Atomic mutagenesis of stop codon nucleotides reveals the chemical prerequisites for release factor-mediated peptide release.

Termination of protein synthesis is triggered by the recognition of a stop codon at the ribosomal A site and is mediated by class I release factors (RFs). Whereas in bacteria, RF1 and RF2 promote termination at UAA/UAG and UAA/UGA stop codons, respectively, eukaryotes only depend on one RF (eRF1) to initiate peptide release at all three stop codons. Based on several structural as well as biochemical studies, interactions between mRNA, tRNA, and rRNA have been proposed to be required for stop codon recognition. In this study, the influence of these interactions was investigated by using chemically modified stop codons. Single functional groups within stop codon nucleotides were substituted to weaken or completely eliminate specific interactions between the respective mRNA and RFs. Our findings provide detailed insight into the recognition mode of bacterial and eukaryotic RFs, thereby revealing the chemical groups of nucleotides that define the identity of stop codons and provide the means to discriminate against noncognate stop codons or UGG sense codons

Patient satisfaction with anaesthesia care: development of a psychometric questionnaire and benchmarking among six hospitals in Switzerland and Austria†‡

Background. We describe the development and comparison of a psychometric questionnaire on patient satisfaction with anaesthesia care among six hospitals. Methods. We used a rigorous protocol: generation of items, construction of the pilot questionnaire, pilot study, statistical analysis (construct validity, factor analysis, reliability analysis), compilation of the final questionnaire, main study, repeated analysis of construct validity and reliability. We compared the mean total problem score and the scores for the dimensions: ‘Information/Involvement in decision‐making', and ‘Continuity of personal care by anaesthetist'. The influence of potential confounding variables was tested (multiple linear regression). Results. The average problem score from all hospitals was 18.6%. Most problems are mentioned in the dimensions ‘Information/Involvement in decision‐making' (mean problem score: 30.9%) and ‘Continuity of personal care by anaesthetist' (mean problem score: 32.2%). The overall assessment of the quality of anaesthesia care was good to excellent in 98.7% of cases. The most important dimension was ‘Information/Involvement in decision‐making'. The mean total problem score was significantly lower for two hospitals than the total mean for all hospitals (significantly higher at two hospitals) (P<0.05). Amongst the confounding variables considered, age, sex, subjective state of health, type of anaesthesia and level of education had an influence on the total problem score and the two dimensions mentioned. There were only marginal differences with and without the influence of the confounding variables for the different hospitals. Conclusions. A psychometric questionnaire on patient satisfaction with anaesthesia care must cover areas such as patient information, involvement in decision‐making, and contact with the anaesthetist. The assessment using summed scores for dimensions is more informative than a global summed rating. There were significant differences between hospitals. Moreover, the high problem scores indicate a great potential for improvement at all hospitals. Br J Anaesth 2002; 89: 863-7

Vitamin D Binding Protein, Total and Free Vitamin D Levels in Different Physiological and Pathophysiological Conditions.

