Rewiring of the ubiquitinated proteome determines ageing in C. elegans.

Ageing is driven by a loss of cellular integrity1. Given the major role of ubiquitin modifications in cell function2, here we assess the link between ubiquitination and ageing by quantifying whole-proteome ubiquitin signatures in Caenorhabditis elegans. We find a remodelling of the ubiquitinated proteome during ageing, which is ameliorated by longevity paradigms such as dietary restriction and reduced insulin signalling. Notably, ageing causes a global loss of ubiquitination that is triggered by increased deubiquitinase activity. Because ubiquitination can tag proteins for recognition by the proteasome3, a fundamental question is whether deficits in targeted degradation influence longevity. By integrating data from worms with a defective proteasome, we identify proteasomal targets that accumulate with age owing to decreased ubiquitination and subsequent degradation. Lowering the levels of age-dysregulated proteasome targets prolongs longevity, whereas preventing their degradation shortens lifespan. Among the proteasomal targets, we find the IFB-2 intermediate filament4 and the EPS-8 modulator of RAC signalling5. While increased levels of IFB-2 promote the loss of intestinal integrity and bacterial colonization, upregulation of EPS-8 hyperactivates RAC in muscle and neurons, and leads to alterations in the actin cytoskeleton and protein kinase JNK. In summary, age-related changes in targeted degradation of structural and regulatory proteins across tissues determine longevity.

Autophagy regulates neuronal excitability by controlling cAMP/protein kinase A signaling at the synapse.

Autophagy provides nutrients during starvation and eliminates detrimental cellular components. However, accumulating evidence indicates that autophagy is not merely a housekeeping process. Here, by combining mouse models of neuron-specific ATG5 deficiency in either excitatory or inhibitory neurons with quantitative proteomics, high-content microscopy, and live-imaging approaches, we show that autophagy protein ATG5 functions in neurons to regulate cAMP-dependent protein kinase A (PKA)-mediated phosphorylation of a synapse-confined proteome. This function of ATG5 is independent of bulk turnover of synaptic proteins and requires the targeting of PKA inhibitory R1 subunits to autophagosomes. Neuronal loss of ATG5 causes synaptic accumulation of PKA-R1, which sequesters the PKA catalytic subunit and diminishes cAMP/PKA-dependent phosphorylation of postsynaptic cytoskeletal proteins that mediate AMPAR trafficking. Furthermore, ATG5 deletion in glutamatergic neurons augments AMPAR-dependent excitatory neurotransmission and causes the appearance of spontaneous recurrent seizures in mice. Our findings identify a novel role of autophagy in regulating PKA signaling at glutamatergic synapses and suggest the PKA as a target for restoration of synaptic function in neurodegenerative conditions with autophagy dysfunction.

[30- and 90-day Lethality in Patients with Stage IV Lung Cancer Depending on the Primary Therapy]. / 30- und 90-Tage-Letalität bei Patienten mit Lungenkarzinom im Stadium IV in Abhängigkeit der Primärtherapie.

Early lethality after initiation of therapy in patients with stage IV lung cancer has rarely been the focus of scientific studies yet. The little time remaining between diagnosis, start of therapy and onset of death, as well as any influencing factors, are of special interest for both, patients and physician. Accordingly, the aim of this work was to analyze the 30- and 90-day morbidity after initiation of systemic therapy and to determine possible factors influencing early lethality. For this purpose, the data of 225 patients with stage IV lung cancer and treatment at the Martha-Maria Halle-Dölau Lung Cancer Center between 01/01/2017 and 05/18/2020 were retrospectively analyzed. Forms of therapy and patient characteristics were analyzed with a frequency distribution and the probability of survival was estimated using the Kaplan-Meier method. The analysis of the early morbidity of all tumor-specifically treated patients showed a morbidity of 8.5 % at day 30 after the start of therapy and a rate of 23.5 % after 90 days. In a direct comparison of the different therapy groups, the patients receiving mono-checkpointinhibition had higher lethality (16.6 % after 30 days and 44.3 % after 90 days). In contrast, the morbidity of patients in the other therapy groups remained below 10 % after 30 days and below 23.3 % after 90 days. A poor general condition, an advanced tumor disease, polymetastasis and a positive history of smoking could be determined as predictors for higher early lethality. In contrast, there was no relevant difference in morbidity between the different tumor entities, gender, PD-L1 and mutation status. With this analysis, very high early lethality, comparable to other studies, could be detected in patients with lung cancer. Relevant differences between the forms of therapy illustrate the importance of individual patient selection for the respective therapy options and the rapid decision to initiate therapy.

