Philipp S. Wild

Phosphorylation of an autophagy receptor restricts pathogenic cytosolic bacterial growth.

Autophagy is the cellular homeostatic pathway that delivers large cytosolic materials for degradation in the lysosome. Recent evidence indicates that autophagy mediates selective removal of protein aggregates, organelles and microbes in cells. Yet, the specificity in targeting a particular substrate to the autophagy pathway remains poorly understood. Here, we show that the mitochondrial protein Nix is a selective autophagy receptor by binding to LC3/GABARAP proteins, ubiquitin‐like modifiers that are required for the growth of autophagosomal membranes. In cultured cells, Nix recruits GABARAP‐L1 to damaged mitochondria through its amino‐terminal LC3‐interacting region. Furthermore, ablation of the Nix:LC3/GABARAP interaction retards mitochondrial clearance in maturing murine reticulocytes. Thus, Nix functions as an autophagy receptor, which mediates mitochondrial clearance after mitochondrial damage and during erythrocyte differentiation.

Autophagy is a catabolic process where cytosolic cellular components are delivered to the lysosome for degradation. Recent studies have indicated the existence of specific receptors, such as p62, which link ubiquitinated targets to autophagosomal degradation pathways. Here we show that NBR1 (neighbor of BRCA1 gene 1) is an autophagy receptor containing LC3- and ubiquitin (Ub)-binding domains. NBR1 is recruited to Ub-positive protein aggregates and degraded by autophagy depending on an LC3-interacting region (LIR) and LC3 family modifiers. Although NBR1 and p62 interact and form oligomers, they can function independently, as shown by autophagosomal clearance of NBR1 in p62-deficient cells. NBR1 was localized to Ub-positive inclusions in patients with liver dysfunction, and depletion of NBR1 abolished the formation of Ub-positive p62 bodies upon puromycin treatment of cells. We propose that NBR1 and p62 act as receptors for selective autophagosomal degradation of ubiquitinated targets.

Chronic kidney disease (CKD) is a significant public health problem, and recent genetic studies have identified common CKD susceptibility variants. The CKDGen consortium performed a meta-analysis of genome-wide association data in 67,093 individuals of European ancestry from 20 predominantly population-based studies in order to identify new susceptibility loci for reduced renal function as estimated by serum creatinine (eGFRcrea), serum cystatin c (eGFRcys) and CKD (eGFRcrea < 60 ml/min/1.73 m(2); n = 5,807 individuals with CKD (cases)). Follow-up of the 23 new genome-wide-significant loci (P < 5 x 10(-8)) in 22,982 replication samples identified 13 new loci affecting renal function and CKD (in or near LASS2, GCKR, ALMS1, TFDP2, DAB2, SLC34A1, VEGFA, PRKAG2, PIP5K1B, ATXN2, DACH1, UBE2Q2 and SLC7A9) and 7 loci suspected to affect creatinine production and secretion (CPS1, SLC22A2, TMEM60, WDR37, SLC6A13, WDR72 and BCAS3). These results further our understanding of the biologic mechanisms of kidney function by identifying loci that potentially influence nephrogenesis, podocyte function, angiogenesis, solute transport and metabolic functions of the kidney.

Significance Selective autophagy of damaged mitochondria (mitophagy) requires protein kinases PINK1 and TBK1, ubiquitin ligase Parkin, and autophagy receptors such as OPTN, driving ubiquitin-labeled mitochondria into autophagosomes. Because all proteins have been genetically linked to either Parkinson’s disease (PINK1 and Parkin) or amyotrophic lateral sclerosis and frontotemporal lobar degeneration (TBK1 and OPTN), it is of great interest to understand their physiological functions. By utilizing quantitative proteomics we show that TBK1 phosphorylates four receptors on several autophagy-relevant sites. Constitutive interaction of TBK1 with OPTN and the ability of OPTN to bind to ubiquitin chains are essential for TBK1 recruitment and activation on mitochondria. TBK1-mediated phosphorylation of OPTN creates a signal amplification loop through combining recruitment and retention of OPTN/TBK1 on ubiquitinated mitochondria.

Continuous synthesis of all cellular components requires their constant turnover in order for a cell to achieve homeostasis. To this end, eukaryotic cells are endowed with two degradation pathways – the ubiquitin-proteasome system and the lysosomal pathway. The latter pathway is partly fed by autophagy, which targets intracellular material in distinct vesicles, termed autophagosomes, to the lysosome. Central to this pathway is a set of key autophagy proteins, including the ubiquitin-like modifier Atg8, that orchestrate autophagosome initiation and biogenesis. In higher eukaryotes, the Atg8 family comprises six members known as the light chain 3 (LC3) or γ-aminobutyric acid (GABA)-receptor-associated protein (GABARAP) proteins. Considerable effort during the last 15 years to decipher the molecular mechanisms that govern autophagy has significantly advanced our understanding of the functioning of this protein family. In this Cell Science at a Glance article and the accompanying poster, we present the current LC3 protein interaction network, which has been and continues to be vital for gaining insight into the regulation of autophagy.

Elevated intraocular pressure (IOP) is an important risk factor in developing glaucoma, and variability in IOP might herald glaucomatous development or progression. We report the results of a genome-wide association study meta-analysis of 18 population cohorts from the International Glaucoma Genetics Consortium (IGGC), comprising 35,296 multi-ancestry participants for IOP. We confirm genetic association of known loci for IOP and primary open-angle glaucoma (POAG) and identify four new IOP-associated loci located on chromosome 3q25.31 within the FNDC3B gene (P = 4.19 × 10(-8) for rs6445055), two on chromosome 9 (P = 2.80 × 10(-11) for rs2472493 near ABCA1 and P = 6.39 × 10(-11) for rs8176693 within ABO) and one on chromosome 11p11.2 (best P = 1.04 × 10(-11) for rs747782). Separate meta-analyses of 4 independent POAG cohorts, totaling 4,284 cases and 95,560 controls, showed that 3 of these loci for IOP were also associated with POAG.

Several methods of noninvasive vascular function testing have been suggested for cardiovascular risk screening in the community. A direct comparison of the different methods and their relation to classical cardiovascular risk factors in a large cohort is missing.In 5000 individuals (mean age, 55.5 ± 10.9 years; age range, 35 to 74 years; women, 49.2%) of the population-based Gutenberg Heart Study, we performed simultaneous measurement of flow-mediated dilation (FMD) and peripheral arterial volume pulse determined by infrared photo (reflection index) and pneumatic plethysmography (PAT) and explored their associations. All function measures were recorded at baseline and after reactive hyperemia induced by 5-minute brachial artery occlusion. Correlations between different measures of vascular function were statistically significant but moderate. The strongest association for hyperemic response variables was observed for PAT ratio and FMD (Spearman r = 0.17; age- and sex-adjusted partial correlation, 0.068). Classical risk factors explained between 15.8% (baseline reflection index) and 58.4% (brachial artery diameter) of the baseline values but only accounted for 3.2% (reflection index), 15.4% (FMD), and 13.9% (PAT ratio) of the variability of reflective hyperemic response. Regression models varied in their relations to classical risk factors for the individual vascular function measures. Consistently associated with different vascular function methods were age, sex, body mass index, and indicators of hypertension. Peripheral tonometry also showed a relation to fasting glucose concentrations.Noninvasive measures of conduit artery and peripheral arterial function are modestly correlated, differ in their relation to classical cardiovascular risk factors, and may thus reflect different pathologies.

Dysfunctional mitochondria show a reduced capacity for fusion and, as mitochondrial fission is maintained, become spatially separated from the intact network. By that mechanism, dysfunctional mitochondria have been proposed to be targeted for selective degradation by mitophagy, thereby providing a quality control system for mitochondria. In yeast, conflicting results concerning the role of mitochondrial dynamics in mitophagy have been reported. Here, we investigate the effects on mitophagy of altering mitochondrial fission and fusion, using biochemical, as well as fluorescence-based, assays. Rapamycin-induced mitophagy was shown to depend upon the autophagy-related proteins Atg11, Atg20 and Atg24, confirming that a selective type of autophagy occurred. Both fragmentation of mitochondria and inhibition of oxidative phosphorylation were not sufficient to trigger mitophagy, and neither deletion of the fission factors Dnm1, Fis1, Mdv1 or Caf4 nor expression of dominant-negative variants of Dnm1 impaired mitophagy. The diminished mitophagy initially observed in a Δfis1 mutant was not due to the absence of Fis1 but rather due to a secondary mutation in WHI2, which encodes a factor reported to function in the general stress response and the Ras-protein kinase A (PKA) signaling pathway. We propose that, in yeast, mitochondrial fission is not a prerequisite for the selective degradation of mitochondria, and that mitophagy is linked to the general stress response and the Ras-PKA signaling pathway.

Ubiquitin chains modify a major subset of the proteome, but detection of ubiquitin signaling dynamics and localization is limited due to a lack of appropriate tools. Here, we employ ubiquitin-binding domain (UBD)-based fluorescent sensors to monitor linear and K63-linked chains in vitro and in vivo. We utilize the UBD in NEMO and ABIN (UBAN) for detection of linear chains, and RAP80 ubiquitin-interacting motif (UIM) and TAB2 Npl4 zinc finger (NZF) domains to detect K63 chains. Linear and K63 sensors decorated the ubiquitin coat surrounding cytosolic Salmonella during bacterial autophagy, whereas K63 sensors selectively monitored Parkin-induced mitophagy and DNA damage responses in fixed and living cells. In addition, linear and K63 sensors could be used to monitor endogenous signaling pathways, as demonstrated by their ability to differentially interfere with TNF- and IL-1-induced NF-κB pathway. We propose that UBD-based biosensors could serve as prototypes to track and trace other chain types and ubiquitin-like signals in vivo.

eQTL analyses are important to improve the understanding of genetic association results. We performed a genome-wide association and global gene expression study to identify functionally relevant variants affecting the risk of coronary artery disease (CAD).In a genome-wide association analysis of 2078 CAD cases and 2953 control subjects, we identified 950 single-nucleotide polymorphisms (SNPs) that were associated with CAD at P<10(-3). Subsequent in silico and wet-laboratory replication stages and a final meta-analysis of 21 428 CAD cases and 38 361 control subjects revealed a novel association signal at chromosome 10q23.31 within the LIPA (lysosomal acid lipase A) gene (P=3.7×10(-8); odds ratio, 1.1; 95% confidence interval, 1.07 to 1.14). The association of this locus with global gene expression was assessed by genome-wide expression analyses in the monocyte transcriptome of 1494 individuals. The results showed a strong association of this locus with expression of the LIPA transcript (P=1.3×10(-96)). An assessment of LIPA SNPs and transcript with cardiovascular phenotypes revealed an association of LIPA transcript levels with impaired endothelial function (P=4.4×10(-3)).The use of data on genetic variants and the addition of data on global monocytic gene expression led to the identification of the novel functional CAD susceptibility locus LIPA, located on chromosome 10q23.31. The respective eSNPs associated with CAD strongly affect LIPA gene expression level, which was related to endothelial dysfunction, a precursor of CAD.

Elevated concentrations of albumin in the urine, albuminuria, are a hallmark of diabetic kidney disease and are associated with an increased risk for end-stage renal disease and cardiovascular events. To gain insight into the pathophysiological mechanisms underlying albuminuria, we conducted meta-analyses of genome-wide association studies and independent replication in up to 5,825 individuals of European ancestry with diabetes and up to 46,061 without diabetes, followed by functional studies. Known associations of variants in CUBN, encoding cubilin, with the urinary albumin-to-creatinine ratio (UACR) were confirmed in the overall sample (P = 2.4 × 10−10). Gene-by-diabetes interactions were detected and confirmed for variants in HS6ST1 and near RAB38/CTSC. Single nucleotide polymorphisms at these loci demonstrated a genetic effect on UACR in individuals with but not without diabetes. The change in the average UACR per minor allele was 21% for HS6ST1 (P = 6.3 × 10–7) and 13% for RAB38/CTSC (P = 5.8 × 10−7). Experiments using streptozotocin-induced diabetic Rab38 knockout and control rats showed higher urinary albumin concentrations and reduced amounts of megalin and cubilin at the proximal tubule cell surface in Rab38 knockout versus control rats. Relative expression of RAB38 was higher in tubuli of patients with diabetic kidney disease compared with control subjects. The loci identified here confirm known pathways and highlight novel pathways influencing albuminuria.

Binding of epidermal growth factor (EGF) to its receptor leads to receptor dimerization, assembly of protein complexes, and activation of signaling networks that control key cellular responses. Despite their fundamental role in cell biology, little is known about protein complexes associated with the EGF receptor (EGFR) before growth factor stimulation. We used a modified membrane yeast two-hybrid system together with bioinformatics to identify 87 candidate proteins interacting with the ligand-unoccupied EGFR. Among them was histone deacetylase 6 (HDAC6), a cytoplasmic lysine deacetylase, which we found negatively regulated EGFR endocytosis and degradation by controlling the acetylation status of alpha-tubulin and, subsequently, receptor trafficking along microtubules. A negative feedback loop consisting of EGFR-mediated phosphorylation of HDAC6 Tyr(570) resulted in reduced deacetylase activity and increased acetylation of alpha-tubulin. This study illustrates the complexity of the EGFR-associated interactome and identifies protein acetylation as a previously unknown regulator of receptor endocytosis and degradation.

While DNA replication and mitosis occur in a sequential manner, precisely how cells maintain their temporal separation and order remains elusive. Here, we unveil a double-negative feedback loop between replication intermediates and an M-phase-specific structure-selective endonuclease, MUS81-SLX4, which renders DNA replication and mitosis mutually exclusive. MUS81 nuclease is constitutively active throughout the cell cycle but requires association with SLX4 for efficient substrate targeting. To preclude toxic processing of replicating chromosomes, WEE1 kinase restrains CDK1 and PLK1-mediated MUS81-SLX4 assembly during S phase. Accordingly, WEE1 inhibition triggers widespread nucleolytic breakage of replication intermediates, halting DNA replication and leading to chromosome pulverization. Unexpectedly, premature entry into mitosis—licensed by unrestrained CDK1 activity during S phase—requires MUS81-SLX4, which inhibits DNA replication. This suggests that ongoing replication assists WEE1 in delaying entry into M phase and, indirectly, in preventing MUS81-SLX4 assembly. Conversely, MUS81-SLX4 activation during mitosis promotes targeted resolution of persistent replication intermediates, which safeguards chromosome segregation.

Abstract Aims To investigate the efficacy and safety of early transition from hospital to ambulatory treatment in low-risk acute PE, using the oral factor Xa inhibitor rivaroxaban. Methods and results We conducted a prospective multicentre single-arm investigator initiated and academically sponsored management trial in patients with acute low-risk PE (EudraCT Identifier 2013-001657-28). Eligibility criteria included absence of (i) haemodynamic instability, (ii) right ventricular dysfunction or intracardiac thrombi, and (iii) serious comorbidities. Up to two nights of hospital stay were permitted. Rivaroxaban was given at the approved dose for PE for ≥3 months. The primary outcome was symptomatic recurrent venous thromboembolism (VTE) or PE-related death within 3 months of enrolment. An interim analysis was planned after the first 525 patients, with prespecified early termination of the study if the null hypothesis could be rejected at the level of α = 0.004 (&amp;lt;6 primary outcome events). From May 2014 through June 2018, consecutive patients were enrolled in seven countries. Of the 525 patients included in the interim analysis, three (0.6%; one-sided upper 99.6% confidence interval 2.1%) suffered symptomatic non-fatal VTE recurrence, a number sufficiently low to fulfil the condition for early termination of the trial. Major bleeding occurred in 6 (1.2%) of the 519 patients comprising the safety population. There were two cancer-related deaths (0.4%). Conclusion Early discharge and home treatment with rivaroxaban is effective and safe in carefully selected patients with acute low-risk PE. The results of the present trial support the selection of appropriate patients for ambulatory treatment of PE.

Genetic determinants of peripheral arterial disease (PAD) remain largely unknown. To identify genetic variants associated with the ankle-brachial index (ABI), a noninvasive measure of PAD, we conducted a meta-analysis of genome-wide association study data from 21 population-based cohorts.Continuous ABI and PAD (ABI ≤0.9) phenotypes adjusted for age and sex were examined. Each study conducted genotyping and imputed data to the ≈2.5 million single nucleotide polymorphisms (SNPs) in HapMap. Linear and logistic regression models were used to test each SNP for association with ABI and PAD using additive genetic models. Study-specific data were combined using fixed effects inverse variance weighted meta-analyses. There were a total of 41 692 participants of European ancestry (≈60% women, mean ABI 1.02 to 1.19), including 3409 participants with PAD and with genome-wide association study data available. In the discovery meta-analysis, rs10757269 on chromosome 9 near CDKN2B had the strongest association with ABI (β=-0.006, P=2.46×10(-8)). We sought replication of the 6 strongest SNP associations in 5 population-based studies and 3 clinical samples (n=16 717). The association for rs10757269 strengthened in the combined discovery and replication analysis (P=2.65×10(-9)). No other SNP associations for ABI or PAD achieved genome-wide significance. However, 2 previously reported candidate genes for PAD and 1 SNP associated with coronary artery disease were associated with ABI: DAB21P (rs13290547, P=3.6×10(-5)), CYBA (rs3794624, P=6.3×10(-5)), and rs1122608 (LDLR, P=0.0026).Genome-wide association studies in more than 40 000 individuals identified 1 genome wide significant association on chromosome 9p21 with ABI. Two candidate genes for PAD and 1 SNP for coronary artery disease are associated with ABI.

<h2>Summary</h2> The host endolysosomal compartment is often manipulated by intracellular bacterial pathogens. <i>Salmonella</i> (<i>Salmonella enterica serovar</i> Typhimurium) secrete numerous effector proteins, including SifA, through a specialized type III secretion system to hijack the host endosomal system and generate the <i>Salmonella</i>-containing vacuole (SCV). To form this replicative niche, <i>Salmonella</i> targets the Rab7 GTPase to recruit host membranes through largely unknown mechanisms. We show that Pleckstrin homology domain-containing protein family member 1 (PLEKHM1), a lysosomal adaptor, is targeted by <i>Salmonella</i> through direct interaction with SifA. By binding the PLEKHM1 PH2 domain, <i>Salmonella</i> utilize a complex containing PLEKHM1, Rab7, and the HOPS tethering complex to mobilize phagolysosomal membranes to the SCV. Depletion of PLEKHM1 causes a profound defect in SCV morphology with multiple bacteria accumulating in enlarged structures and significantly dampens <i>Salmonella</i> proliferation in multiple cell types and mice. Thus, PLEKHM1 provides a critical interface between pathogenic infection and the host endolysosomal system.

Tinnitus is a common symptom reported in otolaryngologic practice. Although the pathophysiology of tinnitus has not been fully understood, clinical studies suggest that psychological symptoms of depression, anxiety, and somatization are increased in tinnitus patients. However, patients seeking medical treatment for tinnitus may be especially vulnerable. Population-based studies reporting on the association between tinnitus and psychological distress are still lacking. The aim of this study was to investigate the correlation of tinnitus with depression, anxiety, or somatization in a large population-based cohort. The Gutenberg Health Study is a population-based cohort study. Participants were asked about the occurrence of tinnitus (yes/no) and how much they were bothered by it. In addition, they completed the PHQ-9, GAD-7, and SSS-8 questionnaires to assess depressive symptoms, anxiety, and somatic symptom disorders. A total of 8539 participants were included in the study cohort. Tinnitus prevalence was 28.0% (2387). The prevalence of depression/anxiety/somatic symptom disorders was significantly higher among participants with tinnitus than among participants without tinnitus (7.9%/5.4%/40.4% participants with tinnitus vs. 4.6%/3.3%/26.9% participants without tinnitus, p-value < 0.0001). Logistic regression results showed that participants with tinnitus were more likely to suffer from depression (OR = 2.033, 95% CI [1.584; 2.601], p-value < 0.0001), anxiety (OR = 1.841, 95% CI [1.228; 2.728], p-value = 0.0027), or somatic symptom disorders (OR = 2.057, 95% CI [1.799; 2.352], p-value < 0.0001). Symptoms of depression, anxiety, and somatic symptom disorders were increased in participants with tinnitus. This must be taken into account when treating these patients.

