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Bacteria employ small regulatory RNAs (sRNA) and/or RNA binding proteins (RBPs) to respond to environmental cues. In Enterobacteriaceae , the FinO-domain containing RBP ProQ associates with numerous sRNAs and mRNAs, impacts sRNA-mediated riboregulation or mRNA stability by binding to 5'- or 3'-untranslated regions as well as to internal stem loop structures. Global RNA-protein interaction studies and sequence comparisons identified a ProQ-like homolog (PA2582/ProQ Pae ) in Pseudomonas aeruginosa ( Pae ). To address the function of ProQ Pae , at first a comparative transcriptome analysis of the Pae strains PAO1 and PAO1Δ proQ was performed. This study revealed more than 100 differentially abundant transcripts, affecting a variety of cellular functions. Among these transcripts were pprA and pprB , encoding the PprA/PprB two component system, psrA , encoding a transcriptional activator of pprB , and oprI , encoding the outer membrane protein OprI. RNA co-purification experiments with Strep-tagged Pae ProQ protein corroborated an association of ProQ Pae with these transcripts. In accordance with the up-regulation of the psrA , pprA , and pprB genes in strain PAO1Δ proQ a phenotypic analysis revealed an increased susceptibility toward the aminoglycosides tobramycin and gentamicin in biofilms. Conversely, the observed down-regulation of the oprI gene in PAO1Δ proQ could be reconciled with a decreased susceptibility toward the synthetic cationic antimicrobial peptide GW-Q6. Taken together, these studies revealed that ProQ Pae is an RBP that impacts antimicrobial resistance in Pae ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Cianciulli Sesso, Resch, Moll, Bläsi and Sonnleitner.)

Aim: To study if functional electrical stimulation (FES) of the peroneal nerve, which activates dorsiflexion, can improve body functions, activities, and participation and could be an effective alternative treatment in individuals with unilateral spastic cerebral palsy (CP)., Method: A randomized cross-over trial was performed in 25 children with unilateral spastic CP (classified in Gross Motor Function Classification System levels I and II) aged 4 to 18 years (median age at inclusion 9 years 8 months, interquartile range = 7 years-13 years 8 months), 15 patients were male. The study consisted of two 12-week blocks of treatment, that is, conventional treatment (ankle foot orthosis [AFO] or adapted shoes) and FES, separated by a 6-week washout period. Outcome measures included the Goal Attainment Scale (GAS), the Cerebral Palsy Quality of Life questionnaire, and a three-dimensional gait analysis., Results: Eighteen patients completed the trial. The proportion of GAS goals achieved was not significantly higher in the FES versus the conventional treatment phase (goal 1 p = 0.065; goal 2 p = 1.00). When walking while stimulated with FES, ankle dorsiflexion during mid-swing decreased over time (p = 0.006, average decrease of 4.8° with FES), with a preserved increased ankle range of motion compared to conventional treatment (p < 0.001, mean range of motion with FES +10.1° compared to AFO). No changes were found in the standard physical examination or regarding satisfaction with orthoses and feelings about the ability to dress yourself. In four patients, FES therapy failed; in 12 patients FES therapy continued after the trial., Interpretation: FES is not significantly worse than AFO; however, patient selection is critical, and a testing period and thorough follow-up are needed., (© 2023 The Authors. Developmental Medicine & Child Neurology published by John Wiley & Sons Ltd on behalf of Mac Keith Press.)

Cachexia is a major cause of morbidity and mortality in individuals with cancer and is characterized by weight loss due to adipose and muscle tissue wasting. Hallmarks of white adipose tissue (WAT) remodeling, which often precedes weight loss, are impaired lipid storage, inflammation and eventually fibrosis. Tissue wasting occurs in response to tumor-secreted factors. Considering that the continuous endothelium in WAT is the first line of contact with circulating factors, we postulated whether the endothelium itself may orchestrate tissue remodeling. Here, we show using human and mouse cancer models that during precachexia, tumors overactivate Notch1 signaling in distant WAT endothelium. Sustained endothelial Notch1 signaling induces a WAT wasting phenotype in male mice through excessive retinoic acid production. Pharmacological blockade of retinoic acid signaling was sufficient to inhibit WAT wasting in a mouse cancer cachexia model. This demonstrates that cancer manipulates the endothelium at distant sites to mediate WAT wasting by altering angiocrine signals., (© 2023. The Author(s).)

Background and Aims: Chronic liver disease is a growing epidemic, leading to fibrosis and cirrhosis. TGF-β is the pivotal profibrogenic cytokine that activates HSC, yet other molecules can modulate TGF-β signaling during liver fibrosis. Expression of the axon guidance molecules semaphorins (SEMAs), which signal through plexins and neuropilins (NRPs), have been associated with liver fibrosis in HBV-induced chronic hepatitis. This study aims at determining their function in the regulation of HSCs., Approach and Results: We analyzed publicly available patient databases and liver biopsies. We used transgenic mice, in which genes are deleted only in activated HSCs to perform ex vivo analysis and animal models. SEMA3C is the most enriched member of the semaphorin family in liver samples from patients with cirrhosis. Higher expression of SEMA3C in patients with NASH, alcoholic hepatitis, or HBV-induced hepatitis discriminates those with a more profibrotic transcriptomic profile. SEMA3C expression is also elevated in different mouse models of liver fibrosis and in isolated HSCs on activation. In keeping with this, deletion of SEMA3C in activated HSCs reduces myofibroblast marker expression. Conversely, SEMA3C overexpression exacerbates TGF-β-mediated myofibroblast activation, as shown by increased SMAD2 phosphorylation and target gene expression. Among SEMA3C receptors, only NRP2 expression is maintained on activation of isolated HSCs. Interestingly, lack of NRP2 in those cells reduces myofibroblast marker expression. Finally, deletion of either SEMA3C or NRP2, specifically in activated HSCs, reduces liver fibrosis in mice., Conclusion: SEMA3C is a novel marker for activated HSCs that plays a fundamental role in the acquisition of the myofibroblastic phenotype and liver fibrosis., (Copyright © 2023 American Association for the Study of Liver Diseases.)

In the opportunistic human pathogen Pseudomonas aeruginosa ( Pae ), c arbon c atabolite r epression (CCR) orchestrates the hierarchical utilization of N and C sources, and impacts virulence, antibiotic resistance and biofilm development. During CCR, the RNA chaperone Hfq and the c atabolite r epression c ontrol protein Crc form assemblies on target mRNAs that impede translation of proteins involved in uptake and catabolism of less preferred C sources. After exhaustion of the preferred C-source, translational repression of target genes is relieved by the regulatory RNA CrcZ, which binds to and acts as a decoy for Hfq. Here, we asked whether Crc action can be modulated to relieve CCR after exhaustion of a preferred carbon source. As Crc does not bind to RNA per se , we endeavored to identify an interacting protein. In vivo co-purification studies, co-immunoprecipitation and biophysical assays revealed that Crc binds to Pae strain O1 protein PA1677. Our structural studies support bioinformatics analyzes showing that PA1677 belongs to the isochorismatase-like superfamily. Ectopic expression of PA 1677 resulted in de-repression of Hfq/Crc controlled target genes, while in the absence of the protein, an extended lag phase is observed during diauxic growth on a preferred and a non-preferred carbon source. This observations indicate that PA1677 acts as an antagonist of Crc that favors synthesis of proteins required to metabolize non-preferred carbon sources. We present a working model wherein PA1677 diminishes the formation of productive Hfq/Crc repressive complexes on target mRNAs by titrating Crc. Accordingly, we propose the name CrcA ( c atabolite r epression c ontrol protein a ntagonist) for PA1677., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Sonnleitner, Bassani, Cianciulli-Sesso, Brear, Lilic, Davidovski, Resch, Luisi, Moll and Bläsi.)

