Lokalisierung von mCLCA5 und mCLCA6 und quantitative mRNA Expressionsanalysen von 15 Kandidatengenen

Cystic Fibrosis (CF) is a fatal autosomal recessive inherited genetic disease in humans caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR encodes for a Cl- channel in secretory epithelia. Its absence or malfunction lead to altered regulation of Cl- secretion across epithelial membranes and a phenotype of severe pulmonary disease, pancreatic insufficiency, meconium ileus in newborns and other conditions. Interestingly, the CF phenotype has large variations. Even dizygous twins sharing most environmental influences show clinical variations. This suggests that factors other than CFTR modulate disease severity. One of these factors is a CFTRindependent, Ca2+-activated Cl- current (CaCC) which was demonstrated to ameliorate disease severity in the intestine, lungs and pancreas of CF mouse models and in part in humans. The molecules responsible for these CaCC may be potential therapeutical targets. However, its molecular identity is still unknown. First aim of the study was to investigate the tissue specific and cellular mRNA expression pattern of two new murine CLCA family members (mCLCA5 and mCLCA6) as important prerequisite for protein and functional analyses. Second aim was to characterize the cellular mRNA expression levels of select CaCC candidates and modulators and possibly transcriptional regulatory changes under conditions of CF. This study used laser capture microdissection (LCM) and immune electron microscopy to determine cellular localizations of mCLCA5 and mCLCA6. It used reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) to quantify and compare the mRNA expression of 15 candidate modulators of CaCC in tissues relevant to CF (stomach, duodenum, jejunum, caecum, colon, pancreas, liver, lung, kidney) between two different CF mouse models and their wild type controls. One mouse model (cftrTgH(neoim)Hgu on BALB/c, DBA/2 and C57BL/6 backgrounds) exhibits residual cftr and a milder phenotype than the other mouse model (cftrtm1Cam on NMRI background) with the complete cftr knockout. Candidates tested were the Ca2+-activated Cl- channels mBEST1, mBEST2, mTTYH3, mTMEM16A, mTMEM16F, mTMEM16K, the Ca2+-activated Cltransporters mCLC-3B and mCLC-4, as well as six murine CLCA gene family members (mCLCA1 to -6), which encode proteins that evoke Ca2+-activated Clcurrents but do not form channels themselves. Additionally the Cl- channel SLC26A9 was tested. Similar to their homologs and orthologs within the CLCA gene family, mCLCA5 and mCLCA6 had their own distinct cellular localizations. mCLCA5 mRNA had a broad expression pattern whereas the protein was found exclusively in cytoplasmic granules of granular layer keratinocytes of stratified squamous epithelia suggesting a role in cornification and an inferior relevance in CF. mCLCA6 mRNA was exclusively expressed in enterocytes suggesting a role in transepithelial anion conductance and fluid secretion which may be of relevance in CF. Of all investigated candidates, only mCLCA6 and mCLCA3 were up-regulated in the caecum and stomach of the cftrTgH(neoim)Hgu DBA/2 mice respectively and only the tweety Cl- channel mTTYH3 was markedly down-regulated throughout the intestinal tract of the cftrtm1Cam NMRI mice. This supports mCLCA6 and mCLCA3 as potential mediators of “alternative” CaCC in CF but argues against a direct compensatory mechanism on transcriptional level in the lungs, pancreas or intestine. On the contrary, mTTYH3 may be linked to the more severe intestinal phenotype suggesting a negative modulatory effect. However, it cannot be excluded that any of the candidates tested are regulated on post-transcriptional level or by modulation of the protein activation status. Thus they may still play a role as modulators in the basic Clsecretory defect of CF and await further analyses.Zystische Fibrose (englisch cystic fibrosis, CF) ist eine tödlich verlaufende, autosomal rezessiv vererbte Erkrankung, die durch Mutationen