Cardiovascular disease (CVD) is the leading cause of death worldwide and considered one of the most environmentally driven diseases. The role of DNA methylation in response to the individual exposure for the development and progression of CVD is still poorly understood and a synthesis of the evidence is lacking.A systematic review of articles examining measurements of DNA cytosine methylation in CVD was conducted in accordance with PRISMA (preferred reporting items for systematic reviews and meta-analyses) guidelines. The search yielded 5,563 articles from PubMed and CENTRAL databases. From 99 studies with a total of 87,827 individuals eligible for analysis, a database was created combining all CpG-, gene- and study-related information. It contains 74,580 unique CpG sites, of which 1452 CpG sites were mentioned in ≥ 2, and 441 CpG sites in ≥ 3 publications. Two sites were referenced in ≥ 6 publications: cg01656216 (near ZNF438) related to vascular disease and epigenetic age, and cg03636183 (near F2RL3) related to coronary heart disease, myocardial infarction, smoking and air pollution. Of 19,127 mapped genes, 5,807 were reported in ≥ 2 studies. Most frequently reported were TEAD1 (TEA Domain Transcription Factor 1) and PTPRN2 (Protein Tyrosine Phosphatase Receptor Type N2) in association with outcomes ranging from vascular to cardiac disease. Gene set enrichment analysis of 4,532 overlapping genes revealed enrichment for Gene Ontology molecular function "DNA-binding transcription activator activity" (q = 1.65 × 10-11) and biological processes "skeletal system development" (q = 1.89 × 10-23). Gene enrichment demonstrated that general CVD-related terms are shared, while "heart" and "vasculature" specific genes have more disease-specific terms as PR interval for "heart" or platelet distribution width for "vasculature." STRING analysis revealed significant protein-protein interactions between the products of the differentially methylated genes (p = 0.003) suggesting that dysregulation of the protein interaction network could contribute to CVD. Overlaps with curated gene sets from the Molecular Signatures Database showed enrichment of genes in hemostasis (p = 2.9 × 10-6) and atherosclerosis (p = 4.9 × 10-4).This review highlights the current state of knowledge on significant relationship between DNA methylation and CVD in humans. An open-access database has been compiled of reported CpG methylation sites, genes and pathways that may play an important role in this relationship.

Selective autophagy is mediated by the interaction of autophagy modifiers and autophagy receptors that also bind to ubiquitinated cargo. Optineurin is an autophagy receptor that plays a role in the clearance of cytosolic Salmonella. The interaction between receptors and modifiers is often relatively weak, with typical values for the dissociation constant in the low micromolar range. The interaction of optineurin with autophagy modifiers is even weaker, but can be significantly enhanced through phosphorylation by the TBK1 {TANK [TRAF (tumour-necrosis-factor-receptor-associated factor)-associated nuclear factor κB activator]-binding kinase 1}. In the present study we describe the NMR and crystal structures of the autophagy modifier LC3B (microtubule-associated protein light chain 3 beta) in complex with the LC3 interaction region of optineurin either phosphorylated or bearing phospho-mimicking mutations. The structures show that the negative charge induced by phosphorylation is recognized by the side chains of Arg¹¹ and Lys⁵¹ in LC3B. Further mutational analysis suggests that the replacement of the canonical tryptophan residue side chain of autophagy receptors with the smaller phenylalanine side chain in optineurin significantly weakens its interaction with the autophagy modifier LC3B. Through phosphorylation of serine residues directly N-terminally located to the phenylalanine residue, the affinity is increased to the level normally seen for receptor-modifier interactions. Phosphorylation, therefore, acts as a switch for optineurin-based selective autophagy.

Many disorders are associated with altered serum protein concentrations, including malnutrition, cancer, and cardiovascular, kidney, and inflammatory diseases. Although these protein concentrations are highly heritable, relatively little is known about their underlying genetic determinants. Through transethnic meta-analysis of European-ancestry and Japanese genome-wide association studies, we identified six loci at genome-wide significance (p < 5 × 10⁻⁸) for serum albumin (<i>HPN-SCN1B</i>, <i>GCKR-FNDC4</i>, <i>SERPINF2-WDR81</i>, <i>TNFRSF11A-ZCCHC2</i>, <i>FRMD5-WDR76</i>, and <i>RPS11-FCGRT</i>, in up to 53,190 European-ancestry and 9,380 Japanese individuals) and three loci for total protein (<i>TNFRS13B</i>, 6q21.3, and <i>ELL2</i>, in up to 25,539 European-ancestry and 10,168 Japanese individuals). We observed little evidence of heterogeneity in allelic effects at these loci between groups of European and Japanese ancestry but obtained substantial improvements in the resolution of fine mapping of potential causal variants by leveraging transethnic differences in the distribution of linkage disequilibrium. We demonstrated a functional role for the most strongly associated serum albumin locus, <i>HPN</i>, for which <i>Hpn</i> knockout mice manifest low plasma albumin concentrations. Other loci associated with serum albumin harbor genes related to ribosome function, protein translation, and proteasomal degradation, whereas those associated with serum total protein include genes related to immune function. Our results highlight the advantages of transethnic meta-analysis for the discovery and fine mapping of complex trait loci and have provided initial insights into the underlying genetic architecture of serum protein concentrations and their association with human disease.

