A dynamic view of hepatitis C virus replication complexes.

Hepatitis C virus (HCV) replicates its genome in a membrane-associated replication complex (RC). Specific membrane alterations, designated membranous webs, represent predominant sites of HCV RNA replication. The principles governing HCV RC and membranous web formation are poorly understood. Here, we used replicons harboring a green fluorescent protein (GFP) insertion in nonstructural protein 5A (NS5A) to study HCV RCs in live cells. Two distinct patterns of NS5A-GFP were observed. (i) Large structures, representing membranous webs, showed restricted motility, were stable over many hours, were partitioned among daughter cells during cell division, and displayed a static internal architecture without detectable exchange of NS5A-GFP. (ii) In contrast, small structures, presumably representing small RCs, showed fast, saltatory movements over long distances. Both populations were associated with endoplasmic reticulum (ER) tubules, but only small RCs showed ER-independent, microtubule (MT)-dependent transport. We suggest that this MT-dependent transport sustains two distinct RC populations, which are both required during the HCV life cycle.

Quality of life after pulmonary embolism: validation of the French version of the PEmb-QoL questionnaire.

Background The PEmb-QoL is a validated 40-item questionnaire to quantify health-related quality of life in patients having experienced pulmonary embolism (PE). It covers six health dimensions: frequency of complaints, activities of daily living limitations, work-related problems, social limitations, intensity of complaints, and emotional complaints. Originally developed in Dutch and English, we sought to prospectively validate the psychometric properties of a French version of the PEmb-QoL.MethodsWe performed a forward and backward translation of the English version of the PEmb-QoL into French. French-speaking consecutive adult patients with an acute, objectively confirmed PE admitted to the emergency department of a Swiss university hospital between 08/2009 and 09/2011 were recruited telephonically. We used standard psychometric tests and criteria to evaluate the acceptability, reliability, and validity of the French version of the PEmb-QoL. We also performed an exploratory factor analysis.ResultsOverall, 102 patients were enrolled in the study. The French version of the PEmb-QoL showed good reliability (internal consistency, item¿total and inter-item correlations), reproducibility (test-retest reliability), and validity (convergent, discriminant) in French-speaking patients with PE. The exploratory factor analysis suggested three underlying dimensions: limitations in daily activity (items 4b-m, 5a-d), symptoms (items 1a-h and 7), and emotional complaints (items 9a-f and j).ConclusionWe successfully validated the French version of the PEmb-QoL questionnaire in patients with PE. Our results show that the PEmb-QoL is a valuable tool for assessing health-related quality of life after PE in French-speaking patients.

SV40-induced expression of calretinin protects mesothelial cells from asbestos cytotoxicity and may be a key factor contributing to mesothelioma pathogenesis.

The calcium-binding protein calretinin has emerged as a useful marker for the identification of mesotheliomas of the epithelioid and mixed types, but its putative role in tumor development has not been addressed previously. Although exposure to asbestos fibers is considered the main cause of mesothelioma, undoubtedly, not all mesothelioma patients have a history of asbestos exposure. The question as to whether the SV40 virus is involved as a possible co-factor is still highly debated. Here we show that increased expression of SV40 early gene products in the mesothelial cell line MeT-5A induces the expression of calretinin and that elevated calretinin levels strongly correlate with increased resistance to asbestos cytotoxicity. Calretinin alone mediates a significant part of this protective effect because cells stably transfected with calretinin cDNA were clearly more resistant to the toxic effects of crocidolite than mock-transfected control cells. Down-regulation of calretinin by antisense methods restored the sensitivity to asbestos toxicity to a large degree. The protective effect observed in clones with higher calretinin expression levels could be eliminated by phosphatidylinositol 3-kinase (PI3K) inhibitors, implying an important role for the PI3K/AKT signaling (survival) pathway in mediating the protective effect. Up-regulation of calretinin, resulting from either asbestos exposure or SV40 oncoproteins, may be a common denominator that leads to increased resistance to asbestos cytotoxicity and thereby contributes to mesothelioma carcinogenesis.

