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Background: Calcific aortic valve disease (CAVD) is characterized by a phenotypic switch of valvular interstitial cells to bone-forming cells. Toll-like receptors (TLRs) are evolutionarily conserved pattern recognition receptors at the interface between innate immunity and tissue repair. Type I interferons (IFNs) are not only crucial for an adequate antiviral response but also implicated in bone formation. We hypothesized that the accumulation of endogenous TLR3 ligands in the valvular leaflets may promote the generation of osteoblast-like cells through enhanced type I IFN signaling. Methods: Human valvular interstitial cells isolated from aortic valves were challenged with mechanical strain or synthetic TLR3 agonists and analyzed for bone formation, gene expression profiles, and IFN signaling pathways. Different inhibitors were used to delineate the engaged signaling pathways. Moreover, we screened a variety of potential lipids and proteoglycans known to accumulate in CAVD lesions as potential TLR3 ligands. Ligand-receptor interactions were characterized by in silico modeling and verified through immunoprecipitation experiments. Biglycan (Bgn), Tlr3 , and IFN-α/β receptor alpha chain (Ifnar1)–deficient mice and a specific zebrafish model were used to study the implication of the biglycan (BGN)-TLR3-IFN axis in both CAVD and bone formation in vivo. Two large-scale cohorts (GERA [Genetic Epidemiology Research on Adult Health and Aging], n=55 192 with 3469 aortic stenosis cases; UK Biobank, n=257 231 with 2213 aortic stenosis cases) were examined for genetic variation at genes implicated in BGN-TLR3-IFN signaling associating with CAVD in humans. Results: Here, we identify TLR3 as a central molecular regulator of calcification in valvular interstitial cells and unravel BGN as a new endogenous agonist of TLR3. Posttranslational BGN maturation by xylosyltransferase 1 (XYLT1) is required for TLR3 activation. Moreover, BGN induces the transdifferentiation of valvular interstitial cells into bone-forming osteoblasts through the TLR3-dependent induction of type I IFNs. It is intriguing that Bgn−/− , Tlr3−/− , and Ifnar1−/− mice are protected against CAVD and display impaired bone formation. Meta-analysis of 2 large-scale cohorts with >300 000 individuals reveals that genetic variation at loci relevant to the XYLT1–BGN–TLR3–interferon-α/β receptor alpha chain (IFNAR) 1 pathway is associated with CAVD in humans. Conclusions: This study identifies the BGN-TLR3-IFNAR1 axis as an evolutionarily conserved pathway governing calcification of the aortic valve and reveals a potential therapeutic target to prevent CAVD. [ABSTRACT FROM AUTHOR]

Open in new tab Download slide Open in new tab Download slide OBJECTIVES Ischaemia and subsequent reperfusion during heart transplantation inevitably result in donor organ injury. Toll-like receptor (TLR)-3 is a pattern recognition receptor activated by viral and endogenous RNA released by injured cells. We hypothesized that ischaemia/reperfusion injury (IRI) leads to RNA release with subsequent TLR3 activation in transplanted hearts. METHODS Human endothelial cells were subjected to IRI and treated with TLR3 agonist polyinosinic–polycytidylic acid or a TLR3/double-stranded RNA complex inhibitor. TLR3 activation was analysed using reporter cells. Gene expression profiles were evaluated via next-generation sequencing. Neutrophil adhesion was assessed in vitro. Syngeneic heart transplantation of wild-type or Tlr3−/− mice was performed following 9 h of cold ischaemia. Hearts were analysed for inflammatory gene expression, cardiac damage, apoptosis and infiltrating leucocytes. RESULTS IRI resulted in RNA release with subsequent activation of TLR3. Treatment with a TLR3 inhibitor abrogated the inflammatory response upon IRI. In parallel, TLR3 stimulation caused activation of the inflammasome. Endothelial IRI resulted in TLR3-dependent adhesion of neutrophils. Tlr3 −/− animals showed reduced intragraft and splenic messenger ribonucleic acid (mRNA) expression of proinflammatory cytokines, resulting in decreased myocardial damage, apoptosis and infiltrating cells. Tlr3 deficiency protected from cardiac damage, apoptosis and leucocyte infiltration after cardiac transplantation. CONCLUSIONS We uncover the release of RNA by injured cells with subsequent activation of TLR3 as a crucial pathomechanism of IRI. Our data indicate that TLR3 represents a novel target in the prevention of IRI in solid organ transplantation. [ABSTRACT FROM AUTHOR]