This review focuses on the biologic importance of the vitamin D binding protein (DBP) with emphasis on its regulation of total and free vitamin D metabolite levels in various clinical conditions. Nearly all DBP is produced in the liver, where its regulation is influenced by estrogen, glucocorticoids and inflammatory cytokines but not by vitamin D itself. DBP is the most polymorphic protein known, and different DBP alleles can have substantial impact on its biologic functions. The three most common alleles-Gc1f, Gc1s, Gc2-differ in their affinity with the vitamin D metabolites and have been variably associated with a number of clinical conditions. Although DBP has a number of biologic functions independent of vitamin D, its major biologic function is that of regulating circulating free and total levels of vitamin D metabolites. 25 hydroxyvitamin D (25(OH)D) is the best studied form of vitamin D as it provides the best measure of vitamin D status. In a normal non-pregnant individual, approximately 0.03% of 25(OH)D is free; 85% is bound to DBP, 15% is bound to albumin. The free hormone hypothesis postulates that only free 25(OH)D can enter cells. This hypothesis is supported by the observation that mice lacking DBP, and therefore with essentially undetectable 25(OH)D levels, do not show signs of vitamin D deficiency unless put on a vitamin D deficient diet. Similar observations have recently been described in a family with a DBP mutation. This hypothesis also applies to other protein bound lipophilic hormones including glucocorticoids, sex steroids, and thyroid hormone. However, tissues expressing the megalin/cubilin complex, such as the kidney, have the capability of taking up 25(OH)D still bound to DBP, but most tissues rely on the free level. Attempts to calculate the free level using affinity constants generated in a normal individual along with measurement of DBP and total 25(OH)D have not accurately reflected directly measured free levels in a number of clinical conditions. In this review, we examine the impact of different clinical conditions as well as different DBP alleles on the relationship between total and free 25(OH)D, using only data in which the free 25(OH)D level was directly measured. The major conclusion is that a number of clinical conditions alter this relationship, raising the question whether measuring just total 25(OH)D might be misleading regarding the assessment of vitamin D status, and such assessment might be improved by measuring free 25(OH)D instead of or in addition to total 25(OH)D

Skeletal muscle proteome analysis underpins multifaceted mitochondrial dysfunction in Friedreich’s ataxia

Friedreich’s ataxia (FRDA) is a severe multisystemic disorder caused by a deficiency of the mitochondrial protein frataxin. While some aspects of FRDA pathology are developmental, the causes underlying the steady progression are unclear. The inaccessibility of key affected tissues to sampling is a main hurdle. Skeletal muscle displays a disease phenotype and may be sampled in vivo to address open questions on FRDA pathophysiology. Thus, we performed a quantitative mass spectrometry-based proteomics analysis in gastrocnemius skeletal muscle biopsies from genetically confirmed FRDA patients (n = 5) and controls. Obtained data files were processed using Proteome Discoverer and searched by Sequest HT engine against a UniProt human reference proteome database. Comparing skeletal muscle proteomics profiles between FRDA and controls, we identified 228 significant differentially expressed (DE) proteins, of which 227 were downregulated in FRDA. Principal component analysis showed a clear separation between FRDA and control samples. Interactome analysis revealed clustering of DE proteins in oxidative phosphorylation, ribosomal elements, mitochondrial architecture control, and fission/fusion pathways. DE findings in the muscle-specific proteomics suggested a shift toward fast-twitching glycolytic fibers. Notably, most DE proteins (169/228, 74%) are target of the transcription factor nuclear factor-erythroid 2. Our data corroborate a mitochondrial biosignature of FRDA, which extends beyond a mere oxidative phosphorylation failure. Skeletal muscle proteomics highlighted a derangement of mitochondrial architecture and maintenance pathways and a likely adaptive metabolic shift of contractile proteins. The present findings are relevant for the design of future therapeutic strategies and highlight the value of skeletal muscle-omics as disease state readout in FRDA

Comparison of two dimensional computer codes on the case study of the flow computation of the river Bolska near Gomilsko

V diplomski nalogi so z dvodimenzijskimi programi PCFLOW2D, MIKE 21 ter MIKE Flood izvedeni izračuni z različnimi pretoki za reko Bolsko pri Gomilskem. Na začetku sta opisana vodotoka Bolska in Trnavca ter računsko območje, ki je bilo modelirano. Izveden je bil tudi terenski ogled območja, da se je nato lažje določilo različne koeficiente. V nadaljevanju so opisane teoretične osnove računanja pri uporabljenih programih ter priprava računskega modela za posamezne programe. Na podlagi zahtevnosti vnosa računskega modela ter dobljenih rezultatov so na koncu podane prednosti in slabosti posameznih programov ter analizirane razlike v izračunanih poljih hitrosti in globinah.In this diploma thesis, I carried out calculations of various flows for the Bolska river below the town of Gomilsko by two-dimensional programmes PCFLOW 2D, MIKE 21, and MIKE Flood. In the beginning, the Bolska and Trnavca watershed and modelled calculation domain are described. I also performed site surveys of the area in order to define various coefficients more effectively. Moreover, the background of the calculation of specific programmes and the preparation of the calculation model for individual programme is briefly described. Based on the complexity of the input of the calculation model and the obtained results, I finally set out the advantages and weaknesses of each programme and analysed the differences of calculated velocity fields and water depths

