Molekularbiologische Untersuchung der Diversität von Mikroorganismen in gefluteten und ungefluteten Pappelmikrokosmen

Auenböden unterliegen aufgrund temporärer Flutungen einem starken Wechsel im Wassergehalt. Als Folge adaptieren sich Pflanzen, wie beispielsweise Pappeln, und Mikroorganismen an diese anoxischen Bedingungen. Ziel dieser Arbeit war es die Auswirkungen von Flutung auf die mikrobiellen Lebensgemeinschaften (Bacteria und Archaea), die mit Pappeln assoziiert sind, in Mikrokosmen zu analysieren. Die Struktur der Lebensgemeinschaften wurde mittels terminaler Restriktionsfragment-Längen-Polymorphismus (T-RFLP)-Analyse, Klonierung und vergleichender Sequenzierung der 16S rRNA-codierenden Gene (16S rDNA) aufgeklärt. Durch Inkubation (90 Tage) von undurchwurzelten Bodenproben aus ungefluteten (bodenfeuchten) und vorgefluteten Pappelmikrokosmen sowie aus feldfrischen Proben wurde ein Überblick der biogeochemischen Prozesse erhalten. Nach Aufschlämmung dieser Proben wurden die vorhandenen Elektronenakzeptoren entsprechend ihrem Redoxpotential sequentiell reduziert. Im Vergleich unterschieden sich die Proben aus den Mikrokosmen von denen der feldfrischen Proben hinsichtlich der Konzentrationen der ermittelten Parameter, insbesondere war die Methanbildung in den feldfrischen Proben wesentlich stärker ausgeprägt. Die bakteriellen Lebensgemeinschaften in Bodenproben aus vorgefluteten und ungefluteten Mikrokosmen veränderten sich kaum mit der Zeit und wurden von Bacillales und Acidobacteria, welche typisch für Böden sind, dominiert. In den feldfrischen Proben waren zusätzlich Actinobacteria und Alphaproteobacteria dominant. Die archaeelle Lebensgemeinschaft zeigte über den untersuchten Zeitraum in allen Ansätzen geringe Veränderungen. Die abundanten Gruppen innerhalb der Archaea zählten zu den nicht kultivierten Crenarchaeota der Linie 1.1b und den Methanosarcinaceae. Zum Ende der Inkubationen wurden mit abnehmender Acetatkonzentration Methanosaetaceae detektiert. Weiterhin wurde der Einfluss der Pappelpflanze auf die mikrobiellen Gemeinschaften durch die Analyse der Kompartimente undurchwurzelter Boden, Rhizosphäre und Rhizoplane in einem weiteren Mikrokosmosexperiment untersucht. Für die bakterielle Gemeinschaft wurde eine Gesamtheit von 281 Klonsequenzen erhalten. Die Anzahl der verschiedenen Sequenzen (<97 % Ähnlichkeit) in den verschiedenen Habitaten repräsentierte jeweils zwischen 16-55 % des gesamten bakteriellen Artenreichtums wie sie mit Chao1 als Indikator abgeschätzt wurde. In Bezug auf die Anzahl der verschiedenen terminalen Restriktionsfragmente zeigten alle Habitate jeweils ca. 20 verschiedene „operational taxonomic units“ (OTUs), mit Ausnahme des Habitats der gefluteten Rhizoplane, welches eine geringere Anzahl an OTUs aufwies. Generell bestätigten sich die mittels Klonierung und T-RFLP-Analyse erzielten Ergebnisse gegenseitig. Die statistische Auswertung der gesamten T-RFLP-Profile mittels Korrespondenzanalyse zeigte, dass sich die bakteriellen Gemeinschaften in den Kompartimenten deutlich voneinander unterschieden und sich durch Flutung veränderten. Beispielsweise traten unter ungefluteten Bedingungen Bacillus spp. vermehrt im undurchwurzelten Boden und in der Rhizosphäre auf. Hingegen waren Bakterien in nächster Verwandtschaft zu Aquaspirillum sp. nur an Pappelwurzeln und in der Rhizosphäre von gefluteten Mikrokosmen abundant. Die archaeelle Gemeinschaft wurde in allen Kompartimenten, sowohl geflutet als auch ungeflutet, zu 99 % von Crenarchaeota-Klonsequenzen der Linie 1.1b dominiert. Die Dominanz crenarchaeotischer Linien in den Bodenaufschlämmungen und allen Kompartimenten der Mikrokosmen weist auf ihre physiologische Bedeutung nicht nur in Böden, sondern auch in der Rhizosphäre und an Wurzeln von Pappelpflanzen hin. Allein zwei Klonsequenzen aus dem Habitat der Rhizoplane wurden bisher unkultivierten Euryarchaeota zugeordnet. Im Gegensatz zu den Bodenaufschlämmungen wurden keine Methanogenen mittels Klonierung detektiert. In dieser Arbeit wurden erstmals in dem System der Pappelmikrokosmen Einblicke in die strukturelle Zusammensetzung der Bacteria und Archaea, auch in Abhängigkeit von Flutung, mit Hilfe molekularbiologischer Methoden erhalten und abundante Mikroorganismen konnten identifiziert werden. Dies stellt eine Basis für gezielte physiologische Fragestellungen in diesem Habitat dar

