The integrity of the G2421-C2395 base pair in the ribosomal E-site is crucial for protein synthesis.

During the elongation cycle of protein biosynthesis, tRNAs traverse through the ribosome by consecutive binding to the 3 ribosomal binding sites (A-, P-, and E- sites). While the ribosomal A- and P-sites have been functionally well characterized in the past, the contribution of the E-site to protein biosynthesis is still poorly understood in molecular terms. Previous studies suggested an important functional interaction of the terminal residue A76 of E-tRNA with the nucleobase of the universally conserved 23S rRNA residue C2394. Using an atomic mutagenesis approach to introduce non-natural nucleoside analogs into the 23S rRNA, we could show that removal of the nucleobase or the ribose 2'-OH at C2394 had no effect on protein synthesis. On the other hand, our data disclose the importance of the highly conserved E-site base pair G2421-C2395 for effective translation. Ribosomes with a disrupted G2421-C2395 base pair are defective in tRNA binding to the E-site. This results in an impaired translation of genuine mRNAs, while homo-polymeric templates are not affected. Cumulatively our data emphasize the importance of E-site tRNA occupancy and in particular the intactness of the 23S rRNA base pair G2421-C2395 for productive protein biosynthesis.

Light-quark and gluon jet discrimination in pp collisions at √s=7 TeV with the ATLAS detector

A likelihood-based discriminant for the identification of quark- and gluon-initiated jets is built and validated using 4.7 fb−1 √ of proton–proton collision data at √s = 7 TeV collected with the ATLAS detector at the LHC. Data sampleswith enriched quark or gluon content are used in the construction and validation of templates of jet properties that are the input to the likelihood-based discriminant. The discriminating power of the jet tagger is established in both data and Monte Carlo samples within a systematic uncertainty of ≈ 10–20 %. In data, light-quark jets can be tagged with an efficiency of ≈ 50% while achieving a gluon-jet mis-tag rate of ≈ 25% in a pT range between 40 GeV and 360 GeV for jets in the acceptance of the tracker. The rejection of gluon-jets found in the data is significantly below what is attainable using a Pythia 6Monte Carlo simulation, where gluon-jet mis-tag rates of 10% can be reached for a 50% selection efficiency of light-quark jets using the same jet properties.

Einfluss des Trampolinsprungtuchs und der Sprunghöhe auf die Vertikalbeschleunigung an der Brustwirbelsäule

Die 2011 für das Trampolinturnen eingeführte, instrumentierte Bewertung der Sprunghöhe hatte die Entwicklung wurfstärkerer Sprungtücher und damit eine Zunahme der Sprunghöhen zur Folge. Inwieweit dabei die Beanspruchung des Bewegungsapparates gestiegen ist, wird durch vergleichende biomechanische Analysen verschiedener Sprünge von Athlet/innen des deutschen Nationalkaders auf dem alten 6x4- und dem neuen 4x4 -Sprungtuch untersucht. Erste Analysen zeigten, dass sich die Zunahme der Sprunghöhe auf dem 4x4-Tuch notwendigerweise in einem größeren Kraftstoß begründet, dieser aber nicht durch eine längere Tuchkontaktzeit, sondern durch eine höhere Kraft zustande kommt (Kredel, Eisele, Schweizer, Kuhn & Riehle, 2014). In der Folge soll nun der Einfluss dieser Kraftzunahme auf Bewegungsparameter und Beanspruchung untersucht werden. Kinematische Merkmale von Körper- und Wirbelsäulensegmenten wurden mit einem Vicon-T40s-System aufgezeichnet, während die Bodenreaktionskraftverläufe mit vier Kis t-ler-Kraftmessplatten erfasst wurden. Eine individualisierte, inversdynamische Modellierung der Tuchkontaktphasen soll Aufschluss über die Beanspruchungsunterschiede relevanter Gelenksstrukturen (Sprung-, Knie-, Hüftgelenk, LWS, BWS) geben. Zur Modellvalidierung dient die mittels Beschleunigungssensor erfasste Brustwirbelsäulenkinematik, deren Ausprägung in Bezug zur Sprunghöhe Gegenstand des aktuellen Untersuchungsteils war. Analysen von 102 Standsprüngen zeigen Zusammenhänge zwischen der Sprunghöhe und sowohl der mittleren (4x4: r(44)=.90, p<.001; 6x4: r(54)=.83, p<.001), als auch der maximalen (4x4: r(44)=.65, p<.001; 6x4: r(54)=.80, p<.001) Vertikalbeschleunigung der Brus twirbelsäule während des Tuchkontakts. Sind die Beschleunigungswerte – und damit die Belastungen – im mittleren Sprunghöhenbereich in ähnlichen Größenordnungen, ist festzuhalten, dass die durch das 4x4-Tuch erreichbaren größeren Sprunghöhen nicht nur einen weiteren Anstieg der mittleren, sondern auch eine weitere Erhöhung der maximalen Vertikalbeschleunigung zur Folge haben (Maximum 4x4: 151.4 m/s2 vs. 6x4: 139.6 m/s2) und somit die Maximalbelastung der Athlet/innen zunimmt. Aktuell wird dieser Befund bei den komplexeren Sprungvarianten verifiziert und die Gelenksbeansp.ruchung modelliert

