A Closer Look on the Polyhydroxybutyrate- (PHB-) Negative Phenotype of Ralstonia eutropha PHB-4

The undefined poly(3-hydroxybutyrate)- (PHB-) negative mutant R. eutropha PHB-4 was generated in 1970 by 1-nitroso-3-nitro-1-methylguanidine (NMG) treatment. Although being scientific relevant, its genotype remained unknown since its isolation except a recent first investigation. In this study, the mutation causing the PHA-negative phenotype of R. eutropha PHB-4 was confirmed independently: sequence analysis of the phaCAB operon identified a G320A mutation in phaC yielding a stop codon, leading to a massively truncated PhaC protein of 106 amino acids (AS) in R. eutropha PHB-4 instead of 589 AS in the wild type. No other mutations were observed within the phaCAB operon. As further mutations probably occurred in the genome of mutant PHB-4 potentially causing secondary effects on the cells' metabolism, the main focus of the study was to perform a 2D PAGE-based proteome analysis in order to identify differences in the proteomes of the wild type and mutant PHB-4. A total of 20 differentially expressed proteins were identified which provide valuable insights in the metabolomic changes of mutant PHB-4. Besides excretion of pyruvate, mutant PHB-4 encounters the accumulation of intermediates such as pyruvate and acetyl-CoA by enhanced expression of the observed protein species: (i) ThiJ supports biosynthesis of cofactor TPP and thereby reinforces the 2-oxoacid dehydrogenase complexes as PDHC, ADHC and OGDHC in order to convert pyruvate at a higher rate and the (ii) 3-isopropylmalate dehydrogenase LeuB3 apparently directs pyruvate to synthesis of several amino acids. Different (iii) acylCoA-transferases enable transfer reactions between organic acid intermediates, and (iv) citrate lyase CitE4 regenerates oxaloacetate from citrate for conversion with acetyl-CoA in the TCC in an anaplerotic reaction. Substantial amounts of reduction equivalents generated in the TCC are countered by (v) synthesis of more ubiquinones due to enhanced synthesis of MenG2 and MenG3, thereby improving the respiratory chain which accepts electrons from NADH and succinate

An ancient pathway combining carbon dioxide fixation with the generation and utilization of a sodium ion gradient for ATP synthesis

Synthesis of acetate from carbon dioxide and molecular hydrogen is considered to be the first carbon assimilation pathway on earth. It combines carbon dioxide fixation into acetyl-CoA with the production of ATP via an energized cell membrane. How the pathway is coupled with the net synthesis of ATP has been an enigma. The anaerobic, acetogenic bacterium Acetobacterium woodii uses an ancient version of this pathway without cytochromes and quinones. It generates a sodium ion potential across the cell membrane by the sodium-motive ferredoxin:NAD oxidoreductase (Rnf). The genome sequence of A. woodii solves the enigma: it uncovers Rnf as the only ion-motive enzyme coupled to the pathway and unravels a metabolism designed to produce reduced ferredoxin and overcome energetic barriers by virtue of electron-bifurcating, soluble enzymes

A generic privacy ontology and its applications to different domains

Privacy is becoming increasingly important due to the advent of e-commerce, but is equally important in other application domains. Domain applications frequently require customers to divulge many personal details about themselves that must be protected carefully in accordance with privacy principles and regulations. Here, we define a privacy ontology to support the provision of privacy and help derive the level of privacy associated with transactions and applications. The privacy ontology provides a framework for developers and service providers to guide and benchmark their applications and systems with regards to the concepts of privacy and the levels and dimensions experienced. Furthermore, it supports users or data subjects with the ability to describe their own privacy requirements and measure them when dealing with other parties that process personal information. The ontology developed captures the knowledge of the domain of privacy and its quality aspects, dimensions and assessment criteria. It is composed of a core ontology, which we call generic privacy ontology and application domain specific extensions, which commit to some of application domain concepts, properties and relationships as well as all of the generic privacy ontology ones. This allows for an evaluation of privacy dimensions in different application domains and we present case studies for two different application domains, namely a restricted B2C e-commerce scenario as well as a restricted hospital scenario from the medical domain