Pancreatic ductal adenocarcinoma (PDAC) frequently metastasizes into the peritoneum, which contributes to poor prognosis. Metastatic spreading is promoted by cancer cell plasticity, yet its regulation by the microenvironment is incompletely understood. Here, we show that the presence of hyaluronan and proteoglycan link protein-1 (HAPLN1) in the extracellular matrix enhances tumor cell plasticity and PDAC metastasis. Bioinformatic analysis showed that HAPLN1 expression is enriched in the basal PDAC subtype and associated with worse overall patient survival. In a mouse model for peritoneal carcinomatosis, HAPLN1-induced immunomodulation favors a more permissive microenvironment, which accelerates the peritoneal spread of tumor cells. Mechanistically, HAPLN1, via upregulation of tumor necrosis factor receptor 2 (TNFR2), promotes TNF-mediated upregulation of Hyaluronan (HA) production, facilitating EMT, stemness, invasion and immunomodulation. Extracellular HAPLN1 modifies cancer cells and fibroblasts, rendering them more immunomodulatory. As such, we identify HAPLN1 as a prognostic marker and as a driver for peritoneal metastasis in PDAC., (© 2023. The Author(s).)

Background: Laminopathies are caused by rare alterations in LMNA , leading to a wide clinical spectrum. Though muscular dystrophy begins at early ages, disease progression is different in each patient. We investigated variability in laminopathy phenotypes by performing a targeted genetic analysis of patients diagnosed with LMNA -related muscular dystrophy to identify rare variants in alternative genes, thereby explaining phenotypic differences. Methods: We analyzed 105 genes associated with muscular diseases by targeted sequencing in 26 pediatric patients of different countries, diagnosed with any LMNA -related muscular dystrophy. Family members were also clinically assessed and genetically analyzed. Results: All patients carried a pathogenic rare variant in LMNA . Clinical diagnoses included Emery-Dreifuss muscular dystrophy (EDMD, 13 patients), LMNA -related congenital muscular dystrophy (L-CMD, 11 patients), and limb-girdle muscular dystrophy 1B (LGMD1B, 2 patients). In 9 patients, 10 additional rare genetic variants were identified in 8 genes other than LMNA . Genotype-phenotype correlation showed additional deleterious rare variants in five of the nine patients (3 L-CMD and 2 EDMD) with severe phenotypes. Conclusion: Analysis f known genes related to muscular diseases in close correlation with personalized clinical assessments may help identify additional rare variants of LMNA potentially associated with early onset or most severe disease progression., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Cesar, Coll, Fiol, Fernandez-Falgueras, Cruzalegui, Iglesias, Moll, Perez-Serra, Martínez-Barrios, Ferrer-Costa, Olmo, Puigmulè, Alcalde, Lopez, Pico, Berrueco, Brugada, Zschaeck, Natera-de Benito, Carrera-García, Exposito-Escudero, Ortez, Nascimento, Brugada, Sarquella-Brugada and Campuzano.)

Introduction: LMNA-related muscular dystrophy is a rare entity that produce "laminopathies" such as Emery-Dreifuss muscular dystrophy (EDMD), limb-girdle muscular dystrophy type 1B (LGMD1B), and LMNA-related congenital muscular dystrophy (L-CMD). Heart failure, malignant arrhythmias, and sudden death may occur. No consensus exists on cardiovascular management in pediatric laminopathies. The aim was to perform an exhaustive cardiologic follow-up in pediatric patients diagnosed with LMNA-related muscular dystrophy. Methods: Baseline cardiac work-up consisted of clinical assessment, transthoracic Doppler echocardiography, 12-lead electrocardiogram, electrophysiological study, and implantation of a long-term implantable cardiac loop recorder (ILR). Results: We enrolled twenty-eight pediatric patients diagnosed with EDMD (13 patients), L-CMD (11 patients), LGMD1B (2 patients), and LMNA-related mild weakness (2 patients). Follow-up showed dilated cardiomyopathy (DCM) in six patients and malignant arrhythmias in five (four concomitant with DCM) detected by the ILR that required implantable cardioverter defibrillator (ICD) implantation. Malignant arrhythmias were detected in 20% of our cohort and early-onset EDMD showed worse cardiac prognosis. Discussion: Patients diagnosed with early-onset EDMD are at higher risk of DCM, while potentially life-threatening arrhythmias without DCM appear earlier in L-CMD patients. Early onset neurologic symptoms could be related with worse cardiac prognosis. Specific clinical guidelines for children are needed to prevent sudden death., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer AF declared a shared affiliation with the authors ID, MG, SQR to the handling editor at the time of review., (Copyright © 2023 Cesar, Campuzano, Cruzalegui, Fiol, Moll, Martínez-Barrios, Zschaeck, Natera-de Benito, Ortez, Carrera, Expósito, Berrueco, Bautista-Rodriguez, Dabaj, Gómez García-de-la-Banda, Quijano-Roy, Brugada, Nascimento and Sarquella-Brugada.)

Fatigue during walking is a common complaint in cerebral palsy (CP). The primary purpose of this study is to investigate muscle fatigue from surface electromyography (sEMG) measurements after a treadmill-based fatigue protocol with increasing incline and speed in children with CP with drop foot. The secondary purpose is to investigate whether changes in sagittal kinematics of hip, knee and ankle occur after fatigue. Eighteen subjects with unilateral spastic CP performed the protocol while wearing their ankle-foot orthosis and scored their fatigue on the OMNI scale of perceived exertion. The median frequency (MF) and root mean square (RMS) were used as sEMG measures for fatigue and linear mixed effects model were applied. The MF was significantly decreased in fatigued condition, especially in the affected leg and in the tibialis anterior and peroneus longus muscle. The RMS did not change significantly in fatigued condition, while the OMNI fatigue score indicated patients felt really fatigued. No changes in sagittal kinematics of hip, knee and ankle were found using statistical non-parametric mapping. In conclusion, the current fatigue protocol seems promising in inducing fatigue in a population with CP with drop foot and it could be used to expand knowledge on muscle fatigue during walking in CP., Competing Interests: No conflicts of interests to declare. The authors don’t have financial relationships with other persons or organizations that might have inappropriately influenced our work presented in this manuscript., (Copyright: © 2022 Moll et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Epithelial ovarian cancer (EOC) is one of the most lethal gynecologic cancers worldwide. EOC cells educate tumor-associated macrophages (TAM) through CD44-mediated cholesterol depletion to generate an immunosuppressive tumor microenvironment (TME). In addition, tumor cells frequently activate Notch1 receptors on endothelial cells (EC) to facilitate metastasis. However, further work is required to establish whether the endothelium also influences the education of recruited monocytes. Here, we report that canonical Notch signaling through RBPJ in ECs is an important player in the education of TAMs and EOC progression. Deletion of Rbpj in the endothelium of adult mice reduced infiltration of monocyte-derived macrophages into the TME of EOC and prevented the acquisition of a typical TAM gene signature; this was associated with stronger cytotoxic activity of T cells and decreased tumor burden. Mechanistically, CXCL2 was identified as a novel Notch/RBPJ target gene that regulated the expression of CD44 on monocytes and subsequent cholesterol depletion of TAMs. Bioinformatic analysis of ovarian cancer patient data showed that increased CXCL2 expression is accompanied by higher expression of CD44 and TAM education. Together, these findings indicate that EOC cells induce the tumor endothelium to secrete CXCL2 to establish an immunosuppressive microenvironment., Significance: Endothelial Notch signaling favors immunosuppression by increasing CXCL2 secretion to stimulate CD44 expression in macrophages, facilitating their education by tumor cells., (©2022 American Association for Cancer Research.)