Protein misfolding results in the accumulation of aggregated β-sheet-rich structures in Parkinson's disease (PD) and Alzheimer's disease. The toxic oligomer hypothesis stipulates that prefibrillar assemblies, such as soluble oligomers or protofibrils, are responsible for the poor prognosis of these diseases. Previous studies demonstrated that a small molecule related to the natural compound orcein, O4, directly binds to amyloid-β fibrils and stabilizes them, accelerating the formation of end-stage mature fibrils. Here we demonstrate a similar phenomenon during O4 treatment of α-synuclein (αsyn) aggregates, the protein responsible for PD pathology. While the drug did not change the kinetics of aggregate formation as measured by the amyloidophilic dye thioflavin T, O4 depleted αsyn oligomers and promoted the formation of sodium dodecyl sulfate and proteinase K resistant aggregates consisting of large fibril clusters. These fibril clusters exhibited reduced toxicity to human neuronal model cells and reduced seeding activity in vitro. The effectiveness of O4 decreased when it was added at later points in the αsyn aggregation pathway, which suggests that the incorporation of O4 into fibril assemblies stabilizes them against chemical, enzymatic, and mechanic degradation. These findings suggest that small molecules, which stabilize amyloid fibrils, can prevent fibril fragmentation and seeding and consequently prevent prion-like replication of misfolded αsyn. Inhibiting prion replication by fibril stabilization could thus be a therapeutic strategy for PD. [ABSTRACT FROM AUTHOR]

Motor functions are often guided by sensory experience, most convincingly illustrated by complex learned behaviors. Key to sensory guidance in motor areas may be the structural and functional organization of sensory inputs and their evoked responses. We study sensory responses in large populations of neurons and neuron-assistive cells in the songbird motor area HVC, an auditory-vocal brain area involved in sensory learning and in adult song production. HVC spike responses to auditory stimulation display remarkable preference for the bird's own song (BOS) compared to other stimuli. Using two-photon calcium imaging in anesthetized zebra finches we measure the spatio-temporal structure of baseline activity and of auditory evoked responses in identified populations of HVC cells. We find strong correlations between calcium signal fluctuations in nearby cells of a given type, both in identified neurons and in astroglia. In identified HVC neurons only, auditory stimulation decorrelates ongoing calcium signals, less for BOS than for other sound stimuli. Overall, calcium transients show strong preference for BOS in identified HVC neurons but not in astroglia, showing diversity in local functional organization among identified neuron and astroglia populations. [ABSTRACT FROM AUTHOR]

