A new method to measure rolling resistance in treadmill cycling.

The purpose of this study was to evaluate a new method of measuring rolling resistance in treadmill cycling and to establish its sensitivity and reproducibility. One participant was asked to keep a bicycle in equilibrium on a treadmill without pedalling at a constant speed of 5.56 m x s(-1), which was held in place in the front by a dynamometer. For each condition, the method consisted of 11 measurements of the force required to hold the cycle at different treadmill slopes (0-10%, increment 1%). The coefficient of rolling resistance was calculated based on the forces applied to the bicycle in equilibrium. To test the sensitivity of the method, the bicycle was successively equipped with three tyre types (700 x 28, 700 x 23, 700 x 22) and inflation pressure was set at 150, 300, 600, 900, and 1100 kPa. To test the reproducibility of the method, a second experimenter repeated all measurements done with the 700 x 23 tyres. The method was sensitive enough to detect an effect of both tyre type and inflation pressure (P < 0.001: two-way ANOVA). The measurement of the coefficient of rolling resistance by two separate experimenters resulted in a small bias of 0.00029 (95% CI, -0.00011 to 0.00068). In conclusion, the new method is sensitive and reliable, as well as being simple and affordable.

Synorogenic extension: Quantitative constraints on the age and displacement of the Zanskar shear zone (northwest Himalaya)

subsequent extension-induced exhumation. Geochronological dating of various Structural, thermobarometric, and geochronological data place limits on the age and tectonic displacement along the Zanskar shear zone, a major north-dipping synorogenic extensional structure separating the high-grade metamorphic sequence of the High Himalayan Crystalline Sequence from the overlying low-grade sedimentary rocks of the Tethyan Himalaya, A complete Barrovian metamorphic succession, from kyanite to biotite zone mineral assemblages, occurs within the I-km-thick Zanskar shear zone. Thermobarometric data indicate a difference In equilibration depths of 12 +/- 3 km between the lower kyanite zone and the garnet zone, which is Interpreted as a minimum estimate for the finite vertical displacement accommodated by the Zanskar shear zone. For the present-day dip of the structure (20 degrees), a simple geometrical model shows that a net slip of 35 +/- 9 km is required to regroup these samples to the same structural level. Because the kyanite to garnet zone rocks represent only part of the Zanskar shear zone, and because its original dip may have been less than the present-day dip, these estimates fur the finite displacement represent minimum values. Field relations and petrographic data suggest that migmatization and associated leucogranite intrusion in the footwall of the Zanskar shear zone occurred as a continuous profess starting at the Barrovian metamorphic peak and lasting throughout the subsequent extension-induced exhumation. Geochronological dataing of various leucogranitic plutons and dikes in the Zanskar shear zone footwall indicates that the main ductile shearing along the structure ended by 19.8 Ma and that extension most likely initiated shortly before 22.2 Ma.

Mechanisms of antifungal resistance in the opportunistic fungus Aspergillus fumigatus

