The FRP1 F-box gene has different functions in sexuality, pathogenicity and metabolism in three fungal pathogens

Plant‐pathogenic fungi employ a variety of infection strategies; as a result, fungi probably rely on different sets of proteins for successful infection. The F‐box protein Frp1, only present in filamentous fungi belonging to the Sordariomycetes, Leotiomycetes and Dothideomycetes, is required for nonsugar carbon catabolism and pathogenicity in the root‐infecting fungus Fusarium oxysporum. To assess the role of Frp1 in other plant‐pathogenic fungi, FRP1 deletion mutants were generated in Fusarium graminearum and Botrytis cinerea, and their phenotypes were analysed. Deletion of FgFRP1 in F. graminearum led to impaired infection of barley roots, but not of aerial plant parts. Deletion of BcFRP1 in B. cinerea did not show any effect on pathogenicity. Sexual reproduction, however, was impaired in both F. graminearum and B. cinerea FRP1 deletion mutants. The mutants of all three fungi displayed different phenotypes when grown on an array of carbon sources. The F. oxysporum and B. cinerea deletion mutants showed opposite growth phenotypes on sugar and nonsugar carbon sources. Replacement of FoFRP1 in F. oxysporum with the B. cinerea BcFRP1 resulted in the restoration of pathogenicity, but also in a switch from impaired growth on nonsugar carbon sources to impaired growth on sugar carbon sources. This effect could be ascribed in part to the B. cinerea BcFRP1 promoter sequence. In conclusion, the function of the F‐box protein Frp1, despite its high sequence conservation, is not conserved between different fungi, leading to differential requirements for pathogenicity and carbon source utilization.     

Thromboelastometry (TEM) findings in disseminated intravascular coagulation in a pig model of endotoxinemia

Standard coagulation tests have a low specificity and sensitivity for diagnosing disseminated intravascular coagulation. The aim of this study was to determine whether whole blood thromboelastometry (TEM) detects lipopolysaccharide (LPS)-induced changes in coagulation. Blood samples from 10 pigs were drawn at baseline, before and at the end of LPS infusion and 2, 3, 4 and 5 h after the start of endotoxinemia. Simultaneous to TEM, standard coagulation tests and extended coagulation analysis including tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1) were performed. Endotoxinemia resulted in a significant acceleration of the nonactivated TEM (NATEM) clotting time 2 h after the end of LPS infusion; in contrast, the changes in international normalized ratio and activated partial thromboplastin time suggested delayed initiation of coagulation. NATEM maximum clot firmness (MCF) and fibrin-based thromboelastometry test (FIBTEM)-MCF decreased significantly from baseline until the last time point (from 64.6 ± 7.8 and 35.1 ± 12.8 mm to 52.8 ± 4.6 and 21.4 ± 11.8 mm, respectively; P = 0.01 for both parameters). A sharp, transient increase of t-PA had no effect on maximum lysis in the NATEM test. PAI-1 increased significantly 3 h after the start of LPS infusion, paralleled by a decrease in maximum lysis. In conclusion, TEM was superior to standard coagulation tests in reflecting initial activation of coagulation during endotoxinemia. TEM further suggested consumption of coagulation substrate; at the same time, inhibition of plasminogen activation was accompanied by improved clot stability. Further investigations are necessary to establish the clinical relevance of these findings.     

FACIL: Fast and Accurate Genetic Code Inference and Logo

MOTIVATION: The intensification of DNA sequencing will increasingly unveil uncharacterized species with potential alternative genetic codes. A total of 0.65% of the DNA sequences currently in Genbank encode their proteins with a variant genetic code, and these exceptions occur in many unrelated taxa. RESULTS: We introduce FACIL (Fast and Accurate genetic Code Inference and Logo), a fast and reliable tool to evaluate nucleic acid sequences for their genetic code that detects alternative codes even in species distantly related to known organisms. To illustrate this, we apply FACIL to a set of mitochondrial genomic contigs of Globobulimina pseudospinescens. This foraminifer does not have any sequenced close relative in the databases, yet we infer its alternative genetic code with high confidence values. Results are intuitively visualized in a Genetic Code Logo. Availability and implementation: FACIL is available as a web-based service at http://www.cmbi.ru.nl/FACIL/ and as a stand-alone program.     

