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The activation of platelets and coagulation at vascular injury sites is crucial for hemostasis but can promote thrombosis and inflammation in vascular pathologies. Here, we delineate an unexpected spatio-temporal control mechanism of thrombin activity that is platelet orchestrated and locally limits excessive fibrin formation after initial hemostatic platelet deposition. During platelet activation, the abundant platelet glycoprotein (GP)V is cleaved by thrombin. We demonstrate, with genetic and pharmacological approaches, that thrombin-mediated shedding of GPV does not primarily regulate platelet activation in thrombus formation but rather has a distinct function after platelet deposition and specifically limits thrombin-dependent generation of fibrin, a crucial mediator of vascular thrombo-inflammation. Genetic or pharmacologic defects in hemostatic platelet function are unexpectedly attenuated by specific blockade of GPV shedding, indicating that the spatio-temporal control of thrombin-dependent fibrin generation also represents a potential therapeutic target to improve hemostasis.

Das Vertrauen in gesellschaftliche Institutionen und die sie tragenden Organisationen, zu denen auch die Freie Wohlfahrtspflege und ihre Spitzenverbände gehören, nimmt stetig ab und droht stellenweise gar zu erodieren. Ist die Freie Wohlfahrtspflege noch in der Lage, zur gesellschaftlichen Integration und zum gesellschaftlichen Zusammenhalt beizutragen?

Autoimmune neuropathy associated with antibodies against pan-neurofascin is a new subtype of nodo-paranodopathy. It is relevant because it is associated with high morbidity and mortality. Affected patients often require intensive care unit treatment for several months, and data on the reversibility and long-term prognosis are limited. The pathogenicity including IgG subclass-associated mechanisms has not been unravelled, nor directly compared to anti-neurofascin-155 IgG4-related pathology. Understanding the underlying pathology might have a direct impact on treatment of these severely affected patients. By a multicentre combined prospective and retrospective approach, we provide clinical data of a large cohort of patients with anti-neurofascin-associated neuropathy (n = 18) including longitudinal titre and neurofilament light chain assessment via Ella® and relate clinical data to in vitro pathogenicity studies of anti-neurofascin antibodies. We assessed antibody binding characteristics and the pathogenic effects of anti-pan-neurofascin versus neurofascin-155 antibodies on living myelinating dorsal root ganglia co-cultures. Additionally, we analysed the IgG subclass profile and the complement binding capacity and effector functions considering the effects of intravenous immunoglobulin preparations via enzyme-linked immunosorbent and cell-based assays. In contrast to chronic neurofascin-155 IgG4-associated neuropathy, anti-pan-neurofascin-associated disease presented with a high morbidity and mortality, but as a monophasic and potentially reversible disorder. During follow-up, antibodies were no longer detectable in 8 of 11 patients. Anti-pan-neurofascin had direct access to the nodes of Ranvier in myelinating cultures titre-dependently, most probably inducing this severe phenotype. Antibody preincubation led to impaired paranode formation, destruction of paranodal architecture and alterations on paranodal myelin and sensory neurons in the cultures, with more severe effects than neurofascin-155 antibodies. Besides IgG4, subclass IgG3 was detected and associated with complement binding and cytotoxic effects in vitro. As a possible correlate of axonal damage in vivo, we detected highly increased serum neurofilament light chain levels (sNF-L), correlating to serum C3a. Still, sNF-L was not identified as a marker for poor prognosis, but rather as an intra- and interindividual marker for acuteness, severity and course, with a strong decrease during recovery. Our data provide evidence that anti-pan-neurofascin antibodies directly attack the node and induce severe and acute, but potentially reversible, nodo-paranodal pathology, possibly involving complement-mediated mechanisms. Screening for autoantibodies thus is crucial to identify this subset of patients who benefit from early antibody-depleting therapy. Titre and sNF-L might serve as valuable follow-up parameters. The prospect of a favourable outcome has high relevance for physicians, patients and relatives during months of critical care.