A rare IL33 loss-of-function mutation reduces blood eosinophil counts and protects from asthma.

IL-33 is a tissue-derived cytokine that induces and amplifies eosinophilic inflammation and has emerged as a promising new drug target for asthma and allergic disease. Common variants at IL33 and IL1RL1, encoding the IL-33 receptor ST2, associate with eosinophil counts and asthma. Through whole-genome sequencing and imputation into the Icelandic population, we found a rare variant in IL33 (NM_001199640:exon7:c.487-1G>C (rs146597587-C), allele frequency = 0.65%) that disrupts a canonical splice acceptor site before the last coding exon. It is also found at low frequency in European populations. rs146597587-C associates with lower eosinophil counts (ß = -0.21 SD, P = 2.5×10-16, N = 103,104), and reduced risk of asthma in Europeans (OR = 0.47; 95%CI: 0.32, 0.70, P = 1.8×10-4, N cases = 6,465, N controls = 302,977). Heterozygotes have about 40% lower total IL33 mRNA expression than non-carriers and allele-specific analysis based on RNA sequencing and phased genotypes shows that only 20% of the total expression is from the mutated chromosome. In half of those transcripts the mutation causes retention of the last intron, predicted to result in a premature stop codon that leads to truncation of 66 amino acids. The truncated IL-33 has normal intracellular localization but neither binds IL-33R/ST2 nor activates ST2-expressing cells. Together these data demonstrate that rs146597587-C is a loss of function mutation and support the hypothesis that IL-33 haploinsufficiency protects against asthma.

Nursing staff's and physicians' acquisition of competences and attitudes to interprofessional education and interprofessional collaboration in pediatrics.

BACKGROUND: Interprofessional education (IPE) is deemed essential for interprofessional collaboration (IPC) in healthcare systems. IPC has positive effects for both patients and healthcare professionals. Especially in pediatrics, IPC is paramount for adequate care of patients and their families though there is a lack of data on the attitudes towards IPE and IPC and acquisition of respective competences in pediatric nursing and medical staff. METHODS: Frequencies of interactions and attitudes towards IPE and IPC, with a focus on acquisition of competences for IPE and IPC, of nurses (N = 79) and physicians (N = 70) in a large pediatric university hospital were evaluated with an online questionnaire. RESULTS: All participants worked as part of interprofessional teams, mostly consisting of nurses and physicians. The majority (94.9% (n = 75) of nurses and 100% (n = 70) of physicians) highly valued IPC. Medical doctors acquired most competences important for IPC during day-to-day work and reported a substantial lack of IPE. Nursing staff on the other hand did report significant interprofessional education during their training as well as ongoing interprofessional learning during day-to-day work. Nurses also appreciated IPE more. CONCLUSIONS: Even though IPC is commonly reported in nurses and physicians working at a large pediatric university hospital there is a lack of structured IPE. A focus should be on IPE for nurses and physicians to enable them to effectively collaborate together. Political and local initiatives for IPE are gaining momentum but still need to be established nationally and internationally.

Three-layered proteomic characterization of a novel ACTN4 mutation unravels its pathogenic potential in FSGS.