Article17 January 2017Open Access Transparent process Dbf4-dependent kinase and the Rtt107 scaffold promote Mus81-Mms4 resolvase activation during mitosis Lissa N Princz Lissa N Princz Max Planck Institute of Biochemistry, DNA Replication and Genome Integrity, Martinsried, Germany Search for more papers by this author Philipp Wild Philipp Wild Institute of Biochemistry, Eidgenössische Technische Hochschule, Zürich, Switzerland Search for more papers by this author Julia Bittmann Julia Bittmann Max Planck Institute of Biochemistry, DNA Replication and Genome Integrity, Martinsried, Germany Search for more papers by this author F Javier Aguado F Javier Aguado Department of Biochemistry and Molecular Biology, Center for Research in Molecular Medicine and Chronic Diseases, Universidade de Santiago de Compostela, Santiago de Compostela, Spain Search for more papers by this author Miguel G Blanco Miguel G Blanco orcid.org/0000-0002-2883-7326 Department of Biochemistry and Molecular Biology, Center for Research in Molecular Medicine and Chronic Diseases, Universidade de Santiago de Compostela, Santiago de Compostela, Spain Search for more papers by this author Joao Matos Joao Matos Institute of Biochemistry, Eidgenössische Technische Hochschule, Zürich, Switzerland Search for more papers by this author Boris Pfander Corresponding Author Boris Pfander [email protected] orcid.org/0000-0003-2180-5054 Max Planck Institute of Biochemistry, DNA Replication and Genome Integrity, Martinsried, Germany Search for more papers by this author Lissa N Princz Lissa N Princz Max Planck Institute of Biochemistry, DNA Replication and Genome Integrity, Martinsried, Germany Search for more papers by this author Philipp Wild Philipp Wild Institute of Biochemistry, Eidgenössische Technische Hochschule, Zürich, Switzerland Search for more papers by this author Julia Bittmann Julia Bittmann Max Planck Institute of Biochemistry, DNA Replication and Genome Integrity, Martinsried, Germany Search for more papers by this author F Javier Aguado F Javier Aguado Department of Biochemistry and Molecular Biology, Center for Research in Molecular Medicine and Chronic Diseases, Universidade de Santiago de Compostela, Santiago de Compostela, Spain Search for more papers by this author Miguel G Blanco Miguel G Blanco orcid.org/0000-0002-2883-7326 Department of Biochemistry and Molecular Biology, Center for Research in Molecular Medicine and Chronic Diseases, Universidade de Santiago de Compostela, Santiago de Compostela, Spain Search for more papers by this author Joao Matos Joao Matos Institute of Biochemistry, Eidgenössische Technische Hochschule, Zürich, Switzerland Search for more papers by this author Boris Pfander Corresponding Author Boris Pfander [email protected] orcid.org/0000-0003-2180-5054 Max Planck Institute of Biochemistry, DNA Replication and Genome Integrity, Martinsried, Germany Search for more papers by this author Author Information Lissa N Princz1, Philipp Wild2, Julia Bittmann1, F Javier Aguado3, Miguel G Blanco3, Joao Matos2 and Boris Pfander *,1 1Max Planck Institute of Biochemistry, DNA Replication and Genome Integrity, Martinsried, Germany 2Institute of Biochemistry, Eidgenössische Technische Hochschule, Zürich, Switzerland 3Department of Biochemistry and Molecular Biology, Center for Research in Molecular Medicine and Chronic Diseases, Universidade de Santiago de Compostela, Santiago de Compostela, Spain *Corresponding author. Tel: +49 89 85783050; Fax: +49 89 85783022; E-mail: [email protected] The EMBO Journal (2017)36:664-678https://doi.org/10.15252/embj.201694831 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract DNA repair by homologous recombination is under stringent cell cycle control. This includes the last step of the reaction, disentanglement of DNA joint molecules (JMs). Previous work has established that JM resolving nucleases are activated specifically at the onset of mitosis. In case of budding yeast Mus81-Mms4, this cell cycle stage-specific activation is known to depend on phosphorylation by CDK and Cdc5 kinases. Here, we show that a third cell cycle kinase, Cdc7-Dbf4 (DDK), targets Mus81-Mms4 in conjunction with Cdc5—both kinases bind to as well as phosphorylate Mus81-Mms4 in an interdependent manner. Moreover, DDK-mediated phosphorylation of Mms4 is strictly required for Mus81 activation in mitosis, establishing DDK as a novel regulator of homologous recombination. The scaffold protein Rtt107, which binds the Mus81-Mms4 complex, interacts with Cdc7 and thereby targets DDK and Cdc5 to the complex enabling full Mus81 activation. Therefore, Mus81 activation in mitosis involves at least three cell cycle kinases, CDK, Cdc5 and DDK. Furthermore, tethering of the kinases in a stable complex with Mus81 is critical for efficient JM resolution. Synopsis DNA joint molecule resolution by the Mus81-Mms4 nucleases is tightly controlled during mitosis. Dbf4-dependent kinase (DDK), best known for its replication roles, now joins previously implicated cell cycle kinases CDK and Cdc5/Plk in this regulation. The budding yeast Cdc7-Dbf4 kinase regulates the Mus81-Mms4 resolvase in mitosis. DDK and Cdc5 form a complex that targets Mms4 and leads to mitotic Mus81 activation. Scaffold protein Rtt107 promotes DDK and Cdc5 activity towards Mus81-Mms4 and is required for full Mus81 activation. DDK, Cdc5 and Rtt107 target Mms4 interdependently, suggesting that they form a signal amplification loop. Introduction Many DNA transactions are under cell cycle control to adjust them to cell cycle phase-specific features of chromosomes (Branzei & Foiani, 2008). Homologous recombination (HR) is cell cycle-regulated at several steps including the first, DNA end resection, and the last, JM removal (Heyer et al, 2010; Ferretti et al, 2013; Mathiasen & Lisby, 2014; Matos & West, 2014). Given that JMs provide stable linkages between sister chromatids, they will interfere with chromosome segregation and therefore need to be disentangled before sister chromatid separation during mitosis. Accordingly, JM resolvases, such as budding yeast Mus81-Mms4 (Interthal & Heyer, 2000; Schwartz et al, 2012) or Yen1 (Ip et al, 2008), become activated during mitosis (Matos et al, 2011, 2013; Gallo-Fernández et al, 2012; Szakal & Branzei, 2013; Blanco et al, 2014; Eissler et al, 2014). In contrast, the alternative JM removal pathway, JM dissolution by the Sgs1-Top3-Rmi1 complex, is thought to be constantly active throughout the cell cycle (Mankouri et al, 2013; Bizard & Hickson, 2014). The activation of JM resolvases in mitosis therefore leads to a shift in the balance between JM removal pathways, with dissolution being preferred outside of mitosis, but JM resolution becoming increasingly important in mitosis (Matos et al, 2011, 2013; Gallo-Fernández et al, 2012; Dehé et al, 2013; Saugar et al, 2013; Szakal & Branzei, 2013; Wyatt et al, 2013). It has been hypothesized that JM resolvases are downregulated at cell cycle stages other than mitosis in order to counteract crossover-induced loss of heterozygosity or to prevent over-active resolvases from interfering with S phase by, for example, cleaving stalled replication forks (Gallo-Fernández et al, 2012; Szakal & Branzei, 2013; Blanco et al, 2014). Budding yeast Mus81-Mms4 has previously been shown to be targeted by two cell cycle kinases, cyclin-dependent kinase Cdc28 (CDK) and the yeast polo-kinase Cdc5 (Matos et al, 2011, 2013; Gallo-Fernández et al, 2012; Szakal & Branzei, 2013). The corresponding Mms4 phosphorylation events were shown to correlate with and to be required for activation of Mus81-Mms4 in mitosis. In 2014, we showed that in mitosis Mus81-Mms4 also forms a complex with Slx4-Slx1 and the scaffold proteins Dpb11 and Rtt107 (Gritenaite et al, 2014). Interestingly, mass spectrometric analysis of this complex (Gritenaite et al, 2014) revealed that Cdc5 and a third cell cycle kinase Dbf4-Cdc7 (Dbf4-dependent kinase, DDK) are also a stable part of this protein assembly (see Appendix Fig S1A). Here, we investigate the role of DDK in Mus81-Mms4 regulation and find that DDK can phosphorylate Mms4 and that DDK and Cdc5 target Mus81-Mms4 in an interdependent manner. Moreover, we show that Rtt107 promotes the association of both kinases with the Mus81-Mms4 complex. The DDK-dependent regulation of Mus81-Mms4 is critical for Mus81 activity thus revealing DDK as a novel regulator of homologous recombination. Results Mus81-Mms4 is a DDK phosphorylation target The cell cycle regulation of JM resolution by Mus81-Mms4 is intricate and involves phosphorylation by the cell cycle kinases CDK and Cdc5 (Matos et al, 2011, 2013; Gallo-Fernández et al, 2012; Szakal & Branzei, 2013) as well as complex formation with the scaffold proteins Dpb11, Slx4 and Rtt107 (Gritenaite et al, 2014). To study this protein complex, we performed an analysis of Mms43FLAG interactors in mitosis by SILAC-based quantitative mass spectrometry (Gritenaite et al, 2014) and found in addition to Dpb11, Slx4, Rtt107 and Cdc5, also Cdc7 and Dbf4 as specific interactors of Mms4 (Appendix Fig S1A). We verified that Cdc7 binds to Mus81-Mms4 in an Mms43FLAG pull down from mitotic cells analysed by Western blots (Fig 1A). The fact that Mus81-Mms4 binds to DDK suggested that it might be involved in the phosphorylation cascade that occurs on Mms4 and controls Mus81 activity in mitosis. Accordingly, we found that purified DDK was able to phosphorylate both subunits of purified Mus81-Mms4 in vitro (Fig 1B, lane 3). When we furthermore compared the DDK-dependent phosphorylation signal to Mms4 phosphorylation by CDK and Cdc5 (Fig 1B, lanes 2–4), we observed different degrees of phosphorylation shifts indicating that the three kinases phosphorylate Mms4 at distinct sites and/or to different degrees. DDK target sites on other proteins have been studied in detail, and in several cases, DDK was found to target (S/T)(S/T) motifs, where phosphorylation was stimulated by a priming phosphorylation usually on the second (S/T) (Masai et al, 2006; Montagnoli et al, 2006; Randell et al, 2010; Lyons et al, 2013). Intriguingly, Mms4 contains 15 of these motifs and we therefore tested whether these could be targeted by DDK and would depend on priming phosphorylation. We therefore turned to a peptide-based assay where Mms4 phosphorylation states are precisely defined. To this end, we synthesized peptides corresponding to two (S/T)(S/T) motifs of Mms4. We chose two representative motifs: S222, as it harbours a minimal CDK consensus motif (S/T)P, and S134, as it contains a non-(S/T)P consensus for CDK [(S/T)X(K/R)(K/R) (Suzuki et al, 2015)]. For each of these motifs, we generated peptides in three different phosphorylation states: non-phosphorylated, phosphorylated at the second serine and doubly phosphorylated (Fig 1C and Appendix Fig S1B). When using such peptides as substrates in in vitro kinase reactions, we saw that CDK targeted specifically only the second serine in each peptide, although much stronger for S222 than for S134, consistent with these residues matching CDK consensus motifs (Fig 1C). In contrast, DDK showed only little activity towards the non-phosphorylated peptides, but was strongly stimulated when the second residue in the (S/T)(S/T) motif was in a phosphorylated state (Fig 1C). DDK may thus be stimulated by priming phosphorylation in order to efficiently phosphorylate Mms4 on (S/T)(S/T) sites. However, using the full-length protein as a phosphorylation substrate, we did not obtain evidence for a stimulatory effect on DDK by prior CDK phosphorylation (Fig 1B and Appendix Fig S1C), perhaps because over the whole 15 (S/T)(S/T) motifs CDK phosphorylation plays a minor role. We also did not reveal any priming activity of either CDK or DDK for Mms4 phosphorylation by Cdc5 (Fig 1B and Appendix Fig S1D). Overall, the data in Fig 1 thus identify Mus81-Mms4 as an interaction partner and potential substrate of DDK. Figure 1. Dbf4-dependent kinase (DDK) binds to the Mus81-Mms4 complex in mitosis and can phosphorylate Mms4 at (S/T)(S/T) motifs Cdc7 and Cdc5 are specifically enriched in Mms43FLAG co-IPs from cells arrested in mitosis (with nocodazole). Under the same conditions, Mus81-Mms4 associates with scaffold proteins such as Dpb11 and Slx4 (Appendix Fig S1A and Gritenaite et al, 2014). DDK can phosphorylate Mus81-Mms4 in vitro. Purified, immobilized Mus81-Mms4 is incubated in an in vitro kinase assay with purified CDK2/cycAN170 (a model CDK), DDK or Cdc5 (lanes 1–4). Additionally, Mus81-Mms4 is incubated with respective kinases after a non-radioactive priming step with CDK (lanes 5–8). DDK phosphorylates Mms4 peptides at (S/T)(S/T) motifs and is enhanced by priming phosphorylation. Mms4 peptides including (S/T)(S/T) motifs (221/222; 133/134) were synthesized in different phosphorylation states (depicted in left panel) and incubated in an in vitro kinase assay with either CDK or DDK. CDK targets unphosphorylated Mms4 peptides 1 and (to a weaker extent) 4 consistent with its substrate specificity (Mok et al, 2010), while DDK primarily targets Mms4 peptides 2 and 5, which harbour a priming phosphorylation at the C-terminal (S/T) site (see Appendix Fig S1B for in-gel running behaviour of peptides). Download figure Download PowerPoint Mus81-Mms4 is phosphorylated by a mitotic Cdc5-DDK complex DDK is present and active throughout S phase and mitosis until anaphase when the Dbf4 subunit is degraded by APC/CCdc20 (Cheng et al, 1999; Weinreich & Stillman, 1999; Ferreira et al, 2000). We therefore tested at which cell cycle stage DDK would associate with Mus81-Mms4 using cells synchronously progressing through the cell cycle. Figure 2A shows that DDK did not associate with Mus81-Mms4 in S phase, but only once cells had reached mitosis. Strikingly, DDK binding therefore coincided with binding of Cdc5, Slx4 and Dpb11 and most notably the appearance of the hyperphosphorylated form of Mms43FLAG (Fig 2A). Figure 2. DDK and Cdc5 target Mus81-Mms4 in an interdependent manner A. DDK stably associates with Mus81-Mms4 in mitosis, but not in S phase or G1. Mms43FLAG pull down experiment (left panel, as in Fig 1A) from cells arrested in G1 (with alpha-factor) or in cells progressing synchronously through S phase until mitosis (arrest with nocodazole) reveals that DDK binds specifically in mitosis concomitant with the raise in Cdc5 levels and Cdc5 binding to Mus81-Mms4. A nocodazole-arrested untagged strain was used as a control. Right panel shows measurements of DNA content by FACS from the respective samples. B. CDK activity is required for DDK and Cdc5 association with Mus81-Mms4. Mms43FLAG pull down as in (A), but in mitotic WT or cdc28-as1 mutant cells treated with 5 μM 1NM-PP1 for 1 h. Additional Western blots of this experiment are shown in Appendix Fig S5B, including as a control the identical anti-FLAG Western blot. C. Cdc5 activity is required for DDK association with Mus81-Mms4. Mms43FLAG pull down as in (A), but with mitotically arrested WT or cdc5-as1 mutant cells treated with 10 μM CMK for 1 h. D, E. DDK is required for Cdc5 binding to Mus81-Mms4 in mitosis and the mitotic Mms4 phospho-shift. (D) Mms43FLAG pull down using mitotically arrested cells as in (A), but using a bob1-1 background (all samples), where the DDK subunit Cdc7 could be deleted. (E) SILAC-based quantification of Mms43FLAG pull downs in mitotically arrested bob1-1 vs. bob1-1 cdc7Δ cells. Plotted are the H/L ratios of two independent experiments including label switch. F. The Cdc5 binding region on Dbf4 is required for interaction of DDK and Cdc5 with Mus81-Mms4 and for efficient Mms4 phosphorylation. Mms43FLAG pull down as in (A), but using mitotically arrested cells expressing N-terminal truncation mutants of Dbf4 lacking aa2–66 (including a D-box motif) or 2–109 [additionally including the Cdc5 binding site (Miller et al, 2009)]. Download figure Download PowerPoint Given this late timing of the association, we tested in co-immunoprecipitation (co-IP) experiments whether DDK binding to Mus81-Mms4 would depend on CDK or Cdc5 activity. Using analog-sensitive mutant yeast strains for CDK [cdc28-as1 (Bishop et al, 2000)] and for Cdc5 [cdc5-as1 (Snead et al, 2007)], we observed that inhibition of these kinases in mitotically arrested cells strongly reduced the hyperphosphorylation shift of Mms4 (see also Matos et al, 2013) and compromised the association with DDK (Fig 2B and C, and Appendix Fig S2A–C). Notably, both conditions also interfered with Cdc5 binding (Fig 2B and C, and Appendix Fig S2A), suggesting that the association of DDK may follow a similar regulation as Cdc5. Next, we tested whether conversely DDK is involved in Mms4 phosphorylation. To bypass the essential function of DDK in DNA replication, we used the mcm5bob1-1 allele (Hardy et al, 1997), which allowed us to test a cdc7Δ mutant. Using Western blot and SILAC-based mass spectrometry as a read-out of Mms43FLAG co-IPs from cells arrested in mitosis, we found that Cdc5 association with Mus81-Mms4 was strongly reduced in the cdc7Δ mutant strain (Fig 2D and E). Moreover, we observed that Mms43FLAG phosphorylation as indicated by mobility shift was decreased in the absence of DDK, although not to the same extent as upon CDK or Cdc5 inhibition (Fig 2D and Appendix Fig S2C). Additionally, as an alternative way to deregulate DDK, we used the cdc7-1 temperature-sensitive mutant. Even with WT cells, we observed that elevated temperature (38°C) leads to a slight reduction in Cdc5 binding to Mus81-Mms4. However, in cdc7-1 mutant cells, incubation at 38°C leads to the complete disappearance of Cdc5 binding to Mus81-Mms4 (Appendix Fig S2D). Therefore, we conclude from these data that DDK and Cdc5 bind to Mus81-Mms4 in an interdependent fashion. Interestingly, Cdc5 was previously shown to interact with DDK via a non-consensus polo-box binding site within Dbf4 (Miller et al, 2009; Chen & Weinreich, 2010). The proposed model based on genetic experiments suggested that DDK binding antagonizes mitotic functions of Cdc5. However, the catalytic activity of Cdc5 was not inhibited in this complex (Miller et al, 2009) and we reason that DDK may simply target Cdc5 to a specific set of substrates. Since the Cdc5 binding site was mapped to the N-terminal portion of Dbf4 (Miller et al, 2009), we tested whether N-terminal truncations of Dbf4 would affect DDK or Cdc5 association with Mus81-Mms4. While the dbf4-ΔN66 truncation lacking the first 66 amino acids (including a D-box motif) did not influence DDK or Cdc5 binding to Mms43FLAG, the dbf4-ΔN109 truncation, which additionally lacks the Cdc5 binding motif (Miller et al, 2009), showed strongly decreased DDK and Cdc5 binding to Mus81-Mms4 (Fig 2F). Additionally, also mitotic hyperphosphorylation of Mms4 was diminished when DDK and Cdc5 could not interact with each other (Fig 2F). Overall, these data strongly suggest that Cdc5 and DDK interact with and target Mus81-Mms4 in an interdependent manner. Furthermore, it is currently unclear whether collaboration of DDK and Cdc5 is a widespread phenomenon that may affect other Cdc5 substrates as well, given that mitotic phosphorylation of two candidate Cdc5 substrates, Ulp2 and Scc1 (Alexandru et al, 2001), was affected to varying degree by the cdc7Δ mutation (Appendix Fig S2E). Given the known cell cycle regulation of Cdc5 and DDK (Shirayama et al, 1998; Cheng et al, 1999; Weinreich & Stillman, 1999; Ferreira et al, 2000; Mortensen et al, 2005), the limiting factor for the temporal regulation of this complex and its restriction to mitosis is expected to be Cdc5 and not DDK, which is present already throughout S phase. Consistently, we observed that forced expression of Cdc5 (using the galactose-inducible GAL promoter) in cells that were arrested in S phase by hydroxyurea (HU) led to the premature occurrence of Mms4 hyperphosphorylation (Fig EV1A; Matos et al, 2013), suggesting that S-phase DDK is in principle competent for Cdc5 binding and joint substrate phosphorylation. Click here to expand this figure. Figure EV1. Cdc5 restricts Mms4 hyperphosphorylation to mitosis Overexpression of CDC5 in S phase results in premature Mms4 hyperphosphorylation. Western blot analysis of Mms49myc, Cdc5 and Dbf4 from whole-cell extracts (upper panel) and FACS data (lower panel). Cells were arrested in G1 (with alpha-factor), S phase (with HU) or G2/M phase (with nocodazole). After arrest, CDC5GFP overexpression was induced by addition of 2% galactose for the indicated time to cells harbouring an additional copy of GFP-tagged CDC5 under the GAL1 promoter. Samples were run in 7% Tris-acetate gels. Mms4 hyperphosphorylation by CDC5 overexpression in S phase is reduced in HU-treated cells. Western blot analysis of Mms49myc and Cdc5 from precipitated whole-cell extracts (upper panel) and FACS data (lower panel) of cells arrested in G1 (with alpha factor) or G2/M phase (with nocodazole), or released to S phase (with or without HU). CDC5GFP overexpression was induced for 30 min by addition of 2% galactose to cells harbouring an additional copy of GFP-tagged CDC5 under the GAL1 promoter. Note that upon CDC5 overexpression cells are partially defective in bulk replication. Samples were run in 7% Tris-acetate gels. Download figure Download PowerPoint Furthermore, we performed additional experiments that addressed the regulation of Mus81-Mms4 by the DNA damage response. In M-phase-arrested cells, association of DDK and Cdc5 with Mus81-Mms4 was reduced after induction of DNA damage with phleomycin (Appendix Fig S2F), but this treatment was not sufficient to induce a significant reduction in the Mms4 phosphorylation shift. Interestingly, when we forced Cdc5 expression in S-phase cells and compared normal S-phase cells to cells treated with hydroxyurea (HU), we observed that the Mms4 phosphorylation shift was less pronounced in the presence of hydroxyurea (HU) (Fig EV1B). These data are therefore consistent with the current view that DNA damage, specifically the DNA damage checkpoint, negatively influences Mus81 resolution activity (Szakal & Branzei, 2013; Gritenaite et al, 2014). Since DDK is known to be targeted and inhibited by the DNA damage checkpoint (Weinreich & Stillman, 1999; Lopez-Mosqueda et al, 2010; Zegerman & Diffley, 2010), it could become particularly critical to regulate Mms4 phosphorylation after DNA damage. Even though DDK and Cdc5 seem to target Mus81-Mms4 in unison, we tested whether it was possible to resolve differences on the level of individual phosphorylation sites. Therefore, we analysed Mms4 phosphorylation sites in M-phase cells after Cdc5 inhibition (Fig 3A and C) or CDC7 deletion (Fig 3B and D) by SILAC-based mass spectrometry. We also applied two different experimental set-ups that used either endogenously expressed Mus81-Mms4 (Fig 3A and B) or overexpressed Mus81-Mms4 (Fig 3C and D), as the latter set-up allowed much better coverage of Mms4 phosphopeptides in higher order phosphorylation states (peptides harbouring > 1 phosphorylated site). Cdc5 inhibition or lack of DDK led to overlapping, but distinct changes in Mms4 phosphorylation sites, suggesting that each kinase phosphorylates specific sites on Mms4. After Cdc5 inhibition, phosphorylation of many sites was reduced and among those were sites that match to a putative Cdc5 consensus [(D/E/N)X(S/T), blue, Fig 3A and C; Mok et al, 2010]. Overall, CDC7 affected Mms4 phosphorylation less than Cdc5 inhibition, but nonetheless, we found widespread changes in the phosphorylation of (S/T)(S/T) motifs (Fig 3B and D). (S/T)(S/T) motifs were found less abundantly in the doubly phosphorylated state (Fig 3D, red), while conversely these motifs were found more abundantly in the state where only the second (S/T) was singly phosphorylated (Fig 3B and D, yellow), as expected for a substrate–product relation. These data are thus consistent with phosphorylation of the second (S/T) priming for phosphorylation at the preceding (S/T) (Appendix Table S1 and Appendix Fig S3). Figure 3. Analysis of Mms4 phosphorylation sites reveals Cdc5 and DDK target sites, as well as the interdependence between the twoChanges of the abundance of phosphorylated Mms4 peptides after Cdc5 inhibition (as in Fig 2C) (A, C) or in the absence of Cdc7 (B, D) in mitotically arrested cells. A, B. Depicted are SILAC-based intensity ratios of individual MS evidences for peptides of endogenously expressed Mms4. Evidences of non-phosphorylated Mms4 peptides are shown in grey; evidences of phosphorylated peptides are shown in black, yellow, orange or blue. Blue colour indicates putative Cdc5 phosphorylation as defined by the (D/E/N)X(S/T) consensus (and additionally S268, which was also very strongly deregulated upon Cdc5 inhibition). Yellow or orange colours mark singly phosphorylated (S/T)(S/T) motifs, with orange marking p(S/T)(S/T) and yellow marking (S/T)p(S/T). Numbers indicate the phosphorylated residue in the depicted peptide. An asterisk marks peptide evidences that contained measured intensity values exclusively in the heavy or light sample. For doubly phosphorylated peptides, the two phospho-sites are separated by a comma. For singly phosphorylated (S/T)(S/T) motifs, peptide ion fragmentation was in some cases unable to unambiguously identify the phosphorylated residue. In these cases, possible phosphorylation sites are indicated as “a/b”. Note that doubly phosphorylated (S/T)(S/T) sites were not reproducibly identified under conditions of endogenous Mus81-Mms4 expression. C, D. As in panels (A, B) but using Mus81-Mms4 expressed from a high-copy promoter. Depicted are SILAC-based H/L ratios of individual MS evidences for phosphorylated peptides only. Peptides were sorted into categories according to their phosphorylation status: putative DDK target sites ((S/T)(S/T) motifs) were differentiated into the categories p(S/T)p(S/T) (red), p(S/T)(S/T) (orange) or (S/T)p(S/T) (yellow). Phosphorylated peptides matching the Cdc5 consensus site are coloured in blue. All other phosphorylated peptides are marked in grey. Bars depict the mean of the ratios of the respective category. Overall, Mms4 H/L ratio is shown on top. Download figure Download PowerPoint DDK phosphorylation is required for activation of Mus81-Mms4 during mitosis Phosphorylation of Mms4 by CDK and Cdc5 has previously been shown to be required for the upregulation of Mus81-Mms4 activity during mitosis (Matos et al, 2011, 2013; Gallo-Fernández et al, 2012; Szakal & Branzei, 2013). Based on our finding that hyperphosphorylation of Mms4 was impaired in the absence of DDK (Fig 2D and Appendix Fig S2C), we predicted that also Mus81-Mms4 activity would be influenced. Therefore, we tested the activity of endogenous Mus819myc-Mms43FLAG immunopurified from G2/M arrested cells (approx. 5 fmol) on a nicked Holliday junction (nHJ) substrate (500 fmol) using an assay related to those in (Matos et al, 2011, 2013; Gritenaite et al, 2014). Notably, the activity of the endogenous purified Mus81-Mms4 from G2/M cells exceeded the activity of recombinant Mus81-Mms4 (subjected to a dephosphorylation step during the purification), indicating that it is the mitotically activated form (Appendix Fig S4A). Moreover, the activity of endogenous purified Mus81-Mms4 was not influenced by 350 mM NaCl salt washes. This indicates that the presence of accessory, salt-labile factors such as Rtt107 or Cdc5 in the reaction is unlikely to contribute to Mus81 activity (Appendix Fig S4B and C). Importantly, when we used this assay to test immunopurified Mus819myc-Mms43FLAG from mitotic cells lacking DDK (cdc7Δ or dbf4Δ), we observed a reduced activity compared to Mus819myc-Mms43FLAG from WT cells (Fig 4A and Appendix Fig S4D; also observed with an RF substrate, Appendix Fig S4E). In order to exclude that indirect effects of the CDC7 deletion may cause the reduction in Mus81 activity, we furthermore created an Mms4 mutant that specifically lacks candidate DDK phosphorylation sites. We chose to mutate (S/T)(S/T) motifs (SS motifs in particular) and created an mms4-8A mutant that harboured eight S to A exchanges at the N-terminal (S/T) of the motifs (see Appendix Fig S3A). This mutant appeared less phosphorylated in mitosis as judged by a less pronounced phosphorylation shift (Fig 4B). Furthermore, we observed a reduction in the association of DDK and Cdc5 with the Mus81-Mms4-8A complex in pull-down experiments (Fig 4B), suggesting that phosphorylation of Mms4 also plays a role in tethering these kinases. Notably, Mus819myc-Mms43FLAG-8A from mitotic cells showed a moderate but reproducible reduction in resolution activity on nHJ substrates compared to WT Mus819myc-Mms43FLAG (Fig 4C and Appendix Fig S4F). These data thus indicate that DDK targets Mus81-Mms4 and that (S/T)(S/T) phosphorylation events are essential for full activation of Mus81 in mitosis. Figure 4. DDK phosphorylation controls activation of Mus81-Mms4 resolvase activity in mitosis A. DDK is required for mitotic activation of Mus81-Mms4. Resolution assay using a nicked Holliday junction (nHJ) substrate and Mus819myc-Mms43FLAG purified from mitotically arrested bob1-1 (DDK-WT+), bob1-1 dbf4Δ and bob1-1 cdc7Δ strains or untagged control cells. Right panel: quantification of cleavage products. See Appendix Fig S4D for Western blots samples of anti-myc IPs. Left panel: representative gel image. B. A defect in the phosphorylation of Mms4 (S/T)(

Background and Objectives: Tinnitus is a common symptom in medical practice, although data on its prevalence vary. As the underlying pathophysiological mechanism is still not fully understood, hearing loss is thought to be an important risk factor for the occurrence of tinnitus. The aim of this study was to assess tinnitus prevalence in a large German cohort and to determine its dependence on hearing impairment. Materials and Methods: The Gutenberg Health Study (GHS) is a population-based cohort study and representative for the population of Mainz and its district. Participants were asked whether they suffer from tinnitus and how much they are burdened by it. Extensive audiological examinations using bone- and air-conduction were also performed. Results: 4942 participants (mean age: 61.0, 2550 men and 2392 women) were included in the study. The overall prevalence of tinnitus was 26.1%. Men were affected significantly more often than women. The prevalence of tinnitus increased with age, peaking at ages 75 to 79 years. Considering only annoying tinnitus, the prevalence was 9.8%. Logistic regression showed that participants with severe to complete hearing loss (&gt;65 dB) were more likely to have tinnitus. Conclusions: Tinnitus is a common symptom, and given demographic changes, its prevalence is expected to increase.

Atrial fibrillation (AF) is an increasingly common problem in primary care, but little is known about its prevalence and the distribution of AF risk factors in the general population.We determined the prevalence of AF and the distribution of known AF risk factors among persons participating in the population-based Gutenberg Health Study. To this end, we used interview data about the medical diagnosis of AF and electrocardiograms (ECGs) that were performed for the study in 5000 persons aged 35 to 74. The response rate was 60.4%.There were 5000 persons in the study sample (age 52.2 ± 11 years; 50.6% were women). The prevalence of AF, weighted for the age and sex distribution of the general population, was 2.5%. AF was found to be more common in older persons, with a more pronounced increase in men: whereas its prevalence was 0.7% in 35- to 44-year-old men, the corresponding figure for the age group 65- to 74 was as high as 10.6%. Twenty five participants (15.5% of AF cases) received their initial diagnosis of AF on the basis of the study ECG. Compared to persons without AF, persons with AF were older and more commonly male, and they had a higher burden of cardiovascular risk factors. 14.3% of persons with AF had none of the well-established risk factors for AF (systolic blood pressure, antihypertensive medication, increased body-mass-index, heart failure). 42.7% of persons with AF were not taking either anticoagulants or platelet inhibitors.These data indicate that the prevalence of AF in the middle-aged general population is 2.5% overall, and higher in the elderly. AF is thus a significant public health problem, and greater awareness of it is needed.