Objective: MazF is a sequence-specific endoribonuclease-toxin of the MazEF toxin-antitoxin system. MazF cleaves single-stranded ribonucleic acid (RNA) regions at adenine-cytosine-adenine (ACA) sequences in the bacterium Escherichia coli. The MazEF system has been used in various biotechnology and synthetic biology applications. In this study, we infer how ectopic mazF overexpression affects production of heterologous proteins. To this end, we quantified the levels of fluorescent proteins expressed in E. coli from reporters translated from the ACA-containing or ACA-less messenger RNAs (mRNAs). Additionally, we addressed the impact of the 5'-untranslated region of these reporter mRNAs under the same conditions by comparing expression from mRNAs that comprise (canonical mRNA) or lack this region (leaderless mRNA)., Results: Flow cytometry analysis indicates that during mazF overexpression, fluorescent proteins are translated from the canonical as well as leaderless mRNAs. Our analysis further indicates that longer mazF overexpression generally increases the concentration of fluorescent proteins translated from ACA-less mRNAs, however it also substantially increases bacterial population heterogeneity. Finally, our results suggest that the strength and duration of mazF overexpression should be optimized for each experimental setup, to maximize the heterologous protein production and minimize the amount of phenotypic heterogeneity in bacterial populations, which is unfavorable in biotechnological processes., (© 2022. The Author(s).)

Blood vessel formation is dependent on metabolic adaption in endothelial cells. Glucose and fatty acids are essential substrates for ATP and biomass production; however, the metabolism of other substrates remains poorly understood. Ketone bodies are important nutrients for cardiomyocytes during starvation or consumption of carbohydrate-restrictive diets. This raises the question whether cardiac endothelial cells would not only transport ketone bodies but also consume some of these to achieve their metabolic needs. Here, we report that cardiac endothelial cells are able to oxidize ketone bodies and that this enhances cell proliferation, migration, and vessel sprouting. Mechanistically, this requires succinyl-CoA:3-oxoacid-CoA transferase, a key enzyme of ketone body oxidation. Targeted metabolite profiling revealed that carbon from ketone bodies got incorporated into tricarboxylic acid cycle intermediates as well as other metabolites fueling biomass production. Elevation of ketone body levels by a high-fat, low-carbohydrate ketogenic diet transiently increased endothelial cell proliferation in mouse hearts. Notably, in a mouse model of heart hypertrophy, ketogenic diet prevented blood vessel rarefication. This suggests a potential beneficial role of dietary intervention in heart diseases., (© 2022 The Authors. Published under the terms of the CC BY 4.0 license.)

Background: Spastic cerebral palsy is the most common cause of motor disability in children. It often leads to foot drop or equinus, interfering with walking. Ankle-foot orthoses (AFOs) are commonly used in these cases. However, AFOs can be too restrictive for mildly impaired patients. Functional electrical stimulation (FES) of the ankle-dorsiflexors is an alternative treatment as it could function as a dynamic functional orthosis. Despite previous research, high level evidence on the effects of FES on activities and participation in daily life is missing. The primary aim of this study is to evaluate whether FES improves the activity and participation level in daily life according to patients, and the secondary aim is to provide evidence of the effect of FES at the level of body functions and activities. Furthermore, we aim to collect relevant information for decisions on its clinical implementation., Methods: A randomized crossover trial will be performed on 25 children with unilateral spastic cerebral palsy. Patients aged between 4 and 18 years, with Gross Motor Functioning Classification System level I or II and unilateral foot drop of central origin, currently treated with AFO or adapted shoes, will be included. All participants will undergo twelve weeks of conventional treatment (AFO/adapted shoes) and 12 weeks of FES treatment, separated by a six-week washout-phase. FES treatment consists of wearing the WalkAide® device, with surface electrodes stimulating the peroneal nerve during swing phase of gait. For the primary objective, the Goal Attainment Scale is used to test whether FES improves activities and participation in daily life. The secondary objective is to prove whether FES is effective at the level of body functions and structures, and activities, including ankle kinematics and kinetics measured during 3D-gait analysis and questionnaire-based frequency of falling. The tertiary objective is to collect relevant information for clinical implementation, including acceptability using the device log file and side effect registration, cost-effectiveness based on quality adjusted life years (QALYs) and clinical characteristics for patient selection., Discussion: We anticipate that the results of this study will allow evidence-based use of FES during walking in children with unilateral spastic cerebral palsy., Trial Registration: ClinicalTrials.gov : NCT03440632 ., (© 2022. The Author(s).)

Bacterial communication via quorum sensing (QS) molecules, as well as toxin-antitoxin (TA) gene modules located on bacterial chromosomes are well-studied mechanisms. Escherichia coli mazEF is a stress-induced TA system mediating cell death requiring a QS extracellular death factor (EDF), the pentapeptide NNWNN. MazF is an endoribonuclease specific for ACA sites. During adverse conditions, the activated MazF generates a stress induced translation machinery, composed of MazF-processed mRNAs and selective ribosomes that specifically translate these processed mRNAs. Moreover, we identified the molecular mechanism underlying the formation of EDF from the zwf mRNA that involves distinct steps comprising the activity of MazF, the trans-translation system as well as the protease ClpPX. Bacterial trans-translation is generally known as a quality control process that rescues stalled translation complexes at the 3'-terminus of non-stop mRNAs. Our results indicate that trans-translation has a similar role in EDF generation from zwf mRNA. However, our data reveal that the trans-translation system may also provide a regulatory mechanism to attenuate EDF generation in the single cells. Thereby, the required threshold of EDF molecules is only achieved by the entire bacterial population, as expected for a genuine QS process.

Introduction: Brain abnormalities in cerebral palsy (CP) are known to relate to motor outcome; however, their association with cognitive functioning is less clear., Aim of the Study: 1) To investigate the prognostic value of brain abnormalities for cognitive functioning; 2) To explore the added value of prognostic variables across ICF domains: motor function, epilepsy, gestational age, birthweight and educational level of the parents., Methods: We retrospectively analyzed brain MRI scans of 75 children with CP (GMFCS level I-V, 36% born preterm), as part of a longitudinal study. MRI classification: qualitative classification of brain abnormality pattern and semi-quantitative grading of the extent of damage. Cognitive functioning, measured as non-verbal intelligent quotient (IQ), was dichotomized into 'impaired cognition' (IQ ≤ 70) and 'normal' (IQ > 70). Multivariable logistic regression produced odds ratios (OR) with 95% confidence interval (C.I.) of risk factors for impaired cognition., Results: Overall, 27% of the tested participants had a non-verbal IQ below 70 and 36% of the participants was classified as 'having impaired cognition'. At a young age, a higher degree of white matter damage (OR 1.6, 95% C.I. 0.97-2.67) and a more severe GMFCS level (OR 3.2, 95% C.I. 1.70-5.98) are risk factors for impaired cognition at school-age (4-7 years of age). This model correctly predicts 89% of the cases. Brain damage alone predicts the presence of impaired cognition in 71% of the cases., Interpretation: Brain MRI characteristics and GMFCS level at a young age can each help identify children with CP at risk for impaired cognition at school age and together have a strong predictive value., Competing Interests: Declaration of competing interest None of the authors has any competing interest to declare., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Oil is frequently used as a solvent to inject lipophilic substances into the peritoneum of laboratory animals. Although mineral oil causes chronic peritoneal inflammation, little is known whether other oils are better suited. We show that olive, peanut, corn, or mineral oil causes xanthogranulomatous inflammation with depletion of resident peritoneal macrophages. However, there were striking differences in the severity of the inflammatory response. Peanut and mineral oil caused severe chronic inflammation with persistent neutrophil and monocyte recruitment, expansion of the vasculature, and fibrosis. Corn and olive oil provoked no or only mild signs of chronic inflammation. Mechanistically, the vegetal oils were taken up by macrophages leading to foam cell formation and induction of cell death. Olive oil triggered caspase-3 cleavage and apoptosis, which facilitate the resolution of inflammation. Peanut oil and, to a lesser degree, corn oil, triggered caspase-1 activation and macrophage pyroptosis, which impair the resolution of inflammation. As such, intraperitoneal oil administration can interfere with the outcome of subsequent experiments. As a proof of principle, intraperitoneal peanut oil injection was compared with its oral delivery in a thioglycolate-induced peritonitis model. The chronic peritoneal inflammation due to peanut oil injection impeded the proper recruitment of macrophages and the resolution of inflammation in this peritonitis model. In summary, the data indicate that it is advisable to deliver lipophilic substances, like tamoxifen, by oral gavage instead of intraperitoneal injection. IMPLICATIONS: This work contributes to the reproducibility of animal research by helping to understand some of the undesired effects observed in animal experiments., (©2020 American Association for Cancer Research.)

