Experimental and computational approaches to quantitative proteomics: status quo and outlook.

Proteomics has come a long way from the initial qualitative analysis of proteins present in a given sample at a given time ("cataloguing") to large-scale characterization of proteomes, their interactions and dynamic behavior. Originally enabled by breakthroughs in protein separation and visualization (by two-dimensional gels) and protein identification (by mass spectrometry), the discipline now encompasses a large body of protein and peptide separation, labeling, detection and sequencing tools supported by computational data processing. The decisive mass spectrometric developments and most recent instrumentation news are briefly mentioned accompanied by a short review of gel and chromatographic techniques for protein/peptide separation, depletion and enrichment. Special emphasis is placed on quantification techniques: gel-based, and label-free techniques are briefly discussed whereas stable-isotope coding and internal peptide standards are extensively reviewed. Another special chapter is dedicated to software and computing tools for proteomic data processing and validation. A short assessment of the status quo and recommendations for future developments round up this journey through quantitative proteomics.

TECTONICS OF THE HELVETIC NAPPE SYSTEM (W SWITZERLAND): CONTROL OF STRAIN LOCALIZATION AND BASEMENT-COVER DEFORMATION. Insights from models based on continuum mechanics

The Helvetic nappe system in Western Switzerland is a stack of fold nappes and thrust sheets em-placed at low grade metamorphism. Fold nappes and thrust sheets are also some of the most common features in orogens. Fold nappes are kilometer scaled recumbent folds which feature a weakly deformed normal limb and an intensely deformed overturned limb. Thrust sheets on the other hand are characterized by the absence of overturned limb and can be defined as almost rigid blocks of crust that are displaced sub-horizontally over up to several tens of kilometers. The Morcles and Doldenhom nappe are classic examples of fold nappes and constitute the so-called infra-Helvetic complex in Western and Central Switzerland, respectively. This complex is overridden by thrust sheets such as the Diablerets and Wildhörn nappes in Western Switzerland. One of the most famous example of thrust sheets worldwide is the Glariis thrust sheet in Central Switzerland which features over 35 kilometers of thrusting which are accommodated by a ~1 m thick shear zone. Since the works of the early Alpine geologist such as Heim and Lugeon, the knowledge of these nappes has been steadily refined and today the geometry and kinematics of the Helvetic nappe system is generally agreed upon. However, despite the extensive knowledge we have today of the kinematics of fold nappes and thrust sheets, the mechanical process leading to the emplacement of these nappe is still poorly understood. For a long time geologist were facing the so-called 'mechanical paradox' which arises from the fact that a block of rock several kilometers high and tens of kilometers long (i.e. nappe) would break internally rather than start moving on a low angle plane. Several solutions were proposed to solve this apparent paradox. Certainly the most successful is the theory of critical wedges (e.g. Chappie 1978; Dahlen, 1984). In this theory the orogen is considered as a whole and this change of scale allows thrust sheet like structures to form while being consistent with mechanics. However this theoiy is intricately linked to brittle rheology and fold nappes, which are inherently ductile structures, cannot be created in these models. When considering the problem of nappe emplacement from the perspective of ductile rheology the problem of strain localization arises. The aim of this thesis was to develop and apply models based on continuum mechanics and integrating heat transfer to understand the emplacement of nappes. Models were solved either analytically or numerically. In the first two papers of this thesis we derived a simple model which describes channel flow in a homogeneous material with temperature dependent viscosity. We applied this model to the Morcles fold nappe and to several kilometer-scale shear zones worldwide. In the last paper we zoomed out and studied the tectonics of (i) ductile and (ii) visco-elasto-plastic and temperature dependent wedges. In this last paper we focused on the relationship between basement and cover deformation. We demonstrated that during the compression of a ductile passive margin both fold nappes and thrust sheets can develop and that these apparently different structures constitute two end-members of a single structure (i.e. nappe). The transition from fold nappe to thrust sheet is to first order controlled by the deformation of the basement. -- Le système des nappes helvétiques en Suisse occidentale est un empilement de nappes de plis et de nappes de charriage qui se sont mis en place à faible grade métamorphique. Les nappes de plis et les nappes de charriage sont parmi les objets géologiques les plus communs dans les orogènes. Les nappes de plis sont des plis couchés d'échelle kilométrique caractérisés par un flanc normal faiblement défor-mé, au contraire de leur flanc inverse, intensément déformé. Les nappes de charriage, à l'inverse se caractérisent par l'absence d'un flanc inverse bien défini. Elles peuvent être définies comme des blocs de croûte terrestre qui se déplacent de manière presque rigide qui sont déplacés sub-horizontalement jusqu'à plusieurs dizaines de kilomètres. La nappe de Mordes et