Up-regulation of neutrophil activating protein in Helicobacter pylori under high-salt stress: Structural and phylogenetic comparison with bacterial iron-binding ferritins

It is generally accepted that most gastrointestinal diseases are probably caused by the bacterial pathogen Helicobacter pylori (H. pylori). In this study we have focused on the comparison of protein expression profiles of H. pylori grown under normal and high-salt conditions by a proteomics approach. We have identified about 190 proteins whose expression levels changed after growth at high salt concentration. Among these proteins, neutrophil-activating protein (NapA) was found to be consistently up-regulated under osmotic stress brought by high salts. We have investigated the effect of high salt on secondary and tertiary structures of NapA by circular dichroism spectroscopy followed by analytical ultracentrifugation to monitor the change of quaternary structure of recombinant NapA with increasing salt concentration. The loss of iron-binding activity of NapA coupled with noticeable energetic variation in protein association of NapA as revealed by isothermal titration calorimetry was found under high salt condition. The phylogenetic tree analysis based on sequence comparison of 16 protein sequences encompassing NapA proteins and ferritin of H. pylori and other prokaryotic organisms pointed to the fact that all H. pylori NapA proteins of human origin are more homologous to NapA of Helicobacter genus than to other bacterial NapA. Based on computer modeling, NapA proteins from H. pylori of human isolates are found more similar to ferritin from H. pylori than to NapA from other species of bacteria. Taken together, these results suggested that divergent evolution of NapA and ferritin possessing dissimilar and diverse sequences follows a path distinct from that of convergent evolution of NapA and ferritin with similar dual functionality of iron-binding and ferroxidase activities.     

A Model of the Membrane-bound Cytochrome b5-Cytochrome P450 Complex from NMR and Mutagenesis Data

Microsomal cytochrome b₅ (cytb₅) is a membrane-bound protein that modulates the catalytic activity of its redox partner, cytochrome P4502B4 (cytP450). Here, we report the first structure of full-length rabbit ferric microsomal cytb₅ (16 kDa), incorporated in two different membrane mimetics (detergent micelles and lipid bicelles). Differential line broadening of the cytb₅ NMR resonances and site-directed mutagenesis data were used to characterize the cytb₅ interaction epitope recognized by ferric microsomal cytP450 (56 kDa). Subsequently, a data-driven docking algorithm, HADDOCK (high ambiguity driven biomolecular docking), was used to generate the structure of the complex between cytP4502B4 and cytb₅ using experimentally derived restraints from NMR, mutagenesis, and the double mutant cycle data obtained on the full-length proteins. Our docking and experimental results point to the formation of a dynamic electron transfer complex between the acidic convex surface of cytb₅ and the concave basic proximal surface of cytP4502B4. The majority of the binding energy for the complex is provided by interactions between residues on the C-helix and β-bulge of cytP450 and residues at the end of helix α4 of cytb₅. The structure of the complex allows us to propose an interprotein electron transfer pathway involving the highly conserved Arg-125 on cytP450 serving as a salt bridge between the heme propionates of cytP450 and cytb₅. We have also shown that the addition of a substrate to cytP450 likely strengthens the cytb₅-cytP450 interaction. This study paves the way to obtaining valuable structural, functional, and dynamic information on membrane-bound complexes.     

Evaluation of the suchey–brooks method of age estimation in an Australian subpopulation using computed tomography of the pubic symphyseal surface

Despite the prominent use of the Suchey–Brooks (S–B) method of age estimation in forensic anthropological practice, it is subject to intrinsic limitations, with reports of differential interpopulation error rates between geographical locations. This study assessed the accuracy of the S–B method to a contemporary adult population in Queensland, Australia and provides robust age parameters calibrated for our population. Three‐dimensional surface reconstructions were generated from computed tomography scans of the pubic symphysis of male and female Caucasian individuals aged 15–70 years (n = 195) in Amira® and Rapidform®. Error was analyzed on the basis of bias, inaccuracy and percentage correct classification for left and right symphyseal surfaces. Application of transition analysis and Chi‐square statistics demonstrated 63.9 and 69.7% correct age classification associated with the left symphyseal surface of Australian males and females, respectively, using the S–B method. Using Bayesian statistics, probability density distributions for each S–B phase were calculated, providing refined age parameters for our population. Mean inaccuracies of 6.77 (±2.76) and 8.28 (±4.41) years were reported for the left surfaces of males and females, respectively; with positive biases for younger individuals (<55 years) and negative biases in older individuals. Significant sexual dimorphism in the application of the S–B method was observed; and asymmetry in phase classification of the pubic symphysis was a frequent phenomenon. These results recommend that the S–B method should be applied with caution in medico‐legal death investigations of Queensland skeletal remains and warrant further investigation of reliable age estimation techniques. Am J Phys Anthropol, 2013. © 2013 Wiley Periodicals, Inc.     