The role of the endothelium is not just limited to acting as an inert barrier for facilitating blood transport. Endothelial cells (ECs), through expression of a repertoire of angiocrine molecules, regulate metabolic demands in an organ-specific manner. Insulin flux across the endothelium to muscle cells is a rate-limiting process influencing insulin-mediated lowering of blood glucose. Here, we demonstrate that Notch signaling in ECs regulates insulin transport to muscle. Notch signaling activity was higher in ECs isolated from obese mice compared to non-obese. Sustained Notch signaling in ECs lowered insulin sensitivity and increased blood glucose levels. On the contrary, EC-specific inhibition of Notch signaling increased insulin sensitivity and improved glucose tolerance and glucose uptake in muscle in a high-fat diet-induced insulin resistance model. This was associated with increased transcription of Cav1, Cav2, and Cavin1, higher number of caveolae in ECs, and insulin uptake rates, as well as increased microvessel density. These data imply that Notch signaling in the endothelium actively controls insulin sensitivity and glucose homeostasis and may therefore represent a therapeutic target for diabetes., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

Vascular smooth muscle cell (VSMC) dysfunction is a hallmark of small vessel disease, a common cause of stroke and dementia. Two of the most frequently mutated genes in familial small vessel disease are HTRA1 and NOTCH3. The protease HTRA1 cleaves the NOTCH3 ligand JAG1 implying a mechanistic link between HTRA1 and Notch signaling. Here we report that HTRA1 is essential for VSMC differentiation into the contractile phenotype. Mechanistically, loss of HTRA1 increased JAG1 protein levels and NOTCH3 signaling activity in VSMC. In addition, the loss of HTRA1 enhanced TGFβ-SMAD2/3 signaling activity. Activation of either NOTCH3 or TGFβ signaling resulted in increased transcription of the HES and HEY transcriptional repressors and promoted the contractile VSMC phenotype. However, their combined over-activation led to an additive accumulation of HES and HEY proteins, which repressed the expression of contractile VSMC marker genes. As a result, VSMC adopted an immature phenotype with impaired arterial vasoconstriction in Htra1-deficient mice. These data demonstrate an essential role of HTRA1 in vascular maturation and homeostasis by controlling Notch and TGFβ signaling.

Merkel-cell carcinoma (MCC) is a rare and aggressive neuroendocrine carcinoma named for its Merkel-cell-like ultrastructure. The neuroendocrine Merkel cell was previously believed to be the cell of origin. However, Merkel cells are postmitotic and thus probably not the cell of origin of MCC. It is derived from an epidermal stem cell, which also might represent the cell of origin of MCC. Further putative cells of origin are dermal stem cells and pre/pro‑B cells, the latter showing some similar markers (e.g. PAX5).About 80% of MCCs are induced by the integration of DNA of the Merkel cell polyoma virus (MCPyV) into the genome. On the other hand, about 20% of MCCs show UV-induced mutations in numerous genes (e.g. TP53, RB1). In routine histology, MCC appears monomorphic and the diagnosis is confirmed by immunohistochemistry showing CK20 arranged in typical paranuclear plaques, together with the presence of neurofilaments and chromogranin A. Virus-positive and virus-negative MCC are not different histologically.UV-induced and viral neoantigens cause the strong immunogenicity of MCC. Moreover, over the last few years, the presence of PD-1 and PD-L1 has been demonstrated within tumor and immune cells. For the checkpoint inhibitors pembrolizumab and avelumab, responses of about 50% have been shown, independent of virus state. Circulating tumor cells (CTCs) seem to be helpful in tumor tracking. Further immunological and molecular studies are necessary for future individual therapies, also concerning immunocompromised patients.

Despite remarkable progress in melanoma therapy, the exceptional heterogeneity of the disease has prevented the development of reliable companion biomarkers for the prediction or monitoring of therapy responses. Here, we show that difficulties in detecting blood-based markers, like circulating tumor cells (CTC), might arise from the translation of the mutational heterogeneity of melanoma cells towards their surface marker expression. We provide a unique method, which enables the molecular characterization of clinically relevant CTC subsets, as well as circulating tumor DNA (ctDNA), from a single blood sample. The study demonstrates the benefit of a combined analysis of ctDNA and CTC counts in melanoma patients, revealing that CTC subsets and ctDNA provide synergistic real-time information on the mutational status, RNA and protein expression of melanoma cells in individual patients, in relation to clinical outcome.

Background: Merkel cell carcinoma (MCC) is a rare, aggressive skin cancer with increasing incidence and high mortality rates. MCC has recently become the subject of immune checkpoint therapy, but reliable biomarkers for estimating prognosis, risk stratification, and prediction of response are missing., Methods: Circulating tumor cells (CTCs) were detected in peripheral blood from patients with MCC by use of the CellSearch ® system. Moreover, CTCs of selected cases were characterized for Merkel cell polyomavirus (MCPyV), chromosomal aberrations, and programed death ligand 1 (PD-L1) production., Results: Fifty-one patients were tested at first blood draw (baseline), and 16 patients had 2 or 3 consecutive measurements to detect CTCs. At baseline, ≥1 CTC (range, 1-790), >1, or ≥5 CTCs/7.5 mL were detected in 21 (41%), 17 (33%), and 6 (12%) patients, respectively. After a median follow-up of 21.1 months for 50 patients, detection of CTCs correlated with overall survival (≥1, P = 0.030; >1, P < 0.020; and ≥5 CTCs/7.5 mL, P < 0.0001). In multivariate Cox regression analysis, the detection of ≥5 CTCs/7.5 mL adjusted to age and sex compared to that of <5 was associated with a reduced overall survival ( P = 0.001, hazard ratio = 17.8; 95% CI, 4.0-93.0). MCPyV DNA and genomic aberrations frequently found in MCC tissues could also be detected in single CTCs. Analyzed CTCs were PD-L1 negative or only weakly positive., Conclusions: The presence of CTCs is a prognostic factor of impaired clinical outcome, with the potential to monitor the progression of the disease in real time. Molecular characterization of CTCs might provide new insights into the biology of MCC., (© 2018 American Association for Clinical Chemistry.)

The serine protease HTRA1 is involved in several vascular diseases and its expression is often deregulated in cancer. We aimed at identifying how HTRA1 in the vasculature affects tumor growth. Here we report that silencing of HTRA1 in cultured endothelial cells increased migration rate and tube formation, whereas forced HTRA1 expression impaired sprouting angiogenesis. Mechanistically, endothelial HTRA1 expression enhanced Delta/Notch signaling by reducing the amount of the weak Notch ligand JAG1. HTRA1 physically interacted with JAG1 and cleaved it within the intracellular domain, leading to protein degradation. Expression of a constitutive active Notch1 prevented the hypersprouting phenotype upon silencing of HTRA1. In HtrA1-deficient mice, endothelial Notch signaling was diminished and isolated endothelial cells had increased expression of VEGF receptor-2. Growth of syngeneic tumors was strongly impaired in HtrA1 -/- mice. The tumor vasculature was much denser in HtrA1 -/- mice and less covered with mural cells. This chaotic and immature vascular network was poorly functional as indicated by large hypoxic tumor areas and low tumor cell proliferation rates. In summary, inhibition of HTRA1 in the tumor stroma impaired tumor progression by deregulating angiogenesis.