Tartrate-Resistant Acid Phosphatase 5b: a Serum Marker of Bone Resorption

Bone is a physiologically dynamic tissue being constantly regenerated throughout life as a consequence of bone turnover by bone-resorbing osteoclasts and bone-forming osteoblasts. In certain bone diseases, such as osteoporosis, the imbalance in bone turnover leads to bone loss and increased fracture risk. Measurement of bone mineral density (BMD) predicts the risk of fracture, but also biochemical markers of bone metabolism have been suggested to be suitable for prediction of fractures and monitoring the efficacy of antiresorptive treatment. Tartrate-resistant acid phosphatase 5b (TRACP 5b) is an enzyme released from osteoclasts into the circulation, from where it can be detected kinetically or immunologically. Conventional assays for serum total TRACP were spectrophotometric and suffered from interference by other acid phosphatases and non-osteoclastic TRACP 5a isoform. Our aim was to develop novel immunoassays for osteoclastic TRACP 5b. Serum TRACP 5b levels were elevated in individuals with high bone turnover, such as children, postmenopausal women, patients with osteoporosis, Paget’s disease and breast cancer patients with bone metastases. As expected, hormone replacement therapy (HRT) in postmenopausal women decreased the levels of serum TRACP 5b. Surprisingly, the highest TRACP 5b levels were observed in individuals with rare autosomal dominant osteopetrosis type II (ADO2), which is characterized by high BMD and fracture risk with simultaneously elevated levels of deficient osteoclasts. In ADO2 patients, elevated levels of serum TRACP 5b were associated with high fracture frequency. It is likely that serum TRACP 5b reflects the number of inactive osteoclasts in ADO2. Similar results supporting the hypothesis that TRACP 5b would reflect the number of osteoclasts instead of their activity were observed with cultured osteoclasts and in animal models. Novel TRACP 5b immunoassays may prove to be of value either as independent or combinatory tools with other bone metabolic markers and BMD measurements in clinical practice and bone research.

Integrated Materials and Construction Practice for Concrete Pavement: A State-of-the-Practice Manual, 2006

At the heart of all concrete pavement projects is the concrete itself. This manual is intended as both a training tool and a reference to help concrete paving engineers, quality control personnel, specifiers, contractors, suppliers, technicians, and tradespeople bridge the gap between recent research and practice regarding optimizing the performance of concrete for pavements. Specifically, it will help readers do the following:

Bilateral renal artery entrapment by diaphragmatic crura: a rare cause of renovascular hypertension with a specific management.

We present the case of a young man with compression of both renal arteries by the crura of the diaphragm. Correct diagnosis of renal artery entrapment is difficult but crucial. The investigations rely on an high index of suspicion and include Doppler ultrasound and spiral computed tomography angiography, which permits visualization of the diaphragm and its relationships with the aorta. This pathology, unlike common renal artery stenoses, requires surgical decompression and sometimes aortorenal bypass graft.

Wheat germ cell-free expression: Two detergents with a low critical micelle concentration allow for production of soluble HCV membrane proteins.

Membrane proteins are notoriously difficult to express in a soluble form. Here, we use wheat germ cell-free expression in the presence of various detergents to produce the non-structural membrane proteins 2, 4B and 5A of the hepatitis C virus (HCV). We show that lauryl maltose neopentyl glycol (MNG-3) and dodecyl octaethylene glycol ether (C12E8) detergents can yield essentially soluble membrane proteins at detergent concentrations that do not inhibit the cell-free reaction. This finding can be explained by the low critical micelle concentration (CMC) of these detergents, which keeps the monomer concentrations low while at the same time providing the necessary excess of detergent concentration above CMC required for full target protein solubilization. We estimate that a tenfold excess of detergent micelles with respect to the protein concentration is sufficient for solubilization, a number that we propose as a guideline for detergent screening assays.

Differential phosphorylation of MAP1b during postnatal development of the cat brain.

