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Halobacillus halophilus, a moderately halophilic bacterium isolated from salt marshes, produces various compatible solutes to cope with osmotic stress. Glutamate and glutamine are dominant compatible solutes at mild salinities. Glutamine synthetase activity in cell suspensions of Halobacillus halophilus wild type was shown to be salt dependent and chloride modulated. A possible candidate to catalyze glutamine synthesis is glutamine synthetase A2, whose transcription is stimulated by chloride. To address the role of GlnA2 in the biosynthesis of the osmolytes glutamate and glutamine, a deletion mutant (ΔglnA2) was generated and characterized in detail. We compared the pool of compatible solutes and performed transcriptional analyses of the principal genes controlling the solute production in the wild type strain and the deletion mutant. These measurements did not confirm the hypothesized role of GlnA2 in the osmolyte production. Most likely the presence of another, yet to be identified enzyme has the main contribution in the measured activity in crude extracts and probably determines the total chloride-modulated profile. The role of GlnA2 remains to be elucidated.
Introduction: Penile carcinomas are rare tumors throughout Europe. Therefore, little attention is drawn to this disease. That makes it important to study tumor-associated key metrics and relate these to known data on penile neoplasias. Materials and methods: A cohort of 60 well-defined penile invasive carcinomas with known human papillomavirus (HPV) infection status was investigated. Data on tumor type, grading and staging were recorded. Additionally, data on the peri- and intratumoral immune cell infiltrate in a semiquanititave manner applying an HE stain were assessed. Results: Our study showed a significant correlation of immune cell infiltrate and pT stage with overall survival. Therefore, in a subset of tumors, PD-L1 staining was applied. For tumor proportion score (TPS), 26 of 30 samples (87%) were scored >0%. For the immune cell score (IC), 28 of 30 samples (93%) were defined as >0% and for CPS, 29 of 30 samples (97%) scored >0. PD-L1 expression was not associated with overall survival. Conclusion: PD-L1 is expressed in penile carcinomas, providing a rationale for targeted therapy with checkpoint inhibitors. We were able to show that immune reaction appears to be prognostically relevant. These data enhance the need for further studies on the immune cell infiltrate in penile neoplasias and show that PD-L1 expression is existent in our cohort, which may be a potential target for checkpoint inhibitor therapy.
Background: Routine human papillomavirus (HPV) testing is performed in cervival cancer and is required for classification of some head and neck cancers. In penile cancer a statement on HPV association of the carcinoma is required. In most cases p16 immunohistochemistry as a surrogate marker is applied in this setting. Since differing clinical outcomes for HPV positive and HPV negative tumors are described we await HPV testing to be requested more frequently by clinicians, also in the context of HPV vaccination, where other HPV subtypes are expected to emerge.
Method: Therefore, a cohort of archived, formalin-fixed paraffin embedded (FFPE) penile neoplasias was stained for p16 and thereafter tested for HPV infection status via PCR based methods. Additionally to Sanger sequencing, we chose LCD-Array technique (HPV 3.5 LCD-Array Kit, Chipron; LCD-Array) for the detection of HPV in our probes expecting a less time consuming and sensitive HPV test for our probes.
Results: We found that LCD-Array is a sensitive and feasible method for HPV testing in routine diagnostics applicable to FFPE material in our cohort. Our cohort of penile carcinomas and carcinomas in situ was associated with HPV infection in 61% of cases. We detected no significant association between HPV infection status and histomorphological tumor characteristics as well as overall survival.
Conclusions: We showed usability of molecular HPV testing on a cohort of archived penile carcinomas. To the best of our knowledge, this is the first study investigating LCD-Array technique on a cohort of penile neoplasias.
