Investigation of protein-RNA interactions by UV cross-linking and mass spectrometry: methodological improvements toward in vivo applications

Protein–RNA complexes play key roles in a variety of cellular functions such as gene expression and its regulation. One frequently applied method for the investigation of direct protein–RNA interactions is UV induced cross-linking. Mass spectrometry (MS) is increasingly utilized for the identification of proteins and peptides directly interacting with RNA. The major challenges for this application are the generally low yield of the UV induced cross-linking reaction and the lack of suitable tools for MS data analysis. In this work, both issues are adressed. A novel cross-linking pathway involving net loss of H2S from the photoreactive base-analogue 4-thio-uracil was identified, and a novel approach for automated identification of cross-linked peptides from mass spectrometry data was developed

Rapid discrimination and categorisation of briefly flashed stimuli in the pigeon (Columba livia)

Zweifellos ist die Fähigkeit, Teile des visuellen Umfelds möglichst schnell zu analysieren, für Tiere von fundamentaler Bedeutung, da ihr Überleben oft von der Geschwindigkeit abhängt, mit der sie Feinde, Beute, oder andere Objekte erkennen. Diese Studie untersuchte daher die Bedingungen, unter denen rasche visuelle Bildverarbeitung auftritt. In der Literatur finden sich zahlreiche Beispiele für die außergewöhnlichen Diskriminations- und Kategorisierungsfähigkeiten von Tauben (Columba livia). Allerdings ist nach wie vor ungeklärt, wie schnell diese Vögel Bildinformationen extrahieren können, die für eine korrekte Klassifizierung notwendig sind. Ziel der vorliegenden Studie war es daher, die Fähigkeit von Tauben, verschiedene visuelle Klassifikationsaufgaben zu lösen, unter zeitlicher Beschränkung zu testen. Mittels einer modifizierten „Go/No go“-Prozedur wurden verschiedene Arten von Bildern — einfache geometrische Formen (Experiment 1) sowie Fotografien von natürlichen Objekten (Experiment 2) — für Zeitspannen zwischen 100 und 10000 ms präsentiert. Aufgrund dieser zeitlichen Begrenzung war es den Tauben nicht möglich, die Stimuli im Detail zu erforschen. Stattdessen waren sie gezwungen, ihre Kategorieentscheidungen in erster Linie auf der Basis sehr früher Verarbeitungsmechanismen des visuellen Systems zu treffen. Die Tauben konnten die meisten Aufgaben auch bei sehr stark beschränkten Präsentationszeiten lösen. Dennoch wirkten sich verlängerte Präsentationszeiten positiv auf die Diskriminationsleistung aus, wobei das Ausmaß dieses Effekts stark von der Komplexität der zu bewertenden Bilder abhing. Experiment 1 zeigte deutlich, dass die Tauben in den meisten Fällen einfache geometrische Formen selbst bei sehr kurzer Präsentation (100 ms) von einander unterscheiden konnten. Des Weiteren konnte gezeigt werden, dass die Vögel Stimuli, die sich nur in der Form unterschieden, schlechter klassifizieren konnten als Stimuli, die unterschiedliche Farben aufwiesen. Dies wurde durch Unterschiede zwischen den beiden Aufgaben hinsichtlich Lerngeschwindigkeit und absoluter Diskriminationsleistung deutlich, die durch besonders kurze Präsentationszeiten noch weiter verstärkt wurden. Experiment 2 zeigte ein etwas anderes Ergebnis. Zwar konnte das Versuchstier Bilder mit und ohne Menschen bei einer Präsentationszeit von 10000 ms korrekt klassifizieren, doch eine schrittweise Verkürzung der Präsentationszeit beeinträchtigte die Leistung in einem viel stärkeren Ausmaß als dies in Experiment 1 der Fall war. Präsentationszeiten von 250 ms und weniger führten sogar zu einem kompletten Versagen. Die Präsentation neuer Bilder zeigte, dass auch unbekannte Klassenvertreter richtig eingeordnet werden konnten, aber auch diese Fähigkeit war stark vom Zeitfaktor abhängig. Die Grenze, ab der korrekte Klassifizierung möglich war, lag vermutlich irgendwo im Bereich zwischen zwei und sechs Sekunden. Zusammenfassend zeigen die Ergebnisse, dass Tauben mehr Verarbeitungszeit für die Klassifizierung natürlicher Stimuli benötigen, als für das richtige Zuordnen einfacher geometrischer Formen.Animals’ ability to analyse and categorise the visual environment as fast as possible is fundamental, as survival will often depend on the speed with which predators, food, or other relevant objects can be recognised. This study examines the conditions under which rapid visual processing occurs. The outstanding visual discrimination and categorisation abilities of pigeons (Columba livia) are well-documented in the literature. However, it is still unclear how quickly these birds may extract the pictorial information necessary to allow for correct classification. In the present study, the pigeons’ ability to solve a series of visual classification tasks with severe temporal restrictions was tested. With a modified go/no go- procedure, different types of images — simple geometrical stimuli (Experiment 1) and natural images that either showed or lacked human figures (Experiment 2) — were presented for time spans ranging from 100 msecs to 1oooo msecs. Due to this time pressure the pigeons could not extensively explore the stimuli, but were forced to base their category decisions on the very first rapid pass through the visual system. In most of the tasks the birds succeeded even with very brief presentation times. Nevertheless, discrimination performance correlated positively with presentation time, with the extent of this effect strongly depending on stimulus complexity. Experiment 1 provided evidence that the pigeons were, for the most part, able to correctly classify simple geometric stimuli even when flashed very briefly (100 msecs). Furthermore, discrimination of stimuli differing only in shape was found to be more difficult than that of stimuli differing only in colour. This was reflected by differences between the two tasks regarding acquisition speed and accuracy. These differences were further enhanced by very short stimulus durations. Results were somewhat different in Experiment 2. The subject was well able to classify images with and without humans when shown for 10000 msec. Stepwise reduction of presentation time had, however, considerably stronger detrimental effects than was the case in Experiment 1, and reduction to 250 msecs and less even led to complete failure. The presentation of novel stimuli provided evidence that also unfamiliar instances of the classes could be categorised, but also this ability was crucially dependent on the time factor: The limit of minimum presentation time at which correct classification was still possible appeared to lie somewhere between two and six seconds. The results suggest that classification of natural stimuli requires longer presentation times and is thus more prone to failure with very short presentations than is the case with discriminations of simple geometric stimuli

