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.

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.

[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.

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.

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.

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.

Treatment of infected lungs by ex vivo perfusion with high dose antibiotics and autotransplantation: A pilot study in pigs.

The emergence of multi-drug resistant bacteria threatens to end the era of antibiotics. Drug resistant bacteria have evolved mechanisms to overcome antibiotics at therapeutic doses and further dose increases are not possible due to systemic toxicity. Here we present a pilot study of ex vivo lung perfusion (EVLP) with high dose antibiotic therapy followed by autotransplantation as a new therapy of last resort for otherwise incurable multidrug resistant lung infections. Severe Pseudomonas aeruginosa pneumonia was induced in the lower left lungs (LLL) of 18 Mini-Lewe pigs. Animals in the control group (n = 6) did not receive colistin. Animals in the conventional treatment group (n = 6) received intravenous application of 2 mg/kg body weight colistin daily. Animals in the EVLP group (n = 6) had their LLL explanted and perfused ex vivo with a perfusion solution containing 200 µg/ml colistin. After two hours of ex vivo treatment, autotransplantation of the LLL was performed. All animals were followed for 4 days following the initiation of treatment. In the control and conventional treatment groups, the infection-related mortality rate after five days was 66.7%. In the EVLP group, there was one infection-related mortality and one procedure-related mortality, for an overall mortality rate of 33.3%. Moreover, the clinical symptoms of infection were less severe in the EVLP group than the other groups. Ex vivo lung perfusion with very high dose antibiotics presents a new therapeutic option of last resort for otherwise incurable multidrug resistant pneumonia without toxic side effects on other organs.

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.

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.

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.

RNA-Seq analysis of isolated satellite cells in Prmt5 deficient mice.

Satellite cells (SCs) represent a distinct population of stem cells, essential for maintenance, growth and regeneration of adult skeletal muscle. SCs are mononuclear and are located between the basal lamina and the plasma membrane of myofibers. They are typically characterized by presence of the transcription factor paired-box 7 (PAX7) that is widely used as a satellite cell marker. Under normal physiological conditions SCs are quiescent but are activated by insults such as injury, disease or exercise. Once activated, satellite cells proliferate and subsequently differentiate into myoblasts to finally fuse to form new myofibers or with preexisting myofibers to repair or rebuild the skeletal muscle. A minority of SCs retains stem cell characteristics and self-renews to assure future bouts of regeneration throughout most of adult life. While a comprehensive picture of the regulatory events controlling SC fate has not yet been achieved, several factors were recently identified playing important roles in functional processes. One example is the arginine methyltransferase Prmt5 that is known to have multiple roles in germ cells and is involved in the maintenance of ES cell pluripotency. We have previously shown that Prmt5 is required for muscle stem cell proliferation and regenerative myogenesis due to direct epigenetic regulation of the cell cycle inhibitor p21. Here we provide a dataset that investigates the loss of Prmt5 in isolated Pax7(+) primary SCs using the Pax7(CreERT2)/Prmt5(loxP/loxP) knockout mouse model. RNA-Seq raw and analyzed data have been deposited in GEO under accession code GSE66822.

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.

Synchronous recurrence of group B streptococcal late-onset sepsis in twins.

Group B Streptococcus (GBS) remains the leading cause of neonatal sepsis and meningitis in industrialized countries. Whereas the use of intrapartum antibiotic prophylaxis has led to a significant decline in early-onset sepsis, the incidence of late-onset sepsis has remained unchanged. Whether late-onset sepsis usually originates from established mucocutaneous GBS colonization of the infant or whether it results from an acute exogenous GBS infection remains controversial. Here we report on twins who both twice developed GBS sepsis in a strikingly parallel fashion, with both instances originating from a single hypervirulent GBS clone. Factored together, the presentation as cervical soft tissue infection in both cases, the synchronicity of the episodes, and the detection of GBS DNA in breast milk all strongly suggest an enteral mode of transmission with a short incubation period.

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.

Association of a FGFR-4 gene polymorphism with bronchopulmonary dysplasia and neonatal respiratory distress.

BACKGROUND: Bronchopulmonary dysplasia (BPD) is the most common chronic lung disease of premature birth, characterized by impaired alveolar development and inflammation. Pathomechanisms contributing to BPD are poorly understood. However, it is assumed that genetic factors predispose to BPD and other pulmonary diseases of preterm neonates, such as neonatal respiratory distress syndrome (RDS). For association studies, genes upregulated during alveolarization are major candidates for genetic analysis, for example, matrix metalloproteinases (MMPs) and fibroblast growth factors (FGFs) and their receptors (FGFR). OBJECTIVE: Determining genetic risk variants in a Caucasian population of premature neonates with BPD and RDS. Methods. We genotyped 27 polymorphisms within 14 candidate genes via restriction fragment length polymorphism (RFLP): MMP-1, -2, -9, and -12, -16, FGF receptors 2 and 4, FGF-2, -3, -4, -7, and -18, Signal-Regulatory Protein α (SIRPA) and Thyroid Transcription Factor-1 (TTF-1). RESULTS: Five single nucleotide polymorphisms (SNPs) in MMP-9, MMP-12, FGFR-4, FGF-3, and FGF-7 are associated (P < 0.05) with RDS, defined as surfactant application within the first 24 hours after birth. One of them, in FGFR-4 (rs1966265), is associated with both RDS (P = 0.003) and BPD (P = 0.023). CONCLUSION: rs1966265 in FGF receptor 4 is a possible genetic key variant in alveolar diseases of preterm newborns.

Extramedullary haematopoiesis presented as intrathoracic tumour in a patient with alpha-thalassaemia.

The authors report a case of extramedullary haematopoiesis (EMH) presenting as an intrathoracic tumour in a patient with alpha-thalassaemia. CT scan and MRI of the chest were obtained and followed by tumour excision. Compared to beta-thalassaemia, only two cases of EMH in patients with alpha-thalassaemia have been described in the literature. A possible reason for this disparity is discussed.

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.