Expansion-enhanced super-resolution radial fluctuations enable nanoscale molecular profiling of pathology specimens.

Expansion microscopy physically enlarges biological specimens to achieve nanoscale resolution using diffraction-limited microscopy systems1. However, optimal performance is usually reached using laser-based systems (for example, confocal microscopy), restricting its broad applicability in clinical pathology, as most centres have access only to light-emitting diode (LED)-based widefield systems. As a possible alternative, a computational method for image resolution enhancement, namely, super-resolution radial fluctuations (SRRF)2,3, has recently been developed. However, this method has not been explored in pathology specimens to date, because on its own, it does not achieve sufficient resolution for routine clinical use. Here, we report expansion-enhanced super-resolution radial fluctuations (ExSRRF), a simple, robust, scalable and accessible workflow that provides a resolution of up to 25 nm using LED-based widefield microscopy. ExSRRF enables molecular profiling of subcellular structures from archival formalin-fixed paraffin-embedded tissues in complex clinical and experimental specimens, including ischaemic, degenerative, neoplastic, genetic and immune-mediated disorders. Furthermore, as examples of its potential application to experimental and clinical pathology, we show that ExSRRF can be used to identify and quantify classical features of endoplasmic reticulum stress in the murine ischaemic kidney and diagnostic ultrastructural features in human kidney biopsies.

The Role of Lamins in the Nucleoplasmic Reticulum, a Pleiomorphic Organelle That Enhances Nucleo-Cytoplasmic Interplay.

Invaginations of the nuclear membrane occur in different shapes, sizes, and compositions. Part of these pleiomorphic invaginations make up the nucleoplasmic reticulum (NR), while others are merely nuclear folds. We define the NR as tubular invaginations consisting of either both the inner and outer nuclear membrane, or only the inner nuclear membrane. Specifically, invaginations of both the inner and outer nuclear membrane are also called type II NR, while those of only the inner nuclear membrane are defined as type I NR. The formation and structure of the NR is determined by proteins associated to the nuclear membrane, which induce a high membrane curvature leading to tubular invaginations. Here we review and discuss the current knowledge of nuclear invaginations and the NR in particular. An increase in tubular invaginations of the nuclear envelope is associated with several pathologies, such as laminopathies, cancer, (reversible) heart failure, and Alzheimer's disease. Furthermore, viruses can induce both type I and II NR. In laminopathies, the amount of A-type lamins throughout the nucleus is generally decreased or the organization of lamins or lamin-associated proteins is disturbed. Also, lamin overexpression or modulation of lamin farnesylation status impacts NR formation, confirming the importance of lamin processing in NR formation. Virus infections reorganize the nuclear lamina via (de)phosphorylation of lamins, leading to an uneven thickness of the nuclear lamina and in turn lobulation of the nuclear membrane and the formation of invaginations of the inner nuclear membrane. Since most studies on the NR have been performed with cell cultures, we present additional proof for the existence of these structures in vivo, focusing on a variety of differentiated cardiovascular and hematopoietic cells. Furthermore, we substantiate the knowledge of the lamin composition of the NR by super-resolution images of the lamin A/C and B1 organization. Finally, we further highlight the essential role of lamins in NR formation by demonstrating that (over)expression of lamins can induce aberrant NR structures.

Management of Metadata Types in Basic Cardiological Research.

INTRODUCTION: Ensuring scientific reproducibility and compliance with documentation guidelines of funding bodies and journals is a topic of greatly increasing importance in biomedical research. Failure to comply, or unawareness of documentation standards can have adverse effects on the translation of research into patient treatments, as well as economic implications. In the context of the German Research Foundation-funded collaborative research center (CRC) 1002, an IT-infrastructure sub-project was designed. Its goal has been to establish standardized metadata documentation and information exchange benefitting the participating research groups with minimal additional documentation efforts. METHODS: Implementation of the self-developed menoci-based research data platform (RDP) was driven by close communication and collaboration with researchers as early adopters and experts. Requirements analysis and concept development involved in person observation of experimental procedures, interviews and collaboration with researchers and experts, as well as the investigation of available and applicable metadata standards and tools. The Drupal-based RDP features distinct modules for the different documented data and workflow types, and both the development and the types of collected metadata were continuously reviewed and evaluated with the early adopters. RESULTS: The menoci-based RDP allows for standardized documentation, sharing and cross-referencing of different data types, workflows, and scientific publications. Different modules have been implemented for specific data types and workflows, allowing for the enrichment of entries with specific metadata and linking to further relevant entries in different modules. DISCUSSION: Taking the workflows and datasets of the frequently involved experimental service projects as a starting point for (meta-)data types to overcome irreproducibility of research data, results in increased benefits for researchers with minimized efforts. While the menoci-based RDP with its data models and metadata schema was originally developed in a cardiological context, it has been implemented and extended to other consortia at GÃuttingen Campus and beyond in different life science research areas.