Most available knowledge on fungal arginine metabolism is derived from studies on Saccharomyces cerevisiae, in which arginine catabolism is initiated by releasing urea via the arginase reaction. Orthologues of the S. cerevisiae genes encoding the first three enzymes in the arginase pathway were cloned from Kluyveromyces lactis and shown to functionally complement the corresponding deletion in S. cerevisiae. Surprisingly, deletion of the single K. lactis arginase gene KlCAR1 did not completely abolish growth on arginine as nitrogen source. Growth rate of the deletion mutant strongly increased during serial transfer in shake-flask cultures. A combination of RNAseq-based transcriptome analysis and 13C-15N-based flux analysis was used to elucidate the arginase-independent pathway. Isotopic 13C15N-enrichment in γ-aminobutyrate revealed succinate as the entry point in the TCA cycle of the alternative pathway. Transcript analysis combined with enzyme activity measurements indicated increased expression in the Klcar1Δ mutant of a guanidinobutyrase (EC.3.5.3.7), a key enzyme in a new pathway for arginine degradation. Expression of the K. lactis KLLA0F27995g (renamed KlGBU1) encoding guanidinobutyrase enabled S. cerevisiae to use guanidinobutyrate as sole nitrogen source and its deletion in K. lactis almost completely abolish growth on this nitrogen source. Phylogenetic analysis suggests that this enzyme activity is widespread in fungi.
Background: Perioperative anaemia leads to impaired oxygen supply with a risk of vital organ ischaemia. In healthy and fit individuals, anaemia can be compensated by several mechanisms. Elderly patients, however, have less compensatory mechanisms because of multiple co-morbidities and age-related decline of functional reserves. The purpose of the study is to evaluate whether elderly surgical patients may benefit from a liberal red blood cell (RBC) transfusion strategy compared to a restrictive transfusion strategy.
Methods: The LIBERAL Trial is a prospective, randomized, multicentre, controlled clinical phase IV trial randomising 2470 elderly (≥ 70 years) patients undergoing intermediate- or high-risk non-cardiac surgery. Registered patients will be randomised only if Haemoglobin (Hb) reaches ≤9 g/dl during surgery or within 3 days after surgery either to the LIBERAL group (transfusion of a single RBC unit when Hb ≤ 9 g/dl with a target range for the post-transfusion Hb level of 9–10.5 g/dl) or the RESTRICTIVE group (transfusion of a single RBC unit when Hb ≤ 7.5 g/dl with a target range for the post-transfusion Hb level of 7.5–9 g/dl). The intervention per patient will be followed until hospital discharge or up to 30 days after surgery, whichever occurs first. The primary efficacy outcome is defined as a composite of all-cause mortality, acute myocardial infarction, acute ischaemic stroke, acute kidney injury (stage III), acute mesenteric ischaemia and acute peripheral vascular ischaemia within 90 days after surgery. Infections requiring iv antibiotics with re-hospitalisation are assessed as important secondary endpoint. The primary endpoint will be analysed by logistic regression adjusting for age, cancer surgery (y/n), type of surgery (intermediate- or high-risk), and incorporating centres as random effect.
Discussion: The LIBERAL-Trial will evaluate whether a liberal transfusion strategy reduces the occurrence of major adverse events after non-cardiac surgery in the geriatric population compared to a restrictive strategy within 90 days after surgery.
Trial registration: ClinicalTrials.gov (identifier: NCT03369210).
Balloon-borne measurements of CFC-11 (on flights of the DIRAC in situ gas chromatograph and the DESCARTES grab sampler), ClO and O3 were made during the 1999/2000 winter as part of the SOLVE-THESEO 2000 campaign. Here we present the CFC-11 data from nine flights and compare them first with data from other instruments which flew during the campaign and then with the vertical distributions calculated by the SLIMCAT 3-D CTM. We calculate ozone loss inside the Arctic vortex between late January and early March using the relation between CFC-11 and O3 measured on the flights, the peak ozone loss (1200 ppbv) occurs in the 440–470 K region in early March in reasonable agreement with other published empirical estimates. There is also a good agreement between ozone losses derived from three independent balloon tracer data sets used here. The magnitude and vertical distribution of the loss derived from the measurements is in good agreement with the loss calculated from SLIMCAT over Kiruna for the same days.