la nappe du Doldenhorn sont des exemples classiques de nappes de plis et constitue le complexe infra-helvétique en Suisse occidentale et centrale, respectivement. Ce complexe repose sous des nappes de charriages telles les nappes des Diablerets et du Widlhörn en Suisse occidentale. La nappe du Glariis en Suisse centrale se distingue par un déplacement de plus de 35 kilomètres qui s'est effectué à la faveur d'une zone de cisaillement basale épaisse de seulement 1 mètre. Aujourd'hui la géométrie et la cinématique des nappes alpines fait l'objet d'un consensus général. Malgré cela, les processus mécaniques par lesquels ces nappes se sont mises en place restent mal compris. Pendant toute la première moitié du vingtième siècle les géologues les géologues ont été confrontés au «paradoxe mécanique». Celui-ci survient du fait qu'un bloc de roche haut de plusieurs kilomètres et long de plusieurs dizaines de kilomètres (i.e., une nappe) se fracturera de l'intérieur plutôt que de se déplacer sur une surface frictionnelle. Plusieurs solutions ont été proposées pour contourner cet apparent paradoxe. La solution la plus populaire est la théorie des prismes d'accrétion critiques (par exemple Chappie, 1978 ; Dahlen, 1984). Dans le cadre de cette théorie l'orogène est considéré dans son ensemble et ce simple changement d'échelle solutionne le paradoxe mécanique (la fracturation interne de l'orogène correspond aux nappes). Cette théorie est étroitement lié à la rhéologie cassante et par conséquent des nappes de plis ne peuvent pas créer au sein d'un prisme critique. Le but de cette thèse était de développer et d'appliquer des modèles basés sur la théorie de la méca-nique des milieux continus et sur les transferts de chaleur pour comprendre l'emplacement des nappes. Ces modèles ont été solutionnés de manière analytique ou numérique. Dans les deux premiers articles présentés dans ce mémoire nous avons dérivé un modèle d'écoulement dans un chenal d'un matériel homogène dont la viscosité dépend de la température. Nous avons appliqué ce modèle à la nappe de Mordes et à plusieurs zone de cisaillement d'échelle kilométrique provenant de différents orogènes a travers le monde. Dans le dernier article nous avons considéré le problème à l'échelle de l'orogène et avons étudié la tectonique de prismes (i) ductiles, et (ii) visco-élasto-plastiques en considérant les transferts de chaleur. Nous avons démontré que durant la compression d'une marge passive ductile, a la fois des nappes de plis et des nappes de charriages peuvent se développer. Nous avons aussi démontré que nappes de plis et de charriages sont deux cas extrêmes d'une même structure (i.e. nappe) La transition entre le développement d'une nappe de pli ou d'une nappe de charriage est contrôlé au premier ordre par la déformation du socle. -- Le système des nappes helvétiques en Suisse occidentale est un emblement de nappes de plis et de nappes de chaînage qui se sont mis en place à faible grade métamoiphique. Les nappes de plis et les nappes de charriage sont parmi les objets géologiques les plus communs dans les orogènes. Les nappes de plis sont des plis couchés d'échelle kilométrique caractérisés par un flanc normal faiblement déformé, au contraire de leur flanc inverse, intensément déformé. Les nappes de charriage, à l'inverse se caractérisent par l'absence d'un flanc inverse bien défini. Elles peuvent être définies comme des blocs de croûte terrestre qui se déplacent de manière presque rigide qui sont déplacés sub-horizontalement jusqu'à plusieurs dizaines de kilomètres. La nappe de Morcles and la nappe du Doldenhorn sont des exemples classiques de nappes de plis et constitue le complexe infra-helvétique en Suisse occidentale et centrale, respectivement. Ce complexe repose sous des nappes de charriages telles les nappes des Diablerets et du Widlhörn en Suisse occidentale. La nappe du Glarüs en Suisse centrale est certainement l'exemple de nappe de charriage le plus célèbre au monde. Elle se distingue par un déplacement de plus de 35 kilomètres qui s'est effectué à la faveur d'une zone de cisaillement basale épaisse de seulement 1 mètre. La géométrie et la cinématique des nappes alpines fait l'objet d'un consensus général parmi les géologues. Au contraire les processus physiques par lesquels ces nappes sont mises en place reste mal compris. Les sédiments qui forment les nappes alpines se sont déposés à l'ère secondaire et à l'ère tertiaire sur le socle de la marge européenne qui a été étiré durant l'ouverture de l'océan Téthys. Lors de la fermeture de la Téthys, qui donnera naissance aux Alpes, le socle et les sédiments de la marge européenne ont été déformés pour former les nappes alpines. Le but de cette thèse était de développer et d'appliquer des modèles basés sur la théorie de la mécanique des milieux continus et sur les transferts de chaleur pour comprendre l'emplacement des nappes. Ces modèles ont été solutionnés de manière analytique ou numérique. Dans les deux premiers articles présentés dans ce mémoire nous nous sommes intéressés à la localisation de la déformation à l'échelle d'une nappe. Nous avons appliqué le modèle développé à la nappe de Morcles et à plusieurs zones de cisaillement provenant de différents orogènes à travers le monde. Dans le dernier article nous avons étudié la relation entre la déformation du socle et la défonnation des sédiments. Nous avons démontré que nappe de plis et nappes de charriages constituent les cas extrêmes d'un continuum. La transition entre nappe de pli et nappe de charriage est intrinsèquement lié à la déformation du socle sur lequel les sédiments reposent.

