Crystallization and preliminary diffraction studies of native and selenomethionine CcmG (CycY, DsbE)

t Disulfide-bond (Dsb) proteins are a family of redox proteins containing a Cys-X-X-Cys motif. They are essential for disulfide-bond exchange in the bacterial periplasm and are necessary for the correct folding and function of many secreted proteins. CcmG (DsbE) is a reducing Dsb protein required for cytochrome c maturation. Crystals of Bradyrhizobium japonicum CcmG have been obtained that diffract X-rays to 1.14 Angstrom resolution. The crystals are orthorhombic, space group P2(1)2(1)2(1), with unit-cell parameters a = 35.1, b = 48.2, c = 90.2 Angstrom. Selenomethionine CcmG was expressed without using a methionine auxotroph or methionine-pathway inhibition and was purified without reducing agents

Hyper high NA achromatic interferometer for immersion lithography at 193nm

International audienceAn apparatus for immersion interferometric lithography is described here where the interfering beams are created by illuminating a first diffraction grating followed by a second diffraction grating recombining the diffracted beams onto the photo-resist plane. The main advantage of this system is to be achromatic: thus it is possible to use a basic commercial ArF excimer laser as the exposure source. We present here the calculations made to evaluate the different parameters that can influence the depth-of-focus in the immersion configuration. As the set-up is mainly based on the two diffraction gratings, it matters to properly design it. The purpose of this paper is to show the optimization made on the diffraction gratings in taking into account their fabrication process since they are fabricated using the capabilities of the silicon line available in our laboratory. On one hand, calculations have been done to determine the second grating period as a function of the first grating period and the ”immersion NA”. By simply adding a fluid to a “dry” system, we will indeed be able to improve the depth of focus but not the resolution. In playing with the diffraction grating periods, we are able to benefit from the introduction of the immersion fluid. We have performed simulations in order to optimize the grating diffraction efficiency as a function of the etch depth and the fractional linewidth. Finally we report on first results obtained with the achromatic immersion interferometer. The apparatus was used with a 193 nm GAM excimer laser to print resist patterns having a period of 100 nm with excellent contrast

Subclavian thrombosis in a patient with advanced lung cancer: a case report

<p>Abstract</p> <p>Introduction</p> <p>Lung cancer is now considered the most common cause of death among cancer patients. Although target biological regimens have emerged in recent years for non-small cell lung carcinoma, the survival and quality of life of patients with this condition still remain low. The five-year survival rate for all stages of lung cancer is 17% or less.</p> <p>Case presentation</p> <p>We describe the case of a 53-year-old Caucasian woman who was diagnosed with advanced stage IIIa (T2aN₂M₀) non-small cell lung carcinoma (adenocarcinoma) and underwent a complete left upper lobectomy three years ago. After two and a half years of follow-up, she suddenly presented with facial edema and venous distension and was immediately treated for superior vena cava syndrome. Because of a diagnostic check, a major clot was detected in the right subclavian vein. Our patient was informed about treatment options, and she was taken to the catheterization laboratory for percutaneous stenting of the superior vena cava to restore superior vena cava patency.</p> <p>Conclusion</p> <p>Lung cancer has a vast number of complications. Superior vena cava syndrome and thrombosis should be considered upon the presentation of a patient with obstructive symptoms. In this case report, even though we expected the clot to be on the side of the former lesion, it was present on the opposite side. Treatment should also start immediately in these patients with clinical suspicion of thrombosis to avoid further complications, even in cases with a differential diagnosis problem. Finally, although patients with non-small cell lung carcinoma have a high incidence of thromboembolic events, anticoagulant treatment is given only as maintenance therapy after a first event occurs.</p