ABSTRACT Aspergillus fumigatus is one of the most prevalent airbone fungal pathogen and can cause severe fatal invasive aspergillosis in immunocompromised patients. Several antifungal agents are available to treat these infections but with limited success. These agents include polyenes (amphotericin B), echinocandins (caspofungin) and azoles, which constitute the most important class with itraconazole (ITC) and voriconazole as major active compounds. Azole-derived antifungal agents target the ergosterol biosynthesis pathway via the inhibition of the lanosterol 14α-demethylase (cyp51/ERG1 1), a cytochrome P450 responsible for the conversion of lanosterol to ergosterol, which is the main component of cell membrane in fungi. A. fumigatus is also found in the environment as a contaminant of rotting plant or present in composting of organic waste. Among antifungal agents used in the environment for crop protection, the class of azoles is also widely used with propiconazole or prochloraz as examples. However, other agents such as dicarboximide (iprodione), phenylamide (benalaxyl) or strobilurin (azoxystrobin) are also used. Emergence of clinical azole-resistant isolates has been described in several European countries. However the incidence of antifungal resistance has not been yet reported in details in Switzerland. In this study, the status of antifungal resistance was investigated on A. fumigatus isolates collected from Swiss hospitals and from different environmental sites and. tested for their susceptibility to several currently used antifungal agents. The data showed a low incidence of resistance for all tested agents among clinical and environmental isolates. Only two azole-resistant environmental isolates were detected and none among the clinical tested isolates. In general, A. fumigatus was susceptible to all antifungals tested in our study, except to azoxystrobin which was the less active agent against all isolates. Since mechanisms of antifungal resistance have been poorly investigated until now in A. fumigatus, this work was aimed 1) to identify A. fumigatus genes involved in antifungal resistance and 2) to test their involvement in the development of resistance in sampled isolates. Therefore, this work proposed to isolate A. fumigatus genes conferring resistance to a drug-hypersusceptible Saccharomyces cerevisiae strain due to a lack of multidrug transporter genes. Several genes were recovered including three distinct efflux transporters (atrF, atrH and mdrA) and a bZip transcription factor, yapA. The inactivation of each transporter in A. fumigatus indicated that the transporters were involved in the basal level of azole susceptibility. The inactivation of YapA led to a hypersusceptibility to H2O2, thus confirming the involvement of this gene in the oxidative stress response of A. fumigatus. The involvement of the abovementioned transporters genes and of other transporters genes identified by genome analysis in azole resistance was tested by probing their expression in some ITC-resistant isolates. Even if upregulation of some transporters genes was observed in some investigated isolates, the correlation between azole resistance and expression levels of all these transporters genes could not be clearly established for all tested isolates. Given these results, the present work addressed 1) alteration in the expression of cyp51A encoding for the azole target enzyme, and 2) mutation(s) in the cyp51A sequence as potential mechanisms of azote resistance in A. . However, overexpression of cyp51A in the investigated isolates was not linked with azote resistance. Since it was reported that mutation(s) in cyp51A were participating in azote resistance in A. fumigatus, a functional complementation of cyp51A cDNAs from ITC-resistant A. fumigatus strains in S. cerevisiae ergl 1 Δ mutant strain was attempted. Expression in S. cerevisiae allowed the testing of these cDNAs with regards to their functionality and involvement in resistance to specific azote compounds. We could demonstrate that Cyp51A protein with a G54E or M220K mutations conferred resistance to specific azoles in S. cerevisiae, therefore suggesting that these mutations were important for the development of azote resistance in A. fumigatus. In conclusion, this work showed a correlation between ITC resistance and mechanisms involving overexpression of transporters and cyp51A mutations in A. fumigatus isolates. However, azole resistance of some isolates has not been solved and thus it will be necessary to approach the study of resistance