Background: Coronary artery disease (CAD) remains a severe socio-economic burden in the Western world. Coronary obstruction and subsequent myocardial ischemia result in the progressive replacement of contractile myocardium with dysfunctional, fibrotic scar tissue. Post-infarctional remodelling is causal for the concomitant decline of left-ventricular function and the fatal syndrome of heart failure. Available neurohumoral treatment strategies aim at the improvement of symptoms. Despite extensive research, therapeutic options for myocardial regeneration, including (stem)-cell therapy, gene therapy, cellular reprogramming or tissue engineering, remain purely experimental. Thus, there is an urgent clinical need for novel treatment options for inducing myocardial regeneration and improving left-ventricular function in ischemic cardiomyopathy. Shockwave therapy (SWT) is a well-established regenerative tool that is effective for the treatment of chronic tendonitis, long-bone non-union and wound-healing disorders. In preclinical trials, SWT regenerated ischemic myocardium via the induction of angiogenesis and the reduction of fibrotic scar tissue, resulting in improved left-ventricular function. Methods: In this prospective, randomized controlled, single-blind, monocentric study, 80 patients with reduced left-ventricular ejection fraction (LVEF≤ 40%) are subjected to coronary-artery bypass-graft surgery (CABG) surgery and randomized in a 1:1 ratio to receive additional cardiac SWT (intervention group; 40 patients) or CABG surgery with sham treatment (control group; 40 patients). This study aims to evaluate (1) the safety and (2) the efficacy of cardiac SWT as adjunctive treatment during CABG surgery for the regeneration of ischemic myocardium. The primary endpoints of the study represent (1) major cardiac events and (2) changes in left-ventricular function 12 months after treatment. Secondary endpoints include 6-min walk test distance, improvement of symptoms and assessment of quality of life. Discussion: This study aims to investigate the safety and efficacy of cardiac SWT during CABG surgery for myocardial regeneration. The induction of angiogenesis, decrease of fibrotic scar tissue formation and, thus, improvement of left-ventricular function could lead to improved quality of life and prognosis for patients with ischemic heart failure. Thus, it could become the first clinically available treatment strategy for the regeneration of ischemic myocardium alleviating the socio-economic burden of heart failure. Trial registration: ClinicalTrials.gov NCT03859466. Registered on 1 March 2019. [ABSTRACT FROM AUTHOR]

Proteins and peptides use their amino acids as medium for electron-transfer reactions that occur either in single-step superexchange or in multistep hopping processes. Whereas the rate of the single-step electron transfer dramatically decreases with the distance, a hopping process is less distance dependent. Electron hopping is possible if amino acids carry oxidizable side chains, like the phenol group in tyrosine. These side chains become intermediate charge carriers. Because of the weak distance dependency of hopping processes, fast electron transfer over very long distances occurs in multistep reactions, as in the enzyme ribonucleotide reductase. [Copyright &y& Elsevier]

OBJECTIVES Barlow's disease is a specific sub-form of mitral valve (MV) disease, characterized by diffuse excessive tissue and multi segment prolapse. The anterolateral mini-thoracotomy represents the standard access for MV regurgitation in many centres. It still remains unclear which surgical technique provides the best results. Therefore, the aim of this study was to compare operative safety and mid-term outcomes after (i) isolated annuloplasty, (ii) use of additional artificial chordae or (iii) leaflet resection in patients suffering from Barlow's disease undergoing minimally invasive MV repair. METHODS A consecutive series of patients suffering from Barlow′s disease undergoing minimally invasive MV surgery between 2001 and 2020 were analysed (n  = 246). Patients were grouped and analysed according to the used surgical technique. The primary outcome was a modified Mitral Valve Academic Research Consortium combined end-point of mortality, reoperation due to repair failure or reoccurrence of severe mitral regurgitation within 5 years. The secondary outcome included operative success and safety up to 30 days. RESULTS No significant difference was found between the 3 surgical techniques with regard to operative safety (P  = 0.774). The primary outcome did not differ between groups (P  = 0.244). Operative success was achieved in 93.5% and was lowest in the isolated annuloplasty group (77.1%). Conversion to MV replacement was increased in patients undergoing isolated annuloplasty (P  < 0.001). CONCLUSIONS Isolated annuloplasty, use of additional artificial chordae and leaflet resection represent feasible techniques in Barlow patients undergoing minimally invasive MV surgery with comparable 5-year results. In view of the increased conversion rate in the annuloplasty group, the pathology should not be oversimplified. [ABSTRACT FROM AUTHOR]

