β-Glucan Antigenemia Anticipates Diagnosis of Blood Culture-Negative Intraabdominal Candidiasis.

Rationale: Life-threatening intraabdominal candidiasis (IAC) occurs in 30 to 40% of high-risk surgical intensive care unit (ICU) patients. Although early IAC diagnosis is crucial, blood cultures are negative, and the role of Candida score/colonization indexes is not established. Objectives: The aim of this prospective Fungal Infection Network of Switzerland (FUNGINOS) cohort study was to assess accuracy of 1,3-β-d-glucan (BG) antigenemia for diagnosis of IAC. Methods: Four hundred thirty-four consecutive adults with abdominal surgery or acute pancreatitis and ICU stay 72 hours or longer were screened: 89 (20.5%) at high risk for IAC were studied (68 recurrent gastrointestinal tract perforation, 21 acute necrotizing pancreatitis). Diagnostic accuracy of serum BG (Fungitell), Candida score, and colonization indexes was compared. Measurements and Main Results: Fifty-eight of 89 (65%) patients were colonized by Candida; 29 of 89 (33%) presented IAC (27 of 29 with negative blood cultures). Nine hundred twenty-one sera were analyzed (9/patient): median BG was 253 pg/ml (46-9,557) in IAC versus 99 pg/ml (8-440) in colonization (P < 0.01). Sensitivity and specificity of two consecutive BG measurements greater than or equal to 80 pg/ml were 65 and 78%, respectively. In recurrent gastrointestinal tract perforation it was 75 and 77% versus 90 and 38% (Candida score ≥ 3), 79 and 34% (colonization index ≥ 0.5), and 54 and 63% (corrected colonization index ≥ 0.4), respectively. BG positivity anticipated IAC diagnosis (5 d) and antifungal therapy (6 d). Severe sepsis/septic shock and death occurred in 10 of 11 (91%) and 4 of 11 (36%) patients with BG 400 pg/ml or more versus 5 of 18 (28%, P = 0.002) and 1 of 18 (6%, P = 0.05) with BG measurement less than 400 pg/ml. β-Glucan decreased in IAC responding to therapy and increased in nonresponse. Conclusions: BG antigenemia is superior to Candida score and colonization indexes and anticipates diagnosis of blood culture-negative IAC. This proof-of-concept observation in strictly selected high-risk surgical ICU patients deserves investigation of BG-driven preemptive therapy.

Cell-free reconstitution of vacuole membrane fragmentation reveals regulation of vacuole size and number by TORC1.

Size and copy number of organelles are influenced by an equilibrium of membrane fusion and fission. We studied this equilibrium on vacuoles-the lysosomes of yeast. Vacuole fusion can readily be reconstituted and quantified in vitro, but it had not been possible to study fission of the organelle in a similar way. Here we present a cell-free system that reconstitutes fragmentation of purified yeast vacuoles (lysosomes) into smaller vesicles. Fragmentation in vitro reproduces physiological aspects. It requires the dynamin-like GTPase Vps1p, V-ATPase pump activity, cytosolic proteins, and ATP and GTP hydrolysis. We used the in vitro system to show that the vacuole-associated TOR complex 1 (TORC1) stimulates vacuole fragmentation but not the opposing reaction of vacuole fusion. Under nutrient restriction, TORC1 is inactivated, and the continuing fusion activity then dominates the fusion/fission equilibrium, decreasing the copy number and increasing the volume of the vacuolar compartment. This result can explain why nutrient restriction not only induces autophagy and a massive buildup of vacuolar/lysosomal hydrolases, but also leads to a concomitant increase in volume of the vacuolar compartment by coalescence of the organelles into a single large compartment.

Efficacy of daptomycin in the treatment of experimental endocarditis due to susceptible and multidrug-resistant enterococci.