Microtubule-associated protein 1b, previously also referred to as microtubule-associated protein 5 or microtubule-associated protein 1x, is a major component of the juvenile cytoskeleton, and is essential during the early differentiation of neurons. It is required for axonal growth and its function is influenced by phosphorylation. The distribution of microtubule-associated protein 1b in kitten cerebellum and cortex during postnatal development was studied with two monoclonal antibodies. Hybridoma clone AA6 detected a non-phosphorylated site, while clone 125 detected a site phosphorylated by casein-kinase II. On blots, both monoclonal antibodies stained the same two proteins of similar molecular weights, also referred to as microtubule-associated protein 5a and 5b. Antibody 125 detected a phosphorylated epitope on both microtubule-associated protein 1b forms; dephosphorylation by alkaline phosphatase abolished the immunological detection. During development of cat cortex and cerebellum, AA6 stained the perikarya and dendrites of neurons during their early differentiation, and especially labelled newly generated axons. The staining decreased during development, and axonal staining was reduced in adult tissue. In contrast to previous reports which demonstrated that antibodies against phosphorylated microtubule-associated protein 1b label exclusively axons, antibody 125 also localized microtubule-associated protein 1b in cell bodies and dendrites, even in adulthood. Some nuclear staining was observed, indicating that a phosphorylated form of microtubule-associated protein 1b may participate in nuclear function. These results demonstrate that microtubule-associated protein 1b is subject to CK2-type phosphorylation throughout neuronal maturation and suggest that phosphorylation of microtubule-associated protein 1b may participate in juvenile and mature-type microtubule functions throughout development.

Formation continue: comment concilier devoir et besoins [Continuing education: how to reconcile needs and wants]

La formation continue fait à l'évidence partie intégrante de la vie du médecin, elle est non seulement un devoir éthique envers les patients mais également l'expression du besoin de se maintenir «à la page» dans sa pratique quotidienne, conséquence des progrès rapides en médecine, particulièrement en oncologie médicale. Elle peut être également source de plaisir quand il s'agit d'accroître ses connaissances. Ses règles minimales ont été définies depuis plusieurs années par la FMH qui délègue aux sociétés de disciplines son application pratique. En 2008, une révision nécessaire pour différentes raisons a facilité le calcul des crédits. Même si le total des heures de formation est resté le même (50 crédits), il a été partagé par deux : 25 pour la formation spécifique et 25 qui peuvent être acquis dans une autre discipline (révision de mars 2009 du Règlement pour la formation continue, art. 5a). Cette révision n'a pas réjoui toutes les sociétés de spécialistes qui gardent la faculté de revoir à la hausse le minimum jugé nécessaire à leur discipline. La quantité des offres de formation continue pour les médecins pose le problème d'être proprement pléthorique (congrès nationaux et internationaux, e-learning, symposiums locaux, etc.), il n'en va pas de même de leur qualité. Dans le domaine de l'oncologie médicale, les offres sont abondantes dans un contexte de marketing évident : les maisons pharmaceutiques parrainent des réunions avec un orateur mercenaire, prestigieux si possible, invité à vanter un produit spécifique dans un cycle de présentations en différents lieux de Romandie (avec à chaque fois, la possibilité d'inscrire des crédits à l'actif des participants)... Elles soutiennent également, par leur logistique, de miniconférences organisées par les différentes institutions locales et auxquelles les médecins ne participent que de façon sporadique vu leur intérêt souvent très secondaire - il n'est pas rare que l'auditoire médical se résume à cinq ou dix participants. Au final, ces offres dispersées et de qualité discutable monopolisent les ressources qui se raréfient rapidement dans le contexte économique actuel et qui doivent impérativement être utilisées de manière plus judicieuse, notamment en évitant les manifestations répétitives. Devant toutes ces offres, il est souvent difficile pour la société de discipline de séparer le bon grain de l'ivraie et en conséquence d'attribuer de manière objective les crédits de formation. Partant de ce constat, un petit groupe romand de médecins oncologues praticiens installés et des centres universitaires ont réfléchi à l'idée de regrouper au sein d'une seule structure romande l'organisation d'une formation continue qui réponde à la fois aux besoins et à l'exigence de qualité. Ses tâches sont multiples : mettre sur pied annuellement plusieurs demi-journées de formation, préaviser avec un comité scientifique de la qualité de la formation continue distillée sur son territoire de compétence (sans empiéter sur les prérogatives de la commission pour la formation postgraduée de la Société suisse d'oncologie médicale - SSOM) en rapprochant les centres universitaires, les hôpitaux cantonaux et régionaux, et les praticiens. Ainsi est née l'association FoROMe (Formation romande en oncologie médicale). Sa légitimité a été établie par la SSOM et par le Comité pour la formation postgraduée et continue (nouvellement SIWF) de la FMH. Elle est maintenant en mesure de mettre en application les tâches pour lesquelles elle a été constituée. Il est évident que cela n'ira pas sans résistance et que certains diront qu'ils ne voient pas la nécessité d'une structure supplémentaire, que les sociétés de disciplines font très bien leur travail, qu'il s'agit encore là d'une atteinte à la liberté. Cependant les nécessités économiques vont tôt ou tard venir au secours de la logique pour confirmer les changements que cette démarche a permis d'anticiper. A l'avenir, il s'agira d'assurer le bien-fondé de cette initiative et de rester vigilant au bon fonctionnement de cette structure à la satisfaction de nos membres.

