Structural studies of glucose-6-phosphate and NADP+ binding to human glucose-6-phosphate dehydrogenase

Human glucose-6-phosphate dehydrogenase (G6PD) is NADP(+)-dependent and catalyses the first and rate-limiting step of the pentose phosphate shunt. Binary complexes of the human deletion mutant, DeltaG6PD, with glucose-6-phosphate and NADP(+) have been crystallized and their structures solved to 2.9 and 2.5 A, respectively. The structures are compared with the previously determined structure of the Canton variant of human G6PD (G6PD(Canton)) in which NADP(+) is bound at the structural site. Substrate binding in DeltaG6PD is shown to be very similar to that described previously in Leuconostoc mesenteroides G6PD. NADP(+) binding at the coenzyme site is seen to be comparable to NADP(+) binding in L. mesenteroides G6PD, although some differences arise as a result of sequence changes. The tetramer interface varies slightly among the human G6PD complexes, suggesting flexibility in the predominantly hydrophilic dimer-dimer interactions. In both complexes, Pro172 of the conserved peptide EKPxG is in the cis conformation; it is seen to be crucial for close approach of the substrate and coenzyme during the enzymatic reaction. Structural NADP(+) binds in a very similar way in the DeltaG6PD-NADP(+) complex and in G6PD(Canton), while in the substrate complex the structural NADP(+) has low occupancy and the C-terminal tail at the structural NADP(+) site is disordered. The implications of possible interaction between the structural NADP(+) and G6P are considered.published_or_final_versio

Gene Disruption in Scedosporium aurantiacum: Proof of Concept with the Disruption of SODC Gene Encoding a Cytosolic Cu,Zn-Superoxide Dismutase

Scedosporium species are opportunistic pathogens responsible for a large variety of infections in humans. An increasing occurrence was observed in patients with underlying conditions such as immunosuppression or cystic fibrosis. Indeed, the genus Scedosporium ranks the second among the filamentous fungi colonizing the respiratory tracts of the CF patients. To date, there is very scarce information on the pathogenic mechanisms, at least in part because of the limited genetic tools available. In the present study, we successfully developed an efficient transformation and targeted gene disruption approach on the species Scedosporium aurantiacum. The disruption cassette was constructed using double-joint PCR procedure, and resistance to hygromycin B as the selection marker. This proof of concept was performed on the functional gene SODC encoding the Cu,Zn-superoxide dismutase. Disruption of the SODC gene improved susceptibility of the fungus to oxidative stress. This technical advance should open new research areas and help to better understand the biology of Scedosporium species

Gamma evaluation with Octavius 4D phantom for pretreatment of modern radiotherapy treatment techniques

Purpose: In modern radiotherapy techniques such as intensity-modulated radiation therapy (IMRT) and volume modulated arc therapy (VMAT), the quality assurance (QA) process is vital. The goal of the study was to verify the treatment planning dose delivered during delivery of complex treatment plans. The QA standard is to perform patient-specific comparisons between planned doses and doses measured in a phantom. Materials and Methods: Ninety-five complex IMRT and VMAT plans for different pathologies planned using Eclipse treatment planning system (TPS). The Octavius 4D phantom has been used to verify patient specific quality assurance of all VMAT plans calculating with different algorithms. Conclusion: Overall, good agreement was observed between measured and calculated doses in most cases with gamma values above 1 in >95% of measured points in volumetric 3D analysis and 1 in >90% in 2D analysis. The Octavius 4D phantom is an effective and efficient method for patient specific QA

Genomic Organization and Expression of Iron Metabolism Genes in the Emerging Pathogenic Mold