OBJECTIVES: Daptomycin was tested in vitro and in rats with experimental endocarditis against the ampicillin-susceptible and vancomycin-susceptible Enterococcus faecalis JH2-2, the vancomycin-resistant (VanA type) mutant of strain JH2-2 (strain JH2-2/pIP819), and the ampicillin-resistant and vancomycin-resistant (VanB type) Enterococcus faecium D366. METHODS: Rats with catheter-induced aortic vegetations were treated with doses simulating intravenously kinetics in humans of daptomycin (6 mg/kg every 24 h), amoxicillin (2 g every 6 h), vancomycin (1 g every 12 h) or teicoplanin (12 mg/kg every 12 h). Treatment was started 16 h post-inoculation and continued for 2 days. RESULTS: MICs of daptomycin were 1, 1 and 2 mg/L, respectively, for strains JH2-2, JH2-2/pIP819 and D366. In time-kill studies, daptomycin showed rapid (within 2 h) bactericidal activity against all strains. Daptomycin was highly bound to rat serum proteins (89%). In the presence of 50% rat serum, simulating free concentrations, daptomycin killing was maintained but delayed (6-24 h). In vivo, daptomycin treatment resulted in 10 of 12 (83%), 9 of 11 (82%) and 11 of 12 (91%) culture-negative vegetations in rats infected with strains JH2-2, JH2-2/pIP819 and D366, respectively (P < 0.001 compared to controls). Daptomycin efficacy was comparable to that of amoxicillin and vancomycin for susceptible isolates. Daptomycin, however, was significantly (P < 0.05) more effective than teicoplanin against the glycopeptide-susceptible strain JH2-2 and superior to all comparators against resistant isolates. CONCLUSIONS: These results support the use of the newly proposed daptomycin dose of 6 mg/kg every 24 h for treatment of enterococcal infections in humans.

Propagation velocity kinetics and repolarization alternans in a free-behaving sheep model of pacing-induced atrial fibrillation.

AIMS: Experimental models have reported conflicting results regarding the role of dispersion of repolarization in promoting atrial fibrillation (AF). Repolarization alternans, a beat-to-beat alternation in action potential duration, enhances dispersion of repolarization when propagation velocity is involved. METHODS AND RESULTS: In this work, original electrophysiological parameters were analysed to study AF susceptibility in a chronic sheep model of pacing-induced AF. Two pacemakers were implanted, each with a single right atrial lead. Right atrial depolarization and repolarization waves were documented at 2-week intervals. A significant and gradual decrease in the propagation velocity at all pacing rates and in the right atrial effective refractory period (ERP) was observed during the weeks of burst pacing before sustained AF developed when compared with baseline conditions. Right atrial repolarization alternans was observed, but because of the development of 2/1 atrioventricular block with far-field ventricular interference, its threshold could not be precisely measured. Non-sustained AF was not observed at baseline, but appeared during the electrical remodelling in association with a decrease in both ERP and propagation velocity. CONCLUSION: We report here on the feasibility of measuring ERP, atrial repolarization alternans, and propagation velocity kinetics and their potential in predicting susceptibility to AF in a free-behaving model of pacing-induced AF using the standard pacemaker technology.