des CFTR-Gens verursacht wird. CFTR codiert für einen Chloridkanal in sekretorischen Epithelien, dessen Fehlen oder Defekt eine fehlregulierte Chloridströmung und schwerwiegende Lungenerkrankungen, Pankreasinsuffizienz sowie Mekoniumileus bei Neugeborenen hervorrufen kann. Interessanterweise zeigen dizygote Zwillinge, die den identischen CFTR-Defekt tragen und unter gleichen Umweltbedingungen leben, eine hohe klinische und pathologische Variation des CF-Phänotyps. Über den CFTR-Gendefekt hinausgehende genetische Faktoren scheinen den Schweregrad der Erkrankung zu beeinflussen. Zu diesen sogenannten modifier genes gehört eine CFTR-unabhängige, Ca2+-aktivierte Chloridleitfähigkeit (CaCC), die den Darm-, Lungen- und Pankreas-Phänotyp in CFMausmodellen mildert und sich möglicherweise als therapeutisches Ziel eignet. Leider ist deren molekulare Identität und Modulation bislang jedoch ungeklärt. Erstes Ziel der Studie war es, das bislang unbekannte gewebsspezifische und zelluläre mRNA-Expressionsmuster von zwei murinen CLCA Vertretern (mCLCA5 und mCLCA6) zu bestimmen. Zweites Ziel war es die zellulären mRNA Expressionshöhen ausgewählter CaCC-Kandidaten und Modulatoren zu analysieren sowie mögliche regulatorische Veränderungen auf transkriptioneller Ebene unter CF Bedingungen festzustellen. Diese Studie hat mittels Lasermikrodissektion und Immunelektronenmikroskopie die zelluläre Lokalisation von mCLCA5 und mCLCA6 bestimmt. Sie quantifiziert und vergleicht mithilfe der Reversen Transkriptase quantitativen Polymerasekettenreaktion (RT-qPCR) die mRNA-Expression von 14 Vertretern, die potenzielle CaCC- Kandidaten oder -Modulatoren darstellen, in CF-relevanten Geweben (Magen, Duodenum, Jejunum, Zäkum, Kolon, Pankreas, Leber, Lunge, Niere) zwischen zwei verschiedenen CF-Mausmodellen und deren Wildtyp-Kontrollen. Eines der hier untersuchten Mausmodelle (cftrTgH(neoim)Hgu auf BALB/c, DBA/2 und C57BL/6 Hintergründen) zeigt eine residuelle cftr-Aktivität und einen milderen Phänotyp, während das andere Mausmodel (cftrtm1Cam auf NMRI Hintergrund) einen kompletten cftr-Knockout darstellt und einen schwereren Phänotyp zeigt. In den Mausmodellen wurden die Ca2+-aktivierten Chloridkanäle mBEST1, mBEST2, mTTYH3, mTMEM16A, mTMEM16F, mTMEM16K und die Ca2+-aktivierten Chloridtransporter mCLC- 3B und mCLC-4 untersucht. Des Weiteren wurden sechs Mitglieder der CLCA Genfamilie (mCLCA1 bis -6) analysiert, die selbst keine Kanalproteine sind, jedoch Ca2+-aktivierte Chloridströme hervorrufen können. Zusätzlich wurde der Chloridkanal SLC26A9 untersucht. mCLCA5 und mCLCA6 zeigten, ähnlich wie ihre Homologen und Orthologen, jeweils spezifische zelluläre Lokalisationen. mCLCA5 mRNA zeigte ein weites Expressionsmuster, wobei das Protein nur in zytoplasmatischen Granula der Keratinozyten des Stratum granulosum aller Plattenepithelien gefunden wurde, was auf eine Funktion in der Verhornung vermuten lässt, und für CF eine untergeordnete Rolle zu spielen scheint. mCLCA6-mRNA wurde ausschließlich in Enterozyten gefunden. Diese Lokalisation deutet auf eine Funktion bei der transepithelialen Anionenströmung hin, welche für CF von hoher Relevanz sein könnte. Von allen untersuchten Kandidaten waren nur mCLCA6 und mCLCA3 im Zäkum bzw. Magen der cftrTgH(neoim)Hgu DBA/2-Mäuse hochreguliert, wohingegen der Tweety- Chloridkanal mTTYH3 im gesamten Darmtrakt der cftrtm1Cam NMRI-Mäuse herunterreguliert war. Diese Ergebnisse sprechen dafür, dass mCLCA6 und mCLCA3 Mediatoren der „alternativen“ CaCC in CF sein könnten, sprechen jedoch gegen einen direkten kompensatorischen Mechanismus auf transkriptioneller Ebene in Lunge, Pankreas oder Darm, mTTYH3 könnte dagegen sogar mit einem schwerwiegenderen intestinalen Phänotyp im Zusammenhang stehen. Es kann jedoch nicht ausgeschlossen werden, dass einer oder mehrere der anderen untersuchten Kandidaten auf post-transkriptioneller Ebene oder durch Modulation des Proteinaktivierungsstatus reguliert wird und daher trotzdem als Modulator im Erkrankungskomplex CF eine Rolle spielt