Im Beitrag wird diskutiert, ob es sich bei der vorgesehenen Ablösung von Hartz IV durch ein Bürgergeld und den Aufbau einer Kindergrundsicherung eher um einen symbolischen Wandel handelt oder die im Herbst 2021 gewählte Ampel-Koalition einen substanziellen Neuanfang und eine grundlegende Reform des Wohlfahrtsstaates einleitet. Zusammenfassend werden die geplanten Maßnahmen der neuen Bundesregierung als schleichende Transformation einer beitragsfinanzierten Lebensstandardabsicherung zum Grundsicherungsstaat mit einer universalistischen Sozialintegration gedeutet.

Background: Platelets can infiltrate ischemic myocardium and are increasingly recognized as critical regulators of inflammatory processes during myocardial ischemia and reperfusion (I/R). Platelets contain a broad repertoire of microRNAs (miRNAs), which, under certain conditions such as myocardial ischemia, may be transferred to surrounding cells or released into the microenvironment. Recent studies could demonstrate that platelets contribute substantially to the circulating miRNA pool holding the potential for so far undiscovered regulatory functions. The present study aimed to determine the role of platelet-derived miRNAs in myocardial injury and repair following myocardial I/R. Methods: In vivo model of myocardial I/R, multimodal in vivo and ex vivo imaging approaches (light-sheet fluorescence microscopy, positron emission tomography and magnetic resonance imaging, speckle-tracking echocardiography) of myocardial inflammation and remodeling, and next-generation deep sequencing analysis of platelet miRNA expression. Results: In mice with a megakaryocyte/platelet-specific knockout of pre-miRNA processing ribonuclease Dicer , the present study discloses a key role of platelet-derived miRNAs in the tightly regulated cellular processes orchestrating left ventricular remodeling after myocardial I/R following transient left coronary artery ligation. Disruption of the miRNA processing machinery in platelets by deletion of Dicer resulted in increased myocardial inflammation, impaired angiogenesis, and accelerated development of cardiac fibrosis, culminating in an increased infarct size by d7 that persisted through d28 of myocardial I/R. Worsened cardiac remodeling after myocardial infarction in mice with a platelet-specific Dicer deletion resulted in an increased fibrotic scar formation and distinguishably increased perfusion defect of the apical and anterolateral wall at day 28 post-myocardial infarction. Altogether, these observations culminated in an impaired left ventricular function and hampered long-term cardiac recovery after experimental myocardial infarction and reperfusion therapy. Treatment with the P2Y 12 (P2Y purinoceptor 12) antagonist ticagrelor completely reversed increased myocardial damage and adverse cardiac remodeling observed in Dicer Pf4∆/Pf4∆ mice. Conclusions: The present study discloses a critical role of platelet-derived miRNA in myocardial inflammation and structural remodeling processes following myocardial I/R.

Cerebral venous (sinus) thrombosis (CVT) is an unusual manifestation of venous thrombosis causing severe neurological impairment and seizures1,2. Molecular mechanisms underlying CVT, potentially involving pathological platelet activation, are unknown. Here we show that antibody-(INU1-fab)-induced cooperative signaling of two platelet receptors, C-type lectin-like receptor-2 (CLEC-2) and GPIIb/IIIa, triggers within minutes a CVT-like thrombotic syndrome in mice, characterized by tonic–myoclonic seizures, platelet consumption and death. Brain autopsy showed thrombi mainly in the cortical venules, but no intracranial hemorrhages or edema formation. Transcranial intravital microscopy revealed rapidly progressing thrombosis in the superior sagittal sinus, a main site of CVT in humans. Interfering with CLEC-2 signaling or inhibition of GPIIb/IIIa completely blocked platelet activation and CVT. Blocking GPIIb/IIIa after onset of neurological symptoms protected mice from platelet consumption, CVT and death, which was not seen after treatment with heparin. These results point to aberrant platelet activation as a major trigger of CVT and potential target for treatment.

