Deep proteogenomics; high throughput gene validation by multidimensional liquid chromatography and mass spectrometry of proteins from the fungal wheat pathogen Stagonospora nodorum

BACKGROUND: Stagonospora nodorum, a fungal ascomycete in the class dothideomycetes, is a damaging pathogen of wheat. It is a model for necrotrophic fungi that cause necrotic symptoms via the interaction of multiple effector proteins with cultivar-specific receptors. A draft genome sequence and annotation was published in 2007. A second-pass gene prediction using a training set of 795 fully EST-supported genes predicted a total of 10762 version 2 nuclear-encoded genes, with an additional 5354 less reliable version 1 genes also retained. RESULTS: In this study, we subjected soluble mycelial proteins to proteolysis followed by 2D LC MALDI-MS/MS. Comparison of the detected peptides with the gene models validated 2134 genes. 62% of these genes (1324) were not supported by prior EST evidence. Of the 2134 validated genes, all but 188 were version 2 annotations. Statistical analysis of the validated gene models revealed a preponderance of cytoplasmic and nuclear localised proteins, and proteins with intracellularassociated GO terms. These statistical associations are consistent with the source of the peptides used in the study. Comparison with a 6-frame translation of the S. nodorum genome assembly confirmed 905 existing gene annotations (including 119 not previously confirmed) and provided evidence supporting 144 genes with coding exon frameshift modifications, 604 genes with extensions of coding exons into annotated introns or untranslated regions (UTRs), 3 new gene annotations which were supported by tblastn to NR, and 44 potential new genes residing within un-assembled regions of the genome. CONCLUSION: We conclude that 2D LC MALDI-MS/MS is a powerful, rapid and economical tool to aid in the annotation of fungal genomic assemblies

Financial Liberalization and Monetary Policy Cooperation in East Asia

As the countries in East Asia embark on financial liberalization, a key issue that confronts policymakers is the greater complexity of risks that is injected into the financial system. In particular, capital account liberalization may potentially increase the vulnerability of individual countries to external financial shocks. This paper advocates the optimally cascading of financial liberalization that is consistent across three dimensions: extent of domestic financial liberalization; the degree of exchange rate flexibility; and the scope of capital account liberalization. Unless the process of liberalization is properly managed, it could provoke destabilizing capital flows and lead to volatile exchange rates. Smooth responses to fluctuating capital flows require accelerated institutional reforms in individual countries and an upgraded regional financial infrastructure. We argue that informal monetary arrangements, sequenced from simple to more intensive commitments, can go a long way in improving sovereign and regional institutions both to handle ongoing financial liberalization and to promote intra-regional currency stability.

Archiving scientific data

We present an archiving technique for hierarchical data with key structure. Our approach is based on the notion of timestamps whereby an element appearing in multiple versions of the database is stored only once along with a compact description of versions in which it appears. The basic idea of timestamping was discovered by Driscoll et. al. in the context of persistent data structures where one wishes to track the sequences of changes made to a data structure. We extend this idea to develop an archiving tool for XML data that is capable of providing meaningful change descriptions and can also efficiently support a variety of basic functions concerning the evolution of data such as retrieval of any specific version from the archive and querying the temporal history of any element. This is in contrast to diff-based approaches where such operations may require undoing a large number of changes or significant reasoning with the deltas. Surprisingly, our archiving technique does not incur any significant space overhead when contrasted with other approaches. Our experimental results support this and also show that the compacted archive file interacts well with other compression techniques. Finally, another useful property of our approach is that the resulting archive is also in XML and hence can directly leverage existing XML tools

Nanomolar-potency 'co-potentiator' therapy for cystic fibrosis caused by a defined subset of minimal function CFTR mutants.