mechanisms in this fungal species using alternative methodologies. RESUME Aspergillus fumigatus est un champignon opportuniste répandu et est la cause d'aspergilloses invasives le plus souvent fatales chez des patients immunodéprimés. Plusieurs antifongiques sont disponibles afin de traiter ces infections, cependant avec un succès limité. Ces agents incluent les polyènes (amphotericin B), les échinocandines (caspofungin) et les azoles, qui représentent la plus importante classe d'antifongiques avec l'itraconazole (ITC) et le voriconazole comme principaux agents actifs. Les dérivés azolés ciblent la voie de biosynthèse de l'ergostérol via l'inhibition de la lanostérol 14α-demethylase (cyp51/ERG11), un cytochrome P450 impliqué dans la conversion du lanostérol en ergostérol, qui est un composant important de la membrane chez les champignons. A. fumigatus est également répandu dans l'environnement. Parmi les antifongiques employés en agriculture afin de protéger les cultures, les azoles sont aussi largement utilisés. Cependant, d'autres agents tels que les dicarboximides (iprodione), les phenylamides (benalaxyl) et les strobilurines (azoxystrobin) peuvent être également utilisés. L'émergence de souches cliniques résistantes aux azoles a été décrite dans différents pays européens. Cependant, l'incidence d'une telle résistance aux azoles n'a pas encore été reportée en détails en Suisse. Dans ce travail, l'émergence de la résistance aux antifongiques a été étudiée par analyse de souches d'A. fumigatus provenant de milieux hospitaliers en Suisse et de différents sites et leur susceptibilité testée envers plusieurs antifongiques couramment utilisés. Les données obtenues ont montré une faible incidence de la résistance parmi les souches cliniques et environnementales pour les agents testés. Seulement deux souches environnementales résistantes aux azoles ont été détectées et aucune parmi les souches cliniques. Les mécanismes de résistance aux antifongiques ayant été très peu étudiés jusqu'à présent chez A. fumigatus , ce travail a eu aussi pour but 1) d'identifier les gènes d' A. fumigatus impliqués dans la résistance aux antifongiques et 2) de tester leur implication dans la résistance de certaines souches. Ainsi, il a été proposé d'isoler les gènes d' A. fumigatus pouvant conférer une résistance aux antifongiques à une souche de Saccharomyces cerevisiae hypersensible aux antifongiques. Trois transporteurs à efflux (atrF, atrH et mdrA) et un facteur de transcription appartenant à la famille des bZip (YapA) ont ainsi été isolés. L'inactivation, dans une souche d'A. fumigatus, de chacun des ces transporteurs a permis de mettre en évidence leur implication dans la susceptibilité d'A. fumigatus aux antifongiques. L'inactivation de YapA a engendré une hypersusceptibilité à l' H2O2, confirmant ainsi le rôle de ce gène dans la réponse au stress oxydatif chez A . fumigatus. La participation dans la résistance aux antifongiques des gènes codant pour des transporteurs ainsi que d'autres gènes identifiés par analyse du génome a été déterminée en testant leur niveau d'expression dans des souches résistantes à l'ITC. Bien qu'une surexpression de transporteurs ait été observée dans certaines souches, une corrélation entre la résistance à l'ITC et les niveaux d'expression de ces transporteurs n'a pu être clairement établie. Ce présent travail s'est donc porté sur l'étude de 2 autres mécanismes potentiellement impliqués dans la résistance aux azoles : 1) la surexpression de cyp51A codant pour l'enzyme cible et 2) des mutations dans cyp51A. Cependant, la surexpression de cyp51A dans les souches étudiées n'a pas été constatée. L'effet des mutations de cyp51A dans la résistance aux azoles a été testée par complémentation fonctionnelle d'une souche S. cerevisiae déletée dans son gène ERG11. L'expression de ces gènes chez S. cerevisiae a permis de démontrer que les protéines Cyp51Ap contenant une mutation G54E ou M220K pouvaient conférer une résistance spécifique à certains azoles, ainsi suggérant que ces mutations pourraient être importantes dans le développement d'une résistance aux azoles chez A. fumigatus. En conclusion, ce travail a permis de mettre en évidence, dans des souches d'A. fumigatus , une corrélation entre leur résistance à l' ITC et les mécanismes impliquant une surexpression de transporteurs et des mutations dans cyp51A. Cependant, ces mécanismes n'ont pu expliquer la résistance aux azoles de certaines souches et c'est pourquoi de nouvelles approches doivent être envisagées afin d'étudier ces mécanismes.