Consciousness is lost within seconds upon cessation of cerebral blood flow. The brain cannot store oxygen, and interruption of oxidative phosphorylation is fatal within minutes. Yet only rudimentary knowledge exists regarding cortical partial oxygen tension (PO2) dynamics under physiological conditions. Here we introduce Green enhanced Nano-lantern (GeNL), a genetically encoded bioluminescent oxygen indicator for PO2 imaging. In awake behaving mice, we uncover the existence of spontaneous, spatially defined “hypoxic pockets” and demonstrate their linkage to the abrogation of local capillary flow. Exercise reduced the burden of hypoxic pockets by 52% compared with rest. The study provides insight into cortical oxygen dynamics in awake behaving animals and concurrently establishes a tool to delineate the importance of oxygen tension in physiological processes and neurological diseases. [ABSTRACT FROM AUTHOR]

Background: Coronary artery diseases (CAD) remains a severe socio-economic burden in the Western world. Coronary obstruction and subsequent myocardial ischemia result in progressive replacement of contractile myocardium with dysfunctional, fibrotic scar tissue. Post-infarctional remodeling is causal for the concomitant decline of left-ventricular function and the fatal syndrome of heart failure. Available neurohumoral treatment strategies aim at the improvement of symptoms. Despite extensive research, therapeutic options for myocardial regeneration, including (stem)-cell therapy, gene therapy, cellular reprogramming or tissue engineering, remain purely experimental. Thus, there is an urgent clinical need for novel treatment options for inducing myocardial regeneration and improving left-ventricular function in ischemic cardiomyopathy. Shockwave Therapy (SWT) is a well-established regenerative tool that is effective for the treatment of chronic tendonitis, long-bone non-union and wound-healing disorders. In preclinical trials, SWT regenerated ischemic myocardium via the induction of angiogenesis and the reduction of fibrotic scar tissue, resulting in improved left-ventricular function.Methods/design: In this prospective, randomized controlled, single-blind, monocentric study, 80 patients with reduced left-ventricular ejection fraction (LVEF≤ 40%) are subjected to coronary-artery bypass-graft surgery (CABG) surgery and randomized in a 1:1 ratio to receive additional cardiac SWT (intervention group; 40 patients) or CABG surgery with sham treatment (control group; 40 patients). This study aims to evaluate (1) the safety and (2) the efficacy of cardiac SWT as adjunctive treatment during CABG surgery for the regeneration of ischemic myocardium. The primary endpoints of the study represent (1) major cardiac events and (2) changes in left-ventricular function 12 months after treatment. Secondary endpoints include 6-min Walk Test distance, improvement of symptoms and assessment of quality of life.Discussion: This study aims to investigate the safety and efficacy of cardiac SWT during CABG surgery for myocardial regeneration. The induction of angiogenesis, decrease of fibrotic scar tissue formation and, thus, improvement of left-ventricular function could lead to improved quality of life and prognosis for patients with ischemic heart failure. Thus, it could become the first clinically available treatment strategy for the regeneration of ischemic myocardium alleviating the socio-economic burden of heart failure.Trial Registration: ClinicalTrials.gov, ID: NCT03859466. Registered on 1 March 2019. [ABSTRACT FROM AUTHOR]

