Untersuchungen zum kulturellen und molekularbiologischen Nachweis von Mycobacterium avium ssp. paratuberculosis (MAP) aus humanen Darmbioptaten

Mycobacterium avium ssp. paratuberculosis (MAP) ist der Erreger der Paratuberkulose der Wiederkäuer. Bereits seit Anfang des letzten Jahrhunderts wird aufgrund der pathomorphologischen Ähnlichkeiten ein ursächlicher Zusammenhang mit Morbus Crohn (MC), einer chronisch-entzündlichen Darmerkrankung des Menschen, vermutet und seitdem kontrovers diskutiert. Für Milchrinderbestände werden in einzelnen Bundesländern Seroprävalenzdaten für Paratuberkulose von bis zu ca. 80 % angegeben. Abgesehen von einer Erregerübertragung durch direkten Tierkontakt und der weiten Verbreitung des Erregers in der Umwelt, werden Lebensmittel als mögliche Vehikel diskutiert. In zahlreichen Studien wurde MAP-Desoxyribonukleinsäure im Darmgewebe von MC-Patienten und Kontrollen nachgewiesen. Nur in wenigen Untersuchungen wurde parallel die kulturelle Überprüfung der Vermehrungsfähigkeit durchgeführt. Zudem mangelt es den Polymerase-chain-reaction (PCR)-Verfahren an einer diagnostischen Qualitätssicherung mit interner Amplifikationskontrolle (IAK) und die gewählten Marker weisen Kreuzreaktionen mit anderen Mykobakterienspezies auf. In den eigenen Arbeiten wurde daher die kulturelle Anzüchtung unter Einsatz eines zuvor eigens validierten Dekontaminationsverfahrens unter Verwendung von N-Acetyl-L-Cystein-NaOH mit der PCR-Analytik kombiniert. Um die Zuverlässigkeit der Nachweisverfahren zu steigern, wurde eine nested PCR und eine kürzlich entwickelte Triplex Real Time-PCR (SCHÖNENBRÜCHER et al., 2008) inklusive einer internen Amplifikationskontrolle verwendet. So wurden erstmals die drei MAP-spezifischen Marker IS900, ISMav2 und F57 bei humanen Darmbioptaten diagnostisch kombiniert. Für die spätere Subkultivierung und Archivierung der MAP-Stämme wurden in einer vergleichenden Untersuchung preiswerte, einfach zu handhabende und produktive Selektivmedien ermittelt. Es konnten insgesamt 120 Proben von 32 Probanden berücksichtigt werden. Darunter befanden sich Bioptate von 12 MC- und acht Colitis ulcerosa (CU)-Patienten sowie 12 Kontrollprobanden, die nicht an einer chronisch-entzündlichen Darmerkrankung leiden. Darunter erwiesen sich Anreicherungskulturen von vier (33,3 %) MC- und zwei (25,0 %) CU-Patienten sowie von neun (75,0 %) Kontrollprobanden (p = 0,059) als molekularbiologisch MAP-positiv. Die Subkultivierung und Isolierung von MAP gelang aus dem Colon transversum einer Kontrollperson. Bei zwei Colitis ulcerosa-Patienten und einer Kontrollperson konnten vermehrungsfähige Mycobacterium avium ssp. avium-Stämme isoliert werden. Alle Isolate wurden mittels Sequenzanalyse der 16S-ribosomalen RNA (ribonucleic acid) und der 16S-23S „intragenic spacer region” (ITS) bestätigt und für die institutseigene Mykobakterienstammsammlung in Form von Gefrierkulturen und Lyophilisaten archiviert. Nach den Ergebnissen dieser Fall-Kontroll-Studie kann nicht auf ein ausschließliches Vorkommen von MAP bei MC-Patienten geschlossen werden, und die Gewinnung überlebensfähiger MAP gestaltet sich schwierig, möglicherweise aufgrund eines Sphäroblasten-Stadiums. Die PCR-Verfahren weisen zusätzlich auf eine weite Verbreitung von MAP bei Kontrollprobanden hin. Das angewandte Triplex PCR-Verfahren in Kombination mit einer nested PCR und der kulturellen Diagnostik liefert Ergebnisse hoher Aussagekraft über das Vorkommen von MAP in humanen Darmbioptaten. Von den für die MAP-Kultivierung untersuchten Festmedien eignete sich aufgrund der kürzesten Nachweisdauer das Herrold’s Egg Yolk-Schrägmedium (Becton Dickinson®) für einen Direktnachweis am besten. Zur Beurteilung von Einzelkolonien, beispielsweise als Kontaminationskontrolle, ist der Einsatz des Middlebrook 7H10-Agars empfehlenswert (Becton Dickinson®). Um den möglichen kausalen Zusammenhang von MAP und MC zu erhellen, sind weitere Untersuchungen mit einer größeren Probandenzahl und unter Einbeziehung einer genauen Erhebung von anamnestischen, klinischen, genetischen und psychosozialen Daten sowie der MAP-Exposition nötig.The zoonotic potential of Mycobacterium avium ssp. paratuberculosis (MAP), the causative agent of Johne’s disease in human chronic inflammatory bowel disease remains uncertain. The involvement in the pathogenesis of Crohn’s disease (CD) is discussed since the beginning of the last century. The Paratuberculosis data for milk cattle stock show over 80 % prevalence in many countries of the Federal Republic of Germany. Aside from the conceivable transmission of the bacteria to humans by the direct animal contact and the widespread in the environment, food has been considered as possible vector. Previous studies on the occurrence of MAP in gut tissues were based only on one single molecular detection system (e. g. Polymerase-chain-reaction [PCR]). In addition, these PCR-methods lacked of an internal amplification control (IAC) for diagnostic quality assurance and the used markers show cross reactions with other mycobacteria species. Furthermore the examination of the agent’s viability was not considered. For this reasons, the present investigation included the cultivation of MAP, using a previously validated decontamination protocol, and PCR-analysis. A nested PCR system as well as a recently developed real time-PCR method were used to increase the reliability of the PCR results. In total three MAP-specific genes were used. Comparative studies of culture media were done to find appropriate media for subcultures of the mycobacteria isolates In this study the three PCR markers IS900, ISMav2 and F57 were first combined with cultural detection of MAP in human biopsies. Four (33.3 %) of the CD-, two (25.0 %) of the ulcerative colitis (UC) - and nine (75.0 %) of the control patients were positive for MAP with the PCR marker (p = 0.059). MAP could be cultivated out of the colon transversum of one control person. M. avium ssp. avium has been isolated out of the biopsies of two UC-patients as well as of a control person. The cultured strains were verified by amplicon sequencing of the 16S-ribosomal RNA (ribonucleic acid) and the 16S-23S „intragenic spacer region” (ITS). The three markers were sequenced as well. All isolates were preserved by deep freezing with glycerol and by freeze drying (lyophilization). On the basis of the preliminary results it can not be concluded that MAP appeared exclusively in Crohn’s disease patients. In addition, the PCR-systems indicate a frequent occurrence of MAP in control specimens and the recovery of viable MAP has been proven to be difficult. The used triplex real-time PCR system combined with nested PCR and cultural detection of MAP presents reliable results. Because of the shortest time to detection Herrold‘s Egg Yolk Medium (Becton Dickinson®) can be recommended for isolation of MAP out of different samples (e. g. biopsy, feces). The low-priced “Paratuberkulosemedium” (Artelt-ENCLIT®) should be used for sub-cultures. Middlebrook 7H10-agar, prepared on plate, offers potential for the examination of single colonies (e. g. control of pure cultures)