Effect of mutation and genetic background on drug resistance in Mycobacterium tuberculosis.

Bacterial factors may contribute to the global emergence and spread of drug-resistant tuberculosis (TB). Only a few studies have reported on the interactions between different bacterial factors. We studied drug-resistant Mycobacterium tuberculosis isolates from a nationwide study conducted from 2000 to 2008 in Switzerland. We determined quantitative drug resistance levels of first-line drugs by using Bactec MGIT-960 and drug resistance genotypes by sequencing the hot-spot regions of the relevant genes. We determined recent transmission by molecular methods and collected clinical data. Overall, we analyzed 158 isolates that were resistant to isoniazid, rifampin, or ethambutol, 48 (30.4%) of which were multidrug resistant. Among 154 isoniazid-resistant strains, katG mutations were associated with high-level and inhA promoter mutations with low-level drug resistance. Only katG(S315T) (65.6% of all isoniazid-resistant strains) and inhA promoter -15C/T (22.7%) were found in molecular clusters. M. tuberculosis lineage 2 (includes Beijing genotype) was associated with any drug resistance (adjusted odds ratio [OR], 3.0; 95% confidence interval [CI], 1.7 to 5.6; P < 0.0001). Lineage 1 was associated with inhA promoter -15C/T mutations (OR, 6.4; 95% CI, 2.0 to 20.7; P = 0.002). We found that the genetic strain background influences the level of isoniazid resistance conveyed by particular mutations (interaction tests of drug resistance mutations across all lineages; P < 0.0001). In conclusion, M. tuberculosis drug resistance mutations were associated with various levels of drug resistance and transmission, and M. tuberculosis lineages were associated with particular drug resistance-conferring mutations and phenotypic drug resistance. Our study also supports a role for epistatic interactions between different drug resistance mutations and strain genetic backgrounds in M. tuberculosis drug resistance.

The impact of transmission clusters on primary drug resistance in newly diagnosed HIV-1 infection.

OBJECTIVES: To monitor HIV-1 transmitted drug resistance (TDR) in a well defined urban area with large access to antiretroviral therapy and to assess the potential source of infection of newly diagnosed HIV individuals. METHODS: All individuals resident in Geneva, Switzerland, with a newly diagnosed HIV infection between 2000 and 2008 were screened for HIV resistance. An infection was considered as recent when the positive test followed a negative screening test within less than 1 year. Phylogenetic analyses were performed by using the maximum likelihood method on pol sequences including 1058 individuals with chronic infection living in Geneva. RESULTS: Of 637 individuals with newly diagnosed HIV infection, 20% had a recent infection. Mutations associated with resistance to at least one drug class were detected in 8.5% [nucleoside reverse transcriptase inhibitors (NRTIs), 6.3%; non-nucleoside reverse transcriptase inhibitors (NNRTIs), 3.5%; protease inhibitors, 1.9%]. TDR (P-trend = 0.015) and, in particular, NNRTI resistance (P = 0.002) increased from 2000 to 2008. Phylogenetic analyses revealed that 34.9% of newly diagnosed individuals, and 52.7% of those with recent infection were linked to transmission clusters. Clusters were more frequent in individuals with TDR than in those with sensitive strains (59.3 vs. 32.6%, respectively; P < 0.0001). Moreover, 84% of newly diagnosed individuals with TDR were part of clusters composed of only newly diagnosed individuals. CONCLUSION: Reconstruction of the HIV transmission networks using phylogenetic analysis shows that newly diagnosed HIV infections are a significant source of onward transmission, particularly of resistant strains, thus suggesting an important self-fueling mechanism for TDR.

Sensitive and rapid detection of ganciclovir resistance by PCR based MALDI-TOF analysis.

BACKGROUND: Cytomegalovirus (CMV) infection is associated with significant morbidity and mortality in transplant recipients. Resistance against ganciclovir is increasingly observed. According to current guidelines, direct drug resistance testing is not always performed due to high costs and work effort, even when resistance is suspected. OBJECTIVES: To develop a more sensitive, easy applicable and cost-effective assay as proof of concept for direct drug resistance testing in CMV surveillance of post-transplant patients. STUDY DESIGN: Five consecutive plasma samples from a heart transplant patient with a primary CMV infection were analyzed by quantitative real-time polymerase chain reaction (rtPCR) as a surrogate marker for therapy failure, and by direct drug resistance detection assays such as Sanger sequencing and the novel primer extension (PEX) reaction matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) based method. RESULTS: This report demonstrates that PEX reaction followed by MALDI-TOF analysis detects the A594V mutation, encoding ganciclovir resistance, ten days earlier compared to Sanger sequencing and more than 30 days prior to an increase in viral load. CONCLUSION: The greatly increased sensitivity and rapid turnaround-time combined with easy handling and moderate costs indicate that this procedure could make a major contribution to improve transplantation outcomes.

