Kultivierung von Denitrifikanten mit einem neuen nirK-Genotyp

Die Denitrifikation ist im Ökosystem von entscheidender Bedeutung. Besonders im Boden spielen Denitrifikanten durch die Remineralisierung von Nitrat zu molekularen Stickstoff eine entscheidende Rolle. Eines der Markergene der Denitrifikation ist nirK, welches für die kupfer-abhängige Nitrit-Reduktase kodiert. Denitrifikanten sind in ihrer Phylogenie sehr divers. Der Stammbaum von nirK beherbergt Cluster, die von nirK-Umweltsequenzen dominiert sind und keine nirK-Sequenz eines Isolates enthalten. Eines dieser Cluster ist Cluster I, welches im Fokus der vorliegenden Arbeit stand, da es von nirK-Sequenzen bislang unkultivierter Denitrifikanten aus Bodenproben dominiert wird. Mit einer Kultivierungsmethode die nicht spezifisch auf Denitrifikanten ausgerichtet war, sondern einen unspezifischen Ansatz verfolgte, sollte die Isolierung und Kultivierung nirK-enthaltender Organismen dieses Clusters aus Bodenproben ermöglicht werden. Als Probenmaterial dienten Lahn-Wiesenbodenproben. Vor der Kultivierung wurde durch die Erstellung einer nirK-Klonbibliothek die Verteilung der nirK-Sequenzen innerhalb des beprobten Lahn-Wiesenbodens analysiert. Mit der Ausnahme einer nirK-Klonsequenz waren alle nirK-Sequenzen auf die Cluster I und VI verteilt. Die aus Probenmaterial angesetzte Bodensuspension wurde vier Wochen unter oxischen Kultivierungsbedingungen, in Minimalmedium bei einer habitatspezifischen Inkubationstemperatur inkubiert. Acht nirK-enthaltende Isolate 187, 406, 467, 471, 469, 373, 484 und 205 wurden mit dieser Methode kultiviert. Die nirK-Sequenzen dreier Isolate gruppierte in das nirK-Cluster I, während die nirK-Sequenzen weiterer fünf Isolate eine phylogenetische Nähe zu den nirK-Sequenzen der Cluster II, V und VII zeigten. Mit unspezifischen Kultivierungsmethoden gelang es also Kulturen mit einem nirK-Genotyp des Clusters I zu kultivieren. Ferner zeigte sich, dass durch molekularbiologische, kultivierungs-unabhängige Methoden zum Teil andere nirK-Sequenzen nachgewiesen wurden als durch kultivierungs-abhängige Methoden. Phylogenetische Analysen der 16S rRNA-Gen-Sequenzen ergaben die Zuordnung der Isolate zu den Gattungen Bradyrhizobium (Isolat 406, 467, 469 und 471), Bosea (Isolat 187), Mesorhizobium (Isolat 373 und 484) und Devosia (Isolat 205). Die Isolate des nirK-Clusters I wurden hierbei alle als Spezies der Gattung Bradyrhizobium identifiziert. Auch die weiteren Gene der Denitrifikation nap/nar, nor und nos konnten für die Isolate der Gattungen Bradyrhizobium und Bosea nachgewiesen werden, mit Ausnahme des Isolates (471) der Gattung Bradyrhizobium das kein nosZ-Gen aufwies. Für die Isolate der Gattung Mesorhizobium wurden neben nirK die Denitrifikationsgene narG und cnorB bestimmt. Das Isolat 205 der Gattung Devosia wies neben nirK kein weiteres Gen der Denitrifikation auf. Ein phylogenetischer Vergleich der 16S rRNA-Gen-Phylogenie der Isolate mit den Phylogenien der funktionellen Gene nirK, norB und nosZ, zeigte eine Kongruenz der Stammbäume, wobei die nirK-Phylogenie die Gruppierungen am differenziertesten widerspiegelte. Aufgrund der großen phylogenetischen Nähe zwischen den nirK-Sequenzen der Isolate und den nirK-Umweltsequenzen des Clusters I kann man vermuten, dass es sich bei diesen Subcluster um eine Taxon-spezifische Gruppierung von Bradyrhizobium Spezies handelt. Trotz ihrer Identifizierung als Spezies der Gattung Bradyrhizobium wiesen die Isolate des nirK-Clusters I keine phylogenetische Nähe zur Spezies Bradyrhizobium japonicum auf, den einzigen bisher bekannten Denitrifikanten innerhalb dieser Gattung. Es lässt sich also vermuten, dass neben B. japonicum auch andere Bradyrhizobium Spezies in der Lage sind zu denitrifizieren. Die Isolate der Gattungen Bradyrhizobium und Bosea zeigten sowohl auf genomischer als auch auf funktioneller Ebene die Fähigkeit zur N2O-Produktion. Die Isolate der Gattung Mesorhizobium 373 und 484 wiesen zwar auf genomischer Ebene das Potential zur Denitrifikation auf, jedoch wurde auf funktioneller Ebene weder eine Umsetzung des NO3- zu N2O, noch ein Wachstum unter anoxischen Bedingungen mit NO3- als Elektronenakzeptor nachgewiesen. Die denitrifizierenden Spezies der Gattung Mesorhizobium wiesen keine phylogenetische Nähe zu den Isolaten der Gattung Mesorhizobium der vorliegenden Arbeit auf, so dass es sich bei den vorliegenden Isolaten möglicherweise um neue Spezies der Gattung Mesorhizobium handeln könnte. Für das Isolat 205 der Gattung Devosia konnte dem nirK-Gen keine Funktion zugeordnet werden. Durch die Ergebnisse der vorliegenden Arbeit ist es möglich, dass bisher von unkultivierten Organismen aus Bodenhabitaten dominierte nirK-Cluster I näher zu charakterisieren