Penile squamous cell carcinomas are rare tumor entities throughout Europe. Early lymphonodal spread urges for aggressive therapeutic approaches in advanced tumor stages. Therefore, understanding tumor biology and its microenvironment and correlation with known survival data is of substantial interest in order to establish treatment strategies adapted to the individual patient. Fifty-five therapy naïve squamous cell carcinomas, age range between 41 and 85 years with known clinicopathological data, were investigated with the use of tissue microarrays (TMA) regarding the tumor-associated immune cell infiltrate density (ICID). Slides were stained with antibodies against CD3, CD8 and CD20. An image analysis software was applied for evaluation. Data were correlated with clinicopathological characteristics and overall survival. There was a significant increase of ICID in squamous cell carcinomas of the penis in relation to tumor adjacent physiological tissue. Higher CD3-positive ICID was significantly associated with lower tumor stage in our cohort. The ICID was not associated with overall survival. Our data sharpens the view on tumor-associated immune cell infiltrate in penile squamous cell carcinomas with an unbiased digital and automated cell count. Further investigations on the immune cell infiltrate and its prognostic and possible therapeutic impact are needed.
Background In melanoma, preclinical data suggest a possible role of polyunsaturated fatty acids inhibiting cell growth. A new target molecule for free fatty acids, the G protein-coupled receptor GPR40, was identified in melanoma cells.
Objectives The aim of this study was to investigate GPR40 expression in human melanocytic tissues and to evaluate its potential as a prognostic marker.
Methods and Results A total of 114 tissue sections of naevi, primary melanoma and melanoma metastasis were immunohistochemically stained with anti-GPR40. The staining was evaluated, using the immunoreactivity scoring system. Compared to naevi, primary melanoma and melanoma metastasis showed significantly higher levels of GPR40 (P < 0.05). In primary melanoma, GPR40 expression positively correlated with tumour thickness (P = 0.044) and AJCC level (P = 0.017) and in melanoma metastasis with AJCC level (P = 0.035). Primary melanoma patients with high levels of GPR40 had a significantly poorer overall survival (P = 0.004) and shorter disease-free survival (0.040).
Conclusion The present study identified GPR40 as a novel target molecule in melanoma. First evidence for a potential role of the receptor in tumour progression and metastases was found, and it could be demonstrated that GPR40 expression is negatively correlated with patient’s survival.
Spinocerebellar ataxia type 2 (SCA2) and amyotrophic lateral sclerosis (ALS) are neurodegenerative disorders, caused or modified by an unstable CAG-repeat expansion in the SCA2 gene, which encodes a polyglutamine (polyQ) domain expansion in ataxin-2 (ATXN2). ATXN2 is an RNA-binding protein and interacts with the poly(A)-binding protein PABPC1, localizing to ribosomes at the rough endoplasmic reticulum. Under cell stress, ATXN2, PABPC1 and small ribosomal subunits are relocated to stress granules, where mRNAs are protected from translation and from degradation. It is unknown whether ATXN2 associates preferentially with specific mRNAs or how it modulates RNA processing. Here, we investigated the RNA profile of the liver and cerebellum from Atxn2 knockout (Atxn2−/−) mice at two adult ages, employing oligonucleotide microarrays. Prominent increases were observed for Lsm12/Paip1 (>2-fold), translation modulators known as protein interactor/competitor of ATXN2 and for Plin3/Mttp (>1.3-fold), known as apolipoprotein modulators in agreement with the hepatosteatosis phenotype of the Atxn2−/− mice. Consistent modest upregulations were also observed for many factors in the ribosome and the translation/secretion apparatus. Quantitative reverse transcriptase PCR in liver tissue validated >1.2-fold upregulations for the ribosomal biogenesis modulator Nop10, the ribosomal components Rps10, Rps18, Rpl14, Rpl18, Gnb2l1, the translation initiation factors Eif2s2, Eif3s6, Eif4b, Pabpc1 and the rER translocase factors Srp14, Ssr1, Sec61b. Quantitative immunoblots substantiated the increased abundance of NOP10, RPS3, RPS6, RPS10, RPS18, GNB2L1 in SDS protein fractions, and of PABPC1. In mouse embryonal fibroblasts, ATXN2 absence also enhanced phosphorylation of the ribosomal protein S6 during growth stimulation, while impairing the rate of overall protein synthesis rates, suggesting a block between the enhanced translation drive and the impaired execution. Thus, the physiological role of ATXN2 subtly modifies the abundance of cellular translation factors as well as global translation.