The Protein Partners of GTP Cyclohydrolase I in Rat Organs

GTP cyclohydrolase I (GCH1) is the rate-limiting enzyme for tetrahydrobiopterin biosynthesis and has been shown to be a promising therapeutic target in ischemic heart disease, hypertension, atherosclerosis and diabetes. The endogenous GCH1-interacting partners have not been identified. Here, we determined endogenous GCH1-interacting proteins in rat.A pulldown and proteomics approach were used to identify GCH1 interacting proteins in rat liver, brain, heart and kidney. We demonstrated that GCH1 interacts with at least 17 proteins including GTP cyclohydrolase I feedback regulatory protein (GFRP) in rat liver by affinity purification followed by proteomics and validated six protein partners in liver, brain, heart and kidney by immunoblotting. GCH1 interacts with GFRP and very long-chain specific acyl-CoA dehydrogenase in the liver, tubulin beta-2A chain in the liver and brain, DnaJ homolog subfamily A member 1 and fatty aldehyde dehydrogenase in the liver, heart and kidney and eukaryotic translation initiation factor 3 subunit I (EIF3I) in all organs tested. Furthermore, GCH1 associates with mitochondrial proteins and GCH1 itself locates in mitochondria.GCH1 interacts with proteins in an organ dependant manner and EIF3I might be a general regulator of GCH1. Our finding indicates GCH1 might have broader functions beyond tetrahydrobiopterin biosynthesis

Consensus guideline for the diagnosis and treatment of tetrahydrobiopterin (BH4) deficiencies

Background: Tetrahydrobiopterin (BH4) deficiencies comprise a group of six rare neurometabolic disorders characterized by insufficient synthesis of the monoamine neurotransmitters dopamine and serotonin due to a disturbance of BH4 biosynthesis or recycling. Hyperphenylalaninemia (HPA) is the first diagnostic hallmark for most BH4 deficiencies, apart from autosomal dominant guanosine triphosphate cyclohydrolase I deficiency and sepiapterin reductase deficiency. Early supplementation of neurotransmitter precursors and where appropriate, treatment of HPA results in significant improvement of motor and cognitive function. Management approaches differ across the world and therefore these guidelines have been developed aiming to harmonize and optimize patient care. Representatives of the International Working Group on Neurotransmitter related Disorders (iNTD) developed the guidelines according to the SIGN (Scottish Intercollegiate Guidelines Network) methodology by evaluating all available evidence for the diagnosis and treatment of BH4 deficiencies. Conclusion: Although the total body of evidence in the literature was mainly rated as low or very low, these consensus guidelines will help to harmonize clinical practice and to standardize and improve care for BH4 deficient patients

Lack of Renal 11 Beta-Hydroxysteroid Dehydrogenase Type 2 at Birth, a Targeted Temporal Window for Neonatal Glucocorticoid Action in Human and Mice

International audienceBackground Glucocorticoid hormones play a major role in fetal organ maturation. Yet, excessive glucocorticoid exposure in utero can result in a variety of detrimental effects, such as growth retardation and increased susceptibility to the development of hypertension. To protect the fetus, maternal glucocorticoids are metabolized into inactive compounds by placental 11beta-hydroxysteroid dehydrogenase type2 (11βHSD2). This enzyme is also expressed in the kidney, where it prevents illicit occupation of the mineralocorticoid receptor by glucocorticoids. We investigated the role of renal 11βHSD2 in the control of neonatal glucocorticoid metabolism in the human and mouse. Methods Cortisol (F) and cortisone (E) concentrations were measured in maternal plasma, umbilical cord blood and human newborn urine using HPLC. 11βHSD2 activity was indirectly assessed by comparing the F/E ratio between maternal and neonatal plasma (placental activity) and between plasma and urine in newborns (renal activity). Direct measurement of renal 11βHSD2 activity was subsequently evaluated in mice at various developmental stages. Renal 11βHSD2 mRNA and protein expression were analyzed by quantitative RT-PCR and immunohistochemistry during the perinatal period in both species. Results We demonstrate that, at variance with placental 11βHSD2 activity, renal 11βHSD2 activity is weak in newborn human and mouse and correlates with low renal mRNA levels and absence of detectable 11βHSD2 protein. Conclusions We provide evidence for a weak or absent expression of neonatal renal 11βHSD2 that is conserved among species. This temporal and tissue-specific 11βHSD2 expression could represent a physiological window for glucocorticoid action yet may constitute an important predictive factor for adverse outcomes of glucocorticoid excess through fetal programming