Genetic diseases constitute the most important cause for end-stage renal disease in children and adolescents. Mutations in the ACTN4 gene, encoding the actin-binding protein α-actinin-4, are a rare cause of autosomal dominant familial focal segmental glomerulosclerosis (FSGS). Here, we report the identification of a novel, disease-causing ACTN4 mutation (p.G195D, de novo) in a sporadic case of childhood FSGS using next generation sequencing. Proteome analysis by quantitative mass spectrometry (MS) of patient-derived urinary epithelial cells indicated that ACTN4 levels were significantly decreased when compared with healthy controls. By resolving the peptide bearing the mutated residue, we could proof that the mutant protein is less abundant when compared with the wild-type protein. Further analyses revealed that the decreased stability of p.G195D is associated with increased ubiquitylation in the vicinity of the mutation site. We next defined the ACTN4 interactome, which was predominantly composed of cytoskeletal modulators and LIM domain-containing proteins. Interestingly, this entire group of proteins, including several highly specific ACTN4 interactors, was globally decreased in the patient-derived cells. Taken together, these data suggest a mechanistic link between ACTN4 instability and proteome perturbations of the ACTN4 interactome. Our findings advance the understanding of dominant effects exerted by ACTN4 mutations in FSGS. This study illustrates the potential of genomics and complementary, high-resolution proteomics analyses to study the pathogenicity of rare gene variants.

Sirt7 stabilizes rDNA heterochromatin through recruitment of DNMT1 and Sirt1.

Maintenance of highly compact heterochromatin at ribosomal DNA (rDNA) segments is essential to prevent homologous recombination between rDNA repeats and for preserving genomic stability and nucleolar architecture. Here, we investigated the role of Sirtuin 7 (Sirt7) in the regulation of rDNA chromatin structure, rDNA repeat stability and nucleolar organization. We found that Sirt7 mediates heterochromatin formation at rRNA genes through recruitment of DNA methyltransferase 1 and another member of the sirtuin family, Sirt1. Lack of Sirt7 leads to nucleolar fragmentation associated with hypomethylation of rDNA and hyperacetylation of histones at rDNA loci resulting in rDNA and genomic instability. Our findings suggest a novel role of Sirt7 in preventing cellular transformation by mediating maintenance of rDNA repeats and nucleolar integrity.

Phosphoproteomic analysis reveals regulatory mechanisms at the kidney filtration barrier.

Diseases of the kidney filtration barrier are a leading cause of ESRD. Most disorders affect the podocytes, polarized cells with a limited capacity for self-renewal that require tightly controlled signaling to maintain their integrity, viability, and function. Here, we provide an atlas of in vivo phosphorylated, glomerulus-expressed proteins, including podocyte-specific gene products, identified in an unbiased tandem mass spectrometry-based approach. We discovered 2449 phosphorylated proteins corresponding to 4079 identified high-confidence phosphorylated residues and performed a systematic bioinformatics analysis of this dataset. We discovered 146 phosphorylation sites on proteins abundantly expressed in podocytes. The prohibitin homology domain of the slit diaphragm protein podocin contained one such site, threonine 234 (T234), located within a phosphorylation motif that is mutated in human genetic forms of proteinuria. The T234 site resides at the interface of podocin dimers. Free energy calculation through molecular dynamic simulations revealed a role for T234 in regulating podocin dimerization. We show that phosphorylation critically regulates formation of high molecular weight complexes and that this may represent a general principle for the assembly of proteins containing prohibitin homology domains.

Factor VII-activating protease is activated in multiple trauma patients and generates anaphylatoxin C5a.