Modified method for determination of sulfur metabolites in plant tissues by stable isotope dilution-based liquid chromatography–electrospray ionization–tandem mass spectrometry

A wide variety of sulfur metabolites play important roles in plant functions. We have developed a precise and sensitive method for the simultaneous measurement of several sulfur metabolites based on liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) and ³⁴S metabolic labeling of sulfur-containing metabolites in Arabidopsis thaliana seedlings. However, some sulfur metabolites were unstable during the extraction procedure. Our proposed method does not allow for the detection of the important sulfur metabolite homocysteine because of its instability during sample extraction. Stable isotope-labeled sulfur metabolites of A. thaliana shoot were extracted and utilized as internal standards for quantification of sulfur metabolites with LC–MS/MS using S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), methionine (Met), glutathione (GSH), and glutathione disulfide (GSSG) as example metabolites. These metabolites were detected using electrospray ionization in positive mode. Standard curves were linear (r² > 0.99) over a range of concentrations (SAM 0.01–2.0 μM, SAH 0.002–0.10 μM, Met 0.05–4.0 μM, GSH 0.17–20.0 μM, GSSG 0.07–20.0 μM), with limits of detection for SAM, SAH, Met, GSH, and GSSG of 0.83, 0.67, 10, 0.56, and 1.1 nM, respectively; and the within-run and between-run coefficients of variation based on quality control samples were less than 8%.     

Pierce's Disease of Grapevines in Taiwan: Isolation,Cultivation and Pathogenicity of Xylella fastidiosa

Characteristic symptoms of Pierce's disease (PD) in grapevines (Vitis vinifera L.) were observed in 2002 in the major grape production fields of central Taiwan. Disease severity in vineyards varied, and all investigated grape cultivars were affected. Diseased tissues were collected from fields for subsequent isolation and characterization of the causal agent of the disease (Xylella fastidiosa). Koch's postulates were fulfilled by artificially inoculating two purified PD bacteria to grape cultivars Kyoho, Honey Red and Golden Muscat. The inoculated plants developed typical leaf‐scorching symptoms, and similar disease severity developed in the three cultivars from which the bacterium was readily re‐isolated, proving that the leaf scorch of grapevines in Taiwan is caused by the fastidious X. fastidiosa. This confirmed PD of grapevines is also the first report from the Asian Continent. Phylogenetic analyses were performed by comparing the 16S rRNA gene and 16S‐23S rRNA internal transcribed spacer region (16S‐23S ITS) of 12 PD strains from Taiwan with the sequences of 13 X. fastidiosa strains from different hosts and different geographical areas. Results showed that the PD strains of Taiwan were closely related to the American X. fastidiosa grape strains but not to the pear strains of Taiwan, suggesting that the X. fastidiosa grape and pear strains of Taiwan may have evolved independently from each other.     

Risk Factors for Renal Scarring and Deterioration of Renal Function in Primary Vesico-Ureteral Reflux Children: A Long-Term Follow-Up Retrospective Cohort Study

Background and Purpose

The aim was to identify the risk factors for renal scarring and deteriorating renal function in children with primary vesico-ureteral reflux (VUR).

Materials and Methods

Patients with primary VUR admitted to the National Cheng Kung University Hospital were retrospectively analyzed. The outcomes were renal scarring, assessed by technetium-99 m dimercaptosuccinic acid scanning, and renal function, assessed by estimated glomerular filtration rate. Univariate and multivariate models were applied to identify the corresponding independent predictors.

Results

A total of 173 patients with primary VUR were recruited. The median age of VUR diagnosis was 10.0 months (IQR: 4.0–43.0 months). After adjusting for confounding factors, it was found that older age of VUR diagnosis (≥5 years vs. <1 year, adjusted OR = 2.78, 95% CI = 1.00–7.70, p = 0.049), higher grade of VUR (high grade [IV–V] vs. none, adjusted OR = 15.17, 95% CI = 5.33–43.19, p<0.0001; low grade [I–III] vs. none, adjusted OR = 5.72, 95% CI = 2.43–13.45, p<0.0001), and higher number of UTI (≥2 vs. 0, adjusted OR = 3.21, 95% CI = 1.06–9.76, p = 0.039) were risk factors for renal scarring, whereas a younger age of VUR diagnosis (≥5 years vs. <1 year, adjusted HR = 0.16, 95% CI: 0.05–0.51, p = 0.002), renal scarring (yes vs. no, adjusted HR = 3.66, 95% CI: 1.32–10.16, p = 0.013), and APN (yes vs. no, adjusted HR = 3.10, 95% CI: 1.05–9.14, p = 0.041) were risk factors for developing chronic kidney disease stage 2 or higher.