Genome analysis and physiological comparison of Alicycliphilus denitrificans strains BC and K601T

The genomes of the Betaproteobacteria Alicycliphilus denitrificans strains BC and K601T have been sequenced to get insight into the physiology of the two strains. Strain BC degrades benzene with chlorate as electron acceptor. The cyclohexanol-degrading denitrifying strain K601T is not able to use chlorate as electron acceptor, while strain BC cannot degrade cyclohexanol. The 16S rRNA sequences of strains BC and K601T are identical and the fatty acid methyl ester patterns of the strains are similar. Basic Local Alignment Search Tool (BLAST) analysis of predicted open reading frames of both strains showed most hits with Acidovorax sp. JS42, a bacterium that degrades nitro-aromatics. The genomes include strain-specific plasmids (pAlide201 in strain K601T and pAlide01 and pAlide02 in strain BC). Key genes of chlorate reduction in strain BC were located on a 120 kb megaplasmid (pAlide01), which was absent in strain K601T. Genes involved in cyclohexanol degradation were only found in strain K601T. Benzene and toluene are degraded via oxygenase-mediated pathways in both strains. Genes involved in the meta-cleavage pathway of catechol are present in the genomes of both strains. Strain BC also contains all genes of the ortho-cleavage pathway. The large number of mono- and dioxygenase genes in the genomes suggests that the two strains have a broader substrate range than known thus far.This research was supported by the Technology Foundation, the Applied Science Division (STW) of the Netherlands Organization for Scientific Research (NWO), project number 08053, the graduate school WIMEK (Wageningen Institute for Environment and Climate Research, which is part of SENSE Research School for Socio-Economic and Natural Sciences of the Environment, www.wimek-new.wur.nl and www.sense.nl), SKB (Dutch Centre for Soil Quality Management and Knowledge Transfer, www.skbodem.nl) and the Consolider project CSD-2007-00055. The research was incorporated in the TRIAS (TRIpartite Approaches 469 toward Soil systems processes) program (http://www.nwo.nl/en/research-and-results/programmes/alw/trias-tripartite-approach-to-soil-system-processes/index. html). Flávia Talarico Saia was supported by a FAPESP (the State of São Paulo Research Foundation) scholarship (2006-01997/5). The work conducted by the DOE JGI is supported by the Office of Science of the United States Department of Energy under contract number DE-AC02-05CH11231. Alfons Stams acknowledges support by an ERC (European Research Counsil) advanced grant (project 323009). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Genome-Wide Identification of Transcriptional Start Sites in the Plant Pathogen Pseudomonas syringae pv. tomato str. DC3000

RNA-Seq has provided valuable insights into global gene expression in a wide variety of organisms. Using a modified RNA-Seq approach and Illumina's high-throughput sequencing technology, we globally identified 5′-ends of transcripts for the plant pathogen Pseudomonas syringae pv. tomato str. DC3000. A substantial fraction of 5′-ends obtained by this method were consistent with results obtained using global RNA-Seq and 5′RACE. As expected, many 5′-ends were positioned a short distance upstream of annotated genes. We also captured 5′-ends within intergenic regions, providing evidence for the expression of un-annotated genes and non-coding RNAs, and detected numerous examples of antisense transcription, suggesting additional levels of complexity in gene regulation in DC3000. Importantly, targeted searches for sequence patterns in the vicinity of 5′-ends revealed over 1200 putative promoters and other regulatory motifs, establishing a broad foundation for future investigations of regulation at the genomic and single gene levels