A Modified RMCE-Compatible Rosa26 Locus for the Expression of Transgenes from Exogenous Promoters

Generation of gain-of-function transgenic mice by targeting the Rosa26 locus has been established as an alternative to classical transgenic mice produced by pronuclear microinjection. However, targeting transgenes to the endogenous Rosa26 promoter results in moderate ubiquitous expression and is not suitable for high expression levels. Therefore, we now generated a modified Rosa26 (modRosa26) locus that combines efficient targeted transgenesis using recombinase-mediated cassette exchange (RMCE) by Flipase (Flp-RMCE) or Cre recombinase (Cre-RMCE) with transgene expression from exogenous promoters. We silenced the endogenous Rosa26 promoter and characterized several ubiquitous (pCAG, EF1α and CMV) and tissue-specific (VeCad, αSMA) promoters in the modRosa26 locus in vivo. We demonstrate that the ubiquitous pCAG promoter in the modRosa26 locus now offers high transgene expression. While tissue-specific promoters were all active in their cognate tissues they additionally led to rare ectopic expression. To achieve high expression levels in a tissue-specific manner, we therefore combined Flp-RMCE for rapid ES cell targeting, the pCAG promoter for high transgene levels and Cre/LoxP conditional transgene activation using well-characterized Cre lines. Using this approach we generated a Cre/LoxP-inducible reporter mouse line with high EGFP expression levels that enables cell tracing in live cells. A second reporter line expressing luciferase permits efficient monitoring of Cre activity in live animals. Thus, targeting the modRosa26 locus by RMCE minimizes the effort required to target ES cells and generates a tool for the use exogenous promoters in combination with single-copy transgenes for predictable expression in mice

The Influence of Radiograph Obliquity on Böhler's and Gissane's Angles in Calcanei

In calcaneal fractures, Böhler's and Gissane's angles are considered important parameters to guide treatment strategy and provide prognostic information during follow-up visits. Therefore, lateral radiographs have to be accurate. The aim of this study was to evaluate the effect of craniocaudal and posteroanterior angular variations (i.e., simulate lower leg malposition) from the true lateral radiograph on Böhler's and Gissane's angles. In this radioanatomical study, 15 embalmed, skeletally mature, human anatomic lower limb specimens were used. Using predefined criteria, a true lateral radiograph (i.e., 0° angular variation) was obtained. Angular variations from this true lateral radiograph were made from –30° to +30° deviation in the craniocaudal and posteroanterior direction at 5° intervals. Böhler's and Gissane angles were independently assessed by 2 experienced trauma surgeons. Böhler's angle decreased with increasing caudal angular variations (maximum –4.3° deviation at –30°). With increasing of the posterior angular variations, Böhler's angle increased (maximum 5.0° deviation at +30°) from the true lateral radiograph, but all deviations were within the measurement error. The deviation of the angle of Gissane was most pronounced in the cranial direction, with the mean angle decreasing by

Genome-wide association analyses identify new Brugada syndrome risk loci and highlight a new mechanism of sodium channel regulation in disease susceptibility

Brugada syndrome (BrS) is a cardiac arrhythmia disorder associated with sudden death in young adults. With the exception of SCN5A, encoding the cardiac sodium channel NaV1.5, susceptibility genes remain largely unknown. Here we performed a genome-wide association meta-analysis comprising 2,820 unrelated cases with BrS and 10,001 controls, and identified 21 association signals at 12 loci (10 new). Single nucleotide polymorphism (SNP)-heritability estimates indicate a strong polygenic influence. Polygenic risk score analyses based on the 21 susceptibility variants demonstrate varying cumulative contribution of common risk alleles among different patient subgroups, as well as genetic associations with cardiac electrical traits and disorders in the general population. The predominance of cardiac transcription factor loci indicates that transcriptional regulation is a key feature of BrS pathogenesis. Furthermore, functional studies conducted on MAPRE2, encoding the microtubule plus-end binding protein EB2, point to microtubule-related trafficking effects on NaV1.5 expression as a new underlying molecular mechanism. Taken together, these findings broaden our understanding of the genetic architecture of BrS and provide new insights into its molecular underpinnings