MorphoGraphX: A platform for quantifying morphogenesis in 4D

Morphogenesis emerges from complex multiscale interactions between genetic and mechanical processes. To understand these processes, the evolution of cell shape, proliferation and gene expression must be quantified. This quantification is usually performed either in full 3D, which is computationally expensive and technically challenging, or on 2D planar projections, which introduces geometrical artifacts on highly curved organs. Here we present MorphoGraphX (www.MorphoGraphX.org), a software that bridges this gap by working directly with curved surface images extracted from 3D data. In addition to traditional 3D image analysis, we have developed algorithms to operate on curved surfaces, such as cell segmentation, lineage tracking and fluorescence signal quantification. The software’s modular design makes it easy to include existing libraries, or to implement new algorithms. Cell geometries extracted with MorphoGraphX can be exported and used as templates for simulation models, providing a powerful platform to investigate the interactions between shape, genes and growth.DOI: http://dx.doi.org/10.7554/eLife.05864.001Author keywordsResearch organism

Privacy Preserving Probabilistic Record Linkage (P3RL): a novel method for linking existing health-related data and maintaining participant confidentiality.

BACKGROUND Record linkage of existing individual health care data is an efficient way to answer important epidemiological research questions. Reuse of individual health-related data faces several problems: Either a unique personal identifier, like social security number, is not available or non-unique person identifiable information, like names, are privacy protected and cannot be accessed. A solution to protect privacy in probabilistic record linkages is to encrypt these sensitive information. Unfortunately, encrypted hash codes of two names differ completely if the plain names differ only by a single character. Therefore, standard encryption methods cannot be applied. To overcome these challenges, we developed the Privacy Preserving Probabilistic Record Linkage (P3RL) method. METHODS In this Privacy Preserving Probabilistic Record Linkage method we apply a three-party protocol, with two sites collecting individual data and an independent trusted linkage center as the third partner. Our method consists of three main steps: pre-processing, encryption and probabilistic record linkage. Data pre-processing and encryption are done at the sites by local personnel. To guarantee similar quality and format of variables and identical encryption procedure at each site, the linkage center generates semi-automated pre-processing and encryption templates. To retrieve information (i.e. data structure) for the creation of templates without ever accessing plain person identifiable information, we introduced a novel method of data masking. Sensitive string variables are encrypted using Bloom filters, which enables calculation of similarity coefficients. For date variables, we developed special encryption procedures to handle the most common date errors. The linkage center performs probabilistic record linkage with encrypted person identifiable information and plain non-sensitive variables. RESULTS In this paper we describe step by step how to link existing health-related data using encryption methods to preserve privacy of persons in the study. CONCLUSION Privacy Preserving Probabilistic Record linkage expands record linkage facilities in settings where a unique identifier is unavailable and/or regulations restrict access to the non-unique person identifiable information needed to link existing health-related data sets. Automated pre-processing and encryption fully protect sensitive information ensuring participant confidentiality. This method is suitable not just for epidemiological research but also for any setting with similar challenges.