Aims As many current approaches for heart regeneration exert unfavourable side effects, the induction of endogenous repair mechanisms in ischaemic heart disease is of particular interest. Recently, exosomes carrying angiogenic miRNAs have been described to improve heart function. However, it remains challenging to stimulate specific release of reparative exosomes in ischaemic myocardium. In the present study, we sought to test the hypothesis that the physical stimulus of shock wave therapy (SWT) causes the release of exosomes. We aimed to substantiate the pro-angiogenic impact of the released factors, to identify the nature of their cargo, and to test their efficacy in vivo supporting regeneration and recovery after myocardial ischaemia. Methods and results Mechanical stimulation of ischaemic muscle via SWT caused extracellular vesicle (EV) release from endothelial cells both in vitro and in vivo. Characterization of EVs via electron microscopy, nanoparticle tracking analysis and flow cytometry revealed specific exosome morphology and size with the presence of exosome markers CD9, CD81, and CD63. Exosomes exhibited angiogenic properties activating protein kinase b (Akt) and extracellular-signal regulated kinase (ERK) resulting in enhanced endothelial tube formation and proliferation. A miRNA array and transcriptome analysis via next-generation sequencing were performed to specify exosome content. miR-19a-3p was identified as responsible cargo, antimir-19a-3p antagonized angiogenic exosome effects. Exosomes and target miRNA were injected intramyocardially in mice after left anterior descending artery ligation. Exosomes resulted in improved vascularization, decreased myocardial fibrosis, and increased left ventricular ejection fraction as shown by transthoracic echocardiography. Conclusion The mechanical stimulus of SWT causes release of angiogenic exosomes. miR-19a-3p is the vesicular cargo responsible for the observed effects. Released exosomes induce angiogenesis, decrease myocardial fibrosis, and improve left ventricular function after myocardial ischaemia. Exosome release via SWT could develop an innovative approach for the regeneration of ischaemic myocardium. [ABSTRACT FROM AUTHOR]

The relevance of perioperative myocardial injury (PMI) after cardiac surgery for 30-day mortality and long-term survival remains to be determined. This study assessed the association of PMI after cardiac surgery, reflected by postoperative troponin release, with 30-day mortality and long-term survival after: 1) coronary artery bypass grafting (CABG); 2) isolated aortic valve replacement (AVR) surgery; and 3) all other cardiac surgeries. A consecutive cohort of 8,292 patients undergoing cardiac surgery with serial perioperative high-sensitivity cardiac troponin T (hs-cTnT) measurements was retrospectively analyzed. The relationship between postoperative hs-cTnT release and 30-day mortality or 5-year mortality was analyzed after adjustment with EuroSCORE II using a Cox proportional hazards model. hs-cTnT thresholds for 30-day and 5-year mortality were determined for isolated CABG (32.3%), AVR (14%), and other cardiac surgery (53.8%). High postoperative hs-cTnT levels were associated with higher 30-day mortality but not 5-year mortality. In CABG, median peak concentration of postoperative hs-cTnT was 1,044 ng/L, in AVR it was 502 ng/L, and in other cardiac surgery it was 1,110 ng/L. hs-cTnT thresholds defining mortality-associated PMI were as follows: for CABG, 2,385 ng/L (170× the upper reference limit of normal in a seemingly healthy population [URL]); for AVR, 568 ng/L (41× URL); and for other cardiac procedures, 1,873 ng/L (134× URL). hs-cTnT levels above the cutoffs resulted in an HR for 30-day mortality for CABG of 12.56 (P < 0.001), for AVR of 4.44 (P = 0.004), and for other cardiac surgery of 3.97 (P < 0.001). PMI reflected by perioperative hs-cTnT release is associated with the expected 30-day mortality but not 5-year mortality. Postoperative hs-cTnT cutoffs to identify survival-relevant PMI are higher than suggested in current definitions. [Display omitted] [ABSTRACT FROM AUTHOR]