Post-translational modifications soften vimentin intermediate filaments.

The mechanical properties of biological cells are determined by the cytoskeleton, a composite biopolymer network consisting of microtubules, actin filaments and intermediate filaments (IFs). By differential expression of cytoskeletal proteins, modulation of the network architecture and interactions between the filaments, cell mechanics may be adapted to varying requirements on the cell. Here, we focus on the intermediate filament protein vimentin and introduce post-translational modifications as an additional, much faster mechanism for mechanical modulation. We study the impact of phosphorylation on filament mechanics by recording force-strain curves using optical traps. Partial phosphorylation softens the filaments. We show that binding of the protein 14-3-3 to phosphorylated vimentin IFs further enhances this effect and speculate that in the cell 14-3-3 may serve to preserve the softening and thereby the altered cell mechanics. We explain our observation by the additional charges introduced during phosphorylation.

Altered atrial cytosolic calcium handling contributes to the development of postoperative atrial fibrillation.

AIMS: Atrial fibrillation (AF) is a commonly occurring arrhythmia after cardiac surgery (postoperative AF, poAF) and is associated with poorer outcomes. Considering that reduced atrial contractile function is a predictor of poAF and that Ca2+ plays an important role in both excitation-contraction coupling and atrial arrhythmogenesis, this study aims to test whether alterations of intracellular Ca2+ handling contribute to impaired atrial contractility and to the arrhythmogenic substrate predisposing patients to poAF. METHODS AND RESULTS: Right atrial appendages were obtained from patients in sinus rhythm undergoing open-heart surgery. Cardiomyocytes were investigated by simultaneous measurement of [Ca2+]i and action potentials (APs, patch-clamp). Patients were followed-up for 6 days to identify those with and without poAF. Speckle-tracking analysis of preoperative echocardiography revealed reduced left atrial contraction strain in poAF patients. At the time of surgery, cellular Ca2+ transients (CaTs) and the sarcoplasmic reticulum (SR) Ca2+ content were smaller in the poAF group. CaT decay was slower in poAF, but the decay of caffeine-induced Ca2+ transients was unaltered, suggesting preserved sodium-calcium exchanger function. In agreement, western blots revealed reduced SERCA2a expression in poAF patients but unaltered phospholamban expression/phosphorylation. Computational modelling indicated that reduced SERCA activity promotes occurrence of CaT and AP alternans. Indeed, alternans of CaT and AP occurred more often and at lower stimulation frequencies in atrial myocytes from poAF patients. Resting membrane potential and AP duration were comparable between both groups at various pacing frequencies (0.25-8 Hz). CONCLUSIONS: Biochemical, functional, and modelling data implicate reduced SERCA-mediated Ca2+ reuptake into the SR as a major contributor to impaired preoperative atrial contractile function and to the pre-existing arrhythmogenic substrate in patients developing poAF.

14-3-3 binding creates a memory of kinase action by stabilizing the modified state of phospholamban.

The cardiac membrane protein phospholamban (PLN) is targeted by protein kinase A (PKA) at Ser16 and by Ca2+/calmodulin-dependent protein kinase II (CaMKII) at Thr17 ß-Adrenergic stimulation and PKA-dependent phosphorylation of Ser16 acutely stimulate the sarcoplasmic reticulum calcium pump (SERCA) by relieving its inhibition by PLN. CaMKII-dependent phosphorylation may lead to longer-lasting SERCA stimulation and may sustain maladaptive Ca2+ handling. Here, we demonstrated that phosphorylation at either Ser16 or Thr17 converted PLN into a target for the phosphoadaptor protein 14-3-3 with different affinities. 14-3-3 proteins were localized within nanometers of PLN and endogenous 14-3-3 coimmunoprecipitated with pentameric PLN from cardiac membranes. Molecular dynamics simulations predicted different molecular contacts for peptides phosphorylated at Ser16 or Thr17 with the binding groove of 14-3-3, resulting in varied binding affinities. 14-3-3 binding protected either PLN phosphosite from dephosphorylation. ß-Adrenergic stimulation of isolated adult cardiomyocytes resulted in the membrane recruitment of endogenous 14-3-3. The exogenous addition of 14-3-3 to ß-adrenergic-stimulated cardiomyocytes led to prolonged SERCA activation, presumably because 14-3-3 protected PLN pentamers from dephosphorylation. Phosphorylation of Ser16 was disrupted by the cardiomyopathy-associated ∆Arg14 mutation, implying that phosphorylation of Thr17 by CaMKII may become crucial for 14-3-3 recruitment to ∆Arg14 PLN. Consistent with PLN acting as a dynamic hub in the control of Ca2+ handling, our results identify 14-3-3 binding to PLN as a contractility-augmenting mechanism.