A pilot test of the new Swiss regulatory procedure for categorizing clinical trials by risk: A randomized controlled trial

BACKGROUND/AIMS Several countries are working to adapt clinical trial regulations to align the approval process to the level of risk for trial participants. The optimal framework to categorize clinical trials according to risk remains unclear, however. Switzerland is the first European country to adopt a risk-based categorization procedure in January 2014. We assessed how accurately and consistently clinical trials are categorized using two different approaches: an approach using criteria set forth in the new law (concept) or an intuitive approach (ad hoc). METHODS This was a randomized controlled trial with a method-comparison study nested in each arm. We used clinical trial protocols from eight Swiss ethics committees approved between 2010 and 2011. Protocols were randomly assigned to be categorized in one of three risk categories using the concept or the ad hoc approach. Each protocol was independently categorized by the trial's sponsor, a group of experts and the approving ethics committee. The primary outcome was the difference in categorization agreement between the expert group and sponsors across arms. Linear weighted kappa was used to quantify agreements, with the difference between kappas being the primary effect measure. RESULTS We included 142 of 231 protocols in the final analysis (concept = 78; ad hoc = 64). Raw agreement between the expert group and sponsors was 0.74 in the concept and 0.78 in the ad hoc arm. Chance-corrected agreement was higher in the ad hoc (kappa: 0.34 (95% confidence interval = 0.10-0.58)) than in the concept arm (0.27 (0.06-0.50)), but the difference was not significant (p = 0.67). LIMITATIONS The main limitation was the large number of protocols excluded from the analysis mostly because they did not fit with the clinical trial definition of the new law. CONCLUSION A structured risk categorization approach was not better than an ad hoc approach. Laws introducing risk-based approaches should provide guidelines, examples and templates to ensure correct application.

Inverse fusion PCR cloning.

Inverse fusion PCR cloning (IFPC) is an easy, PCR based three-step cloning method that allows the seamless and directional insertion of PCR products into virtually all plasmids, this with a free choice of the insertion site. The PCR-derived inserts contain a vector-complementary 5'-end that allows a fusion with the vector by an overlap extension PCR, and the resulting amplified insert-vector fusions are then circularized by ligation prior transformation. A minimal amount of starting material is needed and experimental steps are reduced. Untreated circular plasmid, or alternatively bacteria containing the plasmid, can be used as templates for the insertion, and clean-up of the insert fragment is not urgently required. The whole cloning procedure can be performed within a minimal hands-on time and results in the generation of hundreds to ten-thousands of positive colonies, with a minimal background.

Mechanistic insights into tRNA translocation on the ribosome

The ribosome is a highly conserved cellular complex and constitutes the center of protein biosynthesis. As the ribosome consists to about 2/3 of ribosomal RNA (rRNA), the rRNA is involved in most steps of translation. In order to investigate the role of some defined rRNA residues in different aspects of translation we use the atomic mutagenesis approach. This method allows the site-specific incorporation of unnatural nucleosides into the rRNA in the context of the complete 70S from Thermus aquaticus, and thereby exceeds the possibilities of conventional mutagenesis. We first studied ribosome-stimulated EF-G GTP hydrolysis. Here, we could show that the non-bridging phosphate oxygen of A2662, which is part of the Sarcin-Ricin-Loop, is required for EF-G GTPase activation by the ribosome. EF-G GTPase is a crucial step for tRNA translocation from the A- to the P-site, and from the P- to the E-site, respectively. We furthermore used the atomic mutagenesis approach to more precisely characterize the 23S rRNA functional groups involved in E-site tRNA binding. While the ribosomal A- and P-sites have been functionally well characterized in the past, the contribution of the E-site to protein biosynthesis is still poorly understood in molecular terms. Our data disclose the importance of the highly conserved E-site base pair G2421-C2395 for effective translation. Ribosomes with a disrupted G2421-C2395 base pair are defective in tRNA binding to the E-site. This results in an impaired translation of genuine mRNAs, while homo-polymeric templates are not affected. Cumulatively our data emphasize the importance of E-site tRNA occupancy and in particular the intactness of the 23S rRNA base pair G2421-C2395 for productive protein biosynthesis.