Previously, leaderless mRNAs (lmRNAs) were perceived to make up only a minor fraction of the transcriptome in bacteria. However, advancements in RNA sequencing technology are uncovering vast numbers of lmRNAs, particularly in archaea, Actinobacteria , and extremophiles and thus underline their significance in cellular physiology and regulation. Due to the absence of conventional ribosome binding signals, lmRNA translation initiation is distinct from canonical mRNAs and can therefore be differentially regulated. The ribosome's inherent ability to bind a 5'-terminal AUG can stabilize and protect the lmRNA from degradation or allow ribosomal loading for downstream initiation events. As a result, lmRNAs remain translationally competent during a variety of physiological conditions, allowing them to contribute to multiple regulatory mechanisms. Furthermore, the abundance of lmRNAs can increase during adverse conditions through the upregulation of lmRNA transcription from alternative promoters or by the generation of lmRNAs from canonical mRNAs cleaved by an endonucleolytic toxin. In these ways, lmRNA translation can continue during stress and contribute to regulation, illustrating their importance in the cell. Due to their presence in all domains of life and their ability to be translated by heterologous hosts, lmRNAs appear further to represent ancestral transcripts that might allow us to study the evolution of the ribosome and the translational process.

Background: Nutrients are transported through endothelial cells before being metabolized in muscle cells. However, little is known about the regulation of endothelial transport processes. Notch signaling is a critical regulator of metabolism and angiogenesis during development. Here, we studied how genetic and pharmacological manipulation of endothelial Notch signaling in adult mice affects endothelial fatty acid transport, cardiac angiogenesis, and heart function., Methods: Endothelial-specific Notch inhibition was achieved by conditional genetic inactivation of Rbp-jκ in adult mice to analyze fatty acid metabolism and heart function. Wild-type mice were treated with neutralizing antibodies against the Notch ligand Delta-like 4. Fatty acid transport was studied in cultured endothelial cells and transgenic mice., Results: Treatment of wild-type mice with Delta-like 4 neutralizing antibodies for 8 weeks impaired fractional shortening and ejection fraction in the majority of mice. Inhibition of Notch signaling specifically in the endothelium of adult mice by genetic ablation of Rbp-jκ caused heart hypertrophy and failure. Impaired heart function was preceded by alterations in fatty acid metabolism and an increase in cardiac blood vessel density. Endothelial Notch signaling controlled the expression of endothelial lipase, Angptl4, CD36, and Fabp4, which are all needed for fatty acid transport across the vessel wall. In endothelial-specific Rbp-jκ-mutant mice, lipase activity and transendothelial transport of long-chain fatty acids to muscle cells were impaired. In turn, lipids accumulated in the plasma and liver. The attenuated supply of cardiomyocytes with long-chain fatty acids was accompanied by higher glucose uptake, increased concentration of glycolysis intermediates, and mTOR-S6K signaling. Treatment with the mTOR inhibitor rapamycin or displacing glucose as cardiac substrate by feeding a ketogenic diet prolonged the survival of endothelial-specific Rbp-jκ-deficient mice., Conclusions: This study identifies Notch signaling as a novel regulator of fatty acid transport across the endothelium and as an essential repressor of angiogenesis in the adult heart. The data imply that the endothelium controls cardiomyocyte metabolism and function., (© 2018 American Heart Association, Inc.)

Interferons (IFN) have a direct growth-inhibiting effect on tumor cells through Janus kinase-dependent activation of the transcription factor signal transducer and activator of transcription (STAT1). In vitro, signaling through STAT5 has been demonstrated to counteract this effect and lead to IFN resistance of melanoma cell lines. In 32 patients treated with IFN-α in an adjuvant setting, we investigated paraffin-embedded tumor tissue from primary melanomas and melanoma metastases for expression of STAT3 and STAT5, by immunohistochemistry, and for expression of phosphorylated signaling transduction activating transcription factor (pSTAT)3 and pSTAT5, by immunofluorescence. Tumor cell expression levels of these proteins were correlated with patient characteristics and clinical outcomes. The patient cohort consisted of 12 (37.5%) patients at AJCC stage I/II (primary melanoma) and 20 (62.5%) at stage III/IV (metastatic melanoma). Recurrence was observed for 25 (78.1%) either during or after IFN-α therapy. χ Correlation of staining intensities with clinical data revealed association of pSTAT3 and STAT5 expression with sex (P=0.003 and 0.016, respectively) and of STAT3 with tumor stage (P=0.019). Recurrence of melanoma was found to be associated with high STAT5 expression (P=0.017). Multivariable regression analysis revealed STAT5 expression as an independent factor for predicting progression-free survival (P<0.0001) and overall survival (P=0.022). In summary, high expression of STAT5 correlated with melanoma recurrence and survival of patients treated with IFN-α in the adjuvant setting. Recently, it has been suggested that mutations of Janus kinases are involved in resistance to immune checkpoint blocker treatments implying a possible role of STAT5 for immune checkpoint resistance.

The MazF toxin sequence-specifically cleaves single-stranded RNA upon various stressful conditions, and it is activated as a part of the mazEF toxin-antitoxin module in Escherichia coli. Although autoregulation of mazEF expression through the MazE antitoxin-dependent transcriptional repression has been biochemically characterized, less is known about post-transcriptional autoregulation, as well as how both of these autoregulatory features affect growth of single cells during conditions that promote MazF production. Here, we demonstrate post-transcriptional autoregulation of mazF expression dynamics by MazF cleaving its own transcript. Single-cell analyses of bacterial populations during ectopic MazF production indicated that two-level autoregulation of mazEF expression influences cell-to-cell growth rate heterogeneity. The increase in growth rate heterogeneity is governed by the MazE antitoxin, and tuned by the MazF-dependent mazF mRNA cleavage. Also, both autoregulatory features grant rapid exit from the stress caused by mazF overexpression. Time-lapse microscopy revealed that MazF-mediated cleavage of mazF mRNA leads to increased temporal variability in length of individual cells during ectopic mazF overexpression, as explained by a stochastic model indicating that mazEF mRNA cleavage underlies temporal fluctuations in MazF levels during stress.

Angiogenesis is coordinated by VEGF and Notch signaling. DLL4-induced Notch signaling inhibits tip cell formation and vessel branching. To ensure proper Notch signaling, receptors and ligands are clustered at adherens junctions. However, little is known about factors that control Notch activity by influencing the cellular localization of Notch ligands. Here, we show that the multiple PDZ domain protein (MPDZ) enhances Notch signaling activity. MPDZ physically interacts with the intracellular carboxyterminus of DLL1 and DLL4 and enables their interaction with the adherens junction protein Nectin-2. Inactivation of the MPDZ gene leads to impaired Notch signaling activity and increased blood vessel sprouting in cellular models and the embryonic mouse hindbrain. Tumor angiogenesis was enhanced upon endothelial-specific inactivation of MPDZ leading to an excessively branched and poorly functional vessel network resulting in tumor hypoxia. As such, we identified MPDZ as a novel modulator of Notch signaling by controlling ligand recruitment to adherens junctions., Competing Interests: FT, MA, AF, IM, AM, JR, DS, AF No competing interests declared, (© 2018, Tetzlaff et al.)