Magmatic-dominated fluid evolution in the Jurassic Nambija gold skarn deposits (southeastern Ecuador)

The Jurassic (approximately 145 Ma) Nambija oxidized gold skarns are hosted by the Triassic volcanosedimentary Piuntza unit in the sub-Andean zone of southeastern Ecuador. The skarns consist dominantly of granditic garnet (Ad(20-98)) with subordinate pyroxene (Di(46-92)Hd(17-42)Jo(0-19)) and epidote and are spatially associated with porphyritic quartz-diorite to granodiorite intrusions. Endoskarn is developed at the intrusion margins and grades inwards into a potassic alteration zone. Exoskarn has an outer K- and Na-enriched zone in the volcanosedimentary unit. Gold mineralization is associated with the weakly developed retrograde alteration of the exoskarn and occurs mainly in sulfide-poor vugs and milky quartz veins and veinlets in association with hematite. Fluid inclusion data for the main part of the prograde stage indicate the coexistence of high-temperature (500A degrees C to > 600A degrees C), high-salinity (up to 65 wt.% eq. NaCl), and moderate- to low-salinity aqueous-carbonic fluids interpreted to have been trapped at pressures around 100-120 MPa, corresponding to about 4-km depth. Lower-temperature (510-300A degrees C) and moderate- to low-salinity (23-2 wt.% eq. NaCl) aqueous fluids are recorded in garnet and epidote of the end of the prograde stage. The microthermometric data (Th from 513A degrees C to 318A degrees C and salinity from 1.0 to 23 wt.% eq. NaCl) and delta(18)O values between 6.2aEuro degrees and 11.5aEuro degrees for gold-bearing milky quartz from the retrograde stage suggest that the ore-forming fluid was dominantly magmatic. Pressures during the early retrograde stage were in the range of 50-100 MPa, in line with the evidence for CO(2) effervescence and probable local boiling. The dominance of magmatic low-saline to moderately saline oxidizing fluids during the retrograde stage is consistent with the depth of the skarn system, which could have delayed the ingression of external fluids until relatively low temperatures were reached. The resulting low water-to-rock ratios explain the weak retrograde alteration and the compositional variability of chlorite, essentially controlled by host rock compositions. Gold was precipitated at this stage as a result of cooling and pH increase related to CO(2) effervescence, which both result in destabilization of gold-bearing chloride complexes. Significant ingression of external fluids took place after gold deposition only, as recorded by delta(18)O values of 0.4aEuro degrees to 6.2aEuro degrees for fluids depositing quartz (below 350A degrees C) in sulfide-rich barren veins. Low-temperature (< 300A degrees C) meteoric fluids (delta(18)O(water) between -10.0aEuro degrees and -2.0aEuro degrees) are responsible for the precipitation of late comb quartz and calcite in cavities and veins and indicate mixing with cooler fluids of higher salinities (about 100A degrees C and 25 wt.% eq. NaCl). The latter are similar to low-temperature fluids (202-74.5A degrees C) with delta(18)O values of -0.5aEuro degrees to 3.1aEuro degrees and salinities in the range of 21.1 to 17.3 wt.% eq. CaCl(2), trapped in calcite of late veins and interpreted as basinal brines. Nambija represents a deep equivalent of the oxidized gold skarn class, the presence of CO(2) in the fluids being partly a consequence of the relatively deep setting at about 4-km depth. As in other Au-bearing skarn deposits, not only the prograde stage but also the gold-precipitating retrograde stage is dominated by fluids of magmatic origin.