Background Initial evidence suggests that depressive symptoms are more frequent in patients with atrial fibrillation. Data from the general population are limited. Methods and Results In 10,000 individuals (mean age 56±11 years, 49.4% women) of the population-based Gutenberg Health Study we assessed depression by the Patient Health Questionnaire (PHQ-9) and a history of depression in relation to manifest atrial fibrillation (n = 309 cases). The median (25th/75th percentile) PHQ-9 score of depressive symptoms was 4 (2/6) in atrial fibrillation individuals versus 3 (2/6) individuals without atrial fibrillation, . Multivariable regression analyses of the severity of depressive symptoms in relation to atrial fibrillation in cardiovascular risk factor adjusted models revealed a relation of PHQ-9 values and atrial fibrillation (odds ratio (OR) 1.04, 95% confidence interval (CI) 1.01–1.08; P = 0.023). The association was stronger for the somatic symptom dimension of depression (OR 1.08, 95% CI 1.02–1.15; P = 0.0085) than for cognitive symptoms (OR 1.05, 95% CI 0.98–1.11; P = 0.15). Results did not change markedly after additional adjustment for heart failure, partnership status or the inflammatory biomarker C-reactive protein. Both, self-reported physical health status, very good/good versus fair/bad, (OR 0.54, 95% CI 0.41–0.70; P<0.001) and mental health status (OR 0.61 (0.46–0.82); P = 0.0012) were associated with atrial fibrillation in multivariable-adjusted models. Conclusions In a population-based sample we observed a higher burden of depressive symptoms driven by somatic symptom dimensions in individuals with atrial fibrillation. Depression was associated with a worse perception of physical or mental health status. Whether screening and treatment of depressive symptoms modulates disease progression and outcome needs to be shown.

Homologous recombination (HR) is essential for high-fidelity DNA repair during mitotic proliferation and meiosis. Yet, context-specific modifications must tailor the recombination machinery to avoid (mitosis) or enforce (meiosis) the formation of reciprocal exchanges-crossovers-between recombining chromosomes. To obtain molecular insight into how crossover control is achieved, we affinity purified 7 DNA-processing enzymes that channel HR intermediates into crossovers or noncrossovers from vegetative cells or cells undergoing meiosis. Using mass spectrometry, we provide a global characterization of their composition and reveal mitosis- and meiosis-specific modules in the interaction networks. Functional analyses of meiosis-specific interactors of MutLγ-Exo1 identified Rtk1, Caf120, and Chd1 as regulators of crossing-over. Chd1, which transiently associates with Exo1 at the prophase-to-metaphase I transition, enables the formation of MutLγ-dependent crossovers through its conserved ability to bind and displace nucleosomes. Thus, rewiring of the HR network, coupled to chromatin remodeling, promotes context-specific control of the recombination outcome.

Platelets are the main players in thrombotic diseases, where activated platelets not only mediate thrombus formation but also are involved in multiple interactions with vascular cells, inflammatory components, and the coagulation system. Although in vitro reactivity of platelets provides information on the function of circulating platelets, it is not a full reflection of the in vivo activation state, which may be relevant for thrombotic risk assessment in various disease conditions. Therefore, studying release markers of activated platelets in plasma is of interest. While this type of study has been done for decades, there are several new discoveries that highlight the need for a critical assessment of the available tests and indications for platelet release products. First, new insights have shown that platelets are not only prominent players in arterial vascular disease, but also in venous thromboembolism and atrial fibrillation. Second, knowledge of the platelet proteome has dramatically expanded over the past years, which contributed to an increasing array of tests for proteins released and shed from platelets upon activation. Identification of changes in the level of plasma biomarkers associated with upcoming thromboembolic events allows timely and individualized adjustment of the treatment strategy to prevent disease aggravation. Therefore, biomarkers of platelet activation may become a valuable instrument for acute event prognosis. In this narrative review based on a systematic search of the literature, we summarize the process of platelet activation and release products, discuss the clinical context in which platelet release products have been measured as well as the potential clinical relevance.

Smoking is a well-established risk factor for chronic non-communicable diseases. However, the relationship between cigarette smoking and the risk of developing mental health conditions remains largely elusive. This study examined the relationship between cigarette smoking as well as smoking cessation and prevalent and incident symptoms of depression, anxiety, and sleep disturbance in the general population.In a cohort of 15,010 individuals from the Gutenberg Health Study (aged 35-74 years at enrollment), prevalent (at baseline from 2007 to 2012) and incident symptoms (at follow-up from 2012 to 2017) of depression, anxiety, and sleep disturbance were determined by validated questionnaires and/or medical records. Smoking status, pack-years of smoking in current and former smokers, and years since quitting smoking in former smokers were assessed by a standardized computer-assisted interview.In multivariable logistic regression models with comprehensive adjustment for covariates, smoking status was independently associated with prevalent and incident symptoms of depression (Patient Health Questionnaire-9 ≥ 10), whereas this association was weaker for anxiety (Generalized Anxiety Disorder Scale-2 ≥ 3) and sleep disturbance (Patient Health Questionnaire-9 > 1). Among current and former smokers, smoking ≥30 or ≥10 pack-years, respectively, yielded in general the highest effect estimates. Smoking cessation was weakly associated with the prevalence and incidence of all outcomes, here consistent associations were observed for prevalent symptoms of depression.The observational nature of the study does not allow for causal inferences.The results of the present study suggest that cigarette smoking is positively and that smoking cessation is negatively associated with symptoms of common mental health conditions, in particular of depression.

BackgroundDeep vein thrombosis (DVT) is a multifactorial disease with several outcomes, but current classifications solely stratify it based on recurrence risk.ObjectivesWe aimed to identify DVT phenotypes and assess their relation to recurrent venous thromboembolism (VTE), postthrombotic syndrome, arterial events, and cancer.Patients/MethodsHierarchical clustering was performed on a DVT cohort with a follow-up of up to 5 years using 23 baseline characteristics. Phenotypes were summarized by discriminative characteristics. Hazard ratios (HRs) were calculated using Cox regression; the recurrence risk was adjusted for the anticoagulant therapy duration. The study was carried out in accordance with the Declaration of Helsinki and approved by the medical ethics committee.ResultsIn total, 825 patients were clustered into 4 phenotypes: 1. women using estrogen therapy (n = 112); 2. patients with a cardiovascular risk profile (n = 268); 3. patients with previous VTE (n = 128); and 4. patients without discriminant characteristics (n = 317). Overall, the risks of recurrence, postthrombotic syndrome, arterial events, and cancer were low in phenotype 1 (reference), intermediate in phenotype 4 (HR: 4.6, 1.2, 2.2, 1.8), and high in phenotypes 2 (HR: 6.1, 1.6, 4.5, 2.9) and 3 (HR: 5.7, 2.5, 2.3, 3.7).ConclusionsThis study identified 4 distinct phenotypes among patients with DVT that are not only associated with the increasing recurrence risk but also with outcomes beyond recurrence. Our results thereby highlight the limitations of current risk stratifications that stratify based on the predictors of the recurrence risk only. Overall, risks were lowest in women using estrogen therapy and highest in patients with a cardiovascular risk profile. These findings might inform a more personalized approach to clinical management.

Thrombolytic recombinant tissue plasminogen activator (r-tPA) treatment is the only pharmacologic intervention available in the ischemic stroke acute phase. This treatment is associated with an increased risk of intracerebral hemorrhages, known as hemorrhagic transformations (HTs), which worsen the patient's prognosis.To investigate the association between genetically determined natural hemostatic factors' levels and increased risk of HT after r-tPA treatment.Using data from genome-wide association studies on the risk of HT after r-tPA treatment and data on 7 hemostatic factors (factor [F]VII, FVIII, von Willebrand factor [VWF], FXI, fibrinogen, plasminogen activator inhibitor-1, and tissue plasminogen activator), we performed local and global genetic correlation estimation multitrait analyses and colocalization and 2-sample Mendelian randomization analyses between hemostatic factors and HT.Local correlations identified a genomic region on chromosome 16 with shared covariance: fibrinogen-HT, P = 2.45 × 10-11. Multitrait analysis between fibrinogen-HT revealed 3 loci that simultaneously regulate circulating levels of fibrinogen and risk of HT: rs56026866 (PLXND1), P = 8.80 × 10-10; rs1421067 (CHD9), P = 1.81 × 10-14; and rs34780449, near ROBO1 gene, P = 1.64 × 10-8. Multitrait analysis between VWF-HT showed a novel common association regulating VWF and risk of HT after r-tPA at rs10942300 (ZNF366), P = 1.81 × 10-14. Mendelian randomization analysis did not find significant causal associations, although a nominal association was observed for FXI-HT (inverse-variance weighted estimate [SE], 0.07 [-0.29 to 0.00]; odds ratio, 0.87; 95% CI, 0.75-1.00; raw P = .05).We identified 4 shared loci between hemostatic factors and HT after r-tPA treatment, suggesting common regulatory mechanisms between fibrinogen and VWF levels and HT. Further research to determine a possible mediating effect of fibrinogen on HT risk is needed.

This study aimed to determine if there is an increased risk of incident cardiovascular diseases (CVD) resulting from cumulative night shift work in the German population-based Gutenberg Health Study (GHS).We examined working participants of the GHS at baseline and after five years. Cumulative night shift work in the 10 years before baseline was assessed and categorized as low (1-220 nights ≙ up to 1 year), middle (221-660 nights ≙ 1-3 years), and high (>660 nights ≙ more than 3 years) night shift exposure. Hazard ratios (HR) were estimated for incident "quality-assured CVD events" using Cox proportional hazard models.At baseline, 1092 of 8167 working participants performed night shift work. During the follow-up, 202 incident cardiovascular events occurred. The crude incidence rates for CVD per 1000 person-years were 6.88 [95% confidence interval (CI) 4.80-9.55] for night shift workers and 5.19 (95% CI 4.44-6.04) for day workers. Cumulative incidence curves showed a higher cumulative incidence in workers exposed to night shift work compared to day workers after five years. The adjusted HR for incident CVD events were 1.26 (95% CI 0.68-2.33), 1.37 (95% CI 0.74-2.53) and 1.19 (95% CI 0.67-2.12) for employees in the low, middle and high night shift categories compared to employees without night shift work, respectively.The observed tendencies indicate that night shift work might be negatively associated with cardiovascular health. We expect the continued follow-up will clarify the long-term impact of night shift work.

Echocardiography, the dominant imaging modality for quantification of left ventricular metrics, has undergone continuing development in the past few decades. However, given the lack of population-based data, current guidelines are still based on restricted and small data sets analyzed with methods including expert opinion. This work presents empirically derived reference values from a large-scale, epidemiologic study conducted with state-of-the-art imaging technology and methods.The distribution of echocardiographic measurements of the left ventricle was analyzed in a population-based sample of 5000 mid-Europeans from the Gutenberg Heart Study in Germany. The randomly selected, noninstitutionalized sample provides data on apparently healthy individuals, as well as on those with prevalent disease. Standardized echocardiograms were recorded in a comprehensive data set at a single site with centralized training and certification of sonographers. Sex-specific reference limits and categories indicating the grade of deviation from the reference were calculated, and nomograms were created by quantile regression. Detailed information is given on the association between left ventricular geometry and age.The rapidly evolving echocardiographic technology with persistent improvements in image quality and new measurement conventions require the evaluation of new reference limits for left ventricular metrics. The present investigation formulates reference limits and nomograms from state-of-the-art technology and methods based on a large population-based data set. The distribution of echocardiographic measures of left ventricular geometry presents, in part, nonlinear associations with age, which should be the subject of future investigations.

Vascular reactivity is reflected by blood biomarkers and noninvasive vascular function measurement. The relation of biomarkers to flow-mediated dilation and peripheral arterial tonometry in the general population is little understood. In 5000 individuals (mean age, 56±11 years; age range, 35-74 years; 49% women) of the population-based Gutenberg Health Study we simultaneously assessed 6 biomarkers of cardiovascular function (midregional proadrenomedullin [MR-proADM], midregional pro atrial natriuretic peptide [MR-proANP], N-terminal pro B-type natriuretic peptide, copeptin, C-terminal proendothelin 1, and neopterin) in relation to flow-mediated dilation and peripheral arterial tonometry. Strongest partial correlations (adjusted for age and sex) were observed for baseline pulse amplitude with MR-proADM (r=0.13) and MR-proANP (r=-0.13); hyperemic response variables showed the highest correlation for MR-proADM and peripheral arterial tonometry ratio (r=-0.14). In multivariable linear regression models, strongest associations with baseline vascular function were observed for MR-proANP with baseline pulse amplitude (β per SD increase [99.17%], -0.080 [-0.115 to -0.044]; P<0.0001 after Bonferroni correction for multiple testing) and MR-proADM (-0.044 [-0.070 to -0.017]; P<0.0001), as well as MR-proANP (-0.033 [-0.057 to -0.009]; P=0.0017) and N-terminal pro B-type natriuretic peptide (-0.027 [-0.051 to -0.003]; P=0.015) with brachial artery diameter. For hyperemic response variables, highest associations were seen for peripheral arterial tonometry ratio with MR-proADM (-0.022 [-0.043 to -0.004]; P=0.043), MR-proANP (0.016 [-0.0034 to 0.035]; P=0.18), and C-terminal proendothelin 1 (-0.025 [-0.043 to -0.008]; P=0.00094]. In our large, population-based study, we identified MR-proADM and MR-proANP as circulating biomarkers of vascular function most strongly related to noninvasive measures of conduit artery and peripheral arterial performance. Whether determination of blood biomarkers helps to better understand vascular pathology and may provide prognostic information needs to be investigated in future studies.

Different biological pathways have been related to atrial fibrillation (AF). Novel biomarkers capturing inflammation, oxidative stress, and neurohumoral activation have not been investigated comprehensively in AF.In the population-based Gutenberg Health Study (n = 5000), mean age 56 ± 11 years, 51% males, we measured ten biomarkers representing inflammation (C-reactive protein, fibrinogen), cardiac and vascular function (midregional pro adrenomedullin [MR-proADM], midregional pro atrial natriuretic peptide [MR-proANP], N-terminal pro-B-type natriuretic peptide [Nt-proBNP], sensitive troponin I ultra [TnI ultra], copeptin, and C-terminal pro endothelin-1), and oxidative stress (glutathioneperoxidase-1, myeloperoxidase) in relation to manifest AF (n = 161 cases). Individuals with AF were older, mean age 64.9 ± 8.3, and more often males, 71.4%. In Bonferroni-adjusted multivariable regression analyses strongest associations per standard deviation increase in biomarker concentrations were observed for the natriuretic peptides Nt-proBNP (odds ratio [OR] 2.89, 99.5% confidence interval [CI] 2.14-3.90; P<0.0001), MR-proANP (OR 2.45, 99.5% CI 1.91-3.14; P<0.0001), the vascular function marker MR-proADM (OR 1.54, 99.5% CI 1.20-1.99; P<0.0001), TnI ultra (OR 1.50, 99.5% CI 1.19-1.90; P<0.0001) and. fibrinogen (OR 1.44, 99.5% CI 1.19-1.75; P<0.0001). Based on a model comprising known clinical risk factors for AF, all biomarkers combined resulted in a net reclassification improvement of 0.665 (99.3% CI 0.441-0.888) and an integrated discrimination improvement of >13%.In conclusion, in our large, population-based study, we identified novel biomarkers reflecting vascular function, MR-proADM, inflammation, and myocardial damage, TnI ultra, as related to AF; the strong association of natriuretic peptides was confirmed. Prospective studies need to examine whether risk prediction of AF can be enhanced beyond clinical risk factors using these biomarkers.

Aims and background Midregional proadrenomedullin (MR-proADM) is a protein, which exerts various effects on the cardiovascular system. Recent studies underscored its prognostic implications in patients with acute dyspnea and cardiovascular diseases. Therefore, we aimed to determine the distribution of MR-proADM in the general population and to reveal potential associations of MR-proADM with cardiovascular risk factors and measures of subclinical cardiovascular disease. Methods and results MR-proADM plasma concentrations were determined in individuals of the population-based cohort of the Gutenberg Health Study (N = 5000) using a commercially available fluoroimmunoassay. Individuals were enrolled between April 2007 and October 2008. Subclinical cardiovascular disease was assessed using echocardiographic and functional measures of myocardial and vascular function. The mean age of the study population was 55.5 ± 10.9 years. In the overall population we determined a median MR-proADM plasma concentration of 0.44 nmol/L in men and women. MR-proADM concentrations were elevated in individuals with hypertension, diabetes, dyslipidemia, known cardiovascular disease, heart failure, peripheral artery disease, atrial fibrillation, and history of myocardial infarction and stroke. In men, we observed a positive association of MR-proADM with reduced ejection fraction, intraventricular septal diameter, wall thickness, and echocardiographic measures of diastolic dysfunction. Conclusions In this study, we present age-dependent reference values for MR-proADM in a representative population sample. Elevated MR-proADM plasma concentrations were strongly associated with classical cardiovascular risk factors and manifest cardiovascular diseases. Furthermore, we revealed a gender-specific association with echocardiographic measures of hypertension. MR-proADM seems to be a promising prognostic biomarker for subclinical and manifest cardiovascular disease.

We used a large-scale, high-throughput DNA aptamer-based discovery proteomic platform to identify circulating biomarkers of cardiac remodeling and incident heart failure (HF) in community-dwelling individuals.We evaluated 1895 FHS (Framingham Heart Study) participants (age 55±10 years, 54% women) who underwent proteomic profiling and echocardiography. Plasma levels of 1305 proteins were related to echocardiographic traits and to incident HF using multivariable regression. Statistically significant protein-HF associations were replicated in the HUNT (Nord-Trøndelag Health) study (n=2497, age 63±10 years, 43% women), and results were meta-analyzed. Genetic variants associated with circulating protein levels (pQTLs) were related to echocardiographic traits in the EchoGen (n=30 201) and to incident HF in the CHARGE (n=20 926) consortia.Seventeen proteins associated with echocardiographic traits in cross-sectional analyses (false discovery rate <0.10), and 8 of these proteins had pQTLs associated with echocardiographic traits in EchoGen (P<0.0007). In Cox models adjusted for clinical risk factors, 29 proteins demonstrated associations with incident HF in FHS (174 HF events, mean follow-up 19 [limits, 0.2-23.7] years). In meta-analyses of FHS and HUNT, 6 of these proteins were associated with incident HF (P<3.8×10-5; 3 with higher risk: NT-proBNP [N-terminal proB-type natriuretic peptide], TSP2 [thrombospondin-2], MBL [mannose-binding lectin]; and 3 with lower risk: ErbB1 [epidermal growth factor receptor], GDF-11/8 [growth differentiation factor-11/8], and RGMC [hemojuvelin]). For 5 of the 6 proteins, pQTLs were associated with echocardiographic traits (P<0.0006) in EchoGen, and for RGMC, a protein quantitative trait loci was associated with incident HF (P=0.001).A large-scale proteomics approach identified new predictors of cardiac remodeling and incident HF. Future studies are warranted to elucidate how biological pathways represented by these proteins may mediate cardiac remodeling and HF risk and to assess if these proteins can improve HF risk prediction.

<h2>Summary</h2> The Bloom's helicase ortholog, Sgs1, orchestrates the formation and disengagement of recombination intermediates to enable controlled crossing-over during meiotic and mitotic DNA repair. Whether its enzymatic activity is temporally regulated to implement formation of noncrossovers prior to the activation of crossover-nucleases is unknown. Here, we show that, akin to the Mus81-Mms4, Yen1, and MutLγ-Exo1 nucleases, Sgs1 helicase function is under cell-cycle control through the actions of CDK and Cdc5 kinases. Notably, however, whereas CDK and Cdc5 unleash nuclease function during M phase, they act in concert to stimulate Sgs1 activity during S phase/prophase I. Mechanistically, CDK-mediated phosphorylation enhances the velocity and processivity of Sgs1, which stimulates DNA unwinding <i>in vitro</i> and joint molecule processing <i>in vivo</i>. Subsequent hyper-phosphorylation by Cdc5 appears to reduce the activity of Sgs1, while activating Mus81-Mms4 and MutLγ-Exo1. These findings suggest a concerted mechanism driving orderly formation of noncrossover and crossover recombinants in meiotic and mitotic cells.