A machine-learned analysis of human gene polymorphisms modulating persisting pain points to major roles of neuroimmune processes

Background Human genetic research has implicated functional variants of more than one hundred genes in the modulation of persisting pain. Artificial intelligence and machine-learning techniques may combine this knowledge with results of genetic research gathered in any context, which permits the identification of the key biological processes involved in chronic sensitization to pain. MethodsResultsBased on published evidence, a set of 110 genes carrying variants reported to be associated with modulation of the clinical phenotype of persisting pain in eight different clinical settings was submitted to unsupervised machine-learning aimed at functional clustering. Subsequently, a mathematically supported subset of genes, comprising those most consistently involved in persisting pain, was analysed by means of computational functional genomics in the Gene Ontology knowledgebase. Clustering of genes with evidence for a modulation of persisting pain elucidated a functionally heterogeneous set. The situation cleared when the focus was narrowed to a genetic modulation consistently observed throughout several clinical settings. On this basis, two groups of biological processes, the immune system and nitric oxide signalling, emerged as major players in sensitization to persisting pain, which is biologically highly plausible and in agreement with other lines of pain research. ConclusionsSignificanceThe present computational functional genomics-based approach provided a computational systems-biology perspective on chronic sensitization to pain. Human genetic control of persisting pain points to the immune system as a source of potential future targets for drugs directed against persisting pain. Contemporary machine-learned methods provide innovative approaches to knowledge discovery from previous evidence. We show that knowledge discovery in genetic databases and contemporary machine-learned techniques can identify relevant biological processes involved in Persitent pain.Peer reviewe

Die Kopplung von Kapillarelektrophorese mit Massenspektrometrie f\ufcr die Analytik von Proteinen und Proteomen