Using the gut microbiota as a novel tool for examining colobine primate GI health

Primates of the Colobinae subfamily are highly folivorous. They possess a sacculated foregut and are believed to rely on a specialized gut microbiota to extract sufficient energy from their hard-to-digest diet. Although many colobines are endangered and would benefit from captive breeding programs, maintaining healthy captive populations of colobines can be difficult since they commonly suffer from morbidity and mortality due to gastrointestinal (GI) distress of unknown cause. While there is speculation that this GI distress may be associated with a dysbiosis of the gut microbiota, no study has directly examined the role of the gut microbiota in colobine GI health. In this study, we used high-throughput sequencing to examine the gut microbiota of three genera of colobines housed at the San Diego Zoo: doucs (Pygathrix) (N=7), colobus monkeys (Colobus) (N=4), and langurs (Trachypithecus) (N=5). Our data indicated that GI-healthy doucs, langurs, and colobus monkeys possess a distinct gut microbiota. In addition, GI-unhealthy doucs exhibited a different gut microbiota compared to GI-healthy individuals, including reduced relative abundances of anti-inflammatory Akkermansia. Finally, by comparing samples from wild and captive Asian colobines, we found that captive colobines generally exhibited higher relative abundances of potential pathogens such as Desulfovibrio and Methanobrevibacter compared to wild colobines, implying an increased risk of gut microbial dysbiosis. Together, these results suggest an association between the gut microbiota and GI illness of unknown cause in doucs. Further studies are necessary to corroborate these findings and determine cause-and-effect relationships. Additionally, we found minimal variation in the diversity and composition of the gut microbiota along the colobine GI tract, suggesting that fecal samples may be sufficient for describing the colobine gut microbiota. If these findings can be validated in wild individuals, it will facilitate the rapid expansion of colobine gut microbiome research

Proceedings in Marine Biology

“Proceedings in Marine Biology” is an international journal publishing original research by graduate students on all aspects of marine biology. Subjects covered include: ecological surveys and population studies of oceanic, coastal and shore communities; physiology and experimental biology; taxonomy, morphology and life history of marine animals and plants. Papers are also published on techniques em - ployed at sea for sampling, recording, capture and observation of marine organisms.Zeitschrift zur Kursabschlussreise der Humboldt-Universität zu Berlin (Deutschland) im Bereich Elektronenmikroskopie.Peer Reviewe

Insulin detemir in a twice daily insulin regimen versus a three times daily insulin regimen in the treatment of type 1 diabetes in children: A pilot randomized controlled trial

Article deposited according to agreement with BMC, December 6, 2010.YesFunding provided by the Open Access Authors Fund