Stem cell-specific transcriptional networks are well known to control pluripotency, but constitutive cellular processes such as mRNA splicing and protein synthesis can add complex layers of regulation with poorly understood effects on cell-fate decisions. Here, we show that the RNA binding protein HTATSF1 controls embryonic stem cell differentiation by regulating multiple aspects of RNA processing during ribosome biogenesis. HTATSF1, in a complex with splicing factor SF3B1, controls intron removal from ribosomal protein transcripts and regulates ribosomal RNA transcription and processing, thereby controlling 60S ribosomal abundance and protein synthesis. HTATSF1-dependent protein synthesis is essential for naive pre-implantation epiblast to transition into post-implantation epiblast, a stage with transiently low protein synthesis, and further differentiation toward neuroectoderm. Together, these results identify coordinated regulation of ribosomal RNA and protein synthesis by HTATSF1 and show that this essential mechanism controls protein synthesis during early mammalian embryogenesis., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Aim: To assess the effect of functional electrical stimulation (FES) of ankle dorsiflexors in children and adolescents with spastic cerebral palsy (CP) during walking., Method: A systematic review was performed using the American Academy of Cerebral Palsy and Developmental Medicine methodology and the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Six databases were searched for studies applying interventions to patients aged younger than 20 years. Outcomes were classified according to the International Classification of Functioning, Disability and Health (ICF)., Results: Seven hundred and eighty abstracts were found, 35 articles were fully screened, and 14 articles were used for analysis. Only five articles (three studies) were of level I to III evidence. At ICF participation and activity level, there is limited evidence for a decrease in self-reported frequency of toe-drag and falls. At ICF body structure and function level, there is clear evidence (I-III) that FES increased (active) ankle dorsiflexion angle, strength, and improved selective motor control, balance, and gait kinematics, but decreased walking speed. Adverse events include skin irritation, toleration, and acceptation issues., Interpretation: There are insufficient data supporting functional gain by FES on activity and participation level. However, evidence points towards a role for FES as an alternative to orthoses in children with spastic CP., What This Paper Adds: Effects of functional electrical stimulation (FES) point towards a potential role as an alternative to orthoses for patients with spastic cerebral palsy (CP). Some evidence for a decrease in self-reported frequency of toe-drag and falls with the use of FES in spastic CP. Limited evidence for improvements in activity and participation in patients with spastic CP using FES., (© 2017 Mac Keith Press.)

Bacteria adapt to adverse environmental conditions by altering gene expression patterns. Recently, a novel stress adaptation mechanism has been described that allows Escherichia coli to alter gene expression at the post-transcriptional level. The key player in this regulatory pathway is the endoribonuclease MazF, the toxin component of the toxin-antitoxin module mazEF that is triggered by various stressful conditions. In general, MazF degrades the majority of transcripts by cleaving at ACA sites, which results in the retardation of bacterial growth. Furthermore, MazF can process a small subset of mRNAs and render them leaderless by removing their ribosome binding site. MazF concomitantly modifies ribosomes, making them selective for the translation of leaderless mRNAs. In this study, we employed fluorescent reporter-systems to investigate mazEF expression during stressful conditions, and to infer consequences of the mRNA processing mediated by MazF on gene expression at the single-cell level. Our results suggest that mazEF transcription is maintained at low levels in single cells encountering adverse conditions, such as antibiotic stress or amino acid starvation. Moreover, using the grcA mRNA as a model for MazF-mediated mRNA processing, we found that MazF activation promotes heterogeneity in the grcA reporter expression, resulting in a subpopulation of cells with increased levels of GrcA reporter protein., Competing Interests: The authors declare there are no competing interests.

Hydrocephalus is a common congenital anomaly. LCAM1 and MPDZ ( MUPP1 ) are the only known human gene loci associated with non-syndromic hydrocephalus. To investigate functions of the tight junction-associated protein Mpdz, we generated mouse models. Global Mpdz gene deletion or conditional inactivation in Nestin-positive cells led to formation of supratentorial hydrocephalus in the early postnatal period. Blood vessels, epithelial cells of the choroid plexus, and cilia on ependymal cells, which line the ventricular system, remained morphologically intact in Mpdz -deficient brains. However, flow of cerebrospinal fluid through the cerebral aqueduct was blocked from postnatal day 3 onward. Silencing of Mpdz expression in cultured epithelial cells impaired barrier integrity, and loss of Mpdz in astrocytes increased RhoA activity. In Mpdz -deficient mice, ependymal cells had morphologically normal tight junctions, but expression of the interacting planar cell polarity protein Pals1 was diminished and barrier integrity got progressively lost. Ependymal denudation was accompanied by reactive astrogliosis leading to aqueductal stenosis. This work provides a relevant hydrocephalus mouse model and demonstrates that Mpdz is essential to maintain integrity of the ependyma., (© 2017 The Authors. Published under the terms of the CC BY 4.0 license.)

Merkel cell carcinoma (MCC) is an aggressive skin cancer entity that frequently leads to rapid death due to its high propensity to metastasize. The etiology of most MCC cases is linked to Merkel cell polyomavirus (MCPyV), a virus which is monoclonally integrated in up to 95% of tumors. While there are presently no animal models to study the role of authentic MCPyV infection on transformation, tumorigenesis or metastasis formation, xenograft mouse models employing engrafted MCC-derived cell lines (MCCL) represent a promising approach to study certain aspects of MCC pathogenesis. Here, the two MCPyV-positive MCC cell lines WaGa and MKL-1 were subcutaneously engrafted in scid mice. Engraftment of both MCC cell lines resulted in the appearance of circulating tumor cells and metastasis formation, with WaGa-engrafted mice showing a significantly shorter survival time as well as increased numbers of spontaneous lung metastases compared to MKL-1 mice. Interestingly, explanted tumors compared to parental cell lines exhibit an upregulation of MCPyV sT-Antigen expression in all tumors, with WaGa tumors showing significantly higher sT-Antigen expression than MKL-1 tumors. RNA-Seq analysis of explanted tumors and parental cell lines furthermore revealed that in the more aggressive WaGa tumors, genes involved in inflammatory response, growth factor activity and Wnt signalling pathway are significantly upregulated, suggesting that sT-Antigen is the driver of the observed differences in metastasis formation., (© 2017 UICC.)

Tight junction (TJ) proteins are known to be involved in proliferation and differentiation. These processes are essential for normal skin wound healing. Here, we investigated the TJ proteins claudin-1 and occludin in ex vivo skin wound healing models and tissue samples of acute and chronic human wounds and observed major differences in localization/expression of these proteins, with chronic wounds often showing a loss of the proteins at the wound margins and/or in the regenerating epidermis. Knockdown experiments in primary human keratinocytes showed that decreased claudin-1 expression resulted in significantly impaired scratch wound healing, with delayed migration and reduced proliferation. Activation of AKT pathway was significantly attenuated after claudin-1 knockdown, and protein levels of extracellular signal-related kinase 1/2 were reduced. For occludin, down-regulation had no impact on wound healing in normal scratch assays, but after subjecting the cells to mechanical stress, which is normally present in wounds, wound healing was impaired. For both proteins we show that most of these actions are independent from the formation of barrier-forming TJ structures, thus demonstrating nonbarrier-related functions of TJ proteins in the skin. However, for claudin-1 effects on scratch wound healing were more pronounced when TJs could form. Together, our findings provide evidence for a role of claudin-1 and occludin in epidermal regeneration with potential clinical importance., (Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