The ubiquitous mold is increasingly recognized as an emerging pathogen, especially among patients with underlying disorders such as immunodeficiency or cystic fibrosis (CF). Indeed, it ranks the second among the filamentous fungi colonizing the respiratory tract of CF patients. However, our knowledge about virulence factors of this fungus is still limited. The role of iron-uptake systems may be critical for establishment of infections, notably in the iron-rich environment of the CF lung. Two main strategies are employed by fungi to efficiently acquire iron from their host or from their ecological niche: siderophore production and reductive iron assimilation (RIA) systems. The aim of this study was to assess the existence of orthologous genes involved in iron metabolism in the recently sequenced genome of . At first, a tBLASTn analysis using iron-related proteins as query revealed orthologs of almost all relevant loci in the genome. Whereas the genes putatively involved in RIA were randomly distributed, siderophore biosynthesis and transport genes were organized in two clusters, each containing a non-ribosomal peptide synthetase (NRPS) whose orthologs in have been described to catalyze hydroxamate siderophore synthesis. Nevertheless, comparative genomic analysis of siderophore-related clusters showed greater similarity between and phylogenetically close molds than with species. The expression level of these genes was then evaluated by exposing conidia to iron starvation and iron excess. The expression of several orthologs of genes involved in siderophore-based iron uptake or RIA was significantly induced during iron starvation, and conversely repressed in iron excess conditions. Altogether, these results indicate that possesses the genetic information required for efficient and competitive iron uptake. They also suggest an important role of the siderophore production system in iron uptake by

Solution of the structure of tetrameric human glucose 6-phosphate dehydrogenase by molecular replacement

Recombinant human glucose 6-phosphate dehydrogenase (G6PD) has been crystallized and its structure solved by molecular replacement. Crystals of the natural mutant R459L grow under similar conditions in space groups P212121 and C2221 with eight or four 515-residue molecules in the asymmetric unit, respectively. A non-crystallographic 222 tetramer was found in the C2221 crystal form using a 4 A resolution data set and a dimer of the large beta + alpha domains of the Leuconostoc mesenteroides enzyme as a search model. This tetramer was the only successful search model for the P212121 crystal form using data to 3 A. Crystals of the deletion mutant DeltaG6PD grow in space group F222 with a monomer in the asymmetric unit; 2.5 A resolution data have been collected. Comparison of the packing of tetramers in the three space groups suggests that the N-terminal tail of the enzyme prevents crystallization with exact 222 molecular symmetry.published_or_final_versio

Effect of voriconazole on Candida tropicalis biofilms: Relation with ERG genes expression

Candida tropicalis has emerged as the third most prevalent fungal pathogens and its ability to form biofilms has been considered one of the most important virulence factors, since biofilms represent high tolerance to antifungal agents. However, the mechanisms of C. tropicalis biofilm resistance to antifungals remain poorly understood. Thus, the main aim of this work was to infer about the effect of voriconazole on the formation and control of C. tropicalis biofilms and disclose its relationship with ERG genes' expression. Planktonic cells tolerance of several C. tropicalis clinical isolates to voriconazole was determined through of antifungal susceptibility test, and the effect of this azole against C. tropicalis biofilm formation and pre-formed biofilms was evaluated by cultivable cells determination and total biomass quantification. ERG genes expression was analyzed by quantitative real-time polymerase chain reaction. This work showed that C. tropicalis resistance to voriconazole is strain dependent and that voriconazole was able to partially control biofilm formation, but was unable to eradicate C. tropicalis pre-formed biofilms. Moreover, C. tropicalis biofilms resistance to voriconazole seems to be associated with alterations of sterol content in the cell membrane, resulting in ERG genes overexpression. Voriconazole is unable to control C. tropicalis biofilms, and the overexpression of ERG genes is a possible mechanism of biofilm resistance.TheauthorsthanktheFCTfortheStrategic Project of the UID/BIO/04469/2013 unit, FCT and European Union funds (FEDER/COMPETE) for the project RECI/BBBEBI/0179/2012 (FCOMP-01-0124-FEDER-027462). We also would like to acknowledge Pfizer , S.A. for the kindly donation of voriconazole

Draft Genome Sequence of the Pathogenic Fungus Scedosporium apiospermum

This is the final version of the article. Available from the publisher via the DOI in this record.The first genome of one species of the Scedosporium apiospermum complex, responsible for localized to severe disseminated infections according to the immune status of the host, will contribute to a better understanding of the pathogenicity of these fungi and also to the discovery of the mechanisms underlying their low susceptibility to current antifungals.This work was supported by a grant (RF20120600725) from the association Vaincre la Mucoviscidose (France), which is gratefully acknowledged