Zusammenfassung Im Zuge der Corona-Pandemie wurden der Zugang zu Leistungen der Grundsicherung vereinfacht und die Zugangsvoraussetzungen zur Gewährung von Sozialleistungen sowie die Disziplinierung der Leistungsbeziehenden abgeschwächt. Bislang ist unklar, welche Erfahrungen Leistungsbeziehende mit diesem vereinfachten Zugang gemacht haben. Der Beitrag untersucht dies auf Basis einer quantitativen Erhebung unter Grundsicherungsbeziehenden des Kreises Recklinghausen. Im Fokus stehen die Beurteilung der Interaktion mit den Jobcenter-Beschäftigten seitens der Leistungsbeziehenden, die Zufriedenheit mit dem Hartz IV-System und die Einstellungen zum vereinfachten Zugang zur Grundsicherung im Jahr 2020. Die Befunde zeigen: Leistungsbeziehende nehmen das Jobcenter mehrheitlich nicht als Ort der Angst und Disziplinierung wahr, obgleich Scham ebenso Teil der Realität ist. Positive Erfahrungen mit dem Jobcenter gehen einher mit positiven Effekten auf die Beurteilung des Hartz IV-Systems, was die Bedeutung der Jobcenter als street-level bureaucracy unterstreicht. Eine Verstetigung wesentlicher Elemente des vereinfachten Zugangs wird mehrheitlich befürwortet, allerdings mit der Ausnahme einer dauerhaften Aussetzung von Sanktionen. Der Beitrag diskutiert abschließend sozialpolitische Implikationen für eine neujustierte Grundsicherung. Abstract: Climate of Fear or Respect at Eye Level? Experiences of Hartz IV-Recipients with Jobcentres during the COVID-19 pandemic In the wake of the COVID-19 pandemic, the access to basic security benefits for job seekers was facilitated by promoting unconditional security elements and weakening disciplinary measures for recipients. So far, it is unclear what experiences benefit recipients have had with these new regulations. The article investigates this question on the basis of a quantitative survey of basic security benefit recipients. We focus on the evaluation of the interaction with job centre-employees, the satisfaction with the Hartz IV-system and attitudes towards the pandemic-related regulations. The findings show: benefit recipients do not per se perceive job centres as places of fear and harassment, although feelings of shame are also part of their reality. Positive experiences with job centres show positive effects on the evaluation of the Hartz IV-system, which underlines the important role of job centres as street-level bureaucracies. Making the new regulations permanent is supported by the vast majority, although a permanent renunciation of sanctions is not favoured. Finally, the article discusses social policy implications for a redesigned basic security system.

Zusammenfassung In die deutsche Protestlandschaft haben sich seit dem Herbst 2019 ebenfalls Bäuerinnen und Bauern eingereiht, die u. a. durch Blockaden und Mahnwachen auch Anfang 2021 immer wieder Aufmerksamkeit erregen. Die Forderungen und Anliegen, die mit den Protesten durchgesetzt werden sollen, sind vielfältig und beziehen sich sowohl auf ökonomische Existenzsorgen als auch auf gesellschaftliche Anerkennungsdefizite. Um einen Einblick in die Hintergründe und die zugrundeliegenden Sorgen zu erlangen, wurde eine quantitative Befragung im landwirtschaftlichen Protestmilieu durchgeführt, deren Ergebnisse in diesem Beitrag vorgestellt werden. Nach einer agrarsoziologischen Einordnung, wird der Fokus insbesondere auf die politische Verortung der protestierenden Bäuerinnen und Bauern gelegt.