Available CFTR modulators provide no therapeutic benefit for cystic fibrosis (CF) caused by many loss-of-function mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, including N1303K. We previously introduced the concept of 'co-potentiators' (combination-potentiators) to rescue CFTR function in some minimal function CFTR mutants. Herein, a screen of ~120,000 drug-like synthetic small molecules identified active co-potentiators of pyrazoloquinoline, piperidine-pyridoindole, tetrahydroquinoline and phenylazepine classes, with EC50 down to ~300 nM following initial structure-activity studies. Increased CFTR chloride conductance by up to 8-fold was observed when a co-potentiator (termed 'Class II potentiator') was used with a classical potentiator ('Class I potentiator') such as VX-770 or GLPG1837. To investigate the range of CFTR mutations benefitted by co-potentiators, 14 CF-associated CFTR mutations were studied in transfected cell models. Co-potentiator efficacy was found for CFTR missense, deletion and nonsense mutations in nucleotide binding domain-2 (NBD2), including W1282X, N1303K, c.3700A > G and Q1313X (with corrector for some mutations). In contrast, CFTR mutations G85E, R334W, R347P, V520F, R560T, A561E, M1101K and R1162X showed no co-potentiator activity, even with corrector. Co-potentiator efficacy was confirmed in primary human bronchial epithelial cell cultures generated from a N1303K homozygous CF subject. The Class II potentiators identified here may have clinical benefit for CF caused by mutations in the NBD2 domain of CFTR

Temperature dependent photoluminescence in oxygen ion implanted and rapid thermally annealed ZnO/ZnMgO multiple quantum wells

The authors investigate the effect of oxygen implantation and rapid thermal annealing in ZnO∕ZnMgOmultiple quantum wells using photoluminescence. A blueshift in the photoluminescence is observed in the implanted samples. For a low implantation dose, a significant increase of activation energy and a slight increase of the photoluminescence efficiency are observed. This is attributed to the suppression of the point defect complexes and transformation between defect structures by implantation and subsequent rapid thermal annealing. A high dose of implantation leads to lattice damage and agglomeration of defects leading to large defect clusters, which result to an increase in nonradiative recombination.The authors gratefully acknowledge the Australian Research Council for financial support and Swinburne University of Technology for Strategic Initiative funding. One of the authors X.W. acknowledges partial financial support of the Chinese National Natural Science Foundation 10364004 and the Yunnan Natural Science Foundation 2003E0013M

The Transcription Factor StuA Regulates Central Carbon Metabolism, Mycotoxin Production, and Effector Gene Expression in the Wheat Pathogen Stagonospora nodorum

The Stagonospora nodorum StuA transcription factor gene SnStuA was identified by homology searching in the genome of the wheat pathogen Stagonospora nodorum. Gene expression analysis revealed that SnStuA transcript abundance increased throughout infection and in vitro growth to peak during sporulation. To investigate its role, the gene was deleted by homologous recombination. The growth of the resulting mutants was retarded on glucose compared to the wild-type growth, and the mutants also failed to sporulate. Glutamateas a sole carbon source restored the growth rate defect observed on glucose, although sporulation remained impaired. The SnstuA strains were essentially nonpathogenic, with only minor growth observed around the point of inoculation. The role of SnstuA was investigated using metabolomics, which revealed that this gene's product played a key role in regulating central carbon metabolism, with glycolysis, the TCA cycle, and amino acid synthesis all affected in the mutants. SnStuA was also found to positively regulate the synthesis of the mycotoxin alternariol. Gene expression studies on the recently identified effectors in Stagonospora nodorum found that SnStuA was a positive regulator of SnTox3 but was not required for the expression of ToxA. This study has uncovered a multitude of novel regulatory targets of SnStuA and has highlighted the critical role of this gene product in the pathogenicity of Stagonospora nodorum

SnTox3 Acts in Effector Triggered Susceptibility to Induce Disease on Wheat Carrying the Snn3 Gene