Genome reconstruction and comparative genomics of pneumocystis jirovecii

Pneumocystis jirovecii is a fungus belonging to a basal lineage of the Ascomycotina, the Taphrinomycotina subphylum. It is a parasite specific to humans that dwells primarily in the lung and can cause severe pneumonia in individuals with debilitated immune system. Despite its clinical importance, many aspects of its biology remain poorly understood, at least in part because of the lack of a continuous in vitro cultivation system. The present thesis consists in the genome reconstruction and comparative genomics of P. jirovecii. It is made of three parts: (i) the de novo sequencing of P. jirovecii genome starting from a single broncho- alveolar lavage fluid of a single patient (ii) the de novo sequencing of the genome of the plant pathogen Taphrina deformans, a fungus closely related to P. jirovecii, and (iii) the genome scale comparison of P. jirovecii to other Taphrinomycotina members. Enrichment in P. jirovecii cells by immuno-precipitation, whole DNA random amplification, two complementary high throughput DNA sequencing methods, and in silico sorting and assembly of sequences were used for the de novo reconstruction of P. jirovecii genome from the microbiota of a single clinical specimen. An iterative ad hoc pipeline as well as numerical simulations was used to recover P. jirovecii sequences while purging out contaminants and assembly or amplification chimeras. This strategy produced a 8.1 Mb assembly, which encodes 3,898 genes. Homology searches, mapping on biochemical pathways atlases, and manual validations revealed that this genome lacks (i) most of the enzymes dedicated to the amino acids biosyntheses, and (ii) most virulence factors observed in other fungi, e.g. the glyoxylate shunt pathway and specific peptidases involved in the degradation of the host cell membrane. The same analyses applied to the available genomic sequences from Pneumocystis carinii the species infecting rats and Pneumocystis murina the species infecting mice revealed the same deficiencies. The genome sequencing of T. deformans yielded a 13 Mb assembly, which encodes 5,735 genes. T. deformans possesses enzymes involved plant cell wall degradation, secondary metabolism, the glyoxylate cycle, detoxification, sterol biosynthesis, as well as the biosyntheses of plant hormones such as abscisic acid or indole-3-acetic acid. T. deformans also harbors gene subsets that have counterparts in plant saprophytes or pathogens, which is consistent with its alternate saprophytic and pathogenic lifestyles. Mating genes were also identified. The homothallism of this fungus suggests a mating-type switching mechanism. Comparative analyses indicated that 81% of P. jirovecii genes are shared with eight other Taphrinomycotina members, including T. deformans, P. carinii and P. murina. These genes are mostly involved in housekeeping activities. The genes specific to the Pneumocystis genus represent 8%, and are involved in RNA metabolism and signaling. The signaling is known to be crucial for interaction of Pneumocystis spp with their environment. Eleven percent are unique to P. jirovecii and encode mostly proteins of unknown function. These genes in conjunction with other ones (e.g. the major surface glycoproteins) might govern the interaction of P. jirovecii with its human host cells, and potentially be responsible of the host specificity. P. jirovecii exhibits a reduced genome in size with a low GC content, and most probably scavenges vital compounds such as amino acids and cholesterol from human lungs. Consistently, its genome encodes a large set of transporters (ca. 22% of its genes), which may play a pivotal role in the acquisition of these compounds. All these features are generally observed in obligate parasite of various kingdoms (bacteria, protozoa, fungi). Moreover, epidemiological studies failed to evidence a free-living form of the fungus and Pneumocystis spp were shown to co-evolved with their hosts. Given also the lack of virulence factors, our observations strongly suggest that P. jirovecii is an obligate parasite specialized in the colonization of human lungs, and which causes disease only in individuals with compromised immune system. The same conclusion is most likely true for all other Pneumocystis spp in their respective mammalian host. - Pneumocystis jirovecii est un champignon appartenant à ine branche basale des Ascomycotina, le sous-embranchement des Taphrinomycotina. C'est un parasite spécifique aux humains qui réside principalement dans les poumons, et qui peut causer des pneumonies sévères chez des individus ayant un système immunitaire déficient. En dépit de son importance clinique, de nombreux aspects de sa biologie demeurent,largement méconnus, au moins en partie à cause de l'absence d'un système de culture in vitro continu. Cette thèse traite de la reconstruction du génome et de la génomique comparative de P. jirovecii. Elle comporte trois parties: (i) le séquençage de novo du génome de P. jirovecii à partir d'un lavage broncho-alvéolaire provenant d'un seul patient, (ii) le séquençage de novo du génome d'un champignon pathogène de plante Taphrina deformans qui est phylogénétiquement proche de P. jirovecii, et (iii) la comparaison du génome de P. jirovecii à celui d'autres membres du sous-embranchement des Taphrinomycotina. Un enrichissement en cellules de P. jirovecii par immuno-précipitation, une amplification aléatoire des molécules d'ADN, deux méthodes complémentaires de séquençage à haut débit, un tri in silico et un assemblage des séquences ont été utilisés pour reconstruire de novo le génome de P. jirovecii à partir du microbiote d'un seul échantillon clinique. Un pipeline spécifique ainsi que des simulations numériques ont été utilisés pour récupérer les séquences de P. jirovecii tout en éliminant les séquences contaminants et les chimères d'amplification ou d'assemblage. Cette stratégie a produit un assemblage de 8.1 Mb, qui contient 3898 gènes. Les recherches d'homologies, de cartographie des voies métaboliques et des validations manuelles ont révélé que ce génome est dépourvu (i) de la plupart des enzymes dédiées à la biosynthèse des acides aminés, et (ii) de la plupart des facteurs de virulence observés chez d'autres champignons, par exemple, le cycle du glyoxylate ainsi que des peptidases spécifiques impliquées dans la dégradation de la membrane de la cellule hôte. Les analyses appliquées aux données génomiques disponibles de Pneumocystis carinii, l'espèce infectant les rats, et de Pneumocystis murina, l'espèce infectant les souris, ont révélé les mêmes déficiences. Le séquençage du génome de T. deformans a généré un assemblage de 13.3 Mb qui contient 5735 gènes. T. deformans possède les gènes codant pour les enzymes impliquées dans la dégradation des parois cellulaires des plantes, le métabolisme secondaire, le cycle du glyoxylate, la détoxification, la biosynthèse des stérols ainsi que la biosynthèse d'hormones de plantes telles que l'acide abscissique ou l'acide indole 3-acétique. T. deformans possède également des sous-ensembles de gènes présents exclusivement chez des saprophytes ou des pathogènes de plantes, ce qui est consistent avec son mode de vie alternatif saprophyte et pathogène. Des gènes impliqués dans la conjugaison ont été identifiés. L'homothallisme de ce champignon suggère mécanisme de permutation du type conjuguant. Les analyses comparatives ont démontré que 81% des gènes de P. jirovecii sont présent chez les autres membres du sous-embranchement des Taphrinomycotina. Ces gènes sont essentiellement impliqués dans le métabolisme basai. Les gènes spécifiques au genre Pneumocystis représentent 8%, et sont impliqués dans le métabolisme de l'ARN et la signalisation. La signalisation est connue pour être cruciale pour l'interaction des espèces de Pneumocystis avec leur environnement. Les gènes propres à P. jirovecii représentent 11% et codent en majorité pour des protéines dont la fonction est inconnue. Ces gènes en conjonction avec d'autres (par exemple, les glycoprotéines de surface), pourraient être déterminants dans l'interaction de P. jirovecii avec les cellules de l'hôte humain, et être potentiellement responsable de la spécificité d'hôte. P. jirovecii possède un génome de taille réduite à faible pourcentage en GC et récupère très probablement des composés vitaux comme les acides aminés et le cholestérol à partir des poumons humains. De manière consistante, son génome code pour de nombreux transporteurs (22% de ses gènes), qui pourraient jouer un rôle essentiel dans l'acquisition de ces composés. Ces caractéristiques sont généralement observées chez les parasites obligatoires de plusieurs règnes (bactéries, protozoaires, champignons). De plus, les études épidémiologiques n'ont pas réussi à prouver l'existence d'ime forme vivant librement du champignon. Etant donné également l'absence de facteurs de virulence, nos observations suggèrent que P. jirovecii est un parasite obligatoire spécialisé dans la colonisation des poumons humains, ne causant une maladie que chez des individus ayant un système immunitaire compromis. La même conclusion est très probablement applicable à toutes les autres espèces de Pneumocystis dans leur hôte mammifère respectif.

