Long-Range PCR-Based NGS Applications to Diagnose Mendelian Retinal Diseases

The purpose of this study was to develop a flexible, cost-efficient, next-generation sequencing (NGS) protocol for genetic testing. Long-range polymerase chain reaction (PCR) amplicons of up to 20 kb in size were designed to amplify entire genomic regions for a panel (n = 35) of inherited retinal disease (IRD)-associated loci. Amplicons were pooled and sequenced by NGS. The analysis was applied to 227 probands diagnosed with IRD: (A) 108 previously molecularly diagnosed, (B) 94 without previous genetic testing, and (C) 25 undiagnosed after whole-exome sequencing (WES). The method was validated with 100% sensitivity on cohort A. Long-range PCR-based sequencing revealed likely causative variant(s) in 51% and 24% of proband from cohorts B and C, respectively. Breakpoints of 3 copy number variants (CNVs) could be characterized. Long-range PCR libraries spike-in extended coverage of WES. Read phasing confirmed compound heterozygosity in 5 probands. The proposed sequencing protocol provided deep coverage of the entire gene, including intronic and promoter regions. Our method can be used (i) as a first-tier assay to reduce genetic testing costs, (ii) to elucidate missing heritability cases, (iii) to characterize breakpoints of CNVs at nucleotide resolution, (iv) to extend WES data to non-coding regions by spiking-in long-range PCR libraries, and (v) to help with phasing of candidate variants.     

Production of the tubulin destabilizer disorazol in Sorangium cellulosum: biosynthetic machinery and regulatory genes

Myxobacteria show a high potential for the production of natural compounds that exhibit a wide variety of antibiotic, antifungal, and cytotoxic activities. The genus Sorangium is of special biotechnological interest because it produces almost half of the secondary metabolites isolated from these microorganisms. We describe a transposon-mutagenesis approach to identifying the disorazol biosynthetic gene cluster in Sorangium cellulosum So ce12, a producer of multiple natural products. In addition to the highly effective disorazol-type tubulin destabilizers, S. cellulosum So ce12 produces sorangicins, potent eubacterial RNA polymerase inhibitors, bactericidal sorangiolides, and the antifungal chivosazoles. To obtain a transposon library of sufficient size suitable for the identification of the presumed biosynthetic gene clusters, an efficient transformation method was developed. We present here the first electroporation protocol for a strain of the genus Sorangium. The transposon library was screened for disorazol-negative mutants. This approach led to the identification of the corresponding trans-acyltransferase core biosynthetic gene cluster together with a region in the chromosome that is likely to be involved in disorazol biosynthesis. A third region in the genome harbors another gene that is presumed to be involved in the regulation of disorazol production. A detailed analysis of the biosynthetic and regulatory genes is presented in this paper.     

Nanostructured ordering of fluorescent markers and single proteins on substrates

Highly ordered hexagonal nanopatterns of gold clusters on glass substrates were used as anchoring points for the specific attachment of fluorescence dyes and proteins labeled with fluorescence dyes. Thiol- or disulfide-containing linker molecules were used for the binding to the gold dots. In order to ensure specific binding on the gold dots only, the surface area in between the dots was protected against unspecific adsorption. For the attachment of polar low-molecular-weight fluorescence dyes, an octadecyltrichlorosilane self-assembled monolayer was prepared on the surface in between the gold dots, whereas a layer prepared from star-shaped poly(ethylene oxide-stat-propylene oxide) prepolymers was used to prevent unspecific adsorption of proteins between the gold dots. Fluorescence microscopy proved the specific binding of the dyes as well as of the proteins. Scanning force microscopy studies show that each gold dot is only capable of binding one protein at a time.     

Signal Transduction Pathways as Concurrent Reactive Systems: A Modeling and Simulation Approach Using LSCs and the Play-Engine

Signal transduction pathways describe how cells respond to extracellular signals which are received by receptors at the cell membrane and usually transferred into the nucleus. In this paper we present our approach to model these signal transduction pathways as concurrent reactive systems by means of Life Sequence Charts and to simulate them using the Play-Engine tool. This aspect is part of a bigger approach, where we provide an extendable system to generate models of signal transduction pathways in different modeling languages and to simulate these models with the corresponding simulation tools.     

Grenzenloser Mobile Commerce

Eine wichtige S?ule des Datenschutzrechts sind die Rechte der Betroffenen insbesondere auf Benachrichtigung und Auskunft sowie auf Berichtigung, L?schung und Sperrung personenbezogener Daten. Diese Rechte k?nnen nur dann einen effektiven Schutz der informationellen Selbstbestimmung bewirken, wenn sie gesetzlich normiert sind und gleichzeitig ihre gerichtliche Durchsetzbarkeit gew?hrleistet ist. Der Mobile Commerce überwindet staatliche Grenzen und führt dazu, dass der Umgang mit personenbezogenen Daten zunehmend durch verantwortliche Stellen erfolgt, die ihren Sitz nicht in Deutschland, sondern im Ausland haben. Der Aufsatz zeigt die sich aus dem Internationalen Prozess- und Privatrecht für den Betroffenen ergebenden prozess- und materiell-rechtlichen Konsequenzen auf, wenn die Datenverarbeitung im Ausland erfolgt.     