Da die Anforderungen an die Analytik in Bezug auf Sensitivit\ue4t der Messung und Komplexit\ue4t der Analyten st\ue4ndig zunehmen, wird seit Jahren an Alternativen zur Kopplung von Fl\ufcssigchromatographie mit Massenspektrometrie gesucht. Aus diesem Grund wurde von uns ein Interface zur Kopplung von Kapillarelektrophorese mit Massenspektrometrie mitentwickelt sowie die praktische Anwendbarkeit im Labor bewiesen. Die ersten Forschungsergebnisse mit diesem neuen Interface konnten bereits im Jahr 2011 im h\uf6chstdotierten analytischen Journal \u201eAnalytical Chemistry\u201c von uns pr\ue4sentiert werden. Das Interface und die dadurch m\uf6gliche alternative Ger\ue4tekopplung bestechen durch eine au ferordentliche Sensitivit\ue4tssteigerung und zeigen neue M\uf6glichkeiten f\ufcr die Separierung, Quantifizierung und Strukturaufkl\ue4rung nicht nur von kleinen Biomolek\ufclen wie Peptiden sondern auch von Proteinen auf. Dies hat uns auch veranlasst diese Technik zur Untersuchung von Histonen und deren post-translationalen Modifikationen einzusetzen. Die erhaltenen Ergebnisse wurden in \u201eMolecular & Cellular Proteomics\u201c publiziert, wo wir zeigen konnten, dass es mit dieser neuen Technologie m\uf6glich ist Mikro-Sequenzvarianten von Histonen und deren modifizierte Formen hocheffizient zu charakterisieren und zu quantifizieren. Im Zuge dieser Untersuchung wurden auch eine Anzahl neuer Histonmodifizierungen zum ersten Mal beschrieben. Dies ist insofern von Bedeutung, da bekanntlich das Zusammenspiel verschiedener posttranslationaler Modifikationen an Histonen die Genregulation synergetisch oder antagonistisch beeinflussen kann. Die vielseitige Anwendbarkeit dieser Methode erlaubt es somit epigenetische Studien effizienter durchzuf\ufchren, aber auch sensitiv und rasch Modifizierungen von Proteinen im Detail zu analysieren. Der dritte Forschungsartikel beschreibt die Verwendung von Kapillarelektrophorese gekoppelt an die Massenspektrometrie f\ufcr die relative Quantifizierung zweier Hefeproteome. F\ufcr die elektrophoretische Trennung wurde eine neutral beschichtete Kapillare verwendet, jene Beschichtung die sich im zweiten Forschungsteil als optimal f\ufcr komplexe Peptidgemische herausgestellt hatte. Diese Beschichtung erm\uf6glicht die bestm\uf6gliche Auftrennung der Analyten, f\ufchrt aber zu einer Reduktion der Flie fgeschwindigkeiten innerhalb der Kapillare auf ca. 10 nL/min. Das neue Interface hat sich hierbei als \ue4u ferst hilfreich erwiesen, da es selbst unter diesen ultra low-flow Bedingungen einen stabilen Elektrospray erhalten kann. Das Ergebnis war eine deutlich erh\uf6hte Sensitivit\ue4t, die sich in einer massiven Zunahme an identifizierten und quantifizierten Proteinen niederschlug. Die Streuung der quantitativen Messwerte, die zur Berechnung der Proteinverh\ue4ltnisse dienten, war deutlich geringer, was zu einer pr\ue4ziseren Quantifizierung von Proteinen, insbesondere von niedrig abundanten, f\ufchrte. Zudem konnten wir eine Vielzahl an Phosphopeptiden identifizieren und \uc4nderungen im Grad der Phosphorylierung quantifizieren. Insgesamt haben sich das Interface und die Kopplung von Kapillarelektrophorese mit Massenspektrometrie vorz\ufcglich bew\ue4hrt. Das neue System erlaubt eine sensitive Messung und pr\ue4zise Quantifizierung von Analyten und bietet somit eine echte Alternative zu der konventionellen Kopplung von Fl\ufcssigchromatographie mit Massenspektrometrie mit dem Vorteil, dass die Analysenzeiten sehr einfach der Komplexit\ue4t der Probe angepasst werden k\uf6nnen. M\uf6gliche Reste vorhergehender Proben k\uf6nnen binnen weniger Minuten vollst\ue4ndig aus dem System entfernt werden und beeinflussen somit nicht die Ergebnisse sp\ue4tere Analysen. Das System ist vollst\ue4ndig automatisiert und l\ue4sst die sequentielle Analyse mehrerer Proben zu. Zudem ist der Verbrauch an Probe verschwindend gering, da pro Analyse nur wenige Nanoliter injiziert werden, mit dem Nachteil, dass trotz Isotachophorese nur maximal 30% der Kapillare mit Probe gef\ufcllt werden kann, entsprechend einem Volumen von ca. 200 nL. Eine hohe Probenkonzentration ist deshalb Voraussetzung f\ufcr eine erfolgreiche Analyse. Um hier eine Verbesserung zu erreichen, m\ufcssen f\ufcr die Zukunft M\uf6glichkeiten gefunden werden, die \ue4hnlich wie bei der Fl\ufcssigchromatographie, eine online Konzentrierung der Probe erlauben. Nichtsdestotrotz konnten wir zeigen, dass die Verbindung von Kapillarelektrophorese mit Massenspektrometrie eine ideale Erg\ue4nzung zu konventionellen analytischen Methoden ist, die sowohl den Probendurchsatz als auch die Analysenqualit\ue4t eines jeden Proteinanalytik-Labors steigern kann.Traditionally, LC-MS is the analytical technique of choice for proteomic analyses. However, it is well documented that protein and peptide analysis using