Temporal diffeomorphic free-form deformation: application to motion and strain estimation from 3D echocardiography

This paper presents a new registration algorithm, called Temporal Di eomorphic Free Form Deformation (TDFFD), and its application to motion and strain quanti cation from a sequence of 3D ultrasound (US) images. The originality of our approach resides in enforcing time consistency by representing the 4D velocity eld as the sum of continuous spatiotemporal B-Spline kernels. The spatiotemporal displacement eld is then recovered through forward Eulerian integration of the non-stationary velocity eld. The strain tensor iscomputed locally using the spatial derivatives of the reconstructed displacement eld. The energy functional considered in this paper weighs two terms: the image similarity and a regularization term. The image similarity metric is the sum of squared di erences between the intensities of each frame and a reference one. Any frame in the sequence can be chosen as reference. The regularization term is based on theincompressibility of myocardial tissue. TDFFD was compared to pairwise 3D FFD and 3D+t FFD, bothon displacement and velocity elds, on a set of synthetic 3D US images with di erent noise levels. TDFFDshowed increased robustness to noise compared to these two state-of-the-art algorithms. TDFFD also proved to be more resistant to a reduced temporal resolution when decimating this synthetic sequence. Finally, this synthetic dataset was used to determine optimal settings of the TDFFD algorithm. Subsequently, TDFFDwas applied to a database of cardiac 3D US images of the left ventricle acquired from 9 healthy volunteers and 13 patients treated by Cardiac Resynchronization Therapy (CRT). On healthy cases, uniform strain patterns were observed over all myocardial segments, as physiologically expected. On all CRT patients, theimprovement in synchrony of regional longitudinal strain correlated with CRT clinical outcome as quanti ed by the reduction of end-systolic left ventricular volume at follow-up (6 and 12 months), showing the potential of the proposed algorithm for the assessment of CRT.