Sequential hypothesis testing for Axion Haloscopes

The goal of this paper is to introduce a novel likelihood-based inferential framework for axion haloscopes which is valid under the commonly applied "rescanning" protocol. The proposed method enjoys short data acquisition times and a simple tuning of the detector configuration. Local statistical significance and power are computed analytically, avoiding the need of burdensome simulations. Adequate corrections for the look-elsewhere effect are also discussed. The performance of our inferential strategy is compared with that of a simple method which exploits the geometric probability of rescan. Finally, we exemplify the method with an application to a HAYSTAC type axion haloscope.Comment: 14 pages, 12 figure

Designer rhamnolipid production

Rhamnolipids are biosurfactants featuring surface-active properties that render them suitable for a broad range of applications, e.g., in detergents, food, bioremediation, medicine/pharmacology, and crop science. These properties include their emulsification and foaming capacities and their ability to lower the surface tension. Further, aspects like biocompatibility and environmental friendliness, both features of rhamnolipids [1] are becoming increasingly important. Rhamnolipids thus constitute suitable substitutes for synthetic surfactants produced from fossil resources. Native producers of rhamnolipids are mainly pathogenic bacteria like Pseudomonas aeruginosa. We previously designed and constructed a recombinant Pseudomonas putida KT2440, which synthesizes rhamnolipids by decoupling production from host-intrinsic regulations and cell growth [2]. As most biosurfactants, rhamnolipids are synthesized in mixtures. We here show our approach to alter the native mixture of surfactant molecules to produce specific new-to-nature combinations. The molecular structure (Figure 1) can on the one hand be altered in the hydrophilic moiety by changing the number of rhamnose molecules. We achieved this by using only distinct genes from the native rhamnolipid synthesis pathway. On the other hand, we were also able to change the length of the fatty acids in the hydrophobic part. This chain length is determined by the acyl-transferase (RhlA). Using rhlA genes from different organisms enables our microbial cell factory to synthesize molecules with different chain lengths [3]. The different molecular structures have further been shown to feature diverse physico-chemical properties [4]. Exploiting the natural structural diversity will thus allow for the synthesis of designer rhamnolipids tailormade for specific applications. We thus present a novel approach to use biochemical engineering to create tailormade products for a more sustainable future. Please click Additional Files below to see the full abstract