Preoperative Planning of Periacetabular Osteotomy (PAO)

Pelvic osteotomies improve containment of the femoral head in cases of developmental dysplasia of the hip or in femoroacetabular impingement due to acetabular retroversion. In the evolution of osteotomies, the Ganz Periacetabular Osteotomy (PAO) is among the complex reorientation osteotomies and allows for complete mobilization of the acetabulum without compromising the integrity of the pelvic ring. For the complex reorientation osteotomies, preoperative planning of the required acetabular correction is an important step, due to the need to comprehend the three-dimensional (3D) relationship between acetabulum and femur. Traditionally, planning was performed using conventional radiographs in different projections, reducing the 3D problem to a two-dimensional one. Known disturbance variables, mainly tilt and rotation of the pelvis make assessment by these means approximate at the most. The advent of modern enhanced computation skills and new imaging techniques gave room for more sophisticated means of preoperative planning. Apart from analysis of acetabular geometry on conventional x-rays by sophisticated software applications, more accurate assessment of coverage and congruency and thus amount of correction necessary can be performed on multiplanar CT images. With further evolution of computer-assisted orthopaedic surgery, especially the ability to generate 3D models from the CT data, examiners were enabled to simulate the in vivo situation in a virtual in vitro setting. Based on this ability, different techniques have been described. They basically all employ virtual definition of an acetabular fragment. Subsequently reorientation can be simulated using either 3D calculation of standard parameters of femoroacetabular morphology, or joint contact pressures, or a combination of both. Other techniques employ patient specific implants, templates or cutting guides to achieve the goal of safe periacetabular osteotomies. This chapter will give an overview of the available techniques for planning of periacetabular osteotomy.

Posterior Vertical Bite Reconstructions of Erosively Worn Dentitions and the "Stamp Technique" - A Case Series with a Mean Observation Time of 40 Months

PURPOSE In the present case series, the authors report on seven cases of erosively worn dentitions (98 posterior teeth) which were treated with direct resin composite. MATERIALS AND METHODS In all cases, both arches were restored by using the so-called stamp technique. All patients were treated with standardized materials and protocols. Prior to treatment, a waxup was made on die-cast models to build up the loss of occlusion as well as ensure the optimal future anatomy and function of the eroded teeth to be restored. During treatment, teeth were restored by using templates of silicone (ie, two "stamps," one on the vestibular, one on the oral aspect of each tooth), which were filled with resin composite in order to transfer the planned, future restoration (ie, in the shape of the waxup) from the extra- to the intraoral situation. Baseline examinations were performed in all patients after treatment, and photographs as well as radiographs were taken. To evaluate the outcome, the modified United States Public Health Service criteria (USPHS) were used. RESULTS The patients were re-assessed after a mean observation time of 40 months (40.8 ± 7.2 months). The overall outcome of the restorations was good, and almost exclusively "Alpha" scores were given. Only the marginal integrity and the anatomical form received a "Charlie" score (10.2%) in two cases. CONCLUSION Direct resin composite restorations made with the stamp technique are a valuable treatment option for restoring erosively worn dentitions.

Histone-specific RNA 3' processing in nuclear extracts from mammalian cells.

The mature 3' ends of histone mRNAs are formed by endonucleolytic cleavage of longer precursor transcripts. This process occurs in the nucleus and can be regarded as the equivalent of the polyadenylation reaction involved in 3′-end-generation of all other mRNAs. A sea urchin H3 gene that failed to be properly processed in the Xenopus oocyte system proved particularly useful, because it allowed the identification of a processing component from sea urchins by a complementation assay. Nuclear extracts prepared from cells under various growth conditions have helped to reveal proliferation-dependent changes in the efficiency of histone RNA 3′ processing. RNA substrates for in vitro processing are best prepared by runoff transcription of specific DNA templates with bacterial or phage RNA polymerases. For this purpose, a restriction fragment containing the 3′-terminal region of a histone gene and including the conserved palindrome and spacer motifs is cloned into a polylinker sequence downstream of a strong promoter.