To shed light on nanoscale pathologies in patients with polyneuropathy, we assessed human nerve biopsies by super-resolution fluorescence microscopy. We focused on both physiological protein arrangement and pathological ultrastructural changes at the node of Ranvier, a crucial region of the peripheral myelinated axon. Direct stochastic optical reconstruction microscopy (dSTORM) revealed a ∼190 nm periodic protein arrangement of cytoskeletal proteins and axoglial cell adhesion molecules. Periodic distances increased at the paranodal region of the node of Ranvier in patients with polyneuropathy, both at the axonal cytoskeleton and at the axoglial junction. In-depth image analysis of human nerve biopsies revealed a partial loss of proteins of the axoglial complex (Caspr-1, neurofascin-155) in combination with detachment from the cytoskeletal anchor protein ß2-spectrin. Super-resolution dual-color colocalization data was supported by high-content confocal imaging combined with deep learning-based analysis, indicating that paranodal elongation occurs especially in acute and severe axonal neuropathy, as a possible correlate of Wallerian degeneration and related cytoskeletal damage. Our findings show that super-resolution imaging can identify, quantify and map elongated periodic protein distances in peripheral nerve biopsies for pathophysiological studies and direct implications for diagnostic assessment.Graphical abstractPathological alterations of ultrastructural protein arrangement at the paranodal region of the node of Ranvier in polyneuropathy.Super-resolution microscopy allows for assessing ultrastructural protein arrangement and pathological alterations in patients with polyneuropathy. In human healthy nodes, axoglial and axoskeletal proteins follow a 190 nm periodic arrangement (left). In pathologically altered nodes (right), periodic protein distances of axonal ß2-spectrin elongate, in combination with elongation and partial loss of the axoglial complex of Caspr-1 and neurofascin-155. The axoglial complex itself colocalizes closely even in pathologically altered nodes. A complete loss of the axoglial complex could be the ultrastructural correlate of the detachment of paranodal myelin loops. Scale bar 2 μm.

Das Corona-Virus hat nachdrücklich bewusstgemacht, wie stark die Lebenslagen und auch wirtschaftliche Wertschöpfungen von einer funktionierenden öffentlichen Infrastruktur abhängen. Dazu zählen neben der technischen die soziale Infrastruktur, die sich auf Gesundheits-, Bildungs-, Sozial-, Sport- und Kultureinrichtungen erstreckt. Eine bedarfsgerechte Infrastruktur bestimmt zentral die Lebenssituation vieler Menschen, die gerade im Lockdown auf eine funktionierende Daseinsvorsorge angewiesen sind.

Store-operated Ca(2+) entry (SOCE) is the major route of Ca(2+) influx in platelets. The Ca(2+) sensor stromal interaction molecule 1 (STIM1) triggers SOCE by forming punctate structures with the Ca(2+) channel Orai1 and the inositol trisphosphate receptor (IP(3)R), thereby linking the endo-/sarcoplasmic reticulum to the plasma membrane. Here, we identified the BAR domain superfamily member bridging integrator 2 (BIN2) as an interaction partner of STIM1 and IP(3)R in platelets. Deletion of platelet BIN2 (Bin2(fl/fl,Pf4-Cre) mice) resulted in reduced Ca(2+) store release and Ca(2+) influx in response to all tested platelet agonists. These defects were a consequence of impaired IP(3)R function in combination with defective STIM1-mediated SOC channel activation, while Ca(2+) store content and agonist-induced IP(3) production were unaltered. This severely defective Ca(2+) signaling translated into impaired thrombus formation under flow and a protection of Bin2(fl/fl,Pf4-Cre) mice in models of arterial thrombosis and stroke. Our results establish BIN2 as a central regulator of platelet activation in thrombosis and thrombo-inflammatory disease settings.

Nachdem auf die Bedeutung des Wohnens und des Engagements im Alter eingegangen wird, werden Determinanten wie Zufriedenheit, soziale und räumliche Selektivitäten, Haushaltsstrukturen sowie neue soziotechnische Optionen und nahräumliche Infrastrukturen diskutiert. Abschließend stehen sowohl Herausforderungen für ein „vernetztes“ Wohnen als auch Barrieren bei der Umsetzung im Mittelpunkt.

Visualization of inhibitory synapses requires protocol tailoring for different sample types and imaging techniques, and usually relies on genetic manipulation or the use of antibodies that underperform in tissue immunofluorescence. Starting from an endogenous ligand of gephyrin, a universal marker of the inhibitory synapse, we developed a short peptidic binder and dimerized it, significantly increasing affinity and selectivity. We further tailored fluorophores to the binder, yielding "Sylite"-a probe with outstanding signal-to-background ratio that outperforms antibodies in tissue staining with rapid and efficient penetration, mitigation of staining artifacts, and simplified handling. In super-resolution microscopy Sylite precisely localizes the inhibitory synapse and enables nanoscale measurements. Sylite profiles inhibitory inputs and synapse sizes of excitatory and inhibitory neurons in the midbrain and combined with complimentary tracing techniques reveals the synaptic connectivity.