Sweeping the Space of Admissible Quark Mass Matrices

We propose a new and efficient method of reconstructing quark mass matrices from their eigenvalues and a complete set of mixing observables. By a combination of the principle of NNI (nearest neighbour interaction) bases which are known to cover the general case, and of the polar decomposition theorem that allows to convert arbitrary nonsingular matrices to triangular form, we achieve a parameterization where the remaining freedom is reduced to one complex parameter. While this parameter runs through the domain bounded by a circle with radius R determined by the up-quark masses around the origin in the complex plane one sweeps the space of all mass matrices compatible with the given set of data.Comment: 18 page

Excellent local control and survival after postoperative or definitive radiation therapy for sarcomas of the head and neck

Background: To report our results with postoperative or definitive radiation therapy in head and neck sarcomas. Methods: We performed a retrospective analysis of 26 patients suffering from head and neck sarcomas, who received postoperative or definitive radiation therapy between 2003 and 2012. Median age was 64 years (19-88) and 69 % were male. Tumor locations were skull (including skin) in 31 %,paranasal sinus/orbita in 27 % and neck (including pharynx/larynx) in 42 %. Median tumor size was 4.6 cm (1-12 cm). 22 patients (85 %) presented in primary situation. Stage at presentation (UICC 7th for soft tissue sarcomas) was as follows: Ia: 4 %,IIa:50 %,IIb:15 %,III:31 %. All except one patient suffered from high grade lesions (G2/3 FNCLCC),predominantly angiosarcoma (35 %),MFH (19 %) and synovial sarcoma (15 %). Surgery was performed in 21 pts (81 %),resulting in free margins in 10 (38 %),microscopically positive margins in 6 (23 %) and gross residual disease in 5 (19 %). Median dose to the primary tumor region was 66Gy (45-72Gy) in conventional fractionation, using 3D-CRT in 65 %,IMRT in 27 % and electrons in 8 %. 50 % of the patients also received sequential chemotherapy. Results: Median follow up was 39 months (8-136). We observed three local recurrences, transferring into estimated 3-and 5-year local control rates of 86 %. One additional patient failed distantly, resulting in 3-and 5-year freedom from treatment failure rates of 82 %. Four patients have deceased, transferring into 3-and 5-year overall survival rates of 88 % and 82 %,respectively. Only two of the four deaths were sarcoma related. Maximum acute toxicity (CTCAE 3.0) was grade 1 in 27 % of the patients, grade 2 in 50 % and grade 3 in 23 %. Severe acute toxicity was mainly represented by mucositis and dysphagia. Maximum late toxicity was grade 1 in 31 %,grade 2 in 15 % and grade 3 in 19 % of the patients. Severe late toxicity included skin ulceration (n = 1),dysphagia with persistent tube dependency (n = 1),persistent sinusitis (n = 1) and hearing loss (n = 2). Conclusion: Excellent local control and overall survival rates can be achieved with postoperative or definitive radiation therapy with acceptable acute and late toxicities in patients suffering from sarcomas of the head and neck region