Gasdruckschwingungen und Strömungsgeräusche in Druckbegrenzungsventilen und Rohrleitungen. Pneumatic oszillations and current noises in pressure control valves

Pneumatic oscillations and current noises in pressure control valves Pressure control valves or overflow valves are inserted for the limit of permissible printings in compressor and pump plants. While attaining permissible printing, the valves open and blow off the supported air volume flow into the environment or the supported gas into a pressure‐less receptacle. As a result, the subsequent plant can be protected and the corresponding devices before inadmissibly high printing or a too high gap. Pressure control valves and overflow valves are normally carried out at the same inside diameter of the printing and/or sucking carbine. As a result, redirections and vertebra fields which can lead to strong current noises occur. In order to be able to lower the current noises in pressure control valves the gassing jerk oscillations, the effective sound pressure oscillations and the sound pressure levels of such valves are examined and measured in the following contribution.     

Biosynthesis of volatiles by the myxobacterium Myxococcus xanthus

The volatiles emitted from cell cultures of myxobacterium Myxococcus xanthus were collected by use of a closed-loop stripping apparatus (CLSA) and analyzed by GC-MS. Two new natural products, (S)-9-methyldecan-3-ol ((S)-1) and 9-methyldecan-3-one (2), were identified and synthesized, together with other aliphatic ketones and alcohols, and terpenes. Biosynthesis of the two main components (S)-1 and 2 was examined in feeding experiments carried out with the wild-type strain DK1622 and two mutant strains JD300 and DK11017, which are impaired in the degradation pathway from leucine to isovaleryl-SCoA. Isovaleryl-SCoA is used as a starter, followed by chain elongation with two malonate units. Subsequent use of methyl malonate and decarboxylation leads to (S)-1 and 2. Furthermore, 3,3-dimethylacrylic acid (DMAA) can be used by the mutant strain to form isovaleryl-SCoA, which corroborates recent data on the detection of a novel variety of the mevalonate pathway giving rise to isovaleryl-SCoA from HMGCoA.     

Age-dependent gene expression profile and protein expression in a transgenic rat model of Huntington's disease

Polyglutamine‐induced changes in gene expression have been demonstrated by using several mouse models of Huntington's disease (HD), which express extreme numbers of CAG repeats. We have recently developed a transgenic rat model of HD carrying a truncated huntingtin fragment with 51 CAG repeats, which is in the range seen in adult HD patients. For further evaluation, we have performed microarray analyses on whole brains of transgenic rats at 3 and 12 months of age and correlated it with protein expression by Western blot analysis. We found that genes functionally associated with gene expression and behavior were differently regulated already at 3 months of age, whereas at 12 months of age especially genes related to neurological diseases and cell‐to‐cell signaling and interaction were dysregulated. A detailed analysis of canonical pathways revealed that at 3 months of age genes in calcium signaling and synaptic long term potentation pathways were altered, while at 12 months of age, additionally, expression level of many genes implicated in Huntington's disease signaling, were changed.     

Online help-seeking in communities of practice: Modeling the acceptance of conceptual artifacts

Interactive online help systems are considered to be a fruitful supplement to traditional IT helpdesks, which are often overloaded. They often comprise user-generated FAQ collections playing the role of technology-based conceptual artifacts. Two main questions arise: how the conceptual artifacts should be used, and which factors influence their acceptance in a community of practice (CoP). Firstly, this paper offers a theoretical frame and a usage scenario for technology-based conceptual artifacts against the theoretical background of the academic help-seeking and CoP approach. Each of the two approaches is extensively covered by psychological and educational research literature, however their combination is not yet sufficiently investigated. Secondly, the paper proposes a research model explaining the acceptance of conceptual artifacts. The model includes users' expectations toward the artifact, perceived social influence and users' roles in the CoP as predictors of artifact use intention and actual usage. A correlational study conducted in an academic software users' CoP and involving structural equations modeling validates the model, suggesting thus a research line that is worth further pursuing. For educational practice, the study suggests three ways of supporting knowledge sharing in CoPs, i.e. use of technology-based conceptual artifacts, roles and division of labor, and purposeful communication in CoPs.     

Wenn es persönlich wird in der „personalisierten Medizin“: Aufklärung und Kommunikation aus klinischer Forscher- und Patientenperspektive im empirisch-ethischen Vergleich

Definition of the problem In clinical research and practice, the new paradigm of “personalised medicine” raises questions about necessity, expectations, possibilities and risks. In an ongoing empirical–ethical study, we explore the perspectives of both researchers and patients regarding the implementation of “personalised medicine” in the treatment of locally advanced rectal cancer. This study focuses on ethically relevant aspects in practice, including expectations towards, counseling on, and decision for “personalized” treatment, or research. Methods We conducted interviews (with 19 researchers and physicians and 28 patients) and participant observations during physician–patient consultations (n = 50). Arguments Uncovering differences and similarities in the perspectives of affected patients and physicians will allow potential conflicts in clinical practice to be detected and addressed as early as possible. Conclusions We were able to demonstrate that patients and physicians have different perspectives on “personalised medicine”. This might lead to conflicts in clinical practice that should be addressed as early as possible.