Integrative Modeling of Transcriptional Regulation in Response to Autoimmune Desease Therapies

Die rheumatoide Arthritis (RA) und die Multiple Sklerose (MS) werden allgemein als Autoimmunkrankheiten eingestuft. Zur Behandlung dieser Krankheiten werden immunmodulatorische Medikamente eingesetzt, etwa TNF-alpha-Blocker (z.B. Etanercept) im Falle der RA und IFN-beta-Präparate (z.B. Betaferon und Avonex) im Falle der MS. Bis heute sind die molekularen Mechanismen dieser Therapien weitestgehend unbekannt. Zudem ist ihre Wirksamkeit und Verträglichkeit bei einigen Patienten unzureichend. In dieser Arbeit wurde die transkriptionelle Antwort im Blut von Patienten auf jede dieser drei Therapien untersucht, um die Wirkungsweise dieser Medikamente besser zu verstehen. Dabei wurden Methoden der Netzwerkinferenz eingesetzt, mit dem Ziel, die genregulatorischen Netzwerke (GRNs) der in ihrer Expression veränderten Gene zu rekonstruieren. Ausgangspunkt dieser Analysen war jeweils ein Genexpressions- Datensatz. Daraus wurden zunächst Gene gefiltert, die nach Therapiebeginn hoch- oder herunterreguliert sind. Anschließend wurden die genregulatorischen Regionen dieser Gene auf Transkriptionsfaktor-Bindestellen (TFBS) analysiert. Um schließlich GRN-Modelle abzuleiten, wurde ein neuer Netzwerkinferenz-Algorithmus (TILAR) verwendet. TILAR unterscheidet zwischen Genen und TF und beschreibt die regulatorischen Effekte zwischen diesen durch ein lineares Gleichungssystem. TILAR erlaubt dabei Vorwissen über Gen-TF- und TF-Gen-Interaktionen einzubeziehen. Im Ergebnis wurden komplexe Netzwerkstrukturen rekonstruiert, welche die regulatorischen Beziehungen zwischen den Genen beschreiben, die im Verlauf der Therapien differentiell exprimiert sind. Für die Etanercept-Therapie wurde ein Teilnetz gefunden, das Gene enthält, die niedrigere Expressionslevel bei RA-Patienten zeigen, die sehr gut auf das Medikament ansprechen. Die Analyse von GRNs kann somit zu einem besseren Verständnis Therapie-assoziierter Prozesse beitragen und transkriptionelle Unterschiede zwischen Patienten aufzeigen

Monitoring retinal changes with optical coherence tomography predicts neuronal loss in experimental autoimmune encephalomyelitis.

BACKGROUND:Retinal optical coherence tomography (OCT) is a clinical and research tool in multiple sclerosis, where it has shown significant retinal nerve fiber (RNFL) and ganglion cell (RGC) layer thinning, while postmortem studies have reported RGC loss. Although retinal pathology in experimental autoimmune encephalomyelitis (EAE) has been described, comparative OCT studies among EAE models are scarce. Furthermore, the best practices for the implementation of OCT in the EAE lab, especially with afoveate animals like rodents, remain undefined. We aimed to describe the dynamics of retinal injury in different mouse EAE models and outline the optimal experimental conditions, scan protocols, and analysis methods, comparing these to histology to confirm the pathological underpinnings. METHODS:Using spectral-domain OCT, we analyzed the test-retest and the inter-rater reliability of volume, peripapillary, and combined horizontal and vertical line scans. We then monitored the thickness of the retinal layers in different EAE models: in wild-type (WT) C57Bl/6J mice immunized with myelin oligodendrocyte glycoprotein peptide (MOG35-55) or with bovine myelin basic protein (MBP), in TCR2D2 mice immunized with MOG35-55, and in SJL/J mice immunized with myelin proteolipid lipoprotein (PLP139-151). Strain-matched control mice were sham-immunized. RGC density was counted on retinal flatmounts at the end of each experiment. RESULTS:Volume scans centered on the optic disc showed the best reliability. Retinal changes during EAE were localized in the inner retinal layers (IRLs, the combination of the RNFL and the ganglion cell plus the inner plexiform layers). In WT, MOG35-55 EAE, progressive thinning of IRL started rapidly after EAE onset, with 1/3 of total loss occurring during the initial 2 months. IRL thinning was associated with the degree of RGC loss and the severity of EAE. Sham-immunized SJL/J mice showed progressive IRL atrophy, which was accentuated in PLP-immunized mice. MOG35-55-immunized TCR2D2 mice showed severe EAE and retinal thinning. MBP immunization led to very mild disease without significant retinopathy. CONCLUSIONS:Retinal neuroaxonal damage develops quickly during EAE. Changes in retinal thickness mirror neuronal loss and clinical severity. Monitoring of the IRL thickness after immunization against MOG35-55 in C57Bl/6J mice seems the most convenient model to study retinal neurodegeneration in EAE