Animal experiments and early phase human trials suggest that inhibition of factor XIa (FXIa) safely prevents venous thromboembolism (VTE), and specific murine models of sepsis have shown potential efficacy in alleviating cytokine storm. These latter findings support the role of FXI beyond coagulation. Here, we combine targeted proteomics, machine learning and bioinformatics, to discover associations between FXI activity (FXI:C) and the plasma protein profile of patients with VTE. FXI:C was measured with a modified activated partial prothrombin time (APTT) clotting time assay. Proximity extension assay-based protein profiling was performed on plasma collected from subjects from the Genotyping and Molecular Phenotyping of Venous Thromboembolism (GMP-VTE) Project, collected during an acute VTE event (n = 549) and 12-months after (n = 187). Among 444 proteins investigated, N = 21 and N = 66 were associated with FXI:C during the acute VTE event and at 12 months follow-up, respectively. Seven proteins were identified as FXI:C-associated at both time points. These FXI-related proteins were enriched in immune pathways related to causes of thrombo-inflammation, extracellular matrix interaction, lipid metabolism, and apoptosis. The results of this study offer important new avenues for future research into the multiple properties of FXI, which are of high clinical interest given the current development of FXI inhibitors.

BackgroundMulti‐phenotype analysis of genetically correlated phenotypes can increase the statistical power to detect loci associated with multiple traits, leading to the discovery of novel loci. This is the first study to date to comprehensively analyze the shared genetic effects within different hemostatic traits, and between these and their associated disease outcomes.ObjectivesTo discover novel genetic associations by combining summary data of correlated hemostatic traits and disease events.MethodsSummary statistics from genome wide‐association studies (GWAS) from seven hemostatic traits (factor VII [FVII], factor VIII [FVIII], von Willebrand factor [VWF] factor XI [FXI], fibrinogen, tissue plasminogen activator [tPA], plasminogen activator inhibitor 1 [PAI‐1]) and three major cardiovascular (CV) events (venous thromboembolism [VTE], coronary artery disease [CAD], ischemic stroke [IS]), were combined in 27 multi‐trait combinations using metaUSAT. Genetic correlations between phenotypes were calculated using Linkage Disequilibrium Score Regression (LDSC). Newly associated loci were investigated for colocalization. We considered a significance threshold of 1.85 × 10−9 obtained after applying Bonferroni correction for the number of multi‐trait combinations performed (n = 27).ResultsAcross the 27 multi‐trait analyses, we found 4 novel pleiotropic loci (XXYLT1, KNG1, SUGP1/MAU2, TBL2/MLXIPL) that were not significant in the original individual datasets, were not described in previous GWAS for the individual traits, and that presented a common associated variant between the studied phenotypes.ConclusionsThe discovery of four novel loci contributes to the understanding of the relationship between hemostasis and CV events and elucidate common genetic factors between these traits.

Low birth weight (BW) is associated with increased corneal aberrations in childhood and alterations of corneal geometry in adulthood. Increased corneal aberrations may be a factor contributing to decreased visual function in former low BW newborns in later life. Hence, the aim of this study was to analyze the long-term effect of low BW on corneal aberrations in adulthood. In the German population-based Gutenberg Health Study (GHS) participants (age: 40–80 years) were examined with Scheimpflug imaging (Pentacam HR, Oculus Optikgeräte GmbH, Wetzlar, Germany). The relationship between self-reported BW and the different types of corneal aberrations was analyzed using linear regression analysis as uni- and multivariable analysis with adjustment for potential confounders. The main outcome measures were corneal aberrations defined as astigmatism (Z2−2; Z22), coma (Z3−1; Z31), trefoil (Z3−3; Z33), spherical aberration (Z40) and root-mean square of higher order aberrations (HOA; 3rd up to 8th order; aperture size: 6 mm). Overall, 5,628 participants were included in this analysis (3,004 women, aged 56.0 +/- 10.3 years). In a multivariable analysis lower BW was associated with decreased horizontal trefoil (B = 0.004 [0.001; 0.006] µm/500 g; p=.008); higher spherical aberrations (B=-0.006 [-0.008;-0.003] µm/500 g; p<.001), higher RMS (B=-0.028 [-0.042;-0.014] µm/500 g; p<.001), increased HOA (B=-0.007 [-0.010;-0.003] µm/500 g; p<.001) and increased LOA (B=-0.027 [-0.041;-0.013] µm/500 g; p<.001). No association was observed between birth weight and the other types of corneal aberrations in multivariable model. Our results indicate an association between BW and spherical aberration in adults aged 40 to 80 years. This indicates that low BW may have an association with an altered corneal shape development which may affect optical image quality and, hence, visual function.

Hearing is a basic ability that is needed for participation in daily life. Hearing loss often greatly reduces a person's quality of life. Nevertheless, epidemiological data on the prevalence of hearing disorders in Germany are sparse. This study investigated the prevalence of hearing disorders and the actual provision with hearing aids.The Gutenberg Health Study (GHS) is a representative cohort study carried out at the Department of Medicine of Mainz University to investigate the health of the population of the city of Mainz and the neighboring Mainz‒Bingen district. The GHS participants underwent pure-tone audiometry of each ear independently.Tone audiometry data from a total of 5024 participants were evaluated. The prevalence of hearing loss-regardless of severity-in at least one ear was 40.6% in this study population. The hearing loss was mild in 22.5% of the participants, moderate in 8.3%. Some 2.8% had severe hearing loss. In this group, the women had better hearing than the men (by a mean 4.3 dB). The prevalence of hearing disorders rose with increasing age. The minimum tone audiometry conditions for bilateral hearing aid provision-as defined in the relevant German guideline-were met in 47.7% of the participants. Only 7.7% of the participants already had hearing aids for both ears. The discrepancy between the prevalence of hearing loss and the indication for provision with hearing aids arises from differences in how hearing loss was ascertained and the indications set.The prevalence of hearing loss was high, at 40.6%. Regular hearing tests should be recommended for the general German population, starting at no later than 60 years of age.

Aims: Cigarette smoking is one of the most complex and least understood cardiovascular risk factors. Importantly, differences in the tobacco-related pathophysiology of endothelial dysfunction, an early event in atherogenesis, between circulatory beds remain elusive. Therefore, this study evaluated how smoking impacts endothelial function of conduit and resistance arteries in a large population-based cohort. Methods and results: 15,010 participants (aged 35–74 years) of the Gutenberg Health Study were examined at baseline from 2007 to 2012. Smoking status, pack-years of smoking, and years since quitting smoking were assessed by a computer-assisted interview. Endothelial function of conduit and resistance arteries was determined by flow-mediated dilation (FMD) of the brachial artery, reactive hyperemia index (RHI) using peripheral arterial tonometry, as well as by reflection index (RI) derived from digital photoplethysmography, respectively. Among all subjects, 45.8% had never smoked, 34.7% were former smokers, and 19.4% were current smokers. Mean cumulative smoking exposure was 22.1 ± 18.1 pack-years in current smokers and mean years since quitting was 18.9 ± 12.7 in former smokers. In multivariable linear regression models adjusted for typical confounders, smoking status, pack-years of smoking, and years since quitting smoking were independently associated with RHI and RI, while no association was found for FMD. Overall, no clear dose-dependent associations were observed between variables, whereby higher exposure tended to be associated with pronounced resistance artery endothelial dysfunction. Conclusions: Cigarette smoking is associated with altered endothelial function of resistance, but not conduit arteries. The present results suggest that smoking-induced endothelial dysfunction in different circulatory beds may exhibit a differential picture.

The establishment of stable interactions between chromosomes underpins vital cellular processes such as recombinational DNA repair and bipolar chromosome segregation. On the other hand, timely disengagement of persistent connections is necessary to assure efficient partitioning of the replicated genome prior to cell division. Whereas great progress has been made in defining how cohesin-mediated chromosomal interactions are disengaged as cells prepare to undergo chromosome segregation, little is known about the metabolism of DNA joint molecules (JMs), generated during the repair of chromosomal lesions. Recent work on Mus81 and Yen1/GEN1, two conserved structure-selective endonucleases, revealed unforeseen links between JM-processing and cell cycle progression. Cell cycle kinases and phosphatases control Mus81 and Yen1/GEN1 to restrain deleterious JM-processing during S-phase, while safeguarding chromosome segregation during mitosis.

Venous thromboembolism (VTE) is associated with excessive coagulation activity, which in part can be attributed to activation of contact system. However, the knowledge regarding the impact of contact activation in acute VTE is limited.To unravel the involvement of contact activation in acute VTE.Contact activation was investigated in patients with acute VTE (n = 321) and population controls without a history of VTE (n = 300). For comparison, Factor XI(a) levels, activity, and plasma kallikrein (PKa) activity were determined in plasma samples with an activated partial thromboplastin time- or thrombin generation-based assay (free FXI concentration [FXI:c] and calibrated automated thrombogram:FXIa, respectively) and with enzyme-linked immunosorbent assays for enzyme-inhibitor complexes (FXIa:alpha-1-antitrypsin [α1AT], FXIa:antithrombin [AT], FXIa:C1-inhibitor [C1Inh], and PKa:C1-inh).In patients with VTE, higher FXI:c levels (124 ± 37% vs 114 ± 28%), but lower calibrated automated thrombogram:FXIa levels were apparent. This was accompanied by increased FXIa:α1AT, FXIa:AT, and PKa:C1-inh levels in patients compared with controls (312pM [238-424] vs 203pM [144-288]; 29pM [23-38] vs 23pM [20-30]; 1.9nM [1.2-4.7] vs 1.4nM [0.7-3.5], respectively), whereas FXIa:C1-inh levels did not differ. Logistic regression models showed good discriminatory values for FXI:c and FXIa:α1AT (area under the curve = 0.64 [0.6/0.69] and 0.73 [0.69/0.77], respectively). After a 2-year follow-up, 81 recurrent VTE events or deaths occurred in the patient cohort, for which the baseline levels of FXIa:α1AT and FXIa:C1Inh had a significant prognostic value (Hazard ratios per SD [95% CI], 1.26 [1.10-1.45]; p =.0012 and 1.19 [1.05-1.36]; p =.0082, respectively).Our study revealed elevated FXIa levels and activity in acute VTE, which was also associated with recurrent VTE, suggesting an important risk contribution of FXI activation to VTE. The evidence provided by this study supports the utility of FXIa inhibition in the setting of acute VTE.

Abstract Background This study aimed to compare the quality of life (QoL) reported by childhood cancer survivors (CCS) drawn from a cohort of the German Childhood Cancer Registry with a representative general population sample and, within CCS, to test associations between QoL and health behavior, health risk factors, and physical illness. Methods CCS ( N = 633, age at diagnosis M = 6.34 ( SD = 4.38), age at medical assessment M = 34.92 ( SD = 5.70)) and a general population sample (age-aligned; N = 975) filled out the EORTC QLQ-C30. Comparisons were performed using General linear models (GLMs) (fixed effects: sex/gender, group (CCS vs. general population); covariates: age, education level). CCS underwent an extensive medical assessment (mean time from diagnosis to assessment was 28.07 ( SD = 3.21) years) including an objective diagnosis of health risk factors and physical illnesses (e.g., diabetes and cardiovascular disease). Within CCS, we tested associations between QoL and sociodemographic characteristics, health behavior, health risk factors, and physical illness. Results CCS, especially female CCS, reported both worse functional QoL and higher symptom burden than the general population. Among CCS, better total QoL was related to younger age, higher level of education, being married, and engaging in active sports. Both health risk factors (dyslipidemia and physical inactivity) and manifest physical illnesses (cardiovascular disease) were associated with lower total QoL. Conclusions In all domains, long-term CCS reported worse QoL than the comparison sample. The negative associations with risk factors and physical illnesses indicate an urgent need for long-term surveillance and health promotion.

Abstract Objectives A series of human field studies demonstrated that acute exposure to simulated nocturnal traffic noise is associated with cardiovascular complications and sleep disturbance, including endothelial dysfunction, increased blood pressure, and impaired sleep quality. A pooled analysis of these results remains to be established and is of tremendous interest to consolidate scientific knowledge. Methods We analyzed data from four randomized crossover studies (published between 2013 to 2021 and conducted at the University Medical Center Mainz, Germany). A total of 275 subjects (40.4% women, mean age 43.03 years) were each exposed to one control scenario (regular background noise) and at least to one traffic noise scenario (60 aircraft or train noise events) in their homes during nighttime. After each night, the subjects visited the study center for comprehensive cardiovascular function assessment, including the measurement of endothelial function and hemodynamic and biochemical parameters, as well as sleep-related variables. Results The pooled analysis revealed a significantly impaired endothelial function when comparing the two different noise sequences (0–60 vs. 60–0 simulated noise events, mean difference in flow-mediated dilation −2.00%, 95% CI −2.32; −1.68, p &lt; 0.0001). In concordance, mean arterial pressure was significantly increased after traffic noise exposure (mean difference 2.50 mmHg, 95% CI 0.54; 4.45, p = 0.013). Self-reported sleep quality, the restfulness of sleep, and feeling in the morning were significantly impaired after traffic noise exposure (all p &lt; 0.0001). Discussion Acute exposure to simulated nocturnal traffic noise is associated with endothelial dysfunction, increased mean arterial pressure, and sleep disturbance.

Abstract Background Cigarette smoking is a threat to global human health and a leading cause of the cardiovascular disease (CVD) morbidity and mortality. Importantly, sex-specific differences in smoking-induced arterial stiffness, an early key event in the development of atherosclerotic CVD, remain still elusive. Thus, this study sought out to investigate sex-specific associations between smoking and measures of arterial stiffness. Methods and results Overall, 15,010 participants (7584 men and 7426 women aged 35–74 years) of the Gutenberg Health Study were examined at baseline during 2007–2012. Smoking status, pack-years of smoking, and years since quitting smoking were assessed by a standardized computer-assisted interview. Arterial stiffness and wave reflection were determined by stiffness index (SI) and augmentation index (AI). In the total sample, 45.8% had never smoked, 34.7% were former smokers, and 19.4% were current smokers. Median cumulative smoking exposure was 22.0 pack-years in current male smokers and 16.0 in current female smokers. In general, multivariable linear regression models adjusted for a comprehensive set of confounders revealed that smoking status, pack-years of smoking, and years since quitting smoking were dose-dependently associated with markers of arterial stiffness. In sex-specific analyses, these associations were overall more pronounced in men and SI was stronger related to the male sex, whereas differences between men and women in the case of AI appeared to be less substantial. Discussion The present results indicate that chronic smoking is strongly and dose-dependently associated with increased arterial stiffness in a large population-based cohort regardless of sex but with a stronger association in men.

During the SARS-CoV-2 pandemic, preventive measures like physical distancing, wearing face masks, and hand hygiene have been widely applied to mitigate viral transmission. Beyond increasing vaccination coverage, preventive measures remain urgently needed. The aim of the present project was to assess the effect of protective behavior on SARS-CoV-2 infection risk in the population.Data of the Gutenberg COVID-19 Study (GCS), a prospective cohort study with a representative population-based sample, were analyzed. SARS-CoV-2 infections were identified by sequential sampling of biomaterial, which was analyzed by RT-qPCR and two antibody immunoassays. Self-reported COVID-19 test results were additionally considered. Information on protective behavior including physical distancing, wearing face masks, and hand hygiene was collected via serial questionnaire-based assessments. To estimate adjusted prevalence ratios and hazard ratios, robust Poisson regression and Cox regression were applied.In total, 10,250 participants were enrolled (median age 56.9 [43.3/68.6] years, 50.8% females). Adherence to preventive measures was moderate for physical distancing (48.3%), while the use of face masks (91.5%) and the frequency of handwashing (75.0%) were high. Physical distancing appeared to be a protective factor with respect to SARS-CoV-2 infection risk independent of sociodemographic characteristics and individual pandemic-related behavior (prevalence ratio [PR] = 0.77, 95% confidence interval [CI] 0.62-0.96). A protective association between wearing face masks and SARS-CoV-2 transmission was identified (PR = 0.73, 95% CI 0.55-0.96). However, the protective effect declined after controlling for potential confounding factors (PR = 0.96, 95% CI 0.68-1.36). For handwashing, this investigation did not find a beneficial impact. The adherence to protective behavior was not affected by previous SARS-CoV-2 infection or immunization against COVID-19.The present study suggests primarily a preventive impact of physical distancing of 1.5 m, but also of wearing face masks on SARS-CoV-2 infections, supporting their widespread implementation. The proper fit and use of face masks are crucial for effectively mitigating the spread of SARS-CoV-2 in the population.

Research has described positive psychological outcomes after severe illness, including posttraumatic growth. The aim of the present research was to evaluate a short scale assessing posttraumatic growth within a sample of cancer survivors to provide an efficient instrument for research and care settings.Using data of a registry-based sample of N = 633 childhood cancer survivors (CCS) more than 25 years after diagnosis, we conducted an investigation of a five-item short form of the established Posttraumatic Growth Inventory (PTGI), the PPR-5 (PPR stands for "Posttraumatische Persönliche Reifung", the German expression for posttraumatic growth). We performed a confirmatory factor analysis, tested the PPR-5's internal consistency, and investigated associations with cancer-related, sociodemographic, and mental health variables (assessed using psychometrically tested screening instruments) using group comparisons and correlation analyses within a cross-sectional design.Findings supported a unidimensional structure of the PPR-5. It also showed good reliability (ω = 0.81). CCS especially endorsed Relating to others and Personal strength. The PPR-5's sum score was negatively associated with current depression, anxiety, and sleep disorder symptoms, intake of antidepressants, and lifetime diagnoses of depression and anxiety disorders. It showed positive associations with resilient coping, higher age at diagnosis, partnership, and parenthood.The PPR-5 allows for a brief assessment of posttraumatic growth. As it indicates aspects that support positive psychological adaptation to life as a (cancer) survivor, it could inform research and practice (e.g., as a screening measure, or in psychotherapy/counseling settings).

Introduction Tinnitus is a common symptom in otolaryngologic practice. The pathophysiology behind its development is assumed to be multifactorial and has not yet been fully understood. Resilience is seen as the ability to survive difficult life situations without permanent impairment. Depending on the perspective, it is understood as a dynamic process or a personal characteristic. As such, resilience can promote adaptation to chronic health conditions. The aim of this study was to investigate the influence of resilience on the prevalence of tinnitus in the German population.

Einleitung Tinnitus ist ein häufiges Symptom in der HNO-ärztlichen Praxis. Die Pathophysiologie hinter seiner Entstehung wird als multifaktoriell angenommen und ist noch nicht abschließend geklärt. Resilienz wird als die Fähigkeit angesehen, schwierige Lebenssituationen ohne dauerhafte Beeinträchtigung zu überstehen. Je nach Blickwinkel wird sie als dynamischer Prozess oder persönliches Merkmal verstanden. Als solches kann Resilienz die Anpassung an chronische Gesundheitszustände fördern. Ziel dieser Studie war es zu untersuchen, welchen Einfluss die Resilienz auf die Prävalenz von Tinnitus in der deutschen Bevölkerung hat.

Einleitung Hören gilt als grundlegende Fähigkeit zur Teilnahme am täglichen Leben und Hörverlust beeinträchtigt die Lebensqualität erheblich. Zudem wurde Hörverlust mit Einsamkeit und Depression in Verbindung gebracht und kann Auswirkungen auf die psychosoziale und kognitive Gesundheit haben. Ziel der Studie war es die Prävalenz von Hörstörungen, Einsamkeit und depressiven Symptomen in einer großen deutschen Kohorte zu untersuchen und über den Zusammenhang zu berichten.