Caracterización arqueométrica de la cerámica común producida en la villa romana de Sa Mesquida (Mallorca)

En la villa romana de 5a Mesquida, situada en el oeste de Mallorca, se han localizado hasta la fecha un horno y dos escombreras que indican la producción de cerámica común en el yacimiento. El presente artículo expone los resultados de la caracterización arqueométrica por fluorescencia de rayos x, difracción de rayos X y microscopía óptica mediante lámina delgada. El estudio permite determinar el grupo de referencia de la producción del taller para su posterior utilización en trabajos de difusión y comercio cerámico. A su vez, constituye el primer grupo de referencia sobre cerámica romana producida en la isla. 5e constata también la existencia de algunas contaminaciones y alteraciones que presenta la cerámica calcárea cocida a alta temperatura.

Transient suppression of Shigella flexneri type 3 secretion by a protective O-antigen-specific monoclonal IgA.

Mucosal immunity to the enteric pathogen Shigella flexneri is mediated by secretory IgA (S-IgA) antibodies directed against the O-antigen (O-Ag) side chain of lipopolysaccharide. While secretory antibodies against the O-Ag are known to prevent bacterial invasion of the intestinal epithelium, the mechanisms by which this occurs are not fully understood. In this study, we report that the binding of a murine monoclonal IgA (IgAC5) to the O-Ag of S. flexneri serotype 5a suppresses activity of the type 3 secretion (T3S) system, which is necessary for S. flexneri to gain entry into intestinal epithelial cells. IgAC5's effects on the T3S were rapid (5 to 15 min) and were coincident with a partial reduction in the bacterial membrane potential and a decrease in intracellular ATP levels. Activity of the T3S system returned to normal levels 45 to 90 min following antibody treatment, demonstrating that IgAC5's effects were transient. Nonetheless, these data suggest a model in which the association of IgA with the O-Ag of S. flexneri partially de-energizes the T3S system and temporarily renders the bacterium incapable of invading intestinal epithelial cells. IMPORTANCE: Secretory IgA (S-IgA) serves as the first line of defense against enteric infections. However, despite its well-recognized role in mucosal immunity, relatively little is known at the molecular level about how this class of antibody functions to prevent pathogenic bacteria from penetrating the epithelial barrier. It is generally assumed that S-IgA functions primarily by "immune exclusion," a phenomenon in which the antibody binds to microbial surface antigens and thereby promotes bacterial agglutination, entrapment in mucus, and physical clearance from the gastrointestinal tract via peristalsis. The results of the present study suggest that in addition to serving as a physical barrier, S-IgA may have a direct impact on the ability of microbial pathogens to secrete virulence factors required for invasion of intestinal epithelial cells.