Does training with amplitude modulated tones affect tone-vocoded speech perception?

Temporal-envelope cues are essential for successful speech perception. We asked here whether training on stimuli containing temporal-envelope cues without speech content can improve the perception of spectrally-degraded (vocoded) speech in which the temporal-envelope (but not the temporal fine structure) is mainly preserved. Two groups of listeners were trained on different amplitude-modulation (AM) based tasks, either AM detection or AM-rate discrimination (21 blocks of 60 trials during two days, 1260 trials; frequency range: 4Hz, 8Hz, and 16Hz), while an additional control group did not undertake any training. Consonant identification in vocoded vowel-consonant-vowel stimuli was tested before and after training on the AM tasks (or at an equivalent time interval for the control group). Following training, only the trained groups showed a significant improvement in the perception of vocoded speech, but the improvement did not significantly differ from that observed for controls. Thus, we do not find convincing evidence that this amount of training with temporal-envelope cues without speech content provide significant benefit for vocoded speech intelligibility. Alternative training regimens using vocoded speech along the linguistic hierarchy should be explored

Bromodomain protein BRD4 is a transcriptional repressor of autophagy and lysosomal function

Autophagy is a membrane-trafficking process that directs degradation of cytoplasmic material in lysosomes. The process promotes cellular fidelity, and while the core machinery of autophagy is known, the mechanisms that promote and sustain autophagy are less well defined. Here we report that the epigenetic reader BRD4 and the methyltransferase G9a repress a TFEB/TFE3/MITF-independent transcriptional program that promotes autophagy and lysosome biogenesis. We show that BRD4 knockdown induces autophagy in vitro and in vivo in response to some, but not all, situations. In the case of starvation, a signaling cascade involving AMPK and histone deacetylase SIRT1 displaces chromatin-bound BRD4, instigating autophagy gene activation and cell survival. Importantly, this program is directed independently and also reciprocally to the growth-promoting properties of BRD4 and is potently repressed by BRD4-NUT, a driver of NUT midline carcinoma. These findings therefore identify a distinct and selective mechanism of autophagy regulation