When Escherichia coli encounters stress, the endoribonuclease MazF initiates a post-transcriptional response that results in the reprogramming of protein synthesis. By removing the 3΄-terminus of the 16S rRNA, MazF generates specialized ribosomes that selectively translate mRNAs likewise processed by MazF. Given the energy required for de novo ribosome biosynthesis, we considered the existence of a repair mechanism operating upon stress relief to recycle the modified ribosomes. Here, we show that the stress-ribosomes and the 3΄-terminal 16S rRNA fragment are stable during adverse conditions. Moreover, employing in vitro and in vivo approaches we demonstrate that the RNA ligase RtcB catalyzes the re-ligation of the truncated 16S rRNA present in specialized ribosomes Thereby their ability to translate canonical mRNAs is fully restored. Together, our findings not only provide a physiological function for the RNA ligase RtcB in bacteria but highlight the reversibility of ribosome heterogeneity, a crucial but hitherto undescribed concept for translational regulation., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Endothelial cells (ECs) provide angiocrine factors orchestrating tumor progression. Here, we show that activated Notch1 receptors (N1ICD) are frequently observed in ECs of human carcinomas and melanoma, and in ECs of the pre-metastatic niche in mice. EC N1ICD expression in melanoma correlated with shorter progression-free survival. Sustained N1ICD activity induced EC senescence, expression of chemokines and the adhesion molecule VCAM1. This promoted neutrophil infiltration, tumor cell (TC) adhesion to the endothelium, intravasation, lung colonization, and postsurgical metastasis. Thus, sustained vascular Notch signaling facilitates metastasis by generating a senescent, pro-inflammatory endothelium. Consequently, treatment with Notch1 or VCAM1-blocking antibodies prevented Notch-driven metastasis, and genetic ablation of EC Notch signaling inhibited peritoneal neutrophil infiltration in an ovarian carcinoma mouse model., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Diabetes mellitus is a frequent cause for chronic, difficult-to-treat wounds. New therapies for diabetic wounds are urgently needed and in-vitro or ex-vivo test systems are essential for the initial identification of new active molecules. The aim of this study is to compare in-vitro and ex-vivo test systems for their usability for early drug screening and to investigate the efficacy of a birch bark triterpene extract (TE) that has been proven ex-vivo and clinically to accelerate non-diabetic wound healing (WH), in a diabetic context. We investigated in-vitro models for diabetic WH, i.e. scratch assays with human keratinocytes from diabetic donors or cultured under hyperglycaemic conditions and a newly developed porcine ex-vivo hyperglycaemic WH model for their potential to mimic delayed diabetic WH and for the influence of TE in these test systems. We show that keratinocytes from diabetic donors often fail to exhibit significantly delayed WH. For cells under hyperglycaemic conditions significant decrease is observed but is influenced by choice of medium and presence of supplements. Also, donor age plays a role. Interestingly, hyperglycaemic effects are mainly hyperosmolaric effects in scratch assays. Ex-vivo models under hyperglycaemic conditions show a clear and substantial decrease of WH, and here both glucose and hyperosmolarity effects are involved. Finally, we provide evidence that TE is also beneficial for ex-vivo hyperglycaemic WH, resulting in significantly increased length of regenerated epidermis to 188±16% and 183±11% (SEM; p<0.05) compared to controls when using two different TE formulations. In conclusion, our results suggest that microenvironmental influences are important in WH test systems and that therefore the more complex hyperglycaemic ex-vivo model is more suitable for early drug screening. Limitations of the in-vitro and ex-vivo models are discussed. Furthermore our data recommend TE as a promising candidate for in-vivo testings in diabetic wounds., Competing Interests: This study was supported by Birken AG. However, the company was only involved in study design (for some of the experiments) but not in data collection, analysis, decision to publish or preparation of the manuscript. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials. Therefore they declare that no competing interests exist.

Escherichia coli mazEF is an extensively studied stress-induced toxin-antitoxin (TA) system. The toxin MazF is an endoribonuclease that cleaves RNAs at ACA sites. Thereby, under stress, the induced MazF generates a stress-induced translation machinery (STM), composed of MazF-processed mRNAs and selective ribosomes that specifically translate the processed mRNAs. Here, we further characterized the STM system, finding that MazF cleaves only ACA sites located in the open reading frames of processed mRNAs, while out-of-frame ACAs are resistant. This in-frame ACA cleavage of MazF seems to depend on MazF binding to an extracellular-death-factor (EDF)-like element in ribosomal protein bS1 (bacterial S1), apparently causing MazF to be part of STM ribosomes. Furthermore, due to the in-frame MazF cleavage of ACAs under stress, a bias occurs in the reading of the genetic code causing the amino acid threonine to be encoded only by its synonym codon ACC, ACU, or ACG, instead of by ACA., Importance: The genetic code is a universal characteristic of all living organisms. It defines the set of rules by which nucleotide triplets specify which amino acid will be incorporated into a protein. Our results represent the first existing report on a stress-induced bias in the reading of the genetic code. We found that in E. coli, under stress, the amino acid threonine is encoded only by its synonym codon ACC, ACU, or ACG, instead of by ACA. This is because under stress, MazF generates a stress-induced translation machinery (STM) in which MazF cleaves in-frame ACA sites of the processed mRNAs., (Copyright © 2016 Oron-Gottesman et al.)

Flexible adaptation to environmental stress is vital for bacteria. An energy-efficient post-transcriptional stress response mechanism in Escherichia coli is governed by the toxin MazF. After stress-induced activation the endoribonuclease MazF processes a distinct subset of transcripts as well as the 16S ribosomal RNA in the context of mature ribosomes. As these 'stress-ribosomes' are specific for the MazF-processed mRNAs, the translational program is changed. To identify this 'MazF-regulon' we employed Poly-seq (polysome fractionation coupled with RNA-seq analysis) and analyzed alterations introduced into the transcriptome and translatome after mazF overexpression. Unexpectedly, our results reveal that the corresponding protein products are involved in all cellular processes and do not particularly contribute to the general stress response. Moreover, our findings suggest that translational reprogramming serves as a fast-track reaction to harsh stress and highlight the so far underestimated significance of selective translation as a global regulatory mechanism in gene expression. Considering the reported implication of toxin-antitoxin (TA) systems in persistence, our results indicate that MazF acts as a prime effector during harsh stress that potentially introduces translational heterogeneity within a bacterial population thereby stimulating persister cell formation., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Adjuvant treatment with interferon (IFN)-α-2a improved disease-free survival (DFS) and showed a trend for improving overall survival (OS) in melanoma. This trial was designed to examine whether PEG-IFN is superior to IFN with regard to distant metastasis-free survival (DMFS), DFS and OS., Patients and Methods: In this multicenter, open-label, prospective randomized phase III trial, patients with resected cutaneous melanoma stage IIA(T3a)-IIIB (AJCC 2002) were randomized to receive PEG-IFN (180 μg subcutaneously 1×/week; 24 months) or IFN α-2a (3MIU subcutaneously 3×/week; 24 months). Randomization was stratified for stage, number of metastatic nodes, age and previous IFN treatment. The primary end point was DMFS; secondary end points were OS, DFS, quality of life (QoL) and tolerability., Results: A total of 909 patients were enrolled (451 PEG-IFN versus 458 IFN). Neither 5-year DMFS [PEG-IFN 61.0% versus IFN 67.3%; hazard ratio (HR) 1.16, P = 0.21] nor 5-year OS (PEG-IFN 73.2% versus IFN 75.2%; HR 1.05, P = 0.70) nor 5-year DFS (PEG-IFN 57.3% versus IFN 60.9%; HR 1.09, P = 0.40) showed significant differences. Subgroup analyses in patients ± ulcerated primaries and of different tumor stages did not find differences in DMFS, OS or DFS between the treatment groups. One hundred and eighteen patients (26.2%) in the PEG-IFN and 61 patients (13.3%) in the IFN population did not receive the full dosage and length of treatment due to adverse events (P < 0.001). Leukopenia and elevation of liver enzymes were more common in the PEG-IFN arm (56% versus 23.5% LCP; 19.1% versus 9.4% AST; 33.0% versus 16.5% ALT). QoL was identical for nearly all domains., Conclusion: PEG-IFN did not improve the outcome over IFN. A higher percentage of patients under PEG-IFN discontinued treatment due to toxicity., Clinical Trialsgov Identifier: NCT00204529., (© The Author 2016. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The bacteriostatic aminoglycoside antibiotic kasugamycin inhibits protein synthesis at an initial step without affecting translation elongation. It binds to the mRNA track of the ribosome and prevents formation of the translation initiation complex on canonical mRNAs. In contrast, translation of leaderless mRNAs continues in the presence of the drug in vivo. Previously, we have shown that kasugamycin treatment in E. coli stimulates the formation of protein-depleted ribosomes that are selective for leaderless mRNAs. Here, we provide evidence that prolonged kasugamycin treatment leads to selective synthesis of specific proteins. Our studies indicate that leaderless and short-leadered mRNAs are generated by different molecular mechanisms including alternative transcription and RNA processing. Moreover, we provide evidence for ribosome heterogeneity in response to kasugamycin treatment by alteration of the modification status of the stalk proteins bL7/L12.