This review condenses the knowledge on variable selection methods implemented in R and appropriate for datasets with grouped features. The focus is on regularized regressions identified through a systematic review of the literature, following the Preferred Reporting Items for Systematic Reviews and Meta‐Analyses guidelines. A total of 14 methods are discussed, most of which use penalty terms to perform group variable selection. Depending on how the methods account for the group structure, they can be classified into knowledge and data‐driven approaches. The first encompass group‐level and bi‐level selection methods, while two‐step approaches and collinearity‐tolerant methods constitute the second category. The identified methods are briefly explained and their performance compared in a simulation study. This comparison demonstrated that group‐level selection methods, such as the group minimax concave penalty , are superior to other methods in selecting relevant variable groups but are inferior in identifying important individual variables in scenarios where not all variables in the groups are predictive. This can be better achieved by bi‐level selection methods such as group bridge . Two‐step and collinearity‐tolerant approaches such as elastic net and ordered homogeneity pursuit least absolute shrinkage and selection operator are inferior to knowledge‐driven methods but provide results without requiring prior knowledge. Possible applications in proteomics are considered, leading to suggestions on which method to use depending on existing prior knowledge and research question.

The relation of noninvasive vascular function to sex, sex hormones, and reproductive history in the general population is little understood.We simultaneously assessed flow-mediated dilation (FMD) and peripheral arterial tonometry in 454 women (mean age 40.4±16.1 years, age range 19-78 years) and 100 men (mean age 44.7±15.3 years) in a community-based cohort. Plasma estradiol, progesterone, luteinizing hormone, and follicle stimulating hormones were measured, and menstrual cycle and reproductive history were recorded.Vascular function was blunted in men as compared to women irrespective of menopausal status and adjustment for classical cardiovascular risk factors and hormones. Vascular reactivity changed during the menstrual cycle and correlated with estradiol concentrations for FMD, r=0.13 and inversely with progesterone for pulse amplitude, r=-0.14, and brachial artery diameter, r=-0.10. Multivariable-adjusted regressions showed a relation of estradiol with FMD, β 0.658, 95% confidence interval (CI) 0.084/1.232, P=0.025 in women. Age at menarche (β 0.070, 95% CI 0.039/0.101, P<0.0001) and breastfeeding duration (β -0.006, 95% CI -0.011/-0.001, P=0.036) were related to brachial artery diameter, age at menarche also to FMD (β -0.455, 95% CI -0.886/-0.023, P=0.039).Sex differences in noninvasive conduit and peripheral arterial function with better vascular reactivity in women were not fully explained by female sex hormones and menopausal status. Age at menarche and duration of breastfeeding were also related to vascular function and need further investigation.

We investigated the association between social inequality and participation in a mammography screening program (MSP). Since the German government offers mammography screening free of charge, any effect of social inequality on participation should be due to educational status and not due to the financial burden.The 'Gutenberg Health Study' is a cohort study in the Rhine-Main-region, Germany. A health check-up was performed, and questions about medical history, health behavior, including secondary prevention such as use of mammography, and social status are included. Two indicators of social inequality (equivalence income and educational status), an interaction term of these two, and different covariables were used to explore an association in different logistic regression models.A total of 4,681 women meeting the inclusion criteria were included. Only 6.2% never participated in the MSP. A higher income was associated with higher chances of ever participating in a mammography screening (odds ratios (OR): 1.67 per €1000; 95%CI:1.26-2.25, model 3, adjusted for age, education and an interaction term of income and education). Compared to women with a low educational status, the odds ratios for ever participating in the MSP was lower for the intermediate educational status group (OR = 0.64, 95%CI:0.45-0.91) and for the high educational status group (0.53, 95%CI:0.37-0.76). Results persisted also after controlling for relevant confounders.Despite the absence of financial barriers for participation in the MSP, socioeconomic inequalities still influence participation. It would be interesting to examine whether the educational effect is due to an informed decision.

While chronic exposure to high levels of noise was demonstrated to increase the risk of various cardiovascular diseases, the association between noise annoyance and risk of cardiovascular disease remains still inconsistent. Recently, we showed that noise annoyance is associated with prevalent atrial fibrillation in the general population. However, the association between noise annoyance and risk of incident atrial fibrillation as well as potential sex-differences remain still elusive.15,010 subjects from a German population-based cohort were examined at baseline (2007 to 2012) and follow-up five years later (2012 to 2017) to investigative the association between noise annoyance due to multiple sources and prevalent and incident atrial fibrillation. After multivariable adjustment, the results from logistic regression analyses revealed overall consistent and positive associations between noise annoyance and prevalent and incident atrial fibrillation in men, whereas this association was weaker in women, in particular with respect to incident atrial fibrillation. For instance, industrial noise annoyance was associated with 21% (95% confidence interval (CI) 9-34%) and 18% (8-29%) higher odds of prevalent atrial fibrillation in men and women, respectively. In prospective analysis, this association remained stable in men (odds ratio (OR) 1.25, 1.07-1.44), while in women no association was observed (OR 1.03, 0.89-1.18).The findings suggest that noise annoyance can increase the risk of incident atrial fibrillation in a large population-based cohort and that men may be more sensitive to the adverse effects of noise annoyance with regard to the risk of atrial fibrillation.

Beyond the influence of lifestyle-related risk factors for myocardial infarction (MI), the mechanisms of genetic predispositions for MI remain unclear. We sought to identify and characterize differentially expressed genes in early-onset MI in a translational approach. In an observational case−control study, transcriptomes from 112 early-onset MI individuals showed upregulated G protein-coupled receptor 15 (GPR15) expression in peripheral blood mononuclear cells compared to controls (fold change = 1.4, p = 1.87 × 10−7). GPR15 expression correlated with intima-media thickness (β = 0.8498, p = 0.111), C-reactive protein (β = 0.2238, p = 0.0052), ejection fraction (β = −0.9991, p = 0.0281) and smoking (β = 0.7259, p = 2.79 × 10−10). The relation between smoking and MI was diminished after the inclusion of GPR15 expression as mediator in mediation analysis (from 1.27 (p = 1.9 × 10−5) to 0.46 (p = 0.21)). The DNA methylation of two GPR15 sites was 1%/5% lower in early-onset MI individuals versus controls (p = 2.37 × 10−6/p = 0.0123), with site CpG3.98251219 significantly predicting risk for incident MI (hazard ratio = 0.992, p = 0.0177). The nucleotide polymorphism rs2230344 (C/T) within GPR15 was associated with early-onset MI (odds ratio = 3.61, p = 0.044). Experimental validation showed 6.3-fold increased Gpr15 expression in an ischemic mouse model (p < 0.05) and 4-fold increased Gpr15 expression in cardiomyocytes under ischemic stress (p < 0.001). After the induction of MI, Gpr15gfp/gfp mice showed lower survival (p = 0.042) and deregulated gene expression for response to hypoxia and signaling pathways. Using a translational approach, our data provide evidence that GPR15 is linked to cardiovascular diseases, mediating the adverse effects of smoking.

The tendency of a plasma sample to generate thrombin, a central enzyme in blood coagulation, might be an important indicator of prothrombotic risk linked to cardiovascular disease (CVD), but the presence of platelets may be a critical determinant. Clinical data, laboratory markers and thrombin generation (TG), investigated in both platelet-rich plasma (PRP) and platelet-free plasma (PFP) at 1 pM TF, were available in 407 individuals from the Gutenberg Health Study. Given the well-known effect of anticoagulants on TG, subjects taking anticoagulants (n = 15) have been excluded resulting in 392 subjects for further analysis. Lag time, endogenous thrombin potential (ETP) and peak height were the investigated parameters of a TG curve. Multivariable linear regression analysis was used to identify TG determinants. Mean platelet volume (MPV) and platelet count were both negatively associated to lag time and positively to peak height (MPV, β:6.35 [2.66; 10.0]; platelet count, β:0.111 [0.054; 0.169]) in PRP only. C-reactive protein was positively associated with lag time and ETP in both PRP and PFP, with a stronger effect on ETP in PRP (PRP, β:76.7 [47.5; 106]; PFP, β:34.8 [10.3; 59.2]). After adjustment for fibrinogen, the relation between CRP and ETP was attenuated in PRP and PFP. Of the traditional cardiovascular risk factors (CVRFs), obesity was positively associated to TG in PRP only. Our findings support that TG, particularly in PRP, relates to traditional CVRFs in a representative sample from a population-based study. Assessment of procoagulant activity in a platelet-dependent manner by TG is a promising tool for assessing individual risk for CVD.

The formation of stable interactions between chromosomes of maternal and paternal origin-homologs-is required for their segregation during meiosis. To achieve this, cells take advantage of the recombination machinery, which promotes formation of reciprocal interhomolog exchanges, called crossovers, from the repair of self-inflicted DNA breaks. Important genetic studies led to the identification of key enzymes that control meiotic recombination. However, characterization of their biochemical properties when purified from meiotic cultures has been difficult to achieve. Here, we describe a simple approach to purify and characterize DNA repair enzymes from meiotic yeast cells. First, we provide a protocol to generate large-scale synchronous cultures. Second, we describe a general method to prepare meiotic extracts from which protein complexes can be immunoaffinity-purified. Finally, we detail how the purified material can be used for: (i) mass spectrometry-based analysis of interaction partners and posttranslational modifications, and (ii) monitoring enzymatic activities using synthetic DNA substrates.

Lipids exhibit an essential role in cellular assembly and signaling. Dysregulation of these functions has been linked with many complications including obesity, diabetes, metabolic disorders, cancer and more. Investigating lipid profiles in such conditions can provide insights into cellular functions and possible interventions. Hence the field of lipidomics is expanding in recent years. Even though the role of individual lipids in diseases has been investigated, there is no resource to perform disease enrichment analysis considering the cumulative association of a lipid set. To address this, we have implemented the LipiDisease web server. The tool analyzes millions of records from the PubMed biomedical literature database discussing lipids and diseases, predicts their association and ranks them according to false discovery rates generated by random simulations. The tool takes into account 4270 diseases and 4798 lipids. Since the tool extracts the information from PubMed records, the number of diseases and lipids will be expanded over time as the biomedical literature grows.The LipiDisease webserver can be freely accessed at http://cbdm-01.zdv.uni-mainz.de:3838/piyusmor/LipiDisease/.Supplementary data are available at Bioinformatics online.

ObjectiveThe current study was undertaken to prospectively explore whether having low levels of von Willebrand factor (vWF) antigen and vWF activity reduce the risk for cardiovascular disease and death.MethodsVWF antigen and vWF activity were measured by enzyme‐linked immunosorbent assay and an immunological‐based assay, respectively, in a subsample of 4857 individuals aged between 35 and 74 years old, enrolled between April 2007 and October 2008 in the population‐based Gutenberg Health Study. VWF antigen and activity below the 20th percentile was set as a measure of “low vWF.” Adjusted robust Poisson regression models were used to analyze the relation between low vWF and the incidence of cardiovascular disease (CVD). Consequent adjusted cox regression models as well as cumulative incidence plots were calculated to explore the relation between all‐cause and cardiovascular mortality and low vWF.ResultsVWF activity levels <20th percentile (i.e., <76.2%) were associated with a decreased relative risk for CVD (RR: 0.59, 95% CI: 0.37–0.95), despite adjusting for age and sex. After adjusting for levels of F‐VIII, the association persisted (RR: 0.60, 95% CI: 0.36–0.99). The cumulative incidence plots demonstrated that vWF antigen <20th percentile significantly correlated with decreased cardiovascular mortality. VWF antigen<20th percentile (i.e., <83%) was significantly associated with lower risk of all‐cause mortality, despite adjusting for clinical factors (RR: 61, 95% CI: 0.41–0.91).ConclusionThe study demonstrated that having low vWF activity levels were associated with a lower risk for CVD. Additionally, it revealed a decreased risk of cardiovascular and all‐cause mortality in individuals with low levels of vWF antigen, shining new light on vWF as a potential target for novel therapies.

Previous evidence suggested that non-COVID-19-related medical care was reduced during the first wave of the COVID-19 pandemic, but it remained unclear whether or to which extent this effect lasted beyond the first wave, or existed in a longer time frame. Here, we consider questionnaire data of the Gutenberg-COVID-19 study together with pre-pandemic baseline data of the Gutenberg Health Study concerning the region around Mainz, Germany, to study the effects of the pandemic on the provision of medical care until April 2021. We observed that the proportion of cancelled medical appointments was low and that the fraction of participants with a medical appointment as an indicator for the number of appointments being made was in line with pre-pandemic levels. Appointments were more likely cancelled by the patient (rather than the provider), and more likely cancelled by medical specialists such as dentists or ophthalmologists (rather than GPs). In conclusion, we found some evidence that, at least with regard to realized appointments, the medical system and the provision of medical care were not harmed by the COVID-19 pandemic on a longer time scale.

Abstract Aims Clonal hematopoiesis (CH), defined as the presence of an expanded blood cell clone due to acquired somatic mutations in leukemia driver genes, was shown to be associated with increased mortality in patients with chronic ischemic heart failure with reduced ejection fraction (HFrEF). Mechanistically, circulating monocytes of mutation carriers display increased expression of proinflammatory genes involved in inflammasome and IL-6 signaling. Inherited genetic variants (SNP) in the IL-6 pathway are well known to affect inflammatory activation. Therefore, we investigated whether known SNPs in genes encoding for components of the inflammasome/IL-6 signaling pathway modulate fatal outcomes in HFrEF patients with CH. Methods and Results In a total of 446 patients with chronic HFrEF, peripheral blood or bone marrow mononuclear cells were analyzed for the CH driver mutations DNMT3A and TET2 as well as 48 preselected SNPs affecting genes in the NLRP3 inflammasome/IL-6 signaling pathway. The 103 patients carrying a CH driver mutation demonstrated significantly increased mortality compared to the 343 patients without CH mutations (25,24% vs 13.99% at five years; p=0.0064). We identified three commonly occurring variants known to disrupt IL-6 signaling (rs2228145, rs4129267 and rs4537545), which are in strong linkage disequilibrium and present in more than 50% of CH carriers. Harboring one of those SNPs abrogated the increased mortality risk in patients with HFrEF and CH (p≤0.05 for each SNP). On the contrary, three different SNPs (rs2250417, which is associated with increased IL-18 levels; rs4722172 and rs4845625, which are known to activate IL-6 signaling) were identified to mediate fatal outcomes in patients with HFrEF and CH; p&lt;0.05 for each). None of the assessed SNPs influenced outcomes in patients without DNMT3A or TET2 mutations. Single Cell RNA-sequencing of circulating monocytes of patients with HFrEF revealed increased inflammatory signaling in DNMT3A mutation carriers harboring IL6/IL18 activating SNPs with genes upregulated in pathways such as “cellular response to stress”, “regulation of cell activation” and “cytokine signaling in the immune system”. Conclusion Among CH carriers with HFrEF, inherited variants in loci encoding for genes involved in inflammatory signaling interact with mortality risk. These data not only provide mechanistic insights into inflammatory mechanisms contributing to fatal outcome of HFrEF in CH carriers, but may also inform trials evaluating precision-targeted anti-inflammatory therapy in patients with DNMT3A and TET2 mutations and chronic HFrEF.

Arterial hypertension is considered a risk factor for the development of heart failure. Here we investigate cross-sectional associations of systolic and diastolic blood pressure with subtle functional and morphological changes of left ventricular echocardiographic parameters representing early dysfunction in three representative German population-based studies. We assessed 26,719 individuals without symptomatic heart failure from the Hamburg City Health Study (HCHS, n = 7396, derivation cohort), the Gutenberg Health Study (GHS, 14,715, validation cohort) and the Study of Health in Pomerania (SHIP, 4608, validation cohort). Multivariable linear regression analyses with systolic and diastolic blood pressure as continuous exposure variables were adjusted for common cardiovascular risk factors and antihypertensive medication. Both systolic and diastolic blood pressure were consistently associated with measures of left ventricular hypertrophy (β per standard deviation (SD) for LV mass (g) and systolic blood pressure: 5.09 (p < 0.001); diastolic blood pressure: 2.29 (p < 0.001) in HCHS). Systolic blood pressure correlated with declining diastolic function (β per SD for E/e': 0.29, p < 0.001 in HCHS) and diastolic blood pressure with declining systolic function (β per SD for LVEF, in %: - 0.15; p = 0.041 in HCHS) in all cohorts. Pending further validation, our results from three independent German population samples suggest differential effects of systolic versus diastolic blood pressure on left ventricular structure and function.

(Developmental Cell 39, 740–755, December 19, 2016) During the assembly of the final version of the Supplemental Information, the axes of Figure S7E were inadvertently mislabeled. Figure S7E has now been corrected here and in the article online. The authors apologize for the error and any inconvenience it may have caused.Figure S7. WEE1 Inhibition Causes DNA Damage and EME2-Dependent Premature Entry into M Phase in RPE1-hTERT Cells (original)View Large Image Figure ViewerDownload Hi-res image Download (PPT) A Mechanism for Controlled Breakage of Under-replicated Chromosomes during MitosisDuda et al.Developmental CellDecember 19, 2016In BriefMUS81 nuclease resolves persistent replication intermediates at mitotic onset to safeguard chromosome segregation. Duda et al. uncover dynamic regulation of MUS81 function during the cell cycle to protect S-phase chromosomes from unscheduled breakage and pulverization. This regulation ensures that DNA replication and mitosis occur sequentially and in a mutually exclusive manner. Full-Text PDF Open Archive

Assessment of endothelial function in humans by measuring flow-mediated dilation (FMD) risk-stratifies individuals with established cardiovascular disease, whereas its predictive value is limited in primary prevention. We therefore aimed to establish and evaluate novel markers of FMD at the population level.In order to identify novel targets that were negatively correlated with FMD and investigate their contribution to vascular function, we performed a genome-wide association study (GWAS) of 4175 participants of the population based Gutenberg Health Study. Subsequently, conditional knockout mouse models deleting the gene of interest were generated and characterized. GWAS analysis revealed that single-nucleotide polymorphisms (SNPs) in the tubulin-folding cofactor E (TBCE) gene were negatively correlated with endothelial function and TBCE expression. Vascular smooth muscle cell (VSMC)-targeted TBCE deficiency was associated with endothelial dysfunction, aortic wall hypertrophy, and endoplasmic reticulum (ER) stress-mediated VSMC hyperproliferation in mice, paralleled by calnexin up-regulation and exacerbated by the blood pressure hormone angiotensin II. Treating SMMHC-ERT2-Cre+/-TBCEfl/fl mice with the ER stress modulator tauroursodeoxycholic acid amplified Raptor/Beclin-1-dependent autophagy and reversed vascular dysfunction.TBCE and tubulin homeostasis seem to be novel predictors of vascular function and offer a new drug target to ameliorate ER stress-dependent vascular dysfunction.