Abstract: The hallmarks of Alzheimer’s disease (AD) are characterized by cognitive decline and behavioral changes. The most prominent brain region affected by the progression of AD is the hippocampal formation. The pathogenesis involves a successive loss of hippocampal neurons accompanied by a decline in learning and memory consolidation mainly attributed to an accumulation of senile plaques. The amyloid precursor protein (APP) has been identified as precursor of Aβ-peptides, the main constituents of senile plaques. Until now, little is known about the physiological function of APP within the central nervous system. The allocation of APP to the proteome of the highly dynamic presynaptic active zone (PAZ) highlights APP as a yet unknown player in neuronal communication and signaling. In this study, we analyze the impact of APP deletion on the hippocampal PAZ proteome. The native hippocampal PAZ derived from APP mouse mutants (APP-KOs and NexCreAPP/APLP2-cDKOs) was isolated by subcellular fractionation and immunopurification. Subsequently, an isobaric labeling was performed using TMT6 for protein identification and quantification by high-resolution mass spectrometry. We combine bioinformatics tools and biochemical approaches to address the proteomics dataset and to understand the role of individual proteins. The impact of APP deletion on the hippocampal PAZ proteome was visualized by creating protein-protein interaction (PPI) networks that incorporated APP into the synaptic vesicle cycle, cytoskeletal organization, and calcium-homeostasis. The combination of subcellular fractionation, immunopurification, proteomic analysis, and bioinformatics allowed us to identify APP as structural and functional regulator in a context-sensitive manner within the hippocampal active zone network.
Author Summary: More than 20 years ago, the amyloid precursor protein (APP) was identified as the precursor protein of the Aβ peptide, the main component of senile plaques in brains affected by Alzheimer’s disease. However, little is known about the physiological function of amyloid precursor protein. Allocating APP to the proteome of the structurally and functionally dynamic presynaptic active zone highlights APP as a hitherto unknown player within the presynaptic network. The hippocampus is the most prominent brain region for learning and memory consolidation, and a vulnerable target for neurodegenerative disease, e. g. Alzheimer’s disease. Therefore, our experimental design is focused on the hippocampal neurotransmitter release site. Currently, the underlying mechanism of how APP acts within presynaptic networks is still elusive. Within the scope of this research article, we constructed a network of APP within the presynaptic active zone and how deletion of APP affects these individual networks. We combine bioinformatics tools and biochemical approaches to address the dataset provided by proteomics. Furthermore, we could unravel that APP executes regulatory functions within the synaptic vesicle cycle, cytoskeletal rearrangements and Ca2+-homeostasis. Taken together, our findings offer a new perspective on the physiological function of APP in the central nervous system and may provide a molecular link to the pathogenesis of Alzheimer’s disease.
Synaptic release sites are characterized by exocytosis-competent synaptic vesicles tightly anchored to the presynaptic active zone (PAZ) whose proteome orchestrates the fast signaling events involved in synaptic vesicle cycle and plasticity. Allocation of the amyloid precursor protein (APP) to the PAZ proteome implicated a functional impact of APP in neuronal communication. In this study, we combined state-of-the-art proteomics, electrophysiology and bioinformatics to address protein abundance and functional changes at the native hippocampal PAZ in young and old APP-KO mice. We evaluated if APP deletion has an impact on the metabolic activity of presynaptic mitochondria. Furthermore, we quantified differences in the phosphorylation status after long-term-potentiation (LTP) induction at the purified native PAZ. We observed an increase in the phosphorylation of the signaling enzyme calmodulin-dependent kinase II (CaMKII) only in old APP-KO mice. During aging APP deletion is accompanied by a severe decrease in metabolic activity and hyperphosphorylation of CaMKII. This attributes an essential functional role to APP at hippocampal PAZ and putative molecular mechanisms underlying the age-dependent impairments in learning and memory in APP-KO mice.