PathVisio-MIM: PathVisio plugin for creating and editing Molecular Interaction Maps (MIMs)

MOTIVATION: A plugin for the Java-based PathVisio pathway editor has been developed to help users draw diagrams of bioregulatory networks according to the Molecular Interaction Map (MIM) notation. Together with the core PathVisio application, this plugin presents a simple to use and cross-platform application for the construction of complex MIM diagrams with the ability to annotate diagram elements with comments, literature references and links to external databases. This tool extends the capabilities of the PathVisio pathway editor by providing both MIM-specific glyphs and support for a MIM-specific markup language file format for exchange with other MIM-compatible tools and diagram validation. AVAILABILITY: The PathVisio-MIM plugin is freely available and works with versions of PathVisio 2.0.11 and later on Windows, Mac OS X and Linux. Information about MIM notation and the MIMML format is available at http://discover.nci.nih.gov/mim. The plugin, along with diagram examples, instructions and Java source code, may be downloaded at http://discover.nci.nih.gov/mim/mim_pathvisio.html.     

Binding of human milk to pathogen receptor DC-SIGN varies with bile salt-stimulated lipase (BSSL) gene polymorphism

Objective

Dendritic cells bind an array of antigens and DC-SIGN has been postulated to act as a receptor for mucosal pathogen transmission. Bile salt-stimulated lipase (BSSL) from human milk potently binds DC-SIGN and blocks DC-SIGN mediated trans-infection of CD4⁺ T-lymphocytes with HIV-1. Objective was to study variation in DC-SIGN binding properties and the relation between DC-SIGN binding capacity of milk and BSSL gene polymorphisms.

Study Design

ELISA and PCR were used to study DC-SIGN binding properties and BSSL exon 11 size variation for human milk derived from 269 different mothers distributed over 4 geographical regions.

Results

DC-SIGN binding properties were highly variable for milks derived from different mothers and between samplings from different geographical regions. Differences in DC-SIGN binding were correlated with a genetic polymorphism in BSSL which is related to the number of 11 amino acid repeats at the C-terminus of the protein.

Conclusion

The observed variation in DC-SIGN binding properties among milk samples may have implications for the risk of mucosal transmission of pathogens during breastfeeding.     

Proteomic analysis of the major birch allergen Bet v 1 predicts allergenicity for 15 birch species

Pollen of the European and Asian white birch (Betula pendula and B. platyphylla) causes hay fever in humans. The allergenic potency of other birch species is largely unknown. To identify birch trees with a reduced allergenicity, we assessed the immunochemical characteristics of 15 species and two hybrids, representing four subgenera within the genus Betula, while focusing on the major pollen allergen Bet v 1. Antigenic and allergenic profiles of pollen extracts from these species were evaluated by SDS-PAGE and Western blot using pooled sera of birch-allergic individuals. Tryptic digests of the Bet v 1 bands were analyzed by LC-MS(E) to determine the abundance of various Bet v 1 isoforms. Bet v 1 was the most abundant pollen protein across all birch species. LC-MS(E) confirmed that pollen of all species contained a mixture of multiple Bet v 1 isoforms. Considerable differences in Bet v 1 isoform composition exist between birch species. However, isoforms that are predicted to have a high IgE-reactivity prevailed in pollen of all species. Immunoblotting confirmed that all pollen extracts were similar in immune-reactivity, implying that pollen of all birch species is likely to evoke strong allergic reactions.     

Non-invasive imaging of cysteine cathepsin activity in solid tumors using a 64Cu-labeled activity-based probe

The papain family of cysteine cathepsins are actively involved in multiple stages of tumorigenesis. Because elevated cathepsin activity can be found in many types of human cancers, they are promising biomarkers that can be used to target radiological contrast agents for tumor detection. However, currently there are no radiological imaging agents available for these important molecular targets. We report here the development of positron emission tomography (PET) radionuclide-labeled probes that target the cysteine cathepsins by formation of an enzyme activity-dependent bond with the active site cysteine. These probes contain an acyloxymethyl ketone (AOMK) functional group that irreversibly labels the active site cysteine of papain family proteases attached to a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) tag for labeling with (64)Cu for PET imaging studies. We performed biodistribution and microPET imaging studies in nude mice bearing subcutaneous tumors expressing various levels of cysteine cathepsin activity and found that the extent of probe uptake by tumors correlated with overall protease activity as measured by biochemical methods. Furthermore, probe signals could be reduced by pre-treatment with a general cathepsin inhibitor. We also found that inclusion of a Cy5 tag on the probe increased tumor uptake relative to probes lacking this fluorogenic dye. Overall, these results demonstrate that small molecule activity-based probes carrying radio-tracers can be used to image protease activity in living subjects.     