Severe tissue injury results in early activation of serine protease systems including the coagulation and complement cascade. In this context, little is known about factor VII-activating protease (FSAP), which is activated by substances released from damaged cells such as histones and nucleosomes. Therefore, we have measured FSAP activation in trauma patients and have identified novel FSAP substrates in human plasma. Mass spectrometry-based methods were used to identify FSAP binding proteins in plasma. Anaphylatoxin generation was measured by ELISA, Western blotting, protein sequencing, and chemotaxis assays. Plasma samples from trauma patients were analyzed for FSAP Ag and activity, nucleosomes, C5a, and C3a. Among others, we found complement components C3 and C5 in FSAP coimmunoprecipitates. C3 and C5 were cleaved by FSAP in a dose- and time-dependent manner generating functional C3a and C5a anaphylatoxins. Activation of endogenous FSAP in plasma led to increased C5a generation, but this was not the case in plasma of a homozygous carrier of Marburg I single nucleotide polymorphism with lower FSAP activity. In multiple trauma patients there was a large increase in circulating FSAP activity and nucleosomes immediately after the injury. A high correlation between FSAP activity and C5a was found. These data suggest that activation of FSAP by tissue injury triggers anaphylatoxin generation and thereby modulates the posttraumatic inflammatory response in vivo. A strong link between C5a, nucleosomes, and FSAP activity indicates that this new principle might be important in the regulation of inflammation.

In vitro comparison of two widely used surgical sealants for treating alveolar air leak.

BACKGROUND: Controversies surrounding the efficacy of sealants against alveolar air leak (AAL) are abundant in the literature. We sought to test the widely used sealants, TachoSil (Takeda Pharmaceutical Company Limited, Osaka, Japan) and BioGlue (CryoLife Europa Ltd., Surrey, United Kingdom) in an in vitro model. Materials and METHODS: After creation of a focal superficial defect (40 × 25 mm) in swine lungs (n=40), AAL was assessed with increasing inspired tidal volume (TVi). Upon sealant application in a randomized order, AAL was assessed in the same way until sealant burst. RESULTS: At TVi =400, 500, 600, and 700 mL, BioGlue achieved sealing in 19, 19, 16, and 14 tests, while TachoSil sealed in 19, 14, 4, and no test, respectively. The maximally tolerated pressure of BioGlue was higher than TachoSil (40.3 ± 3.0 vs. 36.0 ± 4.9 cm H2O, p=0.003). Cohesive and adhesive failures were found in 10 and 1 tests of BioGlue, respectively, while all burst failures of TachoSil were adhesive. Concerning elasticity, TachoSil allowed more expansion of the covered defect than BioGlue (6.3 ± 3.9 vs. 1.4 ± 1.0 mm, p<0.001). CONCLUSION: The tested sealants demonstrated high sealing efficacy. While BioGlue was superior in resisting higher ventilation pressure, TachoSil possessed better elasticity.

Genomewide association analysis of respiratory syncytial virus infection in mice.

Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract infection in infants, with about half being infected in their first year of life. Yet only 2 to 3% of infants are hospitalized for RSV infection, suggesting that individual susceptibility contributes to disease severity. Previously, we determined that AKR/J (susceptible) mice developed high lung RSV titers and showed delayed weight recovery, whereas C57BL/6J (resistant) mice demonstrated low lung RSV titers and rapid weight recovery. In addition, we have reported that gene-targeted mice lacking the cystic fibrosis transmembrane conductance regulator (Cftr; ATP-binding cassette subfamily C, member 7) are susceptible to RSV infection. For this report, recombinant backcross and F2 progeny derived from C57BL/6J and AKR/J mice were infected with RSV, their lung titers were measured, and quantitative trait locus (QTL) analysis was performed. A major QTL, designated Rsvs1, was identified on proximal mouse chromosome 6 in both recombinant populations. Microarray analysis comparing lung transcripts of the parental strains during infection identified several candidate genes that mapped to the Rsvs1 interval, including Cftr. These findings add to our understanding of individual RSV susceptibility and strongly support a modifier role for CFTR in RSV infection, a significant cause of respiratory morbidity in infants with cystic fibrosis.

Click-chemistry-derived tetracycline-amino acid conjugates exhibiting exceptional potency and exclusive recognition of the reverse tet repressor.

