. We identified four amino acid residues (Glu(239), Asp(241), Glu(243), and Trp(244)) at the hairpin tip of the P-domain that are critical in the formation of a complex between ERp57 and calreticulin. Although the Glu(239), Asp(241), and Glu(243) mutants did not bind ERp57 efficiently, they fully restored bradykinin-dependent Ca2+ release in crt(-/-) cells. This indicates that binding of ERp57 to calreticulin may not be critical for the chaperone function of calreticulin with respect to the bradykinin receptor.