Effects of noise exposure on young adults with normal audiograms II: Behavioral measures

An estimate of lifetime noise exposure was used as the primary predictor of performance on a range of behavioral tasks: frequency and intensity difference limens, amplitude modulation detection, interaural phase discrimination, the digit triplet speech test, the co-ordinate response speech measure, an auditory localization task, a musical consonance task and a subjective report of hearing ability. One hundred and thirty-eight participants (81 females) aged 18–36 years were tested, with a wide range of self-reported noise exposure. All had normal pure-tone audiograms up to 8 kHz. It was predicted that increased lifetime noise exposure, which we assume to be concordant with noise-induced cochlear synaptopathy, would elevate behavioral thresholds, in particular for stimuli with high levels in a high spectral region. However, the results showed little effect of noise exposure on performance. There were a number of weak relations with noise exposure across the test battery, although many of these were in the opposite direction to the predictions, and none were statistically significant after correction for multiple comparisons. There were also no strong correlations between electrophysiological measures of synaptopathy published previously and the behavioral measures reported here. Consistent with our previous electrophysiological results, the present results provide no evidence that noise exposure is related to significant perceptual deficits in young listeners with normal audiometric hearing. It is possible that the effects of noise-induced cochlear synaptopathy are only measurable in humans with extreme noise exposures, and that these effects always co-occur with a loss of audiometric sensitivity

A proteomics sample metadata representation for multiomics integration and big data analysis

The amount of public proteomics data is rapidly increasing but there is no standardized format to describe the sample metadata and their relationship with the dataset files in a way that fully supports their understanding or reanalysis. Here we propose to develop the transcriptomics data format MAGE-TAB into a standard representation for proteomics sample metadata. We implement MAGE-TAB-Proteomics in a crowdsourcing project to manually curate over 200 public datasets. We also describe tools and libraries to validate and submit sample metadata-related information to the PRIDE repository. We expect that these developments will improve the reproducibility and facilitate the reanalysis and integration of public proteomics datasets.publishedVersio

Expression Atlas update: from tissues to single cells.

Expression Atlas is EMBL-EBI's resource for gene and protein expression. It sources and compiles data on the abundance and localisation of RNA and proteins in various biological systems and contexts and provides open access to this data for the research community. With the increased availability of single cell RNA-Seq datasets in the public archives, we have now extended Expression Atlas with a new added-value service to display gene expression in single cells. Single Cell Expression Atlas was launched in 2018 and currently includes 123 single cell RNA-Seq studies from 12 species. The website can be searched by genes within or across species to reveal experiments, tissues and cell types where this gene is expressed or under which conditions it is a marker gene. Within each study, cells can be visualized using a pre-calculated t-SNE plot and can be coloured by different features or by cell clusters based on gene expression. Within each experiment, there are links to downloadable files, such as RNA quantification matrices, clustering results, reports on protocols and associated metadata, such as assigned cell types

A user guide for the online exploration and visualization of PCAWG data.

Funder: U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)Funder: Ontario Institute for Cancer Research (Institut Ontarien de Recherche sur le Cancer); doi: https://doi.org/10.13039/100012118Funder: EMBL Member States EU FP7 Programme projects EurocanPlatform (260791) CAGEKID (241669)Funder: European Union’s Framework Programme For Research and Innovation Horizon 2020 under the Marie Sklodowska-Curie grant agreement no. 703543Funder: Michael & Susan Dell Foundation; Mary K. Chapman Foundation; CCSG Grant P30 CA016672 (Bioinformatics Shared Resource); ITCR U24 CA199461; GDAN U24 CA210949; GDAN U24 CA210950Funder: European Commission's H2020 Programme, project SOUND, Grant Agreement no 633974Funder: Spanish Government (SEV 2015-0493) BSC-Lenovo Master Collaboration Agreement (2015)The Pan-Cancer Analysis of Whole Genomes (PCAWG) project generated a vast amount of whole-genome cancer sequencing resource data. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2658 cancers across 38 tumor types, we provide a user's guide to the five publicly available online data exploration and visualization tools introduced in the PCAWG marker paper. These tools are ICGC Data Portal, UCSC Xena, Chromothripsis Explorer, Expression Atlas, and PCAWG-Scout. We detail use cases and analyses for each tool, show how they incorporate outside resources from the larger genomics ecosystem, and demonstrate how the tools can be used together to understand the biology of cancers more deeply. Together, the tools enable researchers to query the complex genomic PCAWG data dynamically and integrate external information, enabling and enhancing interpretation