Differential effectiveness of microbially induced resistance against herbivorous insects in Arabidopsis.

Rhizobacteria-induced systemic resistance (ISR) and pathogen-induced systemic acquired resistance (SAR) have a broad, yet partly distinct, range of effectiveness against pathogenic microorganisms. Here, we investigated the effectiveness of ISR and SAR in Arabidopsis against the tissue-chewing insects Pieris rapae and Spodoptera exigua. Resistance against insects consists of direct defense, such as the production of toxins and feeding deterrents and indirect defense such as the production of plant volatiles that attract carnivorous enemies of the herbivores. Wind-tunnel experiments revealed that ISR and SAR did not affect herbivore-induced attraction of the parasitic wasp Cotesia rubecula (indirect defense). By contrast, ISR and SAR significantly reduced growth and development of the generalist herbivore S. exigua, although not that of the specialist P. rapae. This enhanced direct defense against S. exigua was associated with potentiated expression of the defense-related genes PDF1.2 and HEL. Expression profiling using a dedicated cDNA microarray revealed four additional, differentially primed genes in microbially induced S. exigua-challenged plants, three of which encode a lipid-transfer protein. Together, these results indicate that microbially induced plants are differentially primed for enhanced insect-responsive gene expression that is associated with increased direct defense against the generalist S. exigua but not against the specialist P. rapae.

Syndrome des ovaires polykystiques et résistance a l'insuline [Polycystic ovary syndrome and insulin resistance]

Polycystic ovary syndrome is the most common endocrinopathy in women of reproductive age. Insulin resistance is frequently found in affected patients, and probably plays an important physiopathological role. In this paper, we will review the well recognized association between polycystic ovary syndrome and insulin resistance, and discuss the increased risk of glucose intolerance, type 2 diabetes and metabolic syndrome carried by patients diagnosed with this syndrome. We will also suggest a practical strategy for the screening and follow up of the various metabolic complications associated with polycystic ovary syndrome, in light of the rare existing recommendations of the current literature.

Treatment-Naive Individuals Are the Major Source of Transmitted HIV-1 Drug Resistance in Men Who Have Sex With Men in the Swiss HIV Cohort Study.

Background. Human immunodeficiency virus type 1 (HIV-1) transmitted drug resistance (TDR) can compromise antiretroviral therapy (ART) and thus represents an important public health concern. Typically, sources of TDR remain unknown, but they can be characterized with molecular epidemiologic approaches. We used the highly representative Swiss HIV Cohort Study (SHCS) and linked drug resistance database (SHCS-DRDB) to analyze sources of TDR. Methods. ART-naive men who have sex with men with infection date estimates between 1996 and 2009 were chosen for surveillance of TDR in HIV-1 subtype B (N = 1674), as the SHCS-DRDB contains pre-ART genotypic resistance tests for >69% of this surveillance population. A phylogeny was inferred using pol sequences from surveillance patients and all subtype B sequences from the SHCS-DRDB (6934 additional patients). Potential sources of TDR were identified based on phylogenetic clustering, shared resistance mutations, genetic distance, and estimated infection dates. Results. One hundred forty of 1674 (8.4%) surveillance patients carried virus with TDR; 86 of 140 (61.4%) were assigned to clusters. Potential sources of TDR were found for 50 of 86 (58.1%) of these patients. ART-naive patients constitute 56 of 66 (84.8%) potential sources and were significantly overrepresented among sources (odds ratio, 6.43 [95% confidence interval, 3.22-12.82]; P < .001). Particularly large transmission clusters were observed for the L90M mutation, and the spread of L90M continued even after the near cessation of antiretroviral use selecting for that mutation. Three clusters showed evidence of reversion of K103N or T215Y/F. Conclusions. Many individuals harboring viral TDR belonged to transmission clusters with other Swiss patients, indicating substantial domestic transmission of TDR in Switzerland. Most TDR in clusters could be linked to sources, indicating good surveillance of TDR in the SHCS-DRDB. Most TDR sources were ART naive. This, and the presence of long TDR transmission chains, suggests that resistance mutations are frequently transmitted among untreated individuals, highlighting the importance of early diagnosis and treatment.