International nifedipine trial on anti-atherosclerotic therapy (INTACT) - methodologic implications and results of a coronary angiographic follow-up study using computer-assisted film analysis

Animal experiments demonstrated a significant suppressive effect of various calcium channel blockers on the formation of atherosclerotic lesions. Therefore, a prospective, placebo-controlled, randomized, double blind multicenter study was performed to investigate the inhibitory influence of the calcium channel blocker nifedipine (80 mg/day) on the progression of coronary artery disease in man. Study endpoints were changes of coronary morphology documented by coronary angiography with particular respect to the formation of new coronary stenoses. In 348 out of 425 patients included in the study, coronary angiograms were repeated after three years. The angiograms were standardized by induction of a maximal coronary vasodilation with high doses of nitrates and by using absolutely identical angiographic projections. Quantitative analysis of coronary cineangiograms was performed with the computer-assisted contour detection system CAAS. Parameters were mean and minimal diameter of all segments and minimal stenosis diameter, percent diameter stenosis, length and plaque area of all stenoses. Continuous intake of study medication was registered in 282 patients, 134 on nifedipine and 148 patients on placebo. In these patients, a total of 3808 coronary segments with 893 stenoses (≥ 20% diameter reduction in at least one angiographic projection) were compared on the baseline and follow-up cineangiograms. The changes in all angiographic parameters analyzed averaged over all patients by considering all angiographic projections analyzed, indicated significant progression of the disease (p < 0.006). The average changes in all parameters were even about three times more profound, when in the individual patients only the respective projections indicating the maximal changes were considered for the calculation (p < 0.001). However, with neither of these two analysis modes, the differences in progression between the treatment groups were statistically significant. In the follow-up angiograms, a total of 196 new coronary lesions (185 stenoses, 11 occlusions) were found at previously normal arterial sites. In patients on nifedipine, an average of only 0.58 new lesions per patient were detected versus 0,80 lesions per patient on placebo (-27%; p=0.031). INTACT is the first prospective angiographic trial on the progression of coronary artery disease using computer-assisted quantitative coronary angiography in such a high number of patients. All parameters analyzed indicated significant progression of coronary artery sclerosis. Nifedipine had no influen

Privacy support and evaluation on an ontological basis

This work is concerned with user perceived privacy and how clients (which we call data subjects here) can be empowered to control their own data consistently with their own interests. To support building and evaluation of privacy-aware applications, we describe a privacy ontology, how the privacy principles relate to that and how they are influenced by the core concepts as well as by each other. We use this influence of the privacy principles to evaluate the level of privacy for a particular transaction, when applying and extending the core concepts for an application domain

Alternative approach to tree-structured web log representation and mining

More recent approaches to web log data representation aim to capture the user navigational patterns with respect to the overall structure of the web site. One such representation is tree-structured log files which is the focus of this work. Most existing methods for analyzing such data are based on the use of frequent subtree mining techniques to extract frequent user activity and navigational paths. In this paper we evaluate the use of other standard data mining techniques enabled by a recently proposed structure preserving flat data representation for tree-structured data. The initially proposed framework was adjusted to better suit the web log mining task. Experimental evaluation is performed on two real world web log datasets and comparisons are made with an existing state-of-the art classifier for tree-structured data. The results show the great potential of the method in enabling the application of a wider range of data mining/analysis techniques to tree-structured web log data