A click-chemistry-based synthesis of biologically active doxycycline-amino acid conjugates is described. Starting from 9-aminodoxycycline derivatives and complementary functionalized amino acids, ligation was accomplished by copper(I)-catalyzed azide-alkyne [3+2] cycloaddition (CuAAC). The final products were tested in a variety of TetR and revTetR systems, and the C-terminally linked phenylalanine conjugate 12 c exhibited high selectivity for revTetR over TetR. Besides the unique property of the specific effector 12 c to effectively differentiate TetR and its reverse phenotype, the test compound proved to be almost devoid of any antibacterial activity; this will be highly beneficial for future applications to control gene expression in bacterial systems.

Exposure to furosemide as the strongest risk factor for nephrocalcinosis in preterm infants.

BACKGROUND: The objective of this study was to determine which of the many risk factors for nephrocalcinosis (NC) in preterm infants are most relevant. METHODS: In 55 neonates born before 32 completed weeks of gestation, parameters relevant to NC were analyzed. Median birthweight was 1010 g (range 500-2070 g). Fifteen (27%) asymptomatic children had ultrasonographic NC. RESULTS: In multivariate analysis the strongest independent risk factor was furosemide therapy above 10 mg per kg bodyweight cumulative dose, with a 48-fold increased risk of NC (odds ratio confidence interval 4.0-585, P < 0.01). The risk of NC was 1.65-fold higher per 100 g lower weight (1.07-2.56, P= 0.02) and 4.5-fold higher per mmol/l of urinary calcium concentration (1.14-17.7, P= 0.03). Many other risk factors were only significant in univariate analysis (gestational age, mechanical ventilation, infection, broncho-pulmonary dysplasia, blood transfusions, intraventricular hemorrhage, surfactant therapy, vasopressors, phenobarbital or caffeine, duration of hospital stay), indicating an indirect effect only. Other parameters of calcium and phosphate homeostasis were not significant, possibly due to standardized supplementation. CONCLUSION: We suggest that in preterm infants, furosemide should be prescribed with caution and close monitoring of calcium excretions is advisable. Some guidelines for infant respiratory distress syndrome now favor calcium-sparing thiazides if diuretics are considered.

Digenic inheritance of mutations in EPHA2 and SLC26A4 in Pendred syndrome.

Enlarged vestibular aqueduct (EVA) is one of the most commonly identified inner ear malformations in hearing loss patients including Pendred syndrome. While biallelic mutations of the SLC26A4 gene, encoding pendrin, causes non-syndromic hearing loss with EVA or Pendred syndrome, a considerable number of patients appear to carry mono-allelic mutation. This suggests faulty pendrin regulatory machinery results in hearing loss. Here we identify EPHA2 as another causative gene of Pendred syndrome with SLC26A4. EphA2 forms a protein complex with pendrin controlling pendrin localization, which is disrupted in some pathogenic forms of pendrin. Moreover, point mutations leading to amino acid substitution in the EPHA2 gene are identified from patients bearing mono-allelic mutation of SLC26A4. Ephrin-B2 binds to EphA2 triggering internalization with pendrin inducing EphA2 autophosphorylation weakly. The identified EphA2 mutants attenuate ephrin-B2- but not ephrin-A1-induced EphA2 internalization with pendrin. Our results uncover an unexpected role of the Eph/ephrin system in epithelial function.

Association of TNF-alpha with severe respiratory syncytial virus infection and bronchial asthma.

Tumor necrosis factor (TNF-)alpha is a proinflammatory cytokine that is important in the innate host defence and thus in the defence of infectious agents. However, in excess it provokes the development of chronic inflammatory diseases. The aim of this study was to test association of TNF with severe RSV bronchiolitis as example of an infectious disease and asthma as representative for a chronic inflammatory condition. The following study populations were genotyped for 4 polymorphisms within TNF-beta (rs909253) and TNF-alpha (rs1799964, rs1799724, rs1800629): 322 asthmatic children, 151 children with severe respiratory syncytial virus (RSV) bronchiolitis and 270 controls. Furthermore, serum TNF-alpha levels were measured by a FlowCytomix Assay. Asthma showed association with two TNF-alpha polymorphisms as well as with TNF haplotypes (p = 0.0050). In contrast, RSV bronchiolitis was associated with TNF haplotypes (p < 0.00001) but not with any single polymorphism. In addition, TNF-alpha serum levels correlated with rs1799724 (p = 0.034). A genetically mediated up-regulation of TNF-alpha expression might provoke a pronounced inflammation of the airways and thus a more severe course of RSV infection as well as the onset of asthma. It remains to be elucidated whether severe RSV bronchiolitis starts TNF-alpha upregulation and is one first step in the direction to asthma later in life, or whether both diseases are independent from each other and supported by TNF-alpha upregulation.