Excellent local control and survival after postoperative or definitive radiation therapy for sarcomas of the head and neck

Background: To report our results with postoperative or definitive radiation therapy in head and neck sarcomas. Methods: We performed a retrospective analysis of 26 patients suffering from head and neck sarcomas, who received postoperative or definitive radiation therapy between 2003 and 2012. Median age was 64 years (19-88) and 69 % were male. Tumor locations were skull (including skin) in 31 %,paranasal sinus/orbita in 27 % and neck (including pharynx/larynx) in 42 %. Median tumor size was 4.6 cm (1-12 cm). 22 patients (85 %) presented in primary situation. Stage at presentation (UICC 7th for soft tissue sarcomas) was as follows: Ia: 4 %,IIa:50 %,IIb:15 %,III:31 %. All except one patient suffered from high grade lesions (G2/3 FNCLCC),predominantly angiosarcoma (35 %),MFH (19 %) and synovial sarcoma (15 %). Surgery was performed in 21 pts (81 %),resulting in free margins in 10 (38 %),microscopically positive margins in 6 (23 %) and gross residual disease in 5 (19 %). Median dose to the primary tumor region was 66Gy (45-72Gy) in conventional fractionation, using 3D-CRT in 65 %,IMRT in 27 % and electrons in 8 %. 50 % of the patients also received sequential chemotherapy. Results: Median follow up was 39 months (8-136). We observed three local recurrences, transferring into estimated 3-and 5-year local control rates of 86 %. One additional patient failed distantly, resulting in 3-and 5-year freedom from treatment failure rates of 82 %. Four patients have deceased, transferring into 3-and 5-year overall survival rates of 88 % and 82 %,respectively. Only two of the four deaths were sarcoma related. Maximum acute toxicity (CTCAE 3.0) was grade 1 in 27 % of the patients, grade 2 in 50 % and grade 3 in 23 %. Severe acute toxicity was mainly represented by mucositis and dysphagia. Maximum late toxicity was grade 1 in 31 %,grade 2 in 15 % and grade 3 in 19 % of the patients. Severe late toxicity included skin ulceration (n = 1),dysphagia with persistent tube dependency (n = 1),persistent sinusitis (n = 1) and hearing loss (n = 2). Conclusion: Excellent local control and overall survival rates can be achieved with postoperative or definitive radiation therapy with acceptable acute and late toxicities in patients suffering from sarcomas of the head and neck region

Renormalization in Quantum Field Theory: An Improved Rigorous Method

The perturbative construction of the S-matrix in the causal spacetime approach of Epstein and Glaser may be interpreted as a method of regularization for divergent Feynman diagrams. The results of any method of regularization must be equivalent to those obtained from the Epstein-Glaser (EG) construction, within the freedom left by the latter. In particular, the conceptually well-defined approach of Bogoliubov, Parasuk, Hepp, and Zimmermann (BPHZ), though conceptually different from EG, meets this requirement. Based on this equivalence we propose a modified BPHZ procedure which provides a significant simplification of the techniques of perturbation theory, and which applies equally well to standard quantum field theory and to chiral theories. We illustrate the proposed method by a number of examples of various orders in perturbation theory. At the level of multi-loop diagrams we find that subdiagrams as classified by Zimmermann's forest formula in BPHZ may be restricted to subdiagrams in the sense of Epstein-Glaser, thus entailing an important reduction of actual computations. Furthermore, the relationship of our approach to the method of dimensional regularization is particularly transparent, without having to invoke analytic continuation to unphysical spacetime dimension, and sheds new light on certain parameters within dimensional regularization.Comment: 19 pages, 3 figure

A53T-alpha-synuclein overexpression impairs dopamine signaling and striatal synaptic plasticity in old mice