A Potential New Pathway for Staphylococcus aureus Dissemination: The Silent Survival of S. aureus Phagocytosed by Human Monocyte-Derived Macrophages

Although considered to be an extracellular pathogen, Staphylococcus aureus is able to invade a variety of mammalian, non-professional phagocytes and can also survive engulfment by professional phagocytes such as neutrophils and monocytes. In both of these cell types S. aureus promptly escapes from the endosomes/phagosomes and proliferates within the cytoplasm, which quickly leads to host cell death. In this report we show that S. aureus interacted with human monocyte-derived macrophages in a very different way to those of other mammalian cells. Upon phagocytosis by macrophages, S. aureus persisted intracellularly in vacuoles for 3–4 days before escaping into the cytoplasm and causing host cell lysis. Until the point of host cell lysis the infected macrophages showed no signs of apoptosis or necrosis and were functional. They were able to eliminate intracellular staphylococci if prestimulated with interferon-γ at concentrations equivalent to human therapeutic doses. S. aureus survival was dependent on the alternative sigma factor B as well as the global regulator agr, but not SarA. Furthermore, isogenic mutants deficient in α-toxin, the metalloprotease aureolysin, protein A, and sortase A were efficiently killed by macrophages upon phagocytosis, although with different kinetics. In particular α-toxin was a key effector molecule that was essential for S. aureus intracellular survival in macrophages. Together, our data indicate that the ability of S. aureus to survive phagocytosis by macrophages is determined by multiple virulence factors in a way that differs considerably from its interactions with other cell types. S. aureus persists inside macrophages for several days without affecting the viability of these mobile cells which may serve as vehicles for the dissemination of infection

Genome-wide association analyses identify new Brugada syndrome risk loci and highlight a new mechanism of sodium channel regulation in disease susceptibility.

Brugada syndrome (BrS) is a cardiac arrhythmia disorder associated with sudden death in young adults. With the exception of SCN5A, encoding the cardiac sodium channel Na1.5, susceptibility genes remain largely unknown. Here we performed a genome-wide association meta-analysis comprising 2,820 unrelated cases with BrS and 10,001 controls, and identified 21 association signals at 12 loci (10 new). Single nucleotide polymorphism (SNP)-heritability estimates indicate a strong polygenic influence. Polygenic risk score analyses based on the 21 susceptibility variants demonstrate varying cumulative contribution of common risk alleles among different patient subgroups, as well as genetic associations with cardiac electrical traits and disorders in the general population. The predominance of cardiac transcription factor loci indicates that transcriptional regulation is a key feature of BrS pathogenesis. Furthermore, functional studies conducted on MAPRE2, encoding the microtubule plus-end binding protein EB2, point to microtubule-related trafficking effects on Na1.5 expression as a new underlying molecular mechanism. Taken together, these findings broaden our understanding of the genetic architecture of BrS and provide new insights into its molecular underpinnings

Perfusable Biohybrid Designs for Bioprinted Skeletal Muscle Tissue

Engineered, centimeter-scale skeletal muscle tissue (SMT) can mimic muscle pathophysiology to study development, disease, regeneration, drug response, and motion. Macroscale SMT requires perfusable channels to guarantee cell survival, and support elements to enable mechanical cell stimulation and uniaxial myofiber formation. Here, stable biohybrid designs of centimeter-scale SMT are realized via extrusion-based bioprinting of an optimized polymeric blend based on gelatin methacryloyl and sodium alginate, which can be accurately coprinted with other inks. A perfusable microchannel network is designed to functionally integrate with perfusable anchors for insertion into a maturation culture template. The results demonstrate that i) coprinted synthetic structures display highly coherent interfaces with the living tissue, ii) perfusable designs preserve cells from hypoxia all over the scaffold volume, iii) constructs can undergo passive mechanical tension during matrix remodeling, and iv) the constructs can be used to study the distribution of drugs. Extrusion-based multimaterial bioprinting with the inks and design realizes in vitro matured biohybrid SMT for biomedical applications.ISSN:2192-2640ISSN:2192-265