Reliability Evaluation of Drivetrains: Challenges for Off‐ Highway Machines

Downtime of mobile machinery used in fields like construction, earthmoving or mining usually leads to an instant halt of an entire process and can even endanger entire operations. To meet the customer’s requirement for high availabilities of their equipment, safeguarding the reliability of overall systems and components is necessary. Since life expectancy of systems and its components strongly depends on the experienced load history, this information needs to be available as accurately as possible to allow reliable lifetime calculation results. Due to the wide range of machines and applications of off‐highway machines, determining representative loads is especially challenging. The challenges, in determining both load cycles for the entire system and local component loads, are discussed in this work, along with approaches to face them. Additionally, a method is described, which allows users to quantitatively calculate life expectancy of technical systems in both the concept phase and the later stages of the product life cycle. In the end, two examples are presented in which exemplary challenges are faced

Rapid Normalization of Epidermal Integrin Expression After Allografting of Human Keratinocytes

Allogeneic keratinocyte grafts have beneficial effects on skin wounds, but the underlying interactions between graft and woundbed remain to be explored in detail. The epidermal integrins play a pivotal role in mediating cell-to-cell and cell-to-matrix interactions. In unwounded epidermis, α2β1-, α3β1-, α6β4-, α5β1-, and αvβ5-integrins are confined to basal cells. During healing of incisional wounds, these integrins are also expressed in suprabasal cells, where they remain detectable even after epidermal integrity is fully reestablished. We examined the integrin subunits α2, α3, α6, α5, and αv in partial thickness burn wounds grafted with allogeneic keratinocytes and asked whether the effect of allogeneic keratinocyte grafts, i.e., fast reepithelialization, is reflected by an accelerated reversion to a normal integrin pattern. Biopsies were taken after wound debridement before grafting and 10 d after transplantation. After 10 d, a stratified epidermis had developed in all cases and integrins were mainly restricted to the basal cell layer of the neo-epidermis. α2-, α3-, α6-, and αv-subunits were present at basal and/or lateral cell borders, duplicating the integrin pattern in normal epidermis. The findings indicate that grafting accelerates the shift of the epidermis from an inflammatory to a regenerative state, as reflected by the reversion of the integrin pattern from a “spread-and-migrate” to the “steady-state” phenotype

Inorganic pyrophosphatase in uncultivable hemotrophic mycoplasmas: identification and properties of the enzyme from Mycoplasma suis