Biotic Factors Affecting Expression of the 2,4-Diacetylphloroglucinol Biosynthesis Gene phlA in Pseudomonas fluorescens Biocontrol Strain CHA0 in the Rhizosphere.

ABSTRACT Production of the polyketide antimicrobial metabolite 2,4-diacetyl-phloroglucinol (DAPG) is a key factor in the biocontrol activity of Pseudomonas fluorescens CHA0. Strain CHA0 carrying a translational phlA'-'lacZ fusion was used to monitor expression of the phl biosynthetic genes in vitro and in the rhizosphere. Expression of the reporter gene accurately reflected actual production of DAPG in vitro and in planta as determined by direct extraction of the antimicrobial compound. In a gnotobiotic system containing a clay and sand-based artificial soil, reporter gene expression was significantly greater in the rhizospheres of two monocots (maize and wheat) compared with gene expression in the rhizospheres of two dicots (bean and cucumber). We observed this host genotype effect on bacterial gene expression also at the level of cultivars. Significant differences were found among six additional maize cultivars tested under gnotobiotic conditions. There was no difference between transgenic maize expressing the Bacillus thuringiensis insecticidal gene cry1Ab and the near-isogenic parent line. Plant age had a significant impact on gene expression. Using maize as a model, expression of the phlA'-'lacZ reporter gene peaked at 24 h after planting of pregerminated seedlings, and dropped to a fourth of that value within 48 h, remaining at that level throughout 22 days of plant growth. Root infection by Pythium ultimum stimulated bacterial gene expression on both cucumber and maize, and this was independent of differences in rhizosphere colonization on these host plants. To our knowledge, this is the first comprehensive evaluation of how biotic factors that commonly confront bacterial inoculants in agricultural systems (host genotype, host age, and pathogen infection) modulate the expression of key biocontrol genes for disease suppression.

Intravenous lacosamide for treatment of status epilepticus.