Follicular penetration has gained increasing interest regarding (i) safety concerns about (environmentally born) xenobiotics available to the hair follicle (HF), e.g. nanomaterials or allergens which should not enter the skin, and (ii) the possibility for non-invasive follicular drug and antigen delivery. However, not much is known about barriers in the HF which have to be surpassed upon uptake and/or penetration into surrounding tissue. Thus, aim of this work was a detailed investigation of this follicular barrier function, as well as particle uptake into the HF of porcine skin which is often used as a model system for human skin for such purposes. We show that follicular tight junctions (TJs) form a continuous barrier from the infundibulum down to the suprabulbar region, complementary to the stratum corneum in the most exposed upper follicular region, but remaining as the only barrier in the less accessible lower follicular regions. In the bulbar region of the HF no TJ barrier was found, demonstrating the importance of freely supplying this hair-forming part with e.g. nutrients or hormones from the dermal microenvironment. Moreover, the dynamic character of the follicular TJ barrier was shown by modulating its permeability using EDTA. After applying polymeric model-nanoparticles (154 nm) to the skin, transmission electron microscopy revealed that the majority of the particles were localized in the upper part of the HF where the double-barrier is present. Only few penetrated deeper, reaching regions where TJs act as the only barrier, and no particles were observed in the bulbar, barrier-less region. Lastly, the equivalent expression and distribution of TJ proteins in human and porcine HF further supports the suitability of porcine skin as a predictive model to study the follicular penetration and further biological effects of dermally applied nanomaterials in humans., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Unlabelled: Eshcerichia coli mazEF is a stress-induced toxin-antitoxin module mediating cell death and requiring a quorum-sensing (QS) extracellular death factor (EDF), the pentapeptide NNWNN. Here we uncovered several distinct molecular mechanisms involved in its generation from the zwf mRNA encoding glucose-6-phosphate dehydrogenase. In particular, we show that, under stress conditions, the endoribonuclease MazF cleaves specific ACA sites, thereby generating a leaderless zwf mRNA which is truncated 30 codons after the EDF-encoding region. Since the nascent ribosome peptide exit tunnel can accommodate up to 40 amino acids, this arrangement allows the localization of the EDF residues inside the tunnel when the ribosome is stalled at the truncation site. Moreover, ribosome stalling activates the trans-translation system, which provides a means for the involvement of ClpPX in EDF generation. Furthermore, the trans-translation is described as a regulatory system that attenuated the generation of EDF, leading to low levels of EDF in the single cell. Therefore, the threshold EDF molecule concentration required is achieved only by the whole population, as expected for QS., Importance: Bacteria communicate with one another via quorum-sensing (QS) signal molecules. QS provides a mechanism for bacteria to monitor each other's presence and to modulate gene expression in response to population density. Previously, we added E. coli pentapeptide EDF to this list of QS molecules. We showed that, under stress conditions, the induced MazF, an endoribonuclease cleaving at ACA sites, generates EDF from zwf. Here we studied the mechanism of EDF generation and asked whether it is related to EDF density dependency. We illustrated that, under stress conditions, multiple distinct complex mechanisms are involved in EDF generation. This includes formation of leaderless truncated zwf mRNA by MazF, configuration of a length corresponding to the nascent ribosome peptide exit tunnel, rescue performed by the trans-translation system, and cleavage by ClpPX protease. trans-Translation is described as a regulatory system attenuating EDF generation and leading to low levels of EDF in the single cell, as expected for QS., (Copyright © 2016 Kumar et al.)

Cerebral cavernous malformations are fragile blood vessel conglomerates in the central nervous system that are caused by mutations in the CCM1/KRIT1, CCM2 or CCM3 genes. The gene products form a protein complex at adherens junctions and loss of either CCM protein disrupts endothelial cell quiescence leading to increased permeability and excessive angiogenesis. We performed a yeast 2-hybrid screen to identify novel proteins directly interacting with KRIT1. The ankyrin repeat and sterile alpha motif domain-containing protein 1B (ANKS1B) was identified as a novel binding partner of KRIT1. Silencing of ANKS1B or the related gene ANKS1A in primary human endothelial cells had no significant effects on cellular proliferation, migration and sprouting angiogenesis. However, silencing of ANKS1B expression disturbed endothelial cell barrier functions leading to increased permeability. Forced ANKS1B expression reduced permeability. This was independent of Rho kinase activity and the presence of KRIT1. Taken together, ANKS1B was identified as a novel KRIT1-interacting protein that selectively controls endothelial permeability but not angiogenesis.

Tight junctions are important for skin barrier function. The tight junction protein claudin 1 (Cldn-1) has been reported to be down-regulated in nonlesional skin of atopic dermatitis (AD) patients. In contrast, we did not observe a significant down-regulation of Cldn-1 in nonlesional skin of the AD cohort used in this study. However, for the first time, a significant down-regulation of Cldn-1 in the upper and lower epidermal layers of lesional skin was detected. In addition, there was a significant up-regulation of Cldn-4 in nonlesional, but not lesional, AD skin. For occludin, no significant alterations were observed. In an AD-like allergic dermatitis mouse model, Cldn-1 down-regulation in eczema was significantly influenced by dermal inflammation, and significantly correlated with hallmarks of eczema (ie, increased keratinocyte proliferation, altered keratinocyte differentiation, increased epidermal thickness, and impaired barrier function). In human epidermal equivalents, the addition of IL-4, IL-13, and IL-31 resulted in a down-regulation of Cldn-1, and Cldn1 knockdown in keratinocytes resulted in abnormal differentiation. In summary, we provide the first evidence that Cldn-1 and Cldn-4 are differentially involved in AD pathogenesis. Our data suggest a role of Cldn-1 in AD eczema formation triggered by inflammation., (Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Reliable models for the determination of skin penetration and permeation are important for the development of new drugs and formulations. The intention of our study was to develop a skin penetration model which (1) is viable and well supplied with nutrients during the period of the experiment (2) is mimicking human skin as far as possible, but still is independent from the problems of supply and heterogeneity, (3) can give information about the penetration into different compartments of the skin and (4) considers specific inter-individual differences in skin thickness. In addition, it should be quick and inexpensive (5) and without ethical implications (6). Using a chemically divers set of four topically approved active pharmaceutical ingredients (APIs), namely diclofenac, metronidazole, tazarotene, and terbinafine, we demonstrated that the model allows reliable determination of drug concentrations in different layers of the viable epidermis and dermis. For APIs susceptible for skin metabolism, the extent of metabolic transformation in epidermis and dermis can be monitored. Furthermore, a high degree of accordance in the ability for discrimination of skin concentrations of the substances in different layers was found in models derived from porcine and human skin. Viability, proliferation, differentiation and markers for skin barrier function were surveyed in the model. This model, which we call 'Hamburg model of skin penetration' is particularly suited to support a rational ranking and selection of dermatological formulations within drug development projects., (Copyright © 2015 Elsevier B.V. All rights reserved.)