Database search engines for bottom‐up proteomics largely ignore peptide fragment ion intensities during the automated scoring of tandem mass spectra against protein databases. Recent advances in deep learning allow the accurate prediction of peptide fragment ion intensities. Using these predictions to calculate additional intensity‐based scores helps to overcome this drawback.Here, we describe a processing workflow termed INFERYS™ rescoring for the intensity‐based rescoring of Sequest HT search engine results in Thermo Scientific™ Proteome Discoverer™ 2.5 software. The workflow is based on the deep learning platform INFERYS capable of predicting fragment ion intensities, which runs on personal computers without the need for graphics processing units. This workflow calculates intensity‐based scores comparing peptide spectrum matches from Sequest HT and predicted spectra. Resulting scores are combined with classical search engine scores for input to the false discovery rate estimation tool Percolator.We demonstrate the merits of this approach by analyzing a classical HeLa standard sample and exemplify how this workflow leads to a better separation of target and decoy identifications, in turn resulting in increased peptide spectrum match, peptide and protein identification numbers. On an immunopeptidome dataset, this workflow leads to a 50% increase in identified peptides, emphasizing the advantage of intensity‐based scores when analyzing low‐intensity spectra or analytes with very similar physicochemical properties that require vast search spaces.Overall, the end‐to‐end integration of INFERYS rescoring enables simple and easy access to a powerful enhancement to classical database search engines, promising a deeper, more confident and more comprehensive analysis of proteomic data from any organism by unlocking the intensity dimension of tandem mass spectra for identification and more confident scoring. [ABSTRACT FROM AUTHOR]

Shockwave therapy (SWT) represents a promising regenerative treatment option for patients with ischemic cardiomyopathy. Although no side-effects have been described upon SWT, potential cellular damage at therapeutic energies has not been addressed so far. In this work, we aimed to define a therapeutic range for shock wave application for myocardial regeneration. We could demonstrate that SWT does not induce cellular damage beneath energy levels of 0.27 mJ/mm2 total flux density. Endothelial cell proliferation, angiogenic gene expression and phosphorylation of AKT and ERK are enhanced in a dose dependent manner until 0.15 mJ/mm2 energy flux density. SWT induces regeneration of ischemic muscle in vivo via expression of angiogenic gene expression, enhanced neovascularization and improved limb perfusion in a dose-dependent manner. Therefore, we provide evidence for a dose-dependent induction of angiogenesis after SWT, as well as the absence of cellular damage upon SWT within the therapeutic range. These data define for the first time a therapeutic range of SWT, a promising regenerative treatment option for ischemic cardiomyopathy. [ABSTRACT FROM AUTHOR]

A peptide assay was developed that allows the measurement of electron-transfer (ET) efficiencies in peptides. It turns out that two-step ET processes are faster than single-step reactions. This requires relay amino acids with appropriate redox potentials. Not only aromatic but also sulfur-containing aliphatic amino acids can act as stepping stones for the charge. With tryptophan, histidine, and cysteine the reaction is a more complex proton-coupled ET. [ABSTRACT FROM AUTHOR]

Lamins are important filaments forming the inner nuclear membrane. Lamin A is processed by zinc metalloproteinase (ZMPSTE24). Failure to cleave a truncated form of prelamin A—also called progerin—causes Hutchinson–Gilford progeria syndrome a well-known premature aging disease. Minor levels of progerin are readily expressed in the blood of healthy individuals due to alternative splicing. Previously, we found an association of increased progerin mRNA with overweight and chronic inflammation (hs-CRP). Here, we aimed to elucidate correlations of ZMPSTE24, lamin A/C and progerin with the inflammatory marker hs-CRP. In this retrospective, cross-sectional study we analyzed blood samples from 110 heart failure patients for quantitative mRNA expression of ZMPSTE24, lamin A/C, progerin and hs-CRP protein. Spearman correlations and linear regression analyses including adjustments for age, gender and ejection fraction showed a significant positive correlation of lnprogerin with lnZMPSTE24 (n = 110; r = 0.33; p = 0.0004) and lnlamin A/C (n = 110; r = 0.82, p < 0.0001), whereas no association was observed between lnlamin A/C and lnZMPSTE24 expression. Further analyses showed a significant positive correlation of lnhs-CRP with lnZMPSTE24 (n = 110; r = 0.21; p = 0.01) and lnlamin A/C (n = 110; r = 0.24; p = 0.03). We conclude that chronic inflammation is associated with increased expression of ZMPSTE24 and lamin A/C mRNA. Both markers also positively correlate with increased expression of the premature aging marker progerin which may be linked to cardiovascular aging. [ABSTRACT FROM AUTHOR]