Free-and open source software for a course on network management: authoring and enactment of scripts based on collaborative learning strategies

This paper describes a Computer-Supported Collaborative Learning (CSCL) case study in engineering education carried out within the context of a network management course. The case study shows that the use of two computing tools developed by the authors and based on Free- and Open-Source Software (FOSS) provide significant educational benefits over traditional engineering pedagogical approaches in terms of both concepts and engineering competencies acquisition. First, the Collage authoring tool guides and supports the course teacher in the process of authoring computer-interpretable representations (using the IMS Learning Design standard notation) of effective collaborative pedagogical designs. Besides, the Gridcole system supports the enactment of that design by guiding the students throughout the prescribed sequence of learning activities. The paper introduces the goals and context of the case study, elaborates onhow Collage and Gridcole were employed, describes the applied evaluation methodology, anddiscusses the most significant findings derived from the case study.

Contrast agent-enhanced, free-breathing, three-dimensional coronary magnetic resonance angiography.

For free-breathing, high-resolution, three-dimensional coronary magnetic resonance angiography (MRA), the use of intravascular contrast agents may be helpful for contrast enhancement between coronary blood and myocardium. In six patients, 0.1 mmol/kg of the intravascular contrast agent MS-325/AngioMARK was given intravenously followed by double-oblique, free-breathing, three-dimensional inversion-recovery coronary MRA with real-time navigator gating and motion correction. Contrast-enhanced, three-dimensional coronary MRA images were compared with images obtained with a T2 prepulse (T2Prep) without exogenous contrast. The contrast-enhanced images demonstrated a 69% improvement in the contrast-to-noise ratio (6.6 +/- 1.1 vs. 11.1 +/- 2.5; P < 0.01) compared with the T2Prep approach. By using the intravascular agent, extensive portions (> 80 mm) of the native left and right coronary system could be displayed consistently with sub-millimeter in-plane resolution. The intravascular contrast agent, MS-325/AngioMARK, leads to a considerable enhancement of the blood/muscle contrast for coronary MRA compared with T2Prep techniques. The clinical value of the agent remains to be defined in a larger patient series. J. Magn. Reson. Imaging 1999;10:790-799.

Chlamydiaceae and Chlamydia-like organisms in free-living small mammals in Europe and Afghanistan.

Few data are available on the occurrence of chlamydial infections in wild small mammals. We investigated the significance of free-living small mammals as reservoirs or transmission hosts for microorganisms of the phylum/class Chlamydiae. We obtained 3,664 tissue samples from 911 animals in Switzerland, Germany, Austria, the Czech Republic, and Afghanistan. Samples included internal organs (n = 3,652) and feces (n = 12) from 679 rodents (order Rodentia) and 232 insectivores (order Soricomorpha) and were tested by three TaqMan® real-time PCRs specific for members of the family Chlamydiaceae and selected Chlamydia-like organisms such as Parachlamydia spp. and Waddlia spp. Only one of 911 (0.11%) animals exhibited a questionable positive result by Chlamydiaceae-specific real-time PCR. Five of 911 animals were positive by specific real-time PCR for Parachlamydia spp. but could not be confirmed by quantitative PCR targeting the Parachlamydia acanthamoebae secY gene (secY qPCR). One of 746 animals (0.13%) was positive by real-time PCR for Waddlia chondrophila. This result was confirmed by Waddlia secY qPCR. This is the first detection of Chlamydia-like organisms in small wildlife in Switzerland. Considering previous negative results for Chlamydiaceae in wild ruminant species from Switzerland, these data suggest that wild small mammals are unlikely to be important carriers or transport hosts for Chamydiaceae and Chlamydia-like organisms.

Culture marked: racist epithets in translations of Mark Twain's Adventures of Huckleberry Finn

Aquest projecte analitza com s’ha plasmat el terme cultural nigger a dues traduccions al castellà de la novel•la Adventures of Huckleberry Finn de Mark Twain, amb l’objectiu d’estudiar de quina manera poden adaptar-se els elements culturals en una traducció. En primer lloc, s’ofereix la contextualització del terme original al text i a la cultura d’origen, seguida de la descripció del terme traduït en el context de la cultura d’arribada. A continuació, mitjançant l’anàlisi comparativa del mecanisme retòric de la repetició i d’elements com ara determinants, adjectius i complements, emprats tant a l’obra original com a les dues traduccions estudiades, aquest projecte pretén demostrar que la conservació dels esmentats recursos estilístics a la traducció pot facilitar la comprensió de les connotacions del marcador cultural nigger i de la seva funció temàtica al text d’arribada.