Balloon-borne measurements of CFC11 (from the DIRAC in situ gas chromatograph and the DESCARTES grab sampler), ClO and O3 were made during the 1999/2000 Arctic winter as part of the SOLVE-THESEO 2000 campaign, based in Kiruna (Sweden). Here we present the CFC11 data from nine flights and compare them first with data from other instruments which flew during the campaign and then with the vertical distributions calculated by the SLIMCAT 3D CTM. We calculate ozone loss inside the Arctic vortex between late January and early March using the relation between CFC11 and O3 measured on the flights. The peak ozone loss (~1200ppbv) occurs in the 440-470K region in early March in reasonable agreement with other published empirical estimates. There is also a good agreement between ozone losses derived from three balloon tracer data sets used here. The magnitude and vertical distribution of the loss derived from the measurements is in good agreement with the loss calculated from SLIMCAT over Kiruna for the same days.
Funktionelle Langzeitergebnisse nach Hüftgelenksprothesenexplantation (Girdlestone-Situation)
(2004)
Studienziel: Ziel der Arbeit war die Identifikation prognostischer Faktoren, die die zu erwartende Funktion einer Girdlestone-Arthroplastik vorhersagen. Methode: Retrospektiv wurde das Kollektiv einer Klinik von 87 Patienten, 90 Hüften analysiert, die zwischen 1983 und 2000 mit Anlage einer Girdlestone-Hüfte behandelt worden waren. Ergebnisse: Hüftscores ergaben durchschnittlich 38.7 Punkte (Harris Hip Score) und 3.5 Punkte (Score nach Merle d’Aubigné und Postel). Die Standzeit der Girdlestone-Hüfte, im Durchschnitt 90.6 Monate, und das funktionelle Ergebnis korrelierten nicht (r = 0.0920; p = 0.6289). D.h. die Funktion einer Girdlestone-Arthroplastik wurde mit zunehmender Zeit nicht schlechter. Die Anzahl der implantierten Hüftprothesen vor Girdlestone-Anlage korrelierte nicht mit dem funktionellen Ergebnis der Girdlestone-Hüfte bei Nachuntersuchung (r = - 0.1400; p = 0.4524). Die Standzeit der letzten explantierten Hüftprothese, im Mittel 44.6 Monate, und die Funktion der Girdlestone-Hüfte korrelierten nicht (r = - 0.0705; p = 0.7065). Lebensalter bei Erstimplantation der Hüftprothese, Lebensalter zum Zeitpunkt der Nachuntersuchung oder Lebensalter bei Anlage der Girdlestone-Hüfte korrelierten nicht mit dem Harris Hip Score (r = - 0.0367; p = 0.8418 sowie r = 0.1527; p = 0.4121 sowie r = - 0.0151; p = 0.9356). Die Notwendigkeit einer Revision der Girdlestone-Hüfte und das Vorhandensein eines Diabetes korrelierten (p = 0.0104). Die Korrelation zwischen verbliebenem Knochenzement und radiologischen Zeichen eines persistierenden Infektes war signifikant (p = 0.0572). Schlussfolgerung: Das Lebensalter des Patienten, der Zeitraum seit Girdlestone-Anlage, die Zahl vorangegangener HTEP-Wechsel waren keine geeigneten prognostischen Merkmale, die Funktion einer Girdlestone-Hüfte vorherzusagen.