A communal catalogue reveals Earth’s multiscale microbial diversity

Our growing awareness of the microbial world’s importance and diversity contrasts starkly with our limited understanding of its fundamental structure. Despite recent advances in DNA sequencing, a lack of standardized protocols and common analytical frameworks impedes comparisons among studies, hindering the development of global inferences about microbial life on Earth. Here we present a meta-analysis of microbial community samples collected by hundreds of researchers for the Earth Microbiome Project. Coordinated protocols and new analytical methods, particularly the use of exact sequences instead of clustered operational taxonomic units, enable bacterial and archaeal ribosomal RNA gene sequences to be followed across multiple studies and allow us to explore patterns of diversity at an unprecedented scale. The result is both a reference database giving global context to DNA sequence data and a framework for incorporating data from future studies, fostering increasingly complete characterization of Earth’s microbial diversity

A communal catalogue reveals Earth's multiscale microbial diversity

Our growing awareness of the microbial world's importance and diversity contrasts starkly with our limited understanding of its fundamental structure. Despite recent advances in DNA sequencing, a lack of standardized protocols and common analytical frameworks impedes comparisons among studies, hindering the development of global inferences about microbial life on Earth. Here we present a meta-analysis of microbial community samples collected by hundreds of researchers for the Earth Microbiome Project. Coordinated protocols and new analytical methods, particularly the use of exact sequences instead of clustered operational taxonomic units, enable bacterial and archaeal ribosomal RNA gene sequences to be followed across multiple studies and allow us to explore patterns of diversity at an unprecedented scale. The result is both a reference database giving global context to DNA sequence data and a framework for incorporating data from future studies, fostering increasingly complete characterization of Earth's microbial diversity.Peer reviewe

Adding 6 months of androgen deprivation therapy to postoperative radiotherapy for prostate cancer: a comparison of short-course versus no androgen deprivation therapy in the RADICALS-HD randomised controlled trial

Background Previous evidence indicates that adjuvant, short-course androgen deprivation therapy (ADT) improves metastasis-free survival when given with primary radiotherapy for intermediate-risk and high-risk localised prostate cancer. However, the value of ADT with postoperative radiotherapy after radical prostatectomy is unclear. Methods RADICALS-HD was an international randomised controlled trial to test the efficacy of ADT used in combination with postoperative radiotherapy for prostate cancer. Key eligibility criteria were indication for radiotherapy after radical prostatectomy for prostate cancer, prostate-specific antigen less than 5 ng/mL, absence of metastatic disease, and written consent. Participants were randomly assigned (1:1) to radiotherapy alone (no ADT) or radiotherapy with 6 months of ADT (short-course ADT), using monthly subcutaneous gonadotropin-releasing hormone analogue injections, daily oral bicalutamide monotherapy 150 mg, or monthly subcutaneous degarelix. Randomisation was done centrally through minimisation with a random element, stratified by Gleason score, positive margins, radiotherapy timing, planned radiotherapy schedule, and planned type of ADT, in a computerised system. The allocated treatment was not masked. The primary outcome measure was metastasis-free survival, defined as distant metastasis arising from prostate cancer or death from any cause. Standard survival analysis methods were used, accounting for randomisation stratification factors. The trial had 80% power with two-sided α of 5% to detect an absolute increase in 10-year metastasis-free survival from 80% to 86% (hazard ratio [HR] 0·67). Analyses followed the intention-to-treat principle. The trial is registered with the ISRCTN registry, ISRCTN40814031, and ClinicalTrials.gov, NCT00541047. Findings Between Nov 22, 2007, and June 29, 2015, 1480 patients (median age 66 years [IQR 61–69]) were randomly assigned to receive no ADT (n=737) or short-course ADT (n=743) in addition to postoperative radiotherapy at 121 centres in Canada, Denmark, Ireland, and the UK. With a median follow-up of 9·0 years (IQR 7·1–10·1), metastasis-free survival events were reported for 268 participants (142 in the no ADT group and 126 in the short-course ADT group; HR 0·886 [95% CI 0·688–1·140], p=0·35). 10-year metastasis-free survival was 79·2% (95% CI 75·4–82·5) in the no ADT group and 80·4% (76·6–83·6) in the short-course ADT group. Toxicity of grade 3 or higher was reported for 121 (17%) of 737 participants in the no ADT group and 100 (14%) of 743 in the short-course ADT group (p=0·15), with no treatment-related deaths. Interpretation Metastatic disease is uncommon following postoperative bed radiotherapy after radical prostatectomy. Adding 6 months of ADT to this radiotherapy did not improve metastasis-free survival compared with no ADT. These findings do not support the use of short-course ADT with postoperative radiotherapy in this patient population