Objectives -  Treatment of established status epilepticus (SE) requires immediate intravenous anticonvulsant therapy. Currently used first-line drugs may cause potentially hazardous side effects. We aimed to assess the efficacy and safety of intravenous lacosamide (LCM) in SE after failure of standard treatment. Methods -  We retrospectively analyzed 39 patients (21 women, 18 men, median age 62 years) from the hospital databases of five neurological departments in Germany, Austria and Switzerland between September 2008 and January 2010 who were admitted in SE and received at least one dose of intravenous LCM. Results -  Types of SE were generalized convulsive (n = 6), complex partial (n = 17) and simple partial (n = 16). LCM was administered after failure of benzodiazepins or other standard drugs in all but one case. Median bolus dose of LCM was 400 mg (range 200-400 mg), which was administered at 40-80 mg/min in those patients where infusion rate was documented. SE stopped after LCM in 17 patients, while 22 patients needed further anticonvulsant treatment. The success rate in patients receiving LCM as first or second drug was 3/5, as third drug 11/19, and as fourth or later drug 3/15. In five subjects, SE could not be terminated at all. No serious adverse events attributed to LCM were documented. Conclusions -  Intravenous LCM may be an alternative treatment for established SE after failure of standard therapy, or when standard agents are considered unsuitable.

Ictal cerebral positron emission tomography (PET) in focal status epilepticus.

The diagnosis of focal status epilepticus (SE) can be challenging, particularly when clinical manifestations leave doubts about its nature, and electroencephalography (EEG) is not conclusive. This work addresses the utility of ictal (18)F-fluorodeoxyglucose ((18)F-FDG) positron emission tomography (PET) in focal SE, which was performed in eight patients in whom SE was finally diagnosed. Clinical, MRI and EEG data were reviewed. (18)F-FDG-PET proved useful: (1) to establish the diagnosis of focal SE, when clinical elements were equivocal or the EEG did not show clear-cut epileptiform abnormalities; (2) to delineate the epileptogenic area in view of possible resective surgery; and (3) when clinical features, MRI and EEG were incongruent regarding the origin of SE. We suggest that ictal (18)F-FDG-PET may represent a valuable diagnostic tool in selected patients with focal SE or frequent focal seizures.

The presence of a CD4+ CD25high CD45RO+ CD127high T-cell population correlates with the clinical status of kidney transplant recipients

Background: Recent data have suggested that a population of CD4+ CD25high T cells, phenotypically characterized by the expression of CD45RO and CD127, is significantly expanded in stable liver and kidney transplant recipients and represents alloreactive T cells. We analyzed this putative new alloreactive cellular marker in various groups of kidney transplant recipients. Patients and methods: Flow cytometry was used to analyze the expression of CD25, CD45RO and CD127 on peripheral CD4+ T cells. Of 73 kidney recipients, 59 had a stable graft function under standard immunosuppressive therapy (IS), 5 had biopsy-proven chronic humoral rejection (CHR), 8 were stable under minimal IS and one was an operationally "tolerant" patient who had discontinued IS for more than 3 years. Sixty-six healthy subjects (HS) were studied as controls. Results: Overall, the alloreactive T cell population was found to be significantly increased in the 73 kidney recipients (mean ± SE: 15.03 ± 1.04% of CD4+ CD25high T cells) compared to HS (5.93 ± 0.39%) (p <0.001). In the 5 patients with CHR, this population was highly expanded (31.33 ± 4.16%), whereas it was comparable to HS in the 8 stable recipients receiving minimal IS (6.12 ± 0.86%), in 4 patients who had been switched to sirolimus (4.21 ± 0.53%) as well as in the unique "tolerant" recipient (4.69%). Intermediate levels (15.84 ± 0.93%) were found in the 55 recipients with stable graft function on standard CNI-based IS. Regulatory T cells, defined as CD4+ CD25high FoxP3+ CD127low, were found to be significantly reduced in all recipients except in those with minimal or no IS, and this reduction was particularly striking in recipients with CHR. Conclusion: After kidney transplantation, an alloreactive T cell population was found to be significantly expanded and it correlates with the clinical status of the recipients. Interestingly, in stable patients with minimal (or no) IS as well as in patients on sirolimus, alloreactive T cells were comparable the healthy controls. Measuring circulating CD4+ CD25high CD45RO+ CD127high T cells may become a useful monitoring tool after transplantation.