Men and women differ substantially regarding height, weight, and body fat. Interestingly, previous work detecting genetic effects for waist-to-hip ratio, to assess body fat distribution, has found that many of these showed sex-differences. However, systematic searches for sex-differences in genetic effects have not yet been conducted. Therefore, we undertook a genome-wide search for sexually dimorphic genetic effects for anthropometric traits including 133,723 individuals in a large meta-analysis and followed promising variants in further 137,052 individuals, including a total of 94 studies. We identified seven loci with significant sex-difference including four previously established (near GRB14/COBLL1, LYPLAL1/SLC30A10, VEGFA, ADAMTS9) and three novel anthropometric trait loci (near MAP3K1, HSD17B4, PPARG), all of which were significant in women, but not in men. Of interest is that sex-difference was only observed for waist phenotypes, but not for height or body-mass-index. We found no evidence for sex-differences with opposite effect direction for men and women. The PPARG locus is of specific interest due to its link to diabetes genetics and therapy. Our findings demonstrate the importance of investigating sex differences, which may lead to a better understanding of disease mechanisms with a potential relevance to treatment options.
Ependymomas encompass a heterogeneous group of central nervous system (CNS) neoplasms that occur along the entire neuroaxis. In recent years, extensive (epi-)genomic profiling efforts have identified several molecular groups of ependymoma that are characterized by distinct molecular alterations and/or patterns. Based on unsupervised visualization of a large cohort of genome-wide DNA methylation data, we identified a highly distinct group of pediatric-type tumors (n = 40) forming a cluster separate from all established CNS tumor types, of which a high proportion were histopathologically diagnosed as ependymoma. RNA sequencing revealed recurrent fusions involving the pleomorphic adenoma gene-like 1 (PLAGL1) gene in 19 of 20 of the samples analyzed, with the most common fusion being EWSR1:PLAGL1 (n = 13). Five tumors showed a PLAGL1:FOXO1 fusion and one a PLAGL1:EP300 fusion. High transcript levels of PLAGL1 were noted in these tumors, with concurrent overexpression of the imprinted genes H19 and IGF2, which are regulated by PLAGL1. Histopathological review of cases with sufficient material (n = 16) demonstrated a broad morphological spectrum of tumors with predominant ependymoma-like features. Immunohistochemically, tumors were GFAP positive and OLIG2- and SOX10 negative. In 3/16 of the cases, a dot-like positivity for EMA was detected. All tumors in our series were located in the supratentorial compartment. Median age of the patients at the time of diagnosis was 6.2 years. Median progression-free survival was 35 months (for 11 patients with data available). In summary, our findings suggest the existence of a novel group of supratentorial neuroepithelial tumors that are characterized by recurrent PLAGL1 fusions and enriched for pediatric patients.
We present an architecture for the integration of shallow and deep NLP components which is aimed at flexible combination of different language technologies for a range of practical current and future applications. In particular, we describe the integration of a high-level HPSG parsing system with different high-performance shallow components, ranging from named entity recognition to chunk parsing and shallow clause recognition. The NLP components enrich a representation of natural language text with layers of new XML meta-information using a single shared data structure, called the text chart. We describe details of the integration methods, and show how information extraction and language checking applications for realworld German text benefit from a deep grammatical analysis.
Bipolar disorder (BD) is a heritable mental illness with complex etiology. While the largest published genome-wide association study identified 64 BD risk loci, the causal SNPs and genes within these loci remain unknown. We applied a suite of statistical and functional fine-mapping methods to these loci, and prioritized 22 likely causal SNPs for BD. We mapped these SNPs to genes, and investigated their likely functional consequences by integrating variant annotations, brain cell-type epigenomic annotations, brain quantitative trait loci, and results from rare variant exome sequencing in BD. Convergent lines of evidence supported the roles of SCN2A, TRANK1, DCLK3, INSYN2B, SYNE1, THSD7A, CACNA1B, TUBBP5, PLCB3, PRDX5, KCNK4, AP001453.3, TRPT1, FKBP2, DNAJC4, RASGRP1, FURIN, FES, YWHAE, DPH1, GSDMB, MED24, THRA, EEF1A2, and KCNQ2 in BD. These represent promising candidates for functional experiments to understand biological mechanisms and therapeutic potential. Additionally, we demonstrated that fine-mapping effect sizes can improve performance and transferability of BD polygenic risk scores across ancestrally diverse populations, and present a high-throughput fine-mapping pipeline (https://github.com/mkoromina/SAFFARI).