Validação de marcadores moleculares para resistência à giberela em genótipos brasileiros de trigo

O objetivo deste trabalho foi validar 19 marcadores microssatélites para resistência do trigo à giberela, em uma população não estruturada. Foram utilizados marcadores moleculares descritos na literatura como flanqueando QTLs de resistência à giberela em trigo, nos cromossomos 3B, 5A e 6B. Foram avaliadas 96 linhagens e cultivares de trigo quanto à severidade da infecção por giberela, em dois anos de avaliação. As linhagens e as cultivares foram genotipadas com 19 marcadores microssatélites. Os dados obtidos foram analisados pelo teste de Tukey e pelas análises de correlação, regressão linear simples e regressão múltipla; também foi estimada a eficiência de seleção dos marcadores moleculares. A severidade da doença variou de 1,95 a 41,3%, na média dos dois anos. Foram validados os QTLs nos três cromossomos avaliados. Os marcadores Xgwm389, Xgwm533, Xbarc180, Xbarc24, Wmc397, Xbarc101 e Wmc398 foram associados significativamente à resistência do trigo à giberela, tendo sido identificados alelos de resistência e de suscetibilidade. Os marcadores Wmc397, Xbarc101 (cromossomo 6B) e Xbarc180 (cromossomo 5A) têm potencial para uso na seleção assistida por marcadores moleculares, para resistência do trigo à giberela.

Local Salmonella immunostimulation recruits vaccine-specific CD8 T cells and increases regression of bladder tumor.

NlmCategory="UNASSIGNED">The efficacy of antitumoral responses can be increased using combinatorial vaccine strategies. We recently showed that vaccination could be optimized by local administration of diverse molecular or bacterial agents to target and augment antitumoral CD8 T cells in the genital mucosa (GM) and increase regression of cervical cancer in an animal model. Non muscle-invasive bladder cancer is another disease that is easily amenable to local therapies. In contrast to data obtained in the GM, in this study we show that intravesical (IVES) instillation of synthetic toll-like receptor (TLR) agonists only modestly induced recruitment of CD8 T cells to the bladder. However, IVES administration of Ty21a, a live bacterial vaccine against typhoid fever, was much more effective and increased the number of total and vaccine-specific CD8 T cells in the bladder approximately 10 fold. Comparison of chemokines induced in the bladder by either CpG (a TLR-9 agonist) or Ty21a highlighted the preferential increase in complement component 5a, CXCL5, CXCL2, CCL8, and CCL5 by Ty21a, suggesting their involvement in the attraction of T cells to the bladder. IVES treatment with Ty21a after vaccination also significantly increased tumor regression compared to vaccination alone, resulting in 90% survival in an orthotopic murine model of bladder cancer expressing a prototype tumor antigen. Our data demonstrate that combining vaccination with local immunostimulation may be an effective treatment strategy for different types of cancer and also highlight the great potential of the Ty21a vaccine, which is routinely used worldwide, in such combinatorial therapies.

Quality of life after pulmonary embolism, validation of the French version of the PEmb-QoL questionnaire

Background: The PEmb-QoL is a validated 40-item questionnaire to quantify health-related quality of life in patients having experienced pulmonary embolism (PE). It covers six health dimensions: frequency of complaints, activities of daily living limitations, work-related problems, social limitations, intensity of complaints, and emotional complaints. Originally developed in Dutch and English, we sought to prospectively validate the psychometric properties of a French version of the PEmb-QoL. Methods: We performed a forward and backward translation of the English version of the PEmb-QoL into French. French-speaking consecutive adult patients with an acute, objectively confirmed PE admitted to the emergency department of a Swiss university hospital between 08/2009 and 09/2011 were recruited telephonically. We used standard psychometric tests and criteria to evaluate the acceptability, reliability, and validity of the French version of the PEmb-QoL. We also performed an exploratory factor analysis. Results: Overall, 102 patients were enrolled in the study. The French version of the PEmb-QoL showed good reliability (internal consistency, item-total and inter-item correlations), reproducibility (test-retest reliability), and validity (convergent, discriminant) in French-speaking patients with PE. The exploratory factor analysis suggested three underlying dimensions: limitations in daily activity (items 4b-m, 5a-d), symptoms (items 1a-h and 7), and emotional complaints (items 9a-f and j). Conclusion: We successfully validated the French version of the PEmb-QoL questionnaire in patients with PE. Our results show that the PEmb-QoL is a valuable tool for assessing health-related quality of life after PE in French-speaking patients.