Impact of biocontrol strain Pseudomonas fluorescens CHA0 on rhizosphere bacteria isolated from barley (Hordeum vulgare L.) with special reference to Cytophaga-like bacteria.

AIMS: To assess the impact of the biocontrol strain Pseudomonas fluorescens CHA0 on a collection of barley rhizosphere bacteria using an agar plate inhibition assay and a plant microcosm, focusing on a CHA0-sensitive member of the Cytophaga-like bacteria (CLB). METHODS AND RESULTS: The effect of strain CHA0 on a collection of barley rhizosphere bacteria, in particular CLB and fluorescent pseudomonads sampled during a growth season, was assessed by a growth inhibition assay. On average, 85% of the bacteria were sensitive in the May sample, while the effect was reduced to around 68% in the July and August samples. In the May sample, around 95% of the CLB and around 45% of the fluorescent pseudomonads were sensitive to strain CHA0. The proportion of CHA0-sensitive CLB and fluorescent pseudomonad isolates decreased during the plant growth season, i.e. in the July and August samples. A particularly sensitive CLB isolate, CLB23, was selected, exposed to strain CHA0 (wild type) and its genetically modified derivatives in the rhizosphere of barley grown in gnotobiotic soil microcosms. Two dry-stress periods were imposed during the experiment. Derivatives of strain CHA0 included antibiotic or exopolysaccharide (EPS) overproducing strains and a dry-stress-sensitive mutant. Despite their inhibitory activity against CLB23 in vitro, neither wild-type strain CHA0, nor any of its derivatives, had a major effect on culturable and total cell numbers of CLB23 during the 23-day microcosm experiment. Populations of all inoculants declined during the two dry-stress periods, with soil water contents below 5% and plants reaching the wilting point, but they recovered after re-wetting the soil. Survival of the dry-stress-sensitive mutant of CHA0 was most affected by the dry periods; however, this did not result in an increased population density of CLB23. CONCLUSIONS: CLB comprise a large fraction of barley rhizosphere bacteria that are sensitive to the biocontrol pseudomonad CHA0 in vitro. However, in plant microcosm experiments with varying soil humidity conditions, CHA0 or its derivatives had no major impact on the survival of the highly sensitive CLB strain, CLB23, during two dry-stress periods and a re-wetting period; all co-existed well in the rhizosphere of barley plants. SIGNIFICANCE AND IMPACT OF THE STUDY: Results indicate a lack of interaction between the biocontrol pseudomonad CHA0 and a sensitive CLB when the complexity increases from agar plate assays to plant microcosm experiments. This suggests the occurrence of low levels of antibiotic production and/or that the two bacterial genera occupy different niches in the rhizosphere.

Stroke initial severity and outcome relative to insurance status in a universal health care system in Switzerland.

BACKGROUND:  Socioeconomic status is thought to have a significant influence on stroke incidence, risk factors and outcome. Its influence on acute stroke severity, stroke mechanisms, and acute recanalisation treatment is less known. METHODS:  Over a 4-year period, all ischaemic stroke patients admitted within 24 h were entered prospectively in a stroke registry. Data included insurance status, demographics, risk factors, time to hospital arrival, initial stroke severity (NIHSS), etiology, use of acute treatments, short-term outcome (modified Rankin Scale, mRS). Private insured patients (PI) were compared with basic insured patients (BI). RESULTS:  Of 1062 consecutive acute ischaemic stroke patients, 203 had PI and 859 had BI. They were 585 men and 477 women. Both populations were similar in age, cardiovascular risk factors and preventive medications. The onset to admission time, thrombolysis rate, and stroke etiology according to TOAST classification were not different between PI and BI. Mean NIHSS at admission was significantly higher for BI. Good outcome (mRS ≤ 2) at 7 days and 3 months was more frequent in PI than in BI. CONCLUSION:  We found better outcome and lesser stroke severity on admission in patients with higher socioeconomic status in an acute stroke population. The reason for milder strokes in patients with better socioeconomic status in a universal health care system needs to be explained.