A global profile of replicative polymerase usage

Three eukaryotic DNA polymerases are essential for genome replication. Polymerase (Pol) α–primase initiates each synthesis event and is rapidly replaced by processive DNA polymerases: Polɛ replicates the leading strand, whereas Polδ performs lagging-strand synthesis. However, it is not known whether this division of labor is maintained across the whole genome or how uniform it is within single replicons. Using Schizosaccharomyces pombe, we have developed a polymerase usage sequencing (Pu-seq) strategy to map polymerase usage genome wide. Pu-seq provides direct replication-origin location and efficiency data and indirect estimates of replication timing. We confirm that the division of labor is broadly maintained across an entire genome. However, our data suggest a subtle variability in the usage of the two polymerases within individual replicons. We propose that this results from occasional leading-strand initiation by Polδ followed by exchange for Polɛ

ATM and Artemis promote homologous recombination of radiation-induced DNA double-strand breaks in G2

Homologous recombination (HR) and non‐homologous end joining (NHEJ) represent distinct pathways for repairing DNA double‐strand breaks (DSBs). Previous work implicated Artemis and ATM in an NHEJ‐dependent process, which repairs a defined subset of radiation‐induced DSBs in G1‐phase. Here, we show that in G2, as in G1, NHEJ represents the major DSB‐repair pathway whereas HR is only essential for repair of ∼15% of X‐ or γ‐ray‐induced DSBs. In addition to requiring the known HR proteins, Brca2, Rad51 and Rad54, repair of radiation‐induced DSBs by HR in G2 also involves Artemis and ATM suggesting that they promote NHEJ during G1 but HR during G2. The dependency for ATM for repair is relieved by depleting KAP‐1, providing evidence that HR in G2 repairs heterochromatin‐associated DSBs. Although not core HR proteins, ATM and Artemis are required for efficient formation of single‐stranded DNA and Rad51 foci at radiation‐induced DSBs in G2 with Artemis function requiring its endonuclease activity. We suggest that Artemis endonuclease removes lesions or secondary structures, which inhibit end resection and preclude the completion of HR or NHEJ

A case of congenital TTP presenting with microganiopathy in adulthood

BACKGROUND: Congenital thrombotic thrombocytopenic purpura (TTP), also known as Upshaw-Schulman Syndrome is a rare inherited deficiency of ADAMTS13. Unlike the more common acquired TTP which is characterized by an acquired inhibitor of ADAMTS13, patients with congenital TTP have an absolute deficiency of ADAMTS13 without an inhibitor. Congenital TTP generally presents in infancy with repeat episodes of acute hemolysis and evidence of microangiopathy, these episodes are usually triggered by illness or physiological stress. Congenital TTP can be effectively treated with plasma infusion either during acute episodes or on a prophylactic schedule to prevent episodes. CASE PRESENTATION: We present a case of a 25 year old Caucasian woman with no know family history of hematological disorders with congenital TTP. She presented with episodes of hemolysis since infancy, but without clear evidence of microangiopathy until the age of 25. At presentation to our center the patient was documented to have thrombocytopenia, elevated creatinine, and schistocytes. She was initially treated with plasma infusion at a rate of 60 ml/hr continuously for a 24 hr period with resolution of her thrombocytopenia and hemolysis. At the time of writing this article she is maintained on a prophylactic schedule of biweekly plasma infusions at 10 mg/kg and is maintaining a normal platelet count with no evidence of hemolysis. CONCLUSION: Congenital TTP is a rare condition, and the above case is atypical as the patient did not present with clear evidence of microangiopathy until adulthood. Although this a rare condition it is important for physicians to be aware of as it, especially the possibility of atypical presentations, as the condition is potentially fatal and effective treatment exists