Molecular Analysis and Organization of the σ(B) Operon in Staphylococcus aureus

The alternative sigma factor σ(B) of Staphylococcus aureus controls the expression of a variety of genes, including virulence determinants and global regulators. Genetic manipulations and transcriptional start point (TSP) analyses showed that the sigB operon is transcribed from at least two differentially controlled promoters: a putative σ(A)-dependent promoter, termed sigB(p1), giving rise to a 3.6-kb transcript covering sa2059-sa2058-rsbU-rsbV-rsbW-sigB, and a σ(B)-dependent promoter, sigB(p3), initiating a 1.6-kb transcript covering rsbV-rsbW-sigB. TSP and promoter-reporter gene fusion experiments indicated that a third promoter, tentatively termed sigB(p2) and proposed to lead to a 2.5-kb transcript, including rsbU-rsbV-rsbW-sigB, might govern the expression of the sigB operon. Environmental stresses, such as heat shock and salt stress, induced a rapid response within minutes from promoters sigB(p1) and sigB(p3). In vitro, the sigB(p1) promoter was active in the early growth stages, while the sigB(p2) and sigB(p3) promoters produced transcripts throughout the growth cycle, with sigB(p3) peaking around the transition state between exponential growth and stationary phase. The amount of sigB transcripts, however, did not reflect the concentration of σ(B) measured in cell extracts, which remained constant over the entire growth cycle. In a guinea pig cage model of infection, sigB transcripts were as abundant 2 and 8 days postinoculation as values found in vitro, demonstrating that sigB is indeed transcribed during the course of infection. Physical interactions between staphylococcal RsbU-RsbV, RsbV-RsbW, and RsbW-σ(B) were inferred from a yeast (Saccharomyces cerevisiae) two-hybrid approach, indicating the presence of a partner-switching mechanism in the σ(B) activation cascade similar to that of Bacillus subtilis. The finding that overexpression of RsbU was sufficient to trigger an immediate and strong activation of σ(B), however, signals a relevant difference in the regulation of σ(B) activation between B. subtilis and S. aureus in the cascade upstream of RsbU

Optimization of a two-plasmid system for the identification of promoters recognized by RNA polymerase containing Staphylococcus aureus alternative sigma factor sigmaB

We optimized a previously established two-plasmid system for the identification of Staphylococcus aureus promoters that are recognized by the alternative transcription factor sigma(B). The method allowed the identification of 18 S. aureus sigma(B)-dependent promoters, 12 of which are reported here for the first time to be sigma(B)-dependent. S1-nuclease mapping of the respective transcriptional start points revealed that all the promoters contained sequences exhibiting high similarity to the consensus sequence of Bacillus subtilis sigma(B)-dependent promoters. The promoters governed expression of genes encoding proteins proposed to be involved in various cellular functions, including the stress response genes and virulence-associated clfA gene for fibrinogen-binding clumping factor. Comparison of the nucleotide sequences upstream of the identified transcription start points identified a sigma(B) consensus promoter (GttTaa-N(12-15)-gGGTAt) that is highly homologous to that of sigma(B) of B. subtilis

Operation of the LHC with Protons at High Luminosity and High Energy

In 2015 the Large Hadron Collider (LHC) entered the first year in its second long Run, after a 2-year shutdown that prepared it for high energy. The first two months of beam operation were dedicated to setting up the nominal cycle for proton-proton operation at 6.5 TeV/beam, and culminated with the first physics with 3 nominal bunches/ring at 13 TeV CoM on 3 June. The year continued with a stepwise intensity ramp up that allowed reaching 2244 bunches/ring for a peak luminosity of ~5·10³³ cm⁻²s^{−1} and a total of just above 4 fb-1 delivered to the high luminosity experiments. Beam operation was shaped by the high intensity effects, e.g. electron cloud and macroparticle-induced fast losses (UFOs), which on a few occasions caused the first beam induced quenches at high energy. This paper describes the operational experience with high intensity and high energy at the LHC, together with the issues that had to be tackled along the way