Abstract Objective Tissue factor pathway inhibitor (TFPI) is a potent anticoagulant protein in the extrinsic coagulation pathway. In the present study, we aim to identify the cardiovascular determinants for total TFPI activity and its association with cardiovascular disease (CVD) and total mortality. Methods Total TFPI activity was assessed in a selection of the population-based Gutenberg Health Study (n = 5,000). Statistical analysis was performed to identify the determinants for total TFPI activity as well as the associations with CVD and mortality. Results Multivariable linear regression analysis identified smoking (β 0.095 [0.054–0.136]) as a positive determinant for total TFPI activity, while diabetes (β –0.072 [–0.134 to –0.009]), obesity (β –0.063 [–0.101 to –0.024]), and history of coronary artery disease (CAD) were negatively associated with total TFPI activity, independent of age, sex, and the remaining cardiovascular risk factors. After adjustment for lipoprotein levels, the association between total TFPI activity levels and obesity and CAD was lost. The analysis additionally revealed a strong positive association between total TFPI activity levels and low-density lipoprotein (β 0.221 [0.204–0.237]). The Cox regression models revealed that a higher total TFPI activity, above 97.5th percentile of the reference group, was associated with an increased mortality risk (hazard ratio = 2.58 [95% confidence interval: 1.49–4.47]), independent of age, sex, and cardiovascular risk profile. Conclusion In the Gutenberg Health Study population-based cohort, the highest percentage of total TFPI correlated with an increased mortality risk. While elevated TFPI may reflect endothelial cell activation, the associations between total TFPI activity and obesity and CAD, points to additional mechanistic interactions.

The current study aims to identify the relationships between coagulation factors and plasma thrombin generation in a large population-based study by comparing individuals with a history of arterial or venous thrombosis to cardiovascular healthy individuals.This study comprised 502 individuals with a history of arterial disease, 195 with history of venous thrombosis and 1402 cardiovascular healthy individuals (reference group) from the population-based Gutenberg Health Study (GHS). Calibrated Automated Thrombography was assessed and coagulation factors were measured by means of BCS XP Systems. To assess the biochemical determinants of TG variables, a multiple linear regression analysis, adjusted for age, sex and antithrombotic therapy, was conducted.The lag time, the time to form the first thrombin, was mainly positively associated with the natural coagulant and anti-coagulant factors in the reference group, i.e. higher factors result in a longer lag time. The same determinants were negative for individuals with a history of arterial or venous thrombosis, with a 10 times higher effect size. Endogenous thrombin potential, or area under the curve, was predominantly positively determined by factor II, VIII, X and IX in all groups. However, the effect sizes of the reported associations were 4 times higher for the arterial and venous disease groups in comparison to the reference group.This large-scale analysis demonstrated a stronger effect of the coagulant and natural anti-coagulant factors on the thrombin potential in individuals with a history of arterial or venous thrombosis as compared to healthy individuals, which implicates sustained alterations in the plasma coagulome in subjects with a history of thrombotic vascular disease, despite intake of antithrombotic therapy.

18,335 eyes of 9,559 participants aged 40 to 80 years were included in the analysis. Median pupil diameter was 4.19 mm in right eyes and 4.12 mm in left eyes. A smaller pupil was associated with older age, hyperopic refractive error, previous cataract surgery, diabetes, obesity, and ACE inhibitor intake, whereas wider pupil was associated with female gender, arterial hypertension, intake of tricyclic antidepressants, and intake of SNRI and tetracyclic antidepressants. Socioeconomic status and smoking were not associated with pupil size.Individuals of older age, after cataract surgery, under therapy with ACE inhibitors and with diabetes have a smaller pupil. This should be taken into account when planning nonmydriatic fundus photography-based screening programs, for instance, for diabetic retinopathy.

Accurate assessment of left ventricular function by determining left ventricular volumes and ejection fraction is important in evaluating the prognoses of patients with heart failure. Two-dimensional (2D) echocardiography suffers from low correlation with reference methods like ventriculography. Three-dimensionally (3D) assessed data have been proved to have better conformity. Endocardial border delineation remains a problem, however, especially in patients with suboptimal recordings. Few data exist on 3D-echocardiographic volumetry with ultrasound contrast agents (UCAs). We evaluated the second-generation UCA LK565 for its boundary-tracing capacities in freehand 3D echocardiography in a phase II clinical trial. Safety and efficacy of the novel contrast agent were also evaluated.Forty patients between the age of 42 and 77 were included in this trial. Left ventricular end-systolic and -diastolic volume (LVESV, LVEDV) and ejection fraction (EF) were determined by either 2D or 3D freehand second harmonic echocardiography with and without use of LK565. Parameters were compared statistically with ventriculography performed in 35 patients. Immune response to LK565 was evaluated by analysing phagocytosis capacity and kinetics of inflammatory cytokines (TNF-alpha, IL-4, IL-10, IFN-gamma). Patients were monitored for adverse events up to 72 h after application of the UCA. Calculated values for left ventricular volumes and ejection fraction correlated best for freehand 3D echocardiography in combination with LK565 (r=0.92 for LVEDV; r=0.96 for LVESV; r=0.94 for EF). Excellent left ventricular contrast enhancement was achieved for approximately 8 min. A reversible saturation of phagocytosis capacity for monocytes and neutrophils set in with a maximum peak at 6h. No significant increase in cytokine expression was observed.LK565 improves feasibility of endocardial border delineation in 3D echocardiography, leading to better correlation of left ventricular volumetry with reference methods. Efficacy and safety of LK565 are equivalent to those of conventional UCAs.

Abstract Airborne transmission by droplets and aerosols is important for the spread of viruses and face masks are a well-established preventive measure, but their effectiveness for mitigating COVID-19 is still under debate. We show that variations in mask efficacy can be explained by different regimes of virus abundance. For SARS-CoV-2, the virus load of infectious individuals can vary by orders of magnitude, but we find that most environments and contacts are in a virus-limited regime where simple surgical masks are highly effective on individual and population-average levels, whereas more advanced masks and other protective equipment are required in potentially virus-rich indoor environments such as medical centers and hospitals. Due to synergistic effects, masks are particularly effective in combination with other preventive measures like ventilation and distancing. One Sentence Summary Face masks are highly effective due to prevailing virus-limited environments in airborne transmission of COVID-19.

Background: Heart failure (HF) is a multifactorial syndrome with pathophysiological complexities still not fully understood. Higher mean platelet volume (MPV), a potential marker of platelet activation, and high concentrations of parathyroid hormone (PTH) have been implicated in the pathogenesis of HF. Aim: This study aims to investigate sex-specifically the association between PTH concentrations and platelet indices in phenotypes of HF. Methods and Results: PTH and platelet indices (MPV and platelet count) were available in 1,896 participants from the MyoVasc study in Mainz, Germany. Multivariable linear regression models, adjusted for age, sex, season, vitamin D status, cardiovascular risk factors, comorbidities, estimated glomerular filtration rate, and medication, were used to assess the associations between platelet indices and PTH. The results showed distinct sex-specific associations between PTH and platelet indices. A positive association between PTH and MPV was found in females with symptomatic HF with reduced ejection fraction (HFrEF) only [β = 0.60 (0.19; 1.00)]. Platelet count was inversely associated with PTH in male HFrEF individuals [β = -7.6 (-15; -0.30)] and in both males and females with HF with preserved ejection fraction (HFpEF). Conclusion: This study reports differential, sex-specific relationships between PTH and platelet indices in HF individuals independent of vitamin D status and clinical profile. Particularly in phenotypes of symptomatic HF, distinct associations were observed, suggesting a sex-specific mechanism involved in the interaction between PTH and platelets.

Aircraft noise has been regarded as one of the major environmental issues related to air transport. Many airports have introduced a variety of measures to reduce its impact. Several air traffic assignment strategies have been proposed in order to allocate noise more wisely. Even though each decision regarding the assignment of aircraft to routes should consider population exposure to noise, none of the air traffic assignment strategies has addressed daily migrations of population and number of people exposed to noise. The aim of this research is to develop a mathematical model and a heuristic algorithm that could assign aircraft to departure and arrival routes so that number of people exposed to noise is as low as possible, taking into account temporal and spatial variations in population in an airport’s vicinity. The approach was demonstrated on Belgrade airport to show the benefits of the proposed model. Numerical example showed that population exposure to noise could be reduced significantly by applying the proposed air traffic assignment model. As a consequence of the proposed air traffic assignment, overall fuel consumption increased by less than 1%.

Previous studies reported significantly altered tryptophan catabolite concentrations in major depression. Thus, tryptophan catabolites were considered as potential biomarkers of depression and their modulators as potential targets for psychopharmacotherapy. However, the results were based mainly on studies with small sample sizes limiting their generalizability. Against this background, we investigated the relationship of peripheral tryptophan catabolites with depression in a population-based sample with n = 3,389 participants (with fasting status ≥ 8 h and C-reactive protein < 10 mg/L). N = 248 had clinically significant depression according to a PHQ-9 score of ≥ 10, n = 1,101 subjects had mild depressive symptoms with PHQ-9 scores between 5 and 9, and n = 2,040 had no depression. After multivariable adjustment, clinically significant depression was associated with lower kynurenine and kynurenic acid. Spearman correlation coefficients of the tryptophan catabolites with the severity of depression were very small (rho ≤ 0.080, p ≤ 0.015). None of the tryptophan catabolites could diagnostically separate depressed from not depressed persons. Concerning linear associations, kynurenine and kynurenic acid were associated only with the severity and the cognitive dimension of depression but not its somatic dimension. Tryptophan catabolites were not associated with persistence or recurrence of depression at the 5 year follow-up. The results replicated the association between kynurenine and kynurenic acid with depression. However, the associations were small raising doubts about their clinical utility. Findings underline the complexity of the relationships between depression and tryptophan catabolites. The search for subgroups of depression with a potentially higher impact of depression might be warranted.

The present study aimed to investigate how depressive symptoms affect bodyweight change (gain and loss), and how this association is intertwined with other psychosocial and biomedical factors in the adult general population. In a population-based, prospective, observational single-center cohort study in the Rhine-Main-Region, Germany (Gutenberg Health Study GHS) with N = 12,220 participants, we analyzed baseline and five year follow-up data with logistic regressions separately for bodyweight gain and loss (vs. stable bodyweight). Overall, 19.8 % of participants gained bodyweight of at least 5 %. More female participants were affected than male participants (23.3 % vs. 16.6 %). Regarding weight loss, overall, 12.4 % lost >5 % of bodyweight; participants were more often female than male (13.0 % vs. 11.8 %). Depressive symptoms at baseline were associated with weight gain (OR = 1.03, 95 % CI = 1.02–1.05). In models controlling for psychosocial and biomedical factors, female gender, younger age, lower socioeconomic status and smoking cessation were associated with weight gain. In weight loss, there was no overall significant effect of depressive symptoms (OR = 1.01 [0.99; 1.03]). Weight loss was associated with female gender, diabetes, less physical activity, and higher BMI at baseline. Only in women, smoking and cancer were associated with weight loss. Depressive symptoms were assessed via self-report. Voluntary weight loss cannot be determined. Significant weight change frequently occurs in middle to old adulthood resulting from a complex interplay of psychosocial and biomedical factors. Associations with age, gender, somatic illness and health behavior (e.g. smoking cessation) provide important information for the prevention of unfavorable weight change.

Abstract Purpose Anti-retinal autoantibodies are assumed to be associated with age-related macular degeneration (AMD). To our knowledge, this is the first evaluation of autoantibodies in human sera of participants with different stages of AMD in a large population-based, observational cohort study in Germany. Methods The Gutenberg Health Study (GHS) is a population-based, observational cohort study in Germany, including 15,010 participants aged between 35 and 74. Amongst others, non-mydriatic fundus photography (Visucam PRO NM™, Carl Zeiss Meditec AG, Jena, Germany) was performed. Fundus images of the first 5000 participants were graded based on the Rotterdam Eye Study classification. Sera of participants with AMD ( n =541) and sera of age-matched participants without AMD ( n =490) were analyzed by antigen-microarrays. Besides descriptive statistics, autoantibody-levels were compared by Mann-Whitney-U test and the associations of level of autoantibodies with AMD were calculated by logistic regression analysis. Likewise, possible associations of the autoantibodies and both clinical and laboratory parameters on AMD subjects were analyzed. Results Autoantibodies against transferrin ( p &lt;0.001) were significantly downregulated in participants with early AMD and soft, distinct drusen (≥63 μm) or pigmentary abnormalities only compared to Controls. Mitogen-activated protein kinase 3 ( p =0.041), glutathione peroxidase 4 ( p =0.048), clusterin ( p =0.045), lysozyme ( p =0.19), protein kinase C substrate 80K-H ( p =0.02), heat shock 70 kDa protein 1A ( p =0.04) and insulin ( p =0.018) show a trend between Control and participants with early AMD and soft, distinct drusen (≥63 μm) or pigmentary abnormalities only. Conclusions This study contributes to a growing knowledge of autoantibodies in association with different AMD stages compared to controls in the context of a large population-based study in Germany. Especially autoantibodies against inflammatory proteins were downregulated in participants with early AMD and soft, distinct drusen (≥63 μm) or pigmentary abnormalities only.

Introduction: Chronotropic incompetence (CI), an important aspect of exercise intolerance, is related to adverse clinical outcomes in heart failure (HF). However, the lack of standardized evaluation of CI results in inconsistent evidence. Aim: To evaluate CI with regard to clinical and medical determinants and relationship to clinical outcomes in HF. Methods: Data from the MyoVasc study (NCT04064450), a prospective cohort on HF, were analyzed. Subjects underwent deep clinical phenotyping, including cardiopulmonary exercise testing on a cycle ergometer. Measures of CI were evaluated, with continuous predicted heart rate reserve (pHRR) emerging as the most clinically relevant definition for operationalizing CI. Clinical and medical determinants of CI were assessed in multivariable linear regression models and outcomes in Cox regression models. Results: The analysis sample included 886 subjects (median age 63 years [interquartile range (IQR) 55; 72]; 29.9% women) who achieved maximal exertion (respiratory exchange ratio&gt;1.0). Symptomatic HF stage C/D was present in 42.8% (379) of the subjects. Sixteen drug groups were identified as CI modulators including β-blockers (β-estimate per SD -0.83 [95% confidence interval -0.95;-0.70], p&lt;0.0001) and antiarrhythmics (β SD -0.93 [-1.30;-0.56], p&lt;0.0001). New York Heart Association functional class, smoking, fatty liver index (FLI), and diabetes mellitus (DM) were the strongest determinants of CI in all subjects independently of sex, age, and previously identified medication. Determinants differed according to left ventricular ejection fraction [LVEF] (≥50 vs &lt;50%), being strongly associated with markers of HF (NT-proBNP) and lung function (FEV1) in HF with reduced EF and more linked to DM or the FLI in HF with preserved EF. In Cox regression analysis adjusted for clinical profile, pHRR independently predicted all-cause death (HR SD 1.86 [1.41;2.45], p&lt;0.0001) and worsening of HF (HR SD 1.37 [1.04;1.81], p=0.025). Conclusions: CI strongly predicted clinical outcomes and revealed relevant differences between HF with preserved and reduced EF. This highlights the relevance of impaired response to exercise in the clinical course of HF with implication for risk stratification and intervention.

Abstract Background It is unclear to which extent persistence of symptoms and/or residual haemodynamic impairment clinical course of pulmonary embolism are associated with worse quality of life (QoL). Aims To study the correlation between symptoms and haemodynamic impairment with QoL during the first year after acute pulmonary embolism (PE). Methods The Follow-Up after acute pulmonary embolism (FOCUS) study prospectively enrolled and followed consecutive adult patients diagnosed with acute symptomatic objectively diagnosed PE. In the present analysis, we considered patients who completed the Pulmonary Embolism QoL (PEmb-QoL) Questionnaire at predefined visits 3 and 12 months after acute PE. The PEmb-QoL score ranges from 0% (best QoL) to 100% (worst QoL). We evaluated at these two time points the correlation between persisting symptoms (group: symptoms), elevation of natriuretic peptides or residual right ventricular dysfunction (group: RVD), or their combination (group: symptoms + RVD) and QoL. Results A total of 617 patients were included; their median age was 62 years, 44% were women; 8% had active cancer, and 21% previous venous thromboembolism. At 3 months, patients with neither symptoms nor RVD (n=302) had the highest quality of life (median score 18%, 25th–75th percentile: 8%–34%), followed by those without symptoms but with RVD (n=255; median score 19%, 25th–75th percentile: 7%–34%), and by those with symptoms only (n=131; median PEmb-QoL 31%, 25th–75th percentile: 18%–49%). Patients with both symptoms and RVD (n=170) had the worst quality of life (median score 38%, 25th–75th percentile: 19%–53%); Figure 1A. At 12 months, we found an overall improvement of PEmb-QoL score. The degree of this QoL improvement varied across groups, being largest for patients who recovered from having symptoms + RVD at 3 months to normalization of at least one at 12 months. The change in QoL from 3 to 12 months was smaller both in patients who had neither symptoms nor RVD and in patients who had no recovery in either symptoms or RVD; Figure 1B. Conclusions Persistent symptoms after PE, especially in patients with elevated biomarkers or residual echocardiographic dysfunction, were the main drivers of QoL at 3 months as well as of the course of QoL over time. Figure 1 Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): University Medical Center of the Johannes Gutenberg University, Mainz, Germany; German Federal Ministry of Education and Research

Purpose: Arterial stiffness (AS) has been reported to vary sex-specifically with significantly lower values in females. However, the pathophysiological mechanisms responsible for better arterial distensibility among women are still not fully understood. The aim of the present analysis was to investigate the association of arterial stiffness, as determined by stiffness index (SI), with female sex hormones, menopausal status and reproductive history. Methods: AS was assessed by digital volume pulse analysis on a PCA2 device (Carefusion/Micro Medical Limited; Rochester, UK) with subsequent calculation of SI in n=441 women. Data on menstrual cycle, reproductive history and medical history were recorded in a personal computer-assisted history. Female sex hormones (plasma estradiol, progesterone, luteinizing hormone (LH), and follicle stimulating hormone (FSH)) were measured by routine laboratory methods. Results: We investigated 277 premenopausal (aged 29.7±9.33 years) and 164 postmenopausal women (aged 57.7±7.5 years); SI was 5.9 m/s (5.4/7.1) and 9.9 m/s (8.6/11.7), respectively (p value <0.0001). Different patterns of correlation were found for SI with sex hormones and reproductive history among pre- and postmenopausal women. SI was moderately correlated with FSH (r=0.22, p=0.014, age-adjusted) and with estradiol (r=-0.21 p=0.046, age-adjusted) in postmenopausal women, whereas no correlation was observed for SI with sex hormones among premenopausal women. In premenopausal women, however, positive correlation was detected between SI and age at menarche (r=0.22, p=0.00075, age-adjusted) and a trend towards an inverse relationship with duration of breastfeeding (r=-0.26, p=0.046, age-adjusted). Results of multivariable regression analysis adjusted for age and traditional cardiovascular risk factors confirmed no relation of SI with the hormonal status in premenopausal women. In contrast, postmenopausal women demonstrated an association of SI with FSH in the fully adjusted model with a β-estimate of 1.36 per 1 SD increase in hormone concentration (95% confidence interval [CI] 0.443-2.27; p-value=0.004), and with LH with a β-estimate of 1.02 (95% CI 0.097-1.95; p-value=0.031). Age at menarche was related with SI in premenopausal women only (β=0.26, 95% CI 0.07-0.45, p=0.0075). Conclusions: Arterial stiffness assessed by digital photoplethysmography is associated with sex hormones in postmenopausal individuals, but not in premenopausal women. The underlying mechanisms, which might be responsible for such association, should be investigated in the further studies.