LC-MS is not without challenges. To overcome these problems, while maintaining or even increasing sensitivity, efforts are going on to couple capillary electrophoresis, as an alternative separation technique, to mass spectrometry. In this context and in the course of this PhD thesis, we investigated in a novel sheathless interface that enables CE-MS coupling via a porous nanospray emitter tip. Our first scientific results applying this novel interface was published in Analytical Chemistry in 2011. In this article, we describe how to optimize the electrospray voltage and the distance between emitter tip and mass analyzer to obtain a stable electrospray at low flow conditions of around 100nL/min. We tested three different capillary coatings for their use in peptide separation of medium complex samples and evaluated the reproducibility of signal intensity and migration time. Using optimized conditions we observed a remarkable high peptide signal intensity caused by extreme narrow peak shapes. This increase and the fact that multiple low molecular mass peptides were lost in LC-MS (they poorly interact with the reversed phase material) resulted in a higher number of peptide identifications when analyzing the same peptide samples with both CE-MS and LC-MS. In the second manuscript published in Molecular & Cellular Proteomics (2013) we describe in detail the capabilities of CE-MS and LC-MS for the analysis of linker and core histones. Histones are small highly basic proteins occurring in all higher eukaryotes in multiple subtypes and are subjected to extensive post-translational modifications that are known to be significant for gene expression and DNA repair. Both the complexity and unique physical and chemical property of this protein family is a challenging task requiring the most advanced separation techniques. Using CE- and LC-MS in a complementary manner, we were able to map plenty of known and also several unknown modification sites when analyzing enzymatically digested histones (bottom-up proteomics). In contrast, when analyzing intact histones (top-down proteomics), CE-MS was able to separate differentially acetylated histone variants to provide a rapid overview of the core histones acetylation and methylation status. In the third part, the ability of CE-MS was investigated to characterize and quantify proteins, peptides and phosphopeptides present in a highly complex sample, the proteome of yeast. Stable isotope labeling by amino acids in cell culture (SILAC) was used for relative quantification of two yeast proteomes. Protein extracts of these two strains were mixed and enzymatically digested. The resulting peptides were pre-separated by RP-HPLC and analyzed by CE-MS. CE-MS analysis was performed using a neutrally coated capillary, a capillary that ensured highest separation efficiency at real low flow conditions (10 nL/min). These real low flow conditions in combination with the novel sheathless interface provided highest sensitivity and yielded a considerably increased number of quantified and identified proteins when compared to a gel based LC-MS approach. The variability in peptide heavy/light ratios was significantly reduced which was especially important for quantification of low abundant proteins and phosphopeptides. The work presented here shows the first quantitative phosphoproteomic approach that combines a SILAC experiment with CE-MS separation. To conclude, coupling of capillary electrophoresis and mass spectrometry via the novel sheathless interface has proven to be a valuable tool in proteomics. CE-MS allows for high sensitive analysis and precise quantification of analytes. Analysis time can be adapted (via different capillary coatings and background electrolytes) according to the complexity of the sample or to the respective analytical question. Traces of sample carryover can be washed out in just a few minutes; the system is fully automated so that multiple samples can be analyzed sequentially; and the sample consume per analysis is in the low nano-liter range. However, the low sample consume can also be considered as a drawback. Despite the fact that the sample load can be increased by applying isotachophoresis, the analyte concentration should be rather high to ensure successful CE-MS analysis. Therefore, the implementation of an online sample pre-concentration technique, such as applied in LC-MS, will be a prerequisite for the widespread application of CE-MS. Nevertheless, CE-MS has proven to complement standard analytical techniques in a perfect manner and allows to increase sample throughput and analysis sensitivity in proteomics laboratoriesby Klaus FaserlAbweichender Titel laut cbersetzung der Verfasserin/des VerfassersEnth. u.a. 3 Ver\uf6ff. d. Verf. aus den Jahren 2011 - 2013 . - Zsfassung in dt. SpracheInnsbruck, Med. Univ., Diss., 201