Risk factors of neonatal respiratory distress following vaginal delivery and caesarean section in the German population.

BACKGROUND: The incidence of caesarean section (CS) is steadily rising world-wide. In particular, CS on maternal demand is performed more frequently. In parts, this might be due to insufficient information of pregnant women about neonatal risks of CS. We sought to specify neonatal outcomes following different modes of delivery, i.e. vaginal delivery, primary CS and secondary CS and to define risk factors for respiratory morbidity and hospitalization. METHODS: We analysed 2073 births (gestational age > 35 weeks) during a two-year period at a tertiary obstetric and neonatal centre in Germany. Statistical analyses were performed for single parameters by SPSS as well as by logistic regression to account for possible confounders. Furthermore, extensive model calculation was done. RESULTS: Respiratory morbidity was increased following primary and secondary CS (p = 0.001). By multiple logistic regression, the strongest effect on respiratory symptoms was seen with gestational age, each week more in utero reducing the risk by an odds ratio (OR) of 0.69 (95% CI: [0.61; 0.79]; p = 1.9 x 10(-8)). Furthermore, a significant interaction between mode of delivery and gestational age was found for the risk of respiratory symptoms (p = 0.0035). CONCLUSION: For every eight newborns delivered by primary CS one more than expected with vaginal delivery is hospitalized. It is highly relevant to recognize that each week of gestational age reduces the risk of respiratory symptoms, especially if primary CS is performed. The higher rate of respiratory morbidity and neonatal admission following CS should be clearly recognized in counselling of pregnant women.

RASSF10 Is a TGFß-Target That Regulates ASPP2 and E-Cadherin Expression and Acts as Tumor Suppressor That Is Epigenetically Downregulated in Advanced Cancer.

The Ras Association Domain Family (RASSF) encodes members of tumor suppressor genes which are frequently inactivated in human cancers. Here, the function and the regulation of RASSF10, that contains a RA (Ras-association) and two coiled domains, was investigated. We utilized mass spectrometry and immuno-precipitation to identify interaction partners of RASSF10. Additionally, we analyzed the up- and downstream pathways of RASSF10 that are involved in its tumor suppressive function. We report that RASSF10 binds ASPP1 (Apoptosis-stimulating protein of p53) and ASPP2 through its coiled-coils. Induction of RASSF10 leads to increased protein levels of ASPP2 and acts negatively on cell cycle progression. Interestingly, we found that RASSF10 is a target of the EMT (epithelial mesenchymal transition) driver TGFß (Transforming growth factor beta) and that negatively associated genes of RASSF10 are significantly over-represented in an EMT gene set collection. We observed a positive correlation of RASSF10 expression and E-cadherin that prevents EMT. Depletion of RASSF10 by CRISPR/Cas9 technology induces the ability of lung cancer cells to proliferate and to invade an extracellular matrix after TGFß treatment. Additionally, knockdown of RASSF10 or ASPP2 induced constitutive phosphorylation of SMAD2 (Smad family member 2). Moreover, we found that epigenetic reduction of RASSF10 levels correlates with tumor progression and poor survival in human cancers. Our study indicates that RASSF10 acts a TGFß target gene and negatively regulates cell growth and invasion through ASPP2. This data suggests that epigenetic loss of RASSF10 contributes to tumorigenesis by promoting EMT induced by TGFß.