Introduction: Atherosclerosis and pulmonary embolism (PE) affect cardiovascular mortality substantially. We aimed to investigate the impact of atherosclerosis on the outcomes of patients with deep venous thrombosis (DVT) and to identify the differences in DVT patients with and without PE. Methods: Patients with DVT with and without symptomatic atherosclerosis (defined as coronary artery disease, myocardial infarction and/or peripheral artery disease) as well as with and without PE under oral anticoagulation were enrolled during January 2011−April 2013 and compared. The impact of symptomatic atherosclerosis on several outcomes was analyzed. Results: Overall, 509 DVT patients (70.0 [56.0−77.0] years, 51.9% females) were included in this study. Among them, 179 (36.3%) had symptomatic atherosclerosis and 204 (40.1%) a concomitant PE. DVT patients with symptomatic atherosclerosis were older (74.0 [IQR 65.0−80.0] vs. 63.0 [48.0−75.0] years, p < 0.0001), more often male (56.4% vs. 43.9%, p = 0.0087) and had a higher prevalence of classical CVRF and a higher Charlson comorbidity index (7.00 [5.00−8.00] vs. 4.00 [2.00−6.00], p < 0.001). Symptomatic atherosclerosis was associated with increased mortality (HR 1.98 [95%CI 1.12−3.49], p = 0.018) and hospitalizations (HR 1.64 [95%CI 1.21−2.21], p = 0.0012) and primary long-term outcome (HR 1.99 [95%CI 1.31−3.04], p = 0.0013) during the 2 years follow-up-period in DVT patients. DVT patients without PE had diabetes mellitus (28.2% vs. 16.3%, p < 0.01) and symptomatic atherosclerosis (42.9% vs. 26.4%, p < 0.001) more often compared to DVT patients with PE, and symptomatic atherosclerosis was associated with isolated DVT (without PE) (OR 2.01 [95%CI 1.28−3.16], p < 0.01). Conclusions: Atherosclerosis was associated with isolated DVT (without PE) and increased mortality in DVT patients under oral anticoagulation. The profile of CVRF and comorbidities differed between DVT patients with and without a concomitant PE. In the case of DVT or PE, patients should be screened for concomitant atherosclerotic disease. Clinical Trial Registration: at clinicaltrials with Unique identifier NCT01809015.

There is evidence that plasma protein profiles differ in the two subtypes of pulmonary embolism (PE), isolated PE (iPE) and deep vein thrombosis (DVT)-associated PE (DVT-PE), in the acute phase. The aim of this study was to determine specific plasma signatures for proteins related to platelets in acute iPE and DVT-PE compared to isolated DVT (iDVT).Within the Genotyping and Molecular Phenotyping of Venous ThromboEmbolism (GMP-VTE) Project, a multicenter prospective cohort study of 693 confirmed VTE cases, a highly sensitive targeted proteomics approach based on dual-antibody proximity extension assay was applied. LASSO-regularized logistic regression analysis selected 33 and 30 of 135 platelet-related candidate proteins in iPE and DVT-PE vs. iDVT, respectively.All regulated proteins were well associated with six prominently released platelet proteins and the majority showed specificity for iPE and DVT-PE compared to iDVT. While iPE-specific proteins were assigned to be predominantly released via shedding mechanisms and extracellular vesicles, granule secretion was identified as a major release mechanism assigned to DVT-associated PE-specific proteins. Network analysis demonstrated three interconnected clusters of specifically regulated proteins in iPE linked to immunoreceptor signaling, pathogen clearance and chemotaxis, whereas for DVT-associated PE one cluster linked to tissue remodeling and leukocyte trafficking. Machine learning-based analysis reveals specific plasma signatures and differential release mechanisms of proteins related to platelets in acute iPE and DVT-associated PE.These data suggest that the platelet protein releasate contributes to the differential regulation of plasma proteins in acute PE compared to iDVT, which may be associated with different platelet activation patterns.

Background Individuals living at-risk-of-poverty have an increased risk of poor mental health. The pandemic and its societal impacts might have negative effects especially on this group widening the gap between rich and poor and also exacerbate gender gaps, which in turn might impact social cohesion. Aim The objective of this longitudinal study was to determine if people living at-risk-of-poverty were more vulnerable to economic and psychosocial impacts of the pandemic and showed poorer mental health. Moreover, gender differences were analyzed. Method We drew data from a sample of N = 10,250 respondents of two time points (T1 starting from October 2020, T2 starting from March 2021) of the Gutenberg COVID-19 Study. We tested for differences between people living at-risk-of-poverty and more affluent respondents regarding economic impacts, psychosocial stressors, as well as depressiveness, anxiety and loneliness, by comparing mean and distributional differences. To test for significant discrepancy, we opted for chi-square- and t -tests. Results The analysis sample compromised N = 8,100 individuals of which 4,2% could be classified as living at-risk-of-poverty. 23% of respondents living at-risk-of-poverty had a decrease in income since the beginning of the pandemic–twice as many as those not living at-risk-of-poverty, who reported more often an increase in income. Less affluent individuals reported a decrease in working hours, while more affluent people reported an increase. Between our survey time points, we found a significant decrease in these economic impacts. Gender differences for economic changes were only found for more affluent women who worked more hours with no change in income. Less affluent respondents were more impacted by psychosocial stressors, depressiveness, anxiety, and loneliness. Gender differences were found particularly with regard to care responsibilities. Discussion Our results indicate a widening in the gap between the rich and the poor at the beginning of the pandemic. Gender differences concerning economic changes affect more affluent women, but women in both income groups are more burdened by care responsibilities, which might indicate a heightened resurgence of gender role in times of crisis. This increase in inequality might have impacted social cohesion.

Die vorliegende Arbeit stellt die Ergebnisse einer Untersuchung über den Zusammenhang mentaler Repräsentationen des elterlichen Erziehungsverhaltens und die psychosoziale Entwicklung von Erwachsenen in einer großen, registerbasierten Stichprobe Langzeitüberlebender von Krebserkrankungen im Kindes- / Jugendalter (N = 951) vor. Diese waren im Kontext der Studien CVSS und PSYNA umfassend medizinisch und psychologisch charakterisiert worden. Der »Fragebogen zum erinnerten elterlichen Erziehungsverhalten« (FEE) umfasst die Dimensionen emotionale Wärme, Kontrolle / Überbehütung und Strafe / Zurückweisung. Bei statistischer Kontrolle der Diagnosegruppe und aktueller psychischer Belastung standen diese in logistischen Regressionsmodellen mit praktisch relevanten Outcomes in Zusammenhang. So war ein größeres Ausmaß erinnerter emotionaler Wärme mit einer Partnerschaft sowie Elternschaft assoziiert, Repräsentationen strafenden / zurückweisenden Elternverhaltens standen in Zusammenhang mit einem niedrigeren Schulabschluss und aktueller Erwerbslosigkeit. Die Ergebnisse weisen darauf hin, dass Eltern eine wichtige Rolle bei der Verarbeitung einer Krebserkrankung im Kindesalter und für positive Langzeit-Outcomes wie soziale Integration, Bildungserfolg und Berufstätigkeit haben.

The Bloom’s helicase ortholog, Sgs1, orchestrates the formation and disengagement of DNA joint molecules to enable controlled crossing-over during meiotic and mitotic DNA repair. Whether its enzymatic activity is temporally regulated to implement formation of noncrossovers prior to the activation of crossover-nucleases is unknown. Here, we show that akin to the Mus81-Mms4, Yen1 and MutLγ-Exo1 nucleases, Sgs1 helicase function is under tight cell cycle control through the actions of CDK and Polo kinase Cdc5. Notably, however, whereas CDK and Cdc5 unleash nuclease function during M-phase, they collaboratively delimit Sgs1 activation to S-phase. Mechanistically, CDK-mediated phosphorylation enhances the velocity and processivity of Sgs1, which stimulates DNA unwinding in vitro and joint molecule processing in vivo. Subsequent hyper-phosphorylation by Cdc5 appears to reduce the activity of Sgs1, while activating Mus81-Mms4 and MutLγ-Exo1. These findings suggest a general mechanism that drives orderly formation of noncrossover and crossover recombinants in meiotic and mitotic cells.

Abstract Venous thromboembolism (VTE) is associated with increased coagulation activity, which in part can be attributed to the contact pathway of coagulation. Evidence from pre-clinical and epidemiological studies suggests that deficiency in factors of contact activation (e.g. coagulation factors (F) XI and FXII) protects against VTE. However, limited information exists regarding the activation of the contact system in the setting of acute VTE. In the current study, patients with confirmed VTE events (n=321) from the VTEval study and controls (n=300) from the population-based PREVENT-it pilot study were included. Plasma samples were collected from patients after confirmed VTE events or controls upon inclusion in the study. FXI as well as FXIa and plasma kallikrein (PKa) levels were assessed in plasma samples from all subjects using an activated thromboplastin time-based assay (FXI:c), a thrombin generation-based assay (CAT:FXIa) and by measuring inhibitory complexes (FXIa:antithrombin (AT), FXIa:alpha-1-antitrypsin (α1AT), FXIa:C1 esterase inhibitor (C1Inh) and PKa:C1Inh) using enzyme-linked immunoassay (ELISA). After a 2-year follow up period, a composite endpoint of recurrent VTE or death was determined. Increased FXI:c levels were determined in VTE patients compared to control individuals (124.08 ± 37.48% vs. 113.55 ± 27.99%), whereas CAT:FXIa levels were reduced in VTE patients (0 pM [IQR, 0-0.56] vs 0.56 pM [IQR, 0-0.88]). Levels of FXIa:α1AT and FXIa:AT inhibitory complexes were increased in VTE patients compared to controls (median[IQR]; 311.8 pM [238.2-424.0] vs. 202.5 pM [143.7 - 287.5] and 29.1 pM [23.4-38.3] vs 23.2 pM [19.7-29.8], respectively). Considering that 86% of the VTE patients were already on anticoagulant treatment (Table 1), investigation of their possible effect on the biomarkers revealed that only the CAT:FXIa was influenced by the presence of anticoagulants. Logistic regression models revealed a good discriminatory value for FXI:c and FXIa:α1AT (AUC=0.64 [0.6/0.69] and AUC=0.67 [0.62/0.71], respectively) to distinguish VTE from controls, whereas the other biomarkers were not able to distinguish between groups. The outcome recurrent VTE or death could be predicted by the inhibitory complexes, but not by the FXI(a) levels (Figure 1). Only the FXIa:α1AT complexes were able to both detect the presence of VTE (OR per SD [95%CI]: 1.28 [1.01-1.63], p=0.04) and predict recurrent VTE or death (HR per SD [95%CI]: 1.40 [1.2-1.62], p&amp;lt;0.0001). In summary, acute VTE is associated with both elevated FXI:c levels and increased activation of FXI and plasma prekallikrein, the latter specifically indicating contact activation. The generation of FXIa during acute VTE and its association with recurrent VTE suggests an important risk contribution of FXI activation. This study has added evidence favouring the utility of FXIa inhibition in the setting of acute VTE. Figure 1 Figure 1. Disclosures Knoeck: Bayer AG: Consultancy. ten Cate: Bayer AG: Other; Pfizer: Other; LEO Pharma: Other; Gideon Pharmaceuticals: Other; Alveron Pharma: Other. Wild: Bayer AG: Other, Research Funding; Boehringer Ingelheim: Other, Research Funding; Novartis Pharma: Other, Research Funding; Sanofi-Aventis: Other, Research Funding; Astra Zeneca: Other, Research Funding; Daiichi Sankyo Europe: Other, Research Funding.

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Introduction: Low molecular weight heparin is frequently used as bridging therapy (BT) during phases with discontinuation of oral anticoagulation (OAC). Hypothesis: To provide sex-specific data on frequency and duration of BT, to explore clinical and sociodemographic factors associated with BT, to assess complications. Methods: We investigated 760 participants from the prospective thrombEVAL study (NCT01809015), treated with OAC in a specialized coagulation service. Data were obtained from medical records, by clinical visits and laboratory data. All information on endpoints was validated via hospital records. Results: A total of 281,934 days of OAC and 13,065 days of BT were analyzed. Men (m) had less bridging episodes (BE) per year, resulting in 6.2days less BT per year (m: 15.4days of BT/y vs. w: 21.6days of BT/y). BT was applied in 2 out of 3 patients receiving surgery, whereas BT and OAC were equally frequent applied during interventional procedures. Quality of OAC did not differ significantly between...

Introduction: The mechanisms leading to myocardial infarction (MI) are still not fully understood. As the orphan G protein-coupled receptor 15 ( GPR15 ) gene has recently been shown to be hypomethylated in smokers as well as to be involved in inflammation, it poses a new candidate gene for cardiovascular disease. Hypothesis: We hypothesized that GPR15 influences the development of MI via smoking and inflammation. Methods: To investigate the effect of smoking and changes in expression over time, monocytic GPR15 mRNA expression was measured in smokers (n = 273), ex-smokers (n = 533) and never-smokers (n = 687) from the Gutenberg Health Study (GHS) via qPCR. In order to assess GPR15 expression after MI, mRNA expression in peripheral blood mononuclear cells was compared between 112 young MI cases (&lt; 50 years) and 112 controls from the GHS using the Affymetrix Exon ST1.0 GeneChip Array. Subsequently, cardiac GPR15 expression in mouse models of MI (n = 8), myocarditis (n = 8), hypertrophy (n = 6) and untreated controls was measured via qPCR. Results: GPR15 expression was significantly increased in smokers compared to never-smokers (16.0 ± 2.4-fold, p &lt; 0.001). Ex-smokers still had significantly elevated GPR15 expression levels compared to never-smokers up to ten years after quitting (3.9 ± 0.9-fold, p &lt; 0.001). Furthermore, GPR15 expression was 1.43-fold higher in young MI patients compared to controls (± 0.09-fold, p = 1.9*10 -7 ). Likewise, mouse Gpr15 expression was significantly elevated in infarcted hearts (6.3 ± 1.1 fold, p &lt; 0.05) and in inflamed hearts (10.2 ± 2.5 fold, p &lt; 0.01) but not in the hypertrophic hearts (1.0 ± 0.3) compared to controls. Conclusion: Consistent with literature on GPR15 methylation, GPR15 expression was increased in smokers, but also after inflammation and ischemia, independently of smoking. In conclusion, GPR15 might play a role in the development of myocardial infarction via smoking and inflammatory mechanisms.

Circulating levels of the pro-inflammatory cytokine Interleukin-18 (IL-18) associate with cardiovascular events. IL-18 GWAS identified the NLRC4 locus, with rs385076 in the 5’UTR exon 2 of NLRC4 as lead SNPs. Data from the ENCODE project indicates accessible DNA and PU.1 transcription factor binding around rs385076. We assessed the hypothesis that rs385076 alleles regulate NLRC4 gene expression by modulating binding of PU.1. To investigate the influence of rs385076 alleles on PU.1 binding, a luciferase reporter gene assay with T or C allele containing plasmids was transfected into human embryonic kidney cells. The cells were treated with 0 / 12.5 / 25 or 50 ng PU.1. Gene expression of 1) total NLRC4, 2) NLRC4 isoforms containing 5’UTR exon 2 and 3) isoforms without exon 2 was quantified by qPCR in 1,500 individuals of the Gutenberg Health Study. Effects of rs385076 T/C allele on NLRC4 isoform levels were calculated using a linear mixed model. In PU.1 treated cells, luciferase activity of the rs385076 C allele was 6-fold (±1.3) higher compared to controls, whereas the T allele showed no significant increase. Total NLRC4 gene expression was decreased in T allele carriers (p=7x10-3). Comparing isoforms containing the exon 2 (decreased, p=2x10-10) to those without (increased, p=7x10-4), expression of NLRC4 isoforms was regulated by T alleles in opposite directions, indicating a switched isoform usage (see figure). In conclusion, our analyses indicate that the rs385076 T allele leads to decreased gene expression of NLRC4 and a switched isoform usage, putatively affected by decreased PU.1 binding. This is consistent with the finding that the T allele associated with lower IL-18 levels.

Background: Heme oxygenase-1 (HO-1) confers protection to the vasculature and suppresses inflammatory properties of monocytes and macrophages. It is unclear how HO-1 activity and expression determine the extent of vascular dysfunction in mice and humans. Methods and results: Decreasing HO activity was parallelled by decreasing aortic HO-1, eNOS and phospho-eNOS (ser1177) protein expression in HO-1 deficient mice, whereas aortic expression of nox2 showed a stepwise increase in HO-1+/- and HO-1-/- mice as compared to HO-1+/+ controls. Aortic superoxide formation increased depending on the extent of HO-1 deficiency and was blunted by the PKC inhibitor chelerythrine, indicating activation of the NADPH oxidase. When subjected to disease models of vascular dysfunction - angiotensin II-infusion (ATII, 0.1mg/kg/d for 7d), streptozotocin-induced diabetes mellitus and aging - HO-1 deficient mice showed an increased vascular dysfunction (shown by isometric tension studies) that was inversely correlated with HO activity. In a primary prevention population based cohort (the Gutenberg Health Study, GHS), we assessed length polymorphisms of the HO-1 promoter region, established a bipolar frequency pattern of allele length (long vs short repeats) in 4937 individuals and found a moderately significant association with flow mediated dilation of the brachial artery (FMD) in individuals with arterial hypertension. Monocytic HO-1 mRNA expression was positively correlated with CD14 expression indicating proinflammatory monocytes (p&lt;0.001) and inversely with FMD in 733 hypertensive individuals of the GHS. ATII-infused HO-1+/+ mice had a significant infiltration of proinflammatory CD11b+Ly6Chi monocytes into the aortic wall, which was sharpely increased in HO-1+/- and HO-1-/- mice, providing a mechanistic link of the monocyte phenotype determined by HO-1 and vascular dysfunction in arterial hypertension. Conclusions: We here present evidence that HO activity and expression and inversely correlates with vascular dysfunction and NADPH oxidase mediated oxidative stress in mice and humans. We conclude, that HO-1 is a regulator of vascular function in hypertension via determining the phenotype of inflammatory circulating and infiltrating monocytes.

Background: Pulmonary disease has consistently been related to increased mortality. We investigated central spirometry variables in relation to total mortality in individuals from the general population without diagnosed lung disease also accounting for cardiac function. Methods: In 15,010 individuals from the general population (mean age 55±11 years, age range 35-74 years, 50.5% men) in the Gutenberg Health Study we performed spirometry and multimodal transthoracic echocardiography. The biomarkers N-terminal pro-B-type natriuretic peptide (Nt-proBNP) and high-sensitive troponin I (TnI) were measured in the first 5000 individuals using commercially available assays. Multivariable Cox regression analyses were computed for the lung function parameters forced expiratory volume in 1 second (FEV1), forced vital capacity (FVC) and the Tiffeneau-Pinelli index (FEV1/FVC ratio) after exclusion of individuals with prevalent pulmonary disease (N=1135). Results: The median FEV1/FVC ratio was 79.1%, 19.5% were current smokers, 4.2% had prevalent heart failure. After a median follow-up of 5.5 years 360 deaths were recorded. Multivariable-adjusted Cox regression analyses for the common cardiovascular risk factors (age, sex, body mass index, current smoking, diabetes mellitus, hypertension, dyslipidemia) and heart failure revealed that an increase of one standard deviation (SD) of FEV1 was associated with a 38% risk reduction (hazard ratio (HR) per SD 0.62 [95% confidence interval (CI): 0.52-0.73]). The association remained statistically highly significant after additional adjustment for ejection fraction, stroke volume, Nt-proBNP or TnI. Comparable results were seen for FVC, whereas no association of FEV1/FVC ratio with mortality could be shown. No significant interaction by heart failure was observed. Conclusions: The central lung function parameters FEV1 and FVC were related to all-cause mortality in individuals without manifest lung disease. Our findings indicate that subtle, subclinical pulmonary impairment constitutes a risk for increased mortality independent of cardiac performance.