JJ-factors for self-interacting dark matter in 20 dwarf spheroidal galaxies

Dwarf spheroidal galaxies are among the most promising targets for indirect dark matter (DM) searches in $\gamma$-rays. The $\gamma$-ray flux from DM annihilation in a dwarf spheroidal galaxy is proportional to the $J$-factor of the source. The $J$-factor of a dwarf spheroidal galaxy is the line-of-sight integral of the DM mass density squared times $\langle \sigma_{\rm ann} v_{\rm rel} \rangle/(\sigma_{\rm ann} v_{\rm rel})_0$, where $\sigma_{\rm ann} v_{\rm rel}$ is the DM annihilation cross-section times relative velocity $v_{\rm rel}=|{\bf v}_{\rm rel}|$, angle brackets denote average over ${\bf v}_{\rm rel}$, and $(\sigma_{\rm ann} v_{\rm rel})_0$ is the $v_{\rm rel}$-independent part of $\sigma_{\rm ann} v_{\rm rel}$. If $\sigma_{\rm ann} v_{\rm rel}$ is constant in $v_{\rm rel}$, $J$-factors only depend on the DM space distribution in the source. However, if $\sigma_{\rm ann} v_{\rm rel}$ varies with $v_{\rm rel}$, as in the presence of DM self-interactions, $J$-factors also depend on the DM velocity distribution, and on the strength and range of the DM self-interaction. Models for self-interacting DM are increasingly important in the study of the small scale clustering of DM, and are compatible with current cosmological observations. Here we derive the $J$-factor of 20 dwarf spheroidal galaxies from stellar kinematic data under the assumption of Yukawa DM self-interactions. $J$-factors are derived through a profile Likelihood approach, assuming either NFW or cored DM profiles. We also compare our results with $J$-factors derived assuming the same velocity for all DM particles in the target galaxy. We find that this common approximation overestimates the $J$-factors by up to one order of magnitude. $J$-factors for a sample of DM particle masses, self-interaction coupling constants and density profiles are provided electronically, ready to be used in other projects.Comment: 10 pages, 3 figures and 2 table

Rare manifestation of a c.290 C\u3eT, p.Gly97Glu VCP mutation

Introduction. The valosin-containing protein (VCP) regulates several distinct cellular processes. Consistent with this, VCP mutations manifest variable clinical phenotypes among and within families and are a diagnostic challenge. Methods. A 60-year-old man who played ice hockey into his 50’s was evaluated by electrodiagnostics, muscle biopsy, and molecular genetics. Results. With long-standing pes cavus and toe walking, our patient developed progressive weakness, cramps, memory loss, and paresthesias at age 52. An axonal sensorimotor neuropathy was found upon repeated testing at age 58. Neuropathic histopathology was present in the quadriceps, and exome sequencing revealed the VCP mutation c.290 C>T, p.Gly97Glu. Conclusions. Our patient reflects the clinical heterogeneity of VCP mutations, as his neurological localization is a spectrum between a lower motor neuron disorder and a hereditary axonal peripheral neuropathy such as CMT2. Our case demonstrates a rare manifestation of the c.290 C>T, pGly97Glu VCP mutation

Leveraging P2P systems to address the test scenario explosion problem

Modern software development is characterised by a strong customer focus and electronic delivery mechanisms which make it very easy for customers to buy and install a vendor’s software. However, it also makes it very easy for customers to buy and install software from competing vendors and as such it is more important than ever for deployed software to be as correct and bug-free as possible. Whilst certain types of testing can be done in the lab with a high degree of confidence that results will hold when the software is deployed in the wild, in reality software systems are subject to influence from whatever environment they end up being deployed in. Varying factors in customer environments can include operating systems, services packs, device drivers, network connectivity, resource usage by other software, and so on. Any variation or combination of these factors can lead to a situation where a system deviates from its expected behaviour. The problem is amplified even further on mobile devices whereby devices can move between different networks, interrupt apps for phone calls, have varying screen sizes, have user interference in the form of turning features on and o↵ to preserve battery power, vendor-specific operating system code, and so on. A conservative calculation indicates that a software system can be subjected to tens of thousands of different scenarios. Even if one were to execute just one test case against each scenario, obtaining any form of realistic coverage is infeasible for even the most resource-rich organisations. Cloud and grid infrastructures have been proposed as solutions to improving the scalability of software testing since 2008. However, we argue that cloud computing systems are too homogenous and are not representative of real-world usage scenarios. We refer to this problem as the Test Scenario Explosion Problem.peer-reviewe