Characterization of template switching and recombination during Moloney murine leukemia virus replication

Retroviruses uniquely co-package two copies of their genomic RNA within each virion. The two copies are used as templates for synthesis of the proviral DNA during the process of reverse transcription. Two template switches are required to complete retroviral DNA synthesis by the retroviral enzyme, reverse transcriptase. With two RNA genomes present in the virion, reverse transcriptase can make template switches utilizing only one of the RNA templates (intramolecular) or utilizing both RNA templates (intermolecular) during the process of reverse transcription. The results presented in this study show that during a single cycle of Moloney murine leukemia virus replication, both nonrecombinant and recombinant proviruses predominantly underwent intramolecular minus- and plus-strand transfers during the process of reverse transcription. This is the first study to examine the nature of the required template switches occurring during MLV replication and these results support the previous findings for SNV, and the hypothesis that the required template switches are ordered events. This study also determined rates for deletion and a rate of recombination for a single cycle of MLV replication. The rates reported here are comparable to the rates previously reported for both SNV and MLV. ^

High quality InAlN single layers lattice-matched to GaN grown by molecular beam epitaxy

We report on properties of high quality ~60 nm thick InAlN layers nearly in-plane lattice-matched to GaN, grown on c-plane GaN-on-sapphire templates by plasma-assisted molecular beam epitaxy. Excellent crystalline quality and low surface roughness are confirmed by X-ray diffraction, transmission electron microscopy, and atomic force microscopy. High annular dark field observations reveal a periodic in-plane indium content variation (8 nm period), whereas optical measurements evidence certain residual absorption below the band-gap. The indium fluctuation is estimated to be +/- 1.2% around the nominal 17% indium content via plasmon energy oscillations assessed by electron energy loss spectroscopy with sub-nanometric spatial resolution.

Selective area growth of a- and c-plane GaN nanocolumns by molecular beam epitaxy using colloidal nanolithography

Selective area growth of a-plane GaN nanocolumns by molecular beam epitaxy was performed for the first time on a-plane GaN templates. Ti masks with 150 nm diameter nanoholes were fabricated by colloidal lithography, an easy, fast and cheap process capable to handle large areas. Even though colloidal lithography does not provide a perfect geometrical arrangement like e-beam lithography, it produces a very homogeneous mask in terms of nanohole diameter and density, and is used here for the first time for the selective area growth of GaN. Selective area growth of a-plane GaN nanocolumns is compared, in terms of anisotropic lateral and vertical growth rates, with GaN nanocolumns grown selectively on the c-plane

One dimensional exciton luminescence induced by extended defects in nonpolar (Al,Ga)N/GaN quantum wells

In this study, we present the optical properties of nonpolar GaN/(Al,Ga)N single quantum wells (QWs) grown on either a- or m-plane GaN templates for Al contents set below 15%. In order to reduce the density of extended defects, the templates have been processed using the epitaxial lateral overgrowth technique. As expected for polarization-free heterostructures, the larger the QW width for a given Al content, the narrower the QW emission line. In structures with an Al content set to 5 or 10%, we also observe emission from excitons bound to the intersection of I1-type basal plane stacking faults (BSFs) with the QW. Similarly to what is seen in bulk material, the temperature dependence of BSF-bound QW exciton luminescence reveals intra-BSF localization. A qualitative model evidences the large spatial extension of the wavefunction of these BSF-bound QW excitons, making them extremely sensitive to potential fluctuations located in and away from BSF. Finally, polarization-dependent measurements show a strong emission anisotropy for BSF-bound QW excitons, which is related to their one-dimensional character and that confirms that the intersection between a BSF and a GaN/(Al,Ga)N QW can be described as a quantum wire.