Conclusions

Our findings expand on the current knowledge of risk factors for renal scarring and deteriorating renal function, and this information can be used to modify the management and treatment of VUR.     

The Influence of Hepatitis B Viral Load and Pre-S Deletion Mutations on Post-Operative Recurrence of Hepatocellular Carcinoma and the Tertiary Preventive Effects by Anti-Viral Therapy

Background

Whether or not hepatitis B virus (HBV) genotypes, mutations, and viral loads determine outcomes for patients with HBV-induced hepatocellular carcinoma (HCC) remains controversial.

Aims

To study the influence of HBV viral factors on prognoses for patients with HBV-induced HCC after resection surgery and investigate if antiviral therapy could counteract the adverse effects of viral factors.

Methods

A total of 333 HBV-related HCC patients who underwent tumor resection were enrolled retrospectively. Serum HBV DNA levels, mutations, anti-viral therapy, and other clinical variables were analyzed for their association with post-operative recurrence.

Results

After a median follow-up of 45.9 months, 208 patients had HCC recurrence after resection. The 5-year overall survival and recurrence-free survival rates were 55.4% and 35.3%, respectively. Multivariate analysis showed indocyanine green retention rate at 15 minutes >10%, gamma-glutamyltransferase (GGT) level >60 U/L, macroscopic and microscopic venous invasion, and the absence of anti-viral therapy were significant risk factors for recurrence. Anti-viral therapy could decrease recurrence in patients with early stage HCC, but the effect was less apparent in those with the Barcelona-Clinic Liver Cancer stage C HCC. For patients without antiviral therapy after resection, serum HBV DNA levels >10⁶ copies/mL, GGT >60 U/L, and macroscopic and microscopic venous invasion were significant risk factors predicting recurrence. Among the 216 patients without anti-viral therapy but with complete HBV surface gene mapping data, 73 were with pre-S deletion mutants. Among patients with higher serum HBV DNA levels, those with pre-S deletion had significantly higher rates of recurrence. Moreover, multivariate analysis showed multi-nodularity, macroscopic venous invasion, cirrhosis, advanced tumor cell differentiation, and pre-S deletion were significant risk factors predictive of recurrence.

Conclusions

Ongoing HBV viral replication and pre-S deletion are crucial for determining post-operative tumor recurrence. Anti-viral therapy can help reduce recurrence and improve prognosis, especially for those with early stage HCC.     

Tolerance to multiple climate stressors: a case study of Douglas‐fir drought and cold hardiness

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New Insights on Wood Dimensional Stability Influenced by Secondary Metabolites: The Case of a Fast-Growing Tropical Species Bagassa guianensis Aubl.

Challenging evaluation of tropical forest biodiversity requires the reporting of taxonomic diversity but also the systematic characterization of wood properties in order to discover new promising species for timber industry. Among wood properties, the dimensional stability is regarded as a major technological characteristic to validate whether a wood species is adapted to commercial uses. Cell structure and organization are known to influence the drying shrinkage making wood density and microfibrils angle markers of choice to predict wood dimensional stability. On the contrary the role of wood extractive content remains unclear. This work focuses on the fast-growing tropical species Bagassa guianensis and we report herein a correlation between heartwood drying shrinkage and extractive content. Chemical extractions and shrinkage experiments were performed on separate wood twin samples to better evaluate correctly how secondary metabolites influence the wood shrinkage behaviour. Extractive content were qualitatively and quantitatively analysed using HPLC and NMR spectroscopy. We found that B guianensis heartwood has a homogeneous low shrinkage along its radius that could not be explained only by its basic density. In fact the low drying shrinkage is correlated to the high extractive content and a corrected model to improve the prediction of wood dimensional stability is presented. Additionally NMR experiments conducted on sapwood and heartwood extracts demonstrate that secondary metabolites biosynthesis occurs in sapwood thus revealing B. guianensis as a Juglans-Type heartwood formation. This work demonstrates that B. guianensis, a fast-growing species associated with high durability and high dimensional stability, is a good candidate for lumber production and commercial purposes.