Development of a PbWO4 Detector for Single-Shot Positron Annihilation Lifetime Spectroscopy at the GBAR Experiment

We have developed a PbWO4 (PWO) detector with a large dynamic range to measure the intensity of a positron beam and the absolute density of the ortho-positronium (o-Ps) cloud it creates. A simulation study shows that a setup based on such detectors may be used to determine the angular distribution of the emission and reflection of o-Ps to reduce part of the uncertainties of the measurement. These will allow to improve the precision in the measurement of the cross-section for the (anti)hydrogen formation by (anti)proton-positronium charge exchange and to optimize the yield of antihydrogen ion which is an essential parameter in the GBAR experiment

Engineering the Controlled Assembly of Filamentous Injectisomes in E. coli K-12 for Protein Translocation into Mammalian Cells.

Bacterial pathogens containing type III protein secretion systems (T3SS) assemble large needle-like protein complexes in the bacterial envelope, called injectisomes, for translocation of protein effectors into host cells. The application of these molecular syringes for the injection of proteins into mammalian cells is hindered by their structural and genomic complexity, requiring multiple polypeptides encoded along with effectors in various transcriptional units (TUs) with intricate regulation. In this work, we have rationally designed the controlled expression of the filamentous injectisomes found in enteropathogenic Escherichia coli (EPEC) in the nonpathogenic strain E. coli K-12. All structural components of EPEC injectisomes, encoded in a genomic island called the locus of enterocyte effacement (LEE), were engineered in five TUs (eLEEs) excluding effectors, promoters and transcriptional regulators. These eLEEs were placed under the control of the IPTG-inducible promoter Ptac and integrated into specific chromosomal sites of E. coli K-12 using a marker-less strategy. The resulting strain, named synthetic injector E. coli (SIEC), assembles filamentous injectisomes similar to those in EPEC. SIEC injectisomes form pores in the host plasma membrane and are able to translocate T3-substrate proteins (e.g., translocated intimin receptor, Tir) into the cytoplasm of HeLa cells reproducing the phenotypes of intimate attachment and polymerization of actin-pedestals elicited by EPEC bacteria. Hence, SIEC strain allows the controlled expression of functional filamentous injectisomes for efficient translocation of proteins with T3S-signals into mammalian cells

Comparative structural and functional analysis of Bunyavirus and Arenavirus cap-snatching Endonucleases

Segmented negative strand RNA viruses of the arena-, bunya- and orthomyxovirus families uniquely carry out viral mRNA transcription by the cap-snatching mechanism. This involves cleavage of host mRNAs close to their capped 5′ end by an endonuclease (EN) domain located in the N-terminal region of the viral polymerase. We present the structure of the cap-snatching EN of Hantaan virus, a bunyavirus belonging to hantavirus genus. Hantaan EN has an active site configuration, including a metal co-ordinating histidine, and nuclease activity similar to the previously reported La Crosse virus and Influenza virus ENs (orthobunyavirus and orthomyxovirus respectively), but is more active in cleaving a double stranded RNA substrate. In contrast, Lassa arenavirus EN has only acidic metal co-ordinating residues. We present three high resolution structures of Lassa virus EN with different bound ion configurations and show in comparative biophysical and biochemical experiments with Hantaan, La Crosse and influenza ENs that the isolated Lassa EN is essentially inactive. The results are discussed in the light of EN activation mechanisms revealed by recent structures of full-length influenza virus polymerase

A Step Forward in Molecular Diagnostics of Lyssaviruses – Results of a Ring Trial among European Laboratories

Rabies is a lethal and notifiable zoonotic disease for which diagnostics have to meet the highest standards. In recent years, an evolution was especially seen in molecular diagnostics with a wide variety of different detection methods published. Therefore, a first international ring trial specifically designed on the use of reverse transcription polymerase chain reaction (RT-PCR) for detection of lyssavirus genomic RNA was organized. The trial focussed on assessment and comparison of the performance of conventional and real-time assays. In total, 16 European laboratories participated. All participants were asked to investigate a panel of defined lyssavirus RNAs, consisting of Rabies virus (RABV) and European bat lyssavirus 1 and 2 (EBLV-1 and -2) RNA samples, with systems available in their laboratory. The ring trial allowed the important conclusion that conventional RT-PCR assays were really robust assays tested with a high concordance between different laboratories and assays. The real-time RT-PCR system by Wakeley et al. (2005) in combination with an intercalating dye, and the combined version by Hoffmann and co-workers (2010) showed good sensitivity for the detection of all RABV samples included in this test panel. Furthermore, all used EBLV-specific assays, real-time RT-PCRs as well as conventional RT-PCR systems, were shown to be suitable for a reliable detection of EBLVs. It has to be mentioned that differences were seen in the performance between both the individual RT-PCR systems and the laboratories. Laboratories which used more than one molecular assay for testing the sample panel always concluded a correct sample result. Due to the markedly high genetic diversity of lyssaviruses, the application of different assays in diagnostics is needed to achieve a maximum of diagnostic accuracy. To improve the knowledge about the diagnostic performance proficiency testing at an international level is recommended before using lyssavirus molecular diagnostics e.g. for confirmatory testing

In vitro and in vivo antifungal profile of a novel and long acting inhaled azole, PC945, on Aspergillus fumigatus infection