The necrotrophic fungus Stagonospora nodorum produces multiple proteinaceous host-selective toxins (HSTs) which act in effector triggered susceptibility. Here, we report the molecular cloning and functional characterization of the SnTox3-encoding gene, designated SnTox3, as well as the initial characterization of the SnTox3 protein. SnTox3 is a 693 bp intron-free gene with little obvious homology to other known genes. The predicted immature SnTox3 protein is 25.8 kDa in size. A 20 amino acid signal sequence as well as a possible pro sequence are predicted. Six cysteine residues are predicted to form disulfide bonds and are shown to be important for SnTox3 activity. Using heterologous expression in Pichia pastoris and transformation into an avirulent S. nodorum isolate, we show that SnTox3 encodes the SnTox3 protein and that SnTox3 interacts with the wheat susceptibility gene Snn3. In addition, the avirulent S. nodorum isolate transformed with SnTox3 was virulent on host lines expressing the Snn3 gene. SnTox3-disrupted mutants were deficient in the production of SnTox3 and avirulent on the Snn3 differential wheat line BG220. An analysis of genetic diversity revealed that SnTox3 is present in 60.1% of a worldwide collection of 923 isolates and occurs as eleven nucleotide haplotypes resulting in four amino acid haplotypes. The cloning of SnTox3 provides a fundamental tool for the investigation of the S. nodorum-wheat interaction, as well as vital information for the general characterization of necrotroph-plant interactions.This work was supported by USDA-ARS CRIS projects 5442-22000-043-00D and 5442-22000-030-00D

Extending the overnight fast : sex differences in acute metabolic responses to breakfast

Fasting for over 24 h is associated with worsening glucose tolerance, but the effect of extending the overnight fast period (a form of time-restricted feeding) on acute metabolic responses and insulin sensitivity is unclear. The aim of this pilot study was to determine the acute impact of an increased fasting period on postprandial glycaemia, insulinemia, and acute insulin sensitivity responses to a standard meal. Twenty-four lean, young, healthy adults (12 males, 12 females) consumed a standard breakfast after an overnight fast of 12, 14, and 16 h. Each fast duration was repeated on three separate occasions (3 × 3) in random order. Postprandial glucose and insulin responses were measured at regular intervals over 2 h and quantified as incremental area under the curve (iAUC). Insulin sensitivity was determined by homeostatic modelling assessment (HOMA). After 2 h, ad libitum food intake at a buffet meal was recorded. In females, but not males, insulin sensitivity improved (HOMA%S +35%, p = 0.016, marginally significant) with longer fast duration (16 h vs. 12 h), but paradoxically, postprandial glycaemia was higher (glucose iAUC +37%, p = 0.002). Overall, males showed no differences in glucose or insulin homeostasis. Both sexes consumed more energy (+28%) at the subsequent meal (16 h vs. 12 h). Delaying the first meal of the day by 4 h by extending the fasting period may have adverse metabolic effects in young, healthy, adult females, but not males

Pretreatment Effects on the Uptake/Retention Kinetics of L-Dopa in Harding-Passey Melanoma

Malignant melanoma cells possess a unique biochemical pathway that converts L-3,4-dihydroxyphenylalanine (L-dopa) to the biopigment melanin. Selective cytotoxic incorporation of exogenous L-dopa into melanoma cells in vivo may provide a means of designing specific chemotherapeutic agents useful in the treatment of this disease. Using the Harding-Passey murine melanotic tumor model, a preferential uptake of [3H]L-dopa by the tumor was characterized. Following pretreatment of the tumor-bearing mice with nonradioactive L-dopa, a significant enhancement (p < 0.01) of [3H]L-dopa incorporation and retention into melanoma for a period of 24h was observed, when compared with the concomitant tissue distribution and clearance of radioactivity in the control animals. This finding suggests that by initial pretreatment of melanoma with nonradioactive L-dopa, the subsequent selective accumulation of [3H]L-dopa in tumor may provide a useful tool in testing new modalities of therapy in malignant melanoma