ZusammenfassungBis zum Ausbruch der globalen Corona-Pandemie wurde als zentrale Herausforderung, neben der Digitalisierung und dem Klimawandel, auf den demografischen Wandel verwiesen. Diese Fokussierung ergab sich daraus, dass im nächsten Jahrzehnt allein in Deutschland die Zahl der Rentner und Pensionäre gegenüber heute um mehr als drei Millionen ansteigen wird, während die erwerbsfähige Bevölkerung schrumpft. Inzwischen hat sich die Situation verändert: Ein Virus hat uns unvorbereitet getroffen und die gesellschaftlichen Auswirkungen sind elementar. Dennoch geht die Alterung der Gesellschaft weiter und bei der Bewältigung dieser Aufgaben können die in der Corona-Krise erworbenen Erfahrungen (etwa mit der Digitalisierung) weiterhelfen.

We present a protocol and workflow to perform live cell dual-color fluorescence crosscorrelation spectroscopy (FCCS) combined with Förster Resonance Energy transfer (FRET) to study membrane receptor dynamics in live cells using modern fluorescence labeling techniques. In dual-color FCCS, where the fluctuations in fluorescence intensity represents the dynamical “fingerprint” of the respective fluorescent biomolecule, we can probe co-diffusion or binding of the receptors. FRET, with its high sensitivity to molecular distances, serves as a well-known “nanoruler” to monitor intramolecular changes. Taken together, conformational changes and key parameters such as local receptor concentrations, and mobility constants become accessible in cellular settings.Quantitative fluorescence approaches are challenging in cells due to high noise levels and the vulnerable sample itself. We will show how to perform the experiments including the calibration steps. We use dual-color labeled β2-adrenergic receptor (β2AR) labeled (eGFP and SNAPtag-TAMRA). We will guide you step-by-step through the data analysis procedure using open-source software and provide templates that are easy to customize.Our guideline enables researchers to unravel molecular interactions of biomolecules in live cells in situ with high reliability despite the limited signal-to-noise levels in live cell experiments. The operational window of FRET and particularly FCCS at low concentrations allows quantitative analysis near-physiological conditions.Link to accompanying video: https://tr240.uni-wuerzburg.de/vippclass/index.php/s/TL8aWmwE9RjGfLE

Platelet aggregate formation is a multistep process involving receptor-mediated, as well as biomechanical, signaling cascades, which are highly dependent on actin dynamics. We have previously shown that actin depolymerizing factor (ADF)/n-cofilin and Twinfilin 2a, members of the ADF homology (ADF-H) protein family, have distinct roles in platelet formation and function. Coactosin-like 1 (Cotl1) is another ADF-H protein that binds actin and was also shown to enhance biosynthesis of pro-inflammatory leukotrienes (LT) in granulocytes. Here, we generated mice lacking Cotl1 in the megakaryocyte lineage (Cotl1−/−) to investigate its role in platelet production and function. Absence of Cotl1 had no impact on platelet counts, platelet activation or cytoskeletal reorganization under static conditions in vitro. In contrast, Cotl1 deficiency markedly affected platelet aggregate formation on collagen and adhesion to immobilized von Willebrand factor at high shear rates in vitro, pointing to an impaired function of the platelet mechanoreceptor glycoprotein (GP) Ib. Furthermore, Cotl1−/−platelets exhibited increased deformability at high shear rates, indicating that the GPIb defect may be linked to altered biomechanical properties of the deficient cells. In addition, we found that Cotl1 deficiency markedly affected platelet LT biosynthesis. Strikingly, exogenous LT addition restored defective aggregate formation of Cotl1−/− platelets at high shear in vitro, indicating a critical role of platelet-derived LT in thrombus formation. In vivo, Cotl1 deficiency translated into prolonged tail bleeding times and protection from occlusive arterial thrombus formation. Together, our results show that Cotl1 in platelets is an integrator of biomechanical and LT signaling in hemostasis and thrombosis.