Prevalence and formation of primary cataracts and persistent hyperplastic tunica vasculosa lentis in the German Pinscher population in Germany.

OBJECTIVE: The objective of this study was to describe the prevalences of primary noncongenital cataracts (CAT) and persistent hyperplastic tunica vasculosa lentis (PHTVL) in the German Pinscher population in Germany and to analyze the mode of inheritance of CAT in this dog breed. ANIMALS STUDIED: All German Pinschers with a certified veterinary ophthalmological diagnosis and born between 1993 and 2010 were included in this study. Examinations were performed between 1997 and 2013 by certified veterinary ophthalmologists of the German panel of the European Eye Scheme for Diagnosis of Inherited Eye Diseases in Animals (DOK). PROCEDURES: Data were reviewed retrospectively for the prevalence of PHTVL, and the prevalence, location and age at diagnosis of CAT. Inheritance of CAT was analyzed using the Singles Method. RESULTS: A total of 779 eye examination reports of 409 dogs were available. Primary noncongenital cataracts were diagnosed in 64 (15.6%), and PHTVL in 13 (3.2%) of the examined dogs. The pedigrees included 168 ophthalmologically examined dogs with 104 CAT-unaffected and 64 CAT-affected dogs. All affected animals were offspring of a frequently used stud-dog or closely related ancestors of this stud-dog. Simple segregation analysis revealed a recessive mode of inheritance for CAT. CONCLUSIONS: An early onset form of CAT with various cortical, posterior polar or multiple locations was the most prevalent manifestation among close relatives in German Pinschers. The pedigree analysis indicated a hereditary component of cataract formation with a monogenic autosomal recessive pattern of inheritance.

Requirement analysis for an electronic laboratory notebook for sustainable data management in biomedical research.

Sustainable data management in biomedical research requires documentation of metadata for all experiments and results. Scientists usually document research data and metadata in laboratory paper notebooks. An electronic laboratory notebook (ELN) can keep metadata linked to research data resulting in a better understanding of the research results, meaning a scientific benefit [1]. Besides other challenges [2], the biggest hurdles for introducing an ELN seem to be usability, file formats, and data entry mechanisms [3] and that many ELNs are assigned to specific research fields such as biology, chemistry, or physics [4]. We aimed to identify requirements for the introduction of ELN software in a biomedical collaborative research center [5] consisting of different scientific fields and to find software fulfilling most of these requirements.

A rare form of persistent right aorta arch in linkage disequilibrium with the DiGeorge critical region on CFA26 in German Pinschers.

Persistent right aortic arch (PRAA) is a congenital vascular ring anomaly common in several dog breeds. In German Pinscher, the disorder is characterized by a left retroesophageal subclavian artery in combination with a ligamentum arteriosum originating at the aberrant left subclavian artery (PRAA-SA-LA). In this study, we genotyped 38 microsatellite markers on canine chromosome 26 (CFA26) in German Pinschers and tested them for linkage and association. We found a chromosome-wide significantly linked genomic region on CFA26, which corresponds to the human DiGeorge syndrome critical region (DGCR). Therefore, we analyzed sequences from 13 genes of DGCR and the canine t-box gene TBX1. We identified a total of 26 polymorphisms in German Pinschers. Three of these SNPs located within TBX1 and one in the mitochondrial ribosomal protein L40 gene (MRPL40) were associated with the PRAA-SA-LA phenotype in German Pinscher. Despite linkage and association between PRAA-SA-LA and the canine DGCR, none of these mutations appeared responsible for PRAA-SA-LA. As the orthologue human region on HSA22q11.2 is known for high susceptibility to genomic rearrangements, we suspect that in German Pinschers, chromosomal aberrations might cause PRAA-SA-LA.

Unusual vascular ring anomaly associated with a persistent right aortic arch and an aberrant left subclavian artery in German pinschers.

The objective of this study was to describe a specific form of persistent right aortic arch (PRAA) in three German pinscher dogs and to analyse the mode of inheritance in the breed. This type of PRAA is characterised by a left retro-oesophageal subclavian artery in combination with a ligamentum arteriosum originating at the aberrant left subclavian artery (PRAA-SA-LA). This rare combination of anomalies has only been reported in two isolated cases in other dog breeds and the occurrence of any form of PRAA was not previously known to occur in the German pinscher. In the present study, 18 cases of this congenital anomaly were ascertained and their high degree of relatedness and inbreeding could be shown through pedigree analysis. Three of the affected dogs underwent further clinical investigations, and post-mortem examination (two cases) and findings at surgery (one case) verified the diagnosis of PRAA-SA-LA. A monogenic autosomal recessive mode of inheritance was not likely.