Intravenous Lacosamide for Treatment of Status Epilepticus after Failing First-Line Therapy

Rationale: Treatment of status epilepticus (SE) usually requires intravenous anticonvulsant therapy. Although there are established drugs of first choice for its treatment, potentially hazardous side effects of these agents are not uncommon. Lacosamide (LCM) is a novel anticonvulsant drug that is available as infusion solution. LCM could be an alternative for treatment of SE when the standard drugs fail or should be avoided. Methods: We retrospectively identified patients from the hospital databases of two German and one Swiss neurological departments (University Hospital Marburg, Klinikum Osnabrueck, University Hospital Lausanne) between September 1st 2008 and May 22nd 2009 who were admitted because of SE and received at least one dose of intravenous LCM for treatment of SE. Results: Seventeen patients (11 female, 6 male) were identified. Median age was 71 years. 3 patients suffered from generalized convulsive SE, 8 patients had significant reduction of awareness with or without subtle motor symptoms, 6 patients had a simple focal status without relevant reduction of awareness. Etiology was acute symptomatic in 5 patients, remote symptomatic without pre-existing epilepsy in 6 patients, remote symptomatic and pre-existing epilepsy in 5 patients, and unknown in 1 patient. LCM was administered after failure of first line therapy in all cases. The first LCM bolus was 400mg in 13 patients and 200mg in 4 patients. LCM administration stopped SE in 7 patients. In 2 of them, LCM was administered immediately after benzodiazepine administration, in the others after failure of benzodiazepines and other first-line and/or second-line drugs. In 3 patients, SE was terminated by other anticonvulsants like Phenytoin, Phenobarbital or Oxcarbazepine. In 5 patients, SE could only be terminated by intubation and application of high-dose Midazolam, Propofol and/or Thiopental. In 2 patients, SE could not be terminated in spite of high doses of barbiturates. There was no serious adverse event documented that could possibly be attributed to LCM Conclusions: Intravenous LCM may be an alternative treatment for SE after failure of benzodiazepins and other established drugs, or when such agents are considered unsuitable.

Characterization of a new clinical yeast species, Candida tunisiensis sp. nov., isolated from a strain collection from Tunisian hospitals.

From a collection of yeast isolates isolated from patients in Tunisian hospitals between September 2006 and July 2010, the yeast strain JEY63 (CBS 12513), isolated from a 50-year-old male that suffered from oral thrush, could not be identified to the species level using conventional methods used in clinical laboratories. These methods include matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), germ tube formation, and the use of CHROMagar Candida and metabolic galleries. Sequence analysis of the nuclear rRNA (18S rRNA, 5.8S rRNA, and 26S rRNA) and internal transcribed spacer regions (ITS1 and ITS2) indicated that the ribosomal DNA sequences of this species were not yet reported. Multiple gene phylogenic analyses suggested that this isolate clustered at the base of the Dipodascaceae (Saccharomycetales, Saccharomycetes, and Ascomycota). JEY63 was named Candida tunisiensis sp. nov. according to several phenotypic criteria and its geographical origin. C. tunisiensis was able to grow at 42°C and does not form chlamydospores and hyphae but could grow as yeast and pseudohyphal forms. C. tunisiensis exhibited most probably a haploid genome with an estimated size of 10 Mb on at least three chromosomes. Using European Committee for Antimicrobial Susceptibility Testing (EUCAST) and Clinical and Laboratory Standards Institute (CLSI) Candida albicans susceptibility breakpoints as a reference, C. tunisiensis was resistant to fluconazole (MIC = 8 μg/ml), voriconazole (MIC = 0.5 μg/ml), itraconazole (MIC = 16 μg/ml), and amphotericin B (MIC = 4 μg/ml) but still susceptible to posaconazole (MIC = 0.008 μg/ml) and caspofungin (MIC = 0.5 μg/ml). In conclusion, MALDI-TOF MS permitted the early selection of an unusual isolate, which was still unreported in molecular databases but could not be unambiguously classified based on phylogenetic approaches.