The profile of PC945, a novel triazole antifungal, designed for administration via inhalation, hasbeen assessed in a range of in vitro and in vivo studies. PC945 was characterized as a potent, tight-binding inhibitor of Aspergillus fumigatus sterol 14α-demethylase (CYP51A and CYP51B)activity.In addition, when A. fumigatus hyphae or human bronchial cells were treated with PC945, and thenwashed, PC945 was found to be quickly absorbed into both target and non-target cells and toproduce persistent antifungal effects. In temporarily neutropenic immunocompromised miceinfected with A. fumigatus intranasally, 50% of the animals survived until day 7 when treatedintranasally with PC945 at 0.56 μg/mouse, while posaconazole showed similar effects (44%) at14 μg/mouse. This profile affirms that topical treatment with PC945 should provide potentantifungal activity in the lung

Fine-Scale Mapping of the 4q24 Locus Identifies Two Independent Loci Associated with Breast Cancer Risk

Background: A recent association study identified a common variant (rs9790517) at 4q24 to be associated with breast cancer risk. Independent association signals and potential functional variants in this locus have not been explored. Methods: We conducted a fine-mapping analysis in 55,540 breast cancer cases and 51,168 controls from the Breast Cancer Association Consortium. Results: Conditional analyses identified two independent association signals among women of European ancestry, represented by rs9790517 [conditional P = 2.51 × 10−4; OR, 1.04; 95% confidence interval (CI), 1.02–1.07] and rs77928427 (P = 1.86 × 10−4; OR, 1.04; 95% CI, 1.02–1.07). Functional annotation using data from the Encyclopedia of DNA Elements (ENCODE) project revealed two putative functional variants, rs62331150 and rs73838678 in linkage disequilibrium (LD) with rs9790517 (r2 ≥ 0.90) residing in the active promoter or enhancer, respectively, of the nearest gene, TET2. Both variants are located in DNase I hypersensitivity and transcription factor–binding sites. Using data from both The Cancer Genome Atlas (TCGA) and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC), we showed that rs62331150 was associated with level of expression of TET2 in breast normal and tumor tissue. Conclusion: Our study identified two independent association signals at 4q24 in relation to breast cancer risk and suggested that observed association in this locus may be mediated through the regulation of TET2. Impact: Fine-mapping study with large sample size warranted for identification of independent loci for breast cancer risk

Upper Palaeolithic settlements in Buran-Kaya 3 (Crimea, Ukraine): new interdisciplinary researches of the layers 5-2, 6-1 and 6-2

Грот Буран Кая 3 (Крым, Украина) содержит уникальную стратиграфию литологических и культурных отложений, включающую индустрии среднего и верхнего палеолита. В частности, в слоях 5-2, 6-1, и 6-2 был обнаружен кремневый и костяной инвентарь, относящийся к эпиграветту. Эти же слои содержат многочисленные фаунистические остатки, а также антропологический материал. В данной статье публикуются некоторые результаты междисциплинарных исследований: техникотипологический анализ кремневого и костяного инвентаря; зооархеологическое изучение останков крупных млекопитающих; исследование технологии нанесения орнамента на кости; анализ палеоантропологических материалов. На основании полученных данных предлагается функциональная интерпретация стоянки, а также производится сравнение культурных характеристик эпиграветта Буран Каи 3 и памятников соседних территорий

Citrobacter rodentium Subverts ATP Flux and Cholesterol Homeostasis in Intestinal Epithelial Cells In Vivo.

The intestinal epithelial cells (IECs) that line the gut form a robust line of defense against ingested pathogens. We investigated the impact of infection with the enteric pathogen Citrobacter rodentium on mouse IEC metabolism using global proteomic and targeted metabolomics and lipidomics. The major signatures of the infection were upregulation of the sugar transporter Sglt4, aerobic glycolysis, and production of phosphocreatine, which mobilizes cytosolic energy. In contrast, biogenesis of mitochondrial cardiolipins, essential for ATP production, was inhibited, which coincided with increased levels of mucosal O2 and a reduction in colon-associated anaerobic commensals. In addition, IECs responded to infection by activating Srebp2 and the cholesterol biosynthetic pathway. Unexpectedly, infected IECs also upregulated the cholesterol efflux proteins AbcA1, AbcG8, and ApoA1, resulting in higher levels of fecal cholesterol and a bloom of Proteobacteria. These results suggest that C. rodentium manipulates host metabolism to evade innate immune responses and establish a favorable gut ecosystem

Novel de novo EEF1A2 missense mutations causing epilepsy and intellectual disability.

BACKGROUND: Exome sequencing has led to the discovery of mutations in novel causative genes for epilepsy. One such gene is EEF1A2, encoding a neuromuscular specific translation elongation factor, which has been found to be mutated de novo in five cases of severe epilepsy. We now report on a further seven cases, each with a different mutation, of which five are newly described. METHODS: New cases were identified and sequenced through the Deciphering Developmental Disabilities project, via direct contact with neurologists or geneticists, or recruited via our website. RESULTS: All the mutations cause epilepsy and intellectual disability, but with a much wider range of severity than previously identified. All new cases share specific subtle facial dysmorphic features. Each mutation occurs at an evolutionarily highly conserved amino acid position indicating strong structural or functional selective pressure. CONCLUSIONS: EEF1A2 should be considered as a causative gene not only in cases of epileptic encephalopathy but also in children with less severe epilepsy and intellectual disability. The emergence of a possible discernible phenotype, a broad nasal bridge, tented upper lip, everted lower lip and downturned corners of the mouth may help in identifying patients with mutations in EEF1A2