BACKGROUND: Mycoplasma suis belongs to a group of highly specialized hemotrophic bacteria that attach to the surface of host erythrocytes. Hemotrophic mycoplasmas are uncultivable and the genomes are not sequenced so far. Therefore, there is a need for the clarification of essential metabolic pathways which could be crucial barriers for the establishment of an in vitro cultivation system for these veterinary significant bacteria.Inorganic pyrophosphatases (PPase) are important enzymes that catalyze the hydrolysis of inorganic pyrophosphate PPi to inorganic phosphate Pi. PPases are essential and ubiquitous metal-dependent enzymes providing a thermodynamic pull for many biosynthetic reactions. Here, we describe the identification, recombinant production and characterization of the soluble (s)PPase of Mycoplasma suis. RESULTS: Screening of genomic M. suis libraries was used to identify a gene encoding the M. suis inorganic pyrophosphatase (sPPase). The M. suis sPPase consists of 164 amino acids with a molecular mass of 20 kDa. The highest identity of 63.7% was found to the M. penetrans sPPase. The typical 13 active site residues as well as the cation binding signature could be also identified in the M. suis sPPase. The activity of the M. suis enzyme was strongly dependent on Mg2+ and significantly lower in the presence of Mn2+ and Zn2+. Addition of Ca2+ and EDTA inhibited the M. suis sPPase activity. These characteristics confirmed the affiliation of the M. suis PPase to family I soluble PPases. The highest activity was determined at pH 9.0. In M. suis the sPPase builds tetramers of 80 kDa which were detected by convalescent sera from experimentally M. suis infected pigs. CONCLUSION: The identification and characterization of the sPPase of M. suis is an additional step towards the clarification of the metabolism of hemotrophic mycoplasmas and, thus, important for the establishment of an in vitro cultivation system. As an antigenic and conserved protein the M. suis sPPase could in future be further analyzed as a diagnostic antigen

a randomized controlled trial

Diurnal carbohydrate and fat distribution modulates glycaemic control in rodents. In humans, the optimal timing of both macronutrients and its effects on glycaemic control after prolonged consumption are not studied in detail. In this cross-over trial, 29 non-obese men were randomized to two four-week diets: (1) carbohydrate-rich meals until 13.30 and fat-rich meals between 16.30 and 22.00 (HC/HF) versus (2) inverse sequence of meals (HF/HC). After each trial period two meal tolerance tests were performed, at 09.00 and 15.40, respectively, according to the previous intervention. On the HF/HC diet, whole-day glucose level was increased by 7.9% (p = 0.026) in subjects with impaired fasting glucose and/or impaired glucose tolerance (IFG/IGT, n = 11), and GLP-1 by 10.2% (p = 0.041) in normal glucose-tolerant subjects (NGT, n = 18). Diet effects on fasting GLP-1 (p = 0.009) and PYY (p = 0.034) levels were observed in IFG/IGT, but not in NGT. Afternoon decline of glucose tolerance was more pronounced in IFG/IGT and associated with a stronger decrease of postprandial GLP-1 and PYY levels, but not with changes of cortisol rhythm. In conclusion, the HF/HC diet shows an unfavourable effect on glycaemic control in IFG/IGT, but not in NGT subjects. Consequently, large, carbohydrate-rich dinners should be avoided, primarily by subjects with impaired glucose metabolism

Reactive oxygen species produced by myeloid cells in psoriasis as a potential biofactor contributing to the development of vascular inflammation.

Psoriasis is an immune-mediated inflammatory skin disease driven by interleukin-17A (IL-17A) and associated with cardiovascular dysfunction. We used a severe psoriasis mouse model of keratinocyte IL-17A overexpression (K14-IL-17Aind/+ , IL-17Aind/+ control mice) to investigate the activity of neutrophils and a potential cellular interconnection between skin and vasculature. Levels of dermal reactive oxygen species (ROS) and their release by neutrophils were measured by lucigenin-/luminol-based assays, respectively. Quantitative RT-PCR determined neutrophilic activity and inflammation-related markers in skin and aorta. To track skin-derived immune cells, we used PhAM-K14-IL-17Aind/+ mice allowing us to mark all cells in the skin by photoconversion of a fluorescent protein to analyze their migration into spleen, aorta, and lymph nodes by flow cytometry. Compared to controls, K14-IL-17Aind/+ mice exhibited elevated ROS levels in the skin and a higher neutrophilic oxidative burst accompanied by the upregulation of several activation markers. In line with these results psoriatic mice displayed elevated expression of genes involved in neutrophil migration (e.g., Cxcl2 and S100a9) in skin and aorta. However, no direct immune cell migration from the psoriatic skin into the aortic vessel wall was observed. Neutrophils of psoriatic mice showed an activated phenotype, but no direct cellular migration from the skin to the vasculature was observed. This suggests that highly active vasculature-invading neutrophils must originate directly from the bone marrow. Hence, the skin-vasculature crosstalk in psoriasis is most likely based on the systemic effects of the autoimmune skin disease, emphasizing the importance of a systemic therapeutic approach for psoriasis patients