Duration of androgen deprivation therapy with postoperative radiotherapy for prostate cancer: a comparison of long-course versus short-course androgen deprivation therapy in the RADICALS-HD randomised trial

Background Previous evidence supports androgen deprivation therapy (ADT) with primary radiotherapy as initial treatment for intermediate-risk and high-risk localised prostate cancer. However, the use and optimal duration of ADT with postoperative radiotherapy after radical prostatectomy remains uncertain. Methods RADICALS-HD was a randomised controlled trial of ADT duration within the RADICALS protocol. Here, we report on the comparison of short-course versus long-course ADT. Key eligibility criteria were indication for radiotherapy after previous radical prostatectomy for prostate cancer, prostate-specific antigen less than 5 ng/mL, absence of metastatic disease, and written consent. Participants were randomly assigned (1:1) to add 6 months of ADT (short-course ADT) or 24 months of ADT (long-course ADT) to radiotherapy, using subcutaneous gonadotrophin-releasing hormone analogue (monthly in the short-course ADT group and 3-monthly in the long-course ADT group), daily oral bicalutamide monotherapy 150 mg, or monthly subcutaneous degarelix. Randomisation was done centrally through minimisation with a random element, stratified by Gleason score, positive margins, radiotherapy timing, planned radiotherapy schedule, and planned type of ADT, in a computerised system. The allocated treatment was not masked. The primary outcome measure was metastasis-free survival, defined as metastasis arising from prostate cancer or death from any cause. The comparison had more than 80% power with two-sided α of 5% to detect an absolute increase in 10-year metastasis-free survival from 75% to 81% (hazard ratio [HR] 0·72). Standard time-to-event analyses were used. Analyses followed intention-to-treat principle. The trial is registered with the ISRCTN registry, ISRCTN40814031, and ClinicalTrials.gov , NCT00541047 . Findings Between Jan 30, 2008, and July 7, 2015, 1523 patients (median age 65 years, IQR 60–69) were randomly assigned to receive short-course ADT (n=761) or long-course ADT (n=762) in addition to postoperative radiotherapy at 138 centres in Canada, Denmark, Ireland, and the UK. With a median follow-up of 8·9 years (7·0–10·0), 313 metastasis-free survival events were reported overall (174 in the short-course ADT group and 139 in the long-course ADT group; HR 0·773 [95% CI 0·612–0·975]; p=0·029). 10-year metastasis-free survival was 71·9% (95% CI 67·6–75·7) in the short-course ADT group and 78·1% (74·2–81·5) in the long-course ADT group. Toxicity of grade 3 or higher was reported for 105 (14%) of 753 participants in the short-course ADT group and 142 (19%) of 757 participants in the long-course ADT group (p=0·025), with no treatment-related deaths. Interpretation Compared with adding 6 months of ADT, adding 24 months of ADT improved metastasis-free survival in people receiving postoperative radiotherapy. For individuals who can accept the additional duration of adverse effects, long-course ADT should be offered with postoperative radiotherapy. Funding Cancer Research UK, UK Research and Innovation (formerly Medical Research Council), and Canadian Cancer Society