BACKGROUND: Parkinson's disease (PD), the second most frequent neurodegenerative disorder at old age, can be caused by elevated expression or the A53T missense mutation of the presynaptic protein alpha-synuclein (SNCA). PD is characterized pathologically by the preferential vulnerability of the dopaminergic nigrostriatal projection neurons. METHODOLOGY/PRINCIPAL FINDINGS: Here, we used two mouse lines overexpressing human A53T-SNCA and studied striatal dysfunction in the absence of neurodegeneration to understand early disease mechanisms. To characterize the progression, we employed young adult as well as old mice. Analysis of striatal neurotransmitter content demonstrated that dopamine (DA) levels correlated directly with the level of expression of SNCA, an observation also made in SNCA-deficient (knockout, KO) mice. However, the elevated DA levels in the striatum of old A53T-SNCA overexpressing mice may not be transmitted appropriately, in view of three observations. First, a transcriptional downregulation of the extraneural DA degradation enzyme catechol-ortho-methytransferase (COMT) was found. Second, an upregulation of DA receptors was detected by immunoblots and autoradiography. Third, extensive transcriptome studies via microarrays and quantitative real-time RT-PCR (qPCR) of altered transcript levels of the DA-inducible genes Atf2, Cb1, Freq, Homer1 and Pde7b indicated a progressive and genotype-dependent reduction in the postsynaptic DA response. As a functional consequence, long term depression (LTD) was absent in corticostriatal slices from old transgenic mice. CONCLUSIONS/SIGNIFICANCE: Taken together, the dysfunctional neurotransmission and impaired synaptic plasticity seen in the A53T-SNCA overexpressing mice reflect early changes within the basal ganglia prior to frank neurodegeneration. As a model of preclinical stages of PD, such insights may help to develop neuroprotective therapeutic approaches

A53T-Alpha-Synuclein Overexpression Impairs Dopamine Signaling and Striatal Synaptic Plasticity in Old Mice

Parkinson's disease (PD), the second most frequent neurodegenerative disorder at old age, can be caused by elevated expression or the A53T missense mutation of the presynaptic protein alpha-synuclein (SNCA). PD is characterized pathologically by the preferential vulnerability of the dopaminergic nigrostriatal projection neurons.Here, we used two mouse lines overexpressing human A53T-SNCA and studied striatal dysfunction in the absence of neurodegeneration to understand early disease mechanisms. To characterize the progression, we employed young adult as well as old mice. Analysis of striatal neurotransmitter content demonstrated that dopamine (DA) levels correlated directly with the level of expression of SNCA, an observation also made in SNCA-deficient (knockout, KO) mice. However, the elevated DA levels in the striatum of old A53T-SNCA overexpressing mice may not be transmitted appropriately, in view of three observations. First, a transcriptional downregulation of the extraneural DA degradation enzyme catechol-ortho-methytransferase (COMT) was found. Second, an upregulation of DA receptors was detected by immunoblots and autoradiography. Third, extensive transcriptome studies via microarrays and quantitative real-time RT-PCR (qPCR) of altered transcript levels of the DA-inducible genes Atf2, Cb1, Freq, Homer1 and Pde7b indicated a progressive and genotype-dependent reduction in the postsynaptic DA response. As a functional consequence, long term depression (LTD) was absent in corticostriatal slices from old transgenic mice.Taken together, the dysfunctional neurotransmission and impaired synaptic plasticity seen in the A53T-SNCA overexpressing mice reflect early changes within the basal ganglia prior to frank neurodegeneration. As a model of preclinical stages of PD, such insights may help to develop neuroprotective therapeutic approaches

Beach litter sources around Nuuk, Greenland: An analysis by UArctic summer school graduate course students

Modeling studies illustrate the potential for long-range transport of plastics into the Arctic, although the degree to which this occurs remains relatively undocumented. We utilised a teaching exercise at a UArctic summer school graduate course in Nuuk, Greenland to conduct a preliminary in-depth analysis of beach litter sources in the Nuup Kangerlua fjord. Students and instructors collected and analysed 1800 litter items weighing 200 kg from one location in the fjord and another at its mouth. The results suggest a predominance of local sources to macrolitter, rather than long-range transport from Europe. Fisheries-related items and rope were common. Packaging which could be identified was largely suspected to be products distributed in Greenland, and soft plastics, which rarely disperse far from its source, were also common. The results suggest local measures to reduce mismanaged waste and emissions from fisheries are important for reducing marine litter in West Greenland.publishedVersio

Genetic Analysis Workshop 15: gene expression analysis and approaches to detecting multiple functional loci

National Institutes of Health (AR44422, N01-AR-7-2232, 5R01-HL049609-14, IR01-AG021917-01A1); Genome Canada and Associations AFP; Polyarctique-Groupe Taitbout; Rhumatisme et Travail; Arthritis Research Campaign; Unversity of Minnesota; Minnesota Supercomputing Institute; National Institute of General Medical Sciences (R01 GM31575