Mesenteric lymph nodes (mLNs) are sentinel sites of enteral immunosurveillance and immune homeostasis. Immune cells from the gastrointestinal tract (GIT) are constantly recruited to the mLNs in steady-state and under inflammatory conditions resulting in the induction of tolerance and immune cells activation, respectively. Surgical dissection and transplantation of lymph nodes (LN) is a technique that has supported seminal work to study LN function and is useful to investigate resident stromal and endothelial cell biology and their cellular interactions in experimental disease models. Here, we provide a detailed protocol of syngeneic mLN transplantation and report assays to analyze effective mLN engraftment in congenic recipients. Transplanted mLNs allow to study T cell activation and proliferation in preclinical mouse models. Donor mLNs proved viable and functional after surgical transplantation and regenerated blood and lymphatic vessels. Immune cells from the host completely colonized the transplanted mLNs within 7-8 weeks after the surgical intervention. After allogeneic hematopoietic cell transplantation (allo-HCT), adoptively transferred allogeneic CD4+ T cells from FVB/N (H-2q) mice homed to the transplanted mLNs in C57BL/6 (H-2b) recipients during the initiation phase of acute graft-versus-host disease (aGvHD). These CD4+ T cells retained full proliferative capacity and upregulated effector and gut homing molecules comparable to those in mLNs from unmanipulated wild-type recipients. Wild type mLNs transplanted into MHCII deficient syngeneic hosts sufficed to activate alloreactive T cells upon allogeneic hematopoietic cell transplantation, even in the absence of MHCII+ CD11c+ myeloid cells. These data support that orthotopically transplanted mLNs maintain physiological functions after transplantation. The technique of LN transplantation can be applied to study migratory and resident cell compartment interactions in mLNs as well as immune reactions from and to the gut under inflammatory and non-inflammatory conditions.

DNMT3A/3L heterotetramers contain two active centers binding CpG sites at 12 bp distance, however their interaction with DNA not containing this feature is unclear. Using randomized substrates, we observed preferential co-methylation of CpG sites with 6, 9 and 12 bp spacing by DNMT3A and DNMT3A/3L. Co-methylation was favored by AT bases between the 12 bp spaced CpG sites consistent with their increased bending flexibility. SFM analyses of DNMT3A/3L complexes bound to CpG sites with 12 bp spacing revealed either single heterotetramers inducing 40° DNA bending as observed in the X-ray structure, or two heterotetramers bound side-by-side to the DNA yielding 80° bending. SFM data of DNMT3A/3L bound to CpG sites spaced by 6 and 9 bp revealed binding of two heterotetramers and 100° DNA bending. Modeling showed that for 6 bp distance between CpG sites, two DNMT3A/3L heterotetramers could bind side-by-side on the DNA similarly as for 12 bp distance, but with each CpG bound by a different heterotetramer. For 9 bp spacing our model invokes a tetramer swap of the bound DNA. These additional DNA interaction modes explain how DNMT3A and DNMT3A/3L overcome their structural preference for CpG sites with 12 bp spacing during the methylation of natural DNA., Graphical Abstract Graphical AbstractNGS methylation analysis with pool of randomized CpG double site substrates and SFM imaging of enzyme-DNA complexes with example substrates revealed novel interaction modes of DNMT3A and DNMT3A/3L with CpG double site substrates.

G-protein-coupled receptors (GPCRs) are hypothesized to possess molecular mobility over a wide temporal range. Until now the temporal range has not been fully accessible due to the crucially limited temporal range of available methods. This in turn, may lead relevant dynamic constants to remain masked. Here, we expand this dynamic range by combining fluorescent techniques using a spot confocal setup. We decipher mobility constants of β

We introduce Sylites - small and versatile fluorogenic affinity probes for high-contrast visualization of inhibitory synapses. Having stoichiometric labeling and exceptional selectivity for neuronal gephyrin, a hallmark protein of the inhibitory post-synapse, Sylites enable superior synapse staining compared with antibodies. Combined with super-resolution microscopy, Sylites allow precise nanoscopic measurements of the synapse. In brain tissue, Sylites reveal the three-dimensional distribution of inhibitory synapses within just an hour.