A mutation in the gene encoding the vaccinia virus 37,000-M(r) protein confers resistance to an inhibitor of virus envelopment and release.

Plaque formation in vaccinia virus is inhibited by the compound N1-isonicotinoyl-N2-3-methyl-4-chlorobenzoylhydrazine (IMCBH). We have isolated a mutant virus that forms wild-type plaques in the presence of the drug. Comparison of wild-type and mutant virus showed that both viruses produced similar amounts of infectious intracellular naked virus in the presence of the drug. In contrast to the mutant, no extracellular enveloped virus was obtained from IMCBH-treated cells infected with wild-type virus. Marker rescue experiments were used to map the mutation conferring IMCBH resistance to the mutant virus. The map position coincided with that of the gene encoding the viral envelope antigen of M(r) 37,000. Sequence analysis of both wild-type and mutant genes showed a single nucleotide change (G to T) in the mutant gene. In the deduced amino acid sequence, the mutation changes the codon for an acidic Asp residue in the wild-type gene to one for a polar noncharged Tyr residue in the mutant.

Matrix metalloproteinase 9 (MMP-9/gelatinase B) proteolytically cleaves ICAM-1 and participates in tumor cell resistance to natural killer cell-mediated cytotoxicity.

Shedding of intercellular adhesion molecule 1 (ICAM-1) is believed to play a role in tumor cell resistance to cell-mediated cytotoxicity. However, the mechanism whereby ICAM-1 is shed from the surface of tumor cells remains unclear. In this study, we have addressed the possibility that matrix metalloproteinases are implicated in ICAM-1 shedding. Our observations suggest a functional relationship between ICAM-1 and matrix metalloproteinase 9 (MMP-9) whereby ICAM-1 provides a cell surface docking mechanism for proMMP-9, which, upon activation, proteolytically cleaves the extracellular domain of ICAM-1 leading to its release from the cell surface. MMP-9-dependent shedding of ICAM-1 is found to augment tumor cell resistance to natural killer (NK) cell-mediated cytotoxicity. Taken together, our observations propose a mechanism for ICAM-1 shedding from the cell surface and provide support for MMP involvement in tumor cell evasion of immune surveillance.

Lentivirus-mediated Bcl-2 expression in betaTC-tet cells improves resistance to hypoxia and cytokine-induced apoptosis while preserving in vitro and in vivo control of insulin secretion.

betaTC-tet cells are conditionally immortalized pancreatic beta cells which can confer long-term correction of hyperglycemia when transplanted in syngeneic streptozocin diabetic mice. The use of these cells for control of type I diabetes in humans will require their encapsulation and transplantation in non-native sites where relative hypoxia and cytokines may threaten their survival. In this study we genetically engineered betaTC-tet cells with the anti-apoptotic gene Bcl-2 using new lentiviral vectors and showed that it protected this cell line against apoptosis induced by hypoxia, staurosporine and a mixture of cytokines (IL-1beta, IFN-gamma and TNF-alpha). We further demonstrated that Bcl-2 expression permitted growth at higher cell density and with shorter doubling time. Expression of Bcl-2, however, did not inter- fere either with the intrinsic mechanism of growth arrest present in the betaTC-tet cells or with their normal glucose dose-dependent insulin secretory activity. Furthermore, Bcl-2 expressing betaTC-tet cells retained their capacity to secrete insulin under mild hypoxia. Finally, transplantation of these cells under the kidney capsule of streptozocin diabetic C3H mice corrected hyperglycemia for several months. These results demonstrate that the murine betaTC-tet cell line can be genetically modified to improve its resistance against different stress-induced apoptosis while preserving its normal physiological function. These modified cells represent an improved source for cell transplantation therapy of type I diabetes.