The flying buttress construct for posterior spinopelvic fixation: a technical note

Background

School screening for adolescent idiopathic scoliosis (AIS) is discussed. The aim of the present study was to describe the point prevalence of AIS and to evaluate the effectiveness of school screening in 12-year- old children.

Methods

Community nurses and physical therapists in the Southern Health region of Norway including about 12000 school children aged 12 years were invited to participate. All participating community nurses and physical therapists fulfilled an educational course to improve their knowledge about AIS and learn the screening procedure including the Adam Forward Bending Test and measurement of gibbus using a scoliometer.

Results

Sub-regions including 4000 school children participated. The prevalence of idiopathic scoliosis defined as a positive Adam Forward Bending Test, gibbus > 7° and primary major curve on radiographs > 10°, was 0.55%. Five children (0.13%) had a major curve > 20°. Bracing was not indicated in any child; all children were post menarche; four had Risser sign of 4, and one with Risser 1 did not have curve progression > 5° at later follow-up. In one of these 5 children however, the major curve progressed to 45° within 7 months after screening and the girl was operated.

Conclusion

The point prevalence of AIS in 12- year old children is in agreement or slightly lower than previous studies. The screening model employed demonstrates acceptable sensitivity and specificity and low referral rates. Screening at the age of 12 years only was not effective for detecting patients with indication for brace treatment.     

High fat diet-induced changes in mouse muscle mitochondrial phospholipids do not impair mitochondrial respiration despite insulin resistance

Background

Type 2 diabetes mellitus and muscle insulin resistance have been associated with reduced capacity of skeletal muscle mitochondria, possibly as a result of increased intake of dietary fat. Here, we examined the hypothesis that a prolonged high-fat diet consumption (HFD) increases the saturation of muscle mitochondrial membrane phospholipids causing impaired mitochondrial oxidative capacity and possibly insulin resistance.

Methodology

C57BL/6J mice were fed an 8-week or 20-week low fat diet (10 kcal%; LFD) or HFD (45 kcal%). Skeletal muscle mitochondria were isolated and fatty acid (FA) composition of skeletal muscle mitochondrial phospholipids was analyzed by thin-layer chromatography followed by GC. High-resolution respirometry was used to assess oxidation of pyruvate and fatty acids by mitochondria. Insulin sensitivity was estimated by HOMA-IR.

Principal Findings

At 8 weeks, mono-unsaturated FA (16∶1n7, 18∶1n7 and 18∶1n9) were decreased (−4.0%, p<0.001), whereas saturated FA (16∶0) were increased (+3.2%, p<0.001) in phospholipids of HFD vs. LFD mitochondria. Interestingly, 20 weeks of HFD descreased mono-unsaturated FA while n-6 poly-unsaturated FA (18∶2n6, 20∶4n6, 22∶5n6) showed a pronounced increase (+4.0%, p<0.001). Despite increased saturation of muscle mitochondrial phospholipids after the 8-week HFD, mitochondrial oxidation of both pyruvate and fatty acids were similar between LFD and HFD mice. After 20 weeks of HFD, the increase in n-6 poly-unsaturated FA was accompanied by enhanced maximal capacity of the electron transport chain (+49%, p = 0.002) and a tendency for increased ADP-stimulated respiration, but only when fuelled by a lipid-derived substrate. Insulin sensitivity in HFD mice was reduced at both 8 and 20 weeks.

Conclusions/Interpretation

Our findings do not support the concept that prolonged HF feeding leads to increased saturation of skeletal muscle mitochondrial phospholipids resulting in a decrease in mitochondrial fat oxidative capacity and (muscle) insulin resistance.