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

Tight cycles in hypergraphs

We apply a recent version of the Strong Hypergraph Regularity Lemma(see [1], [2]) to prove two new results on tight cycles in k-uniform hypergraphs. The first result is an extension of the Erdos-Gallai Theorem for graphs: For every > 0, every sufficiently large k-uniform hypergraph on n vertices with at least edges contains a tight cycle of length @n for any @ 2 [0; 1]. Our second result concerns k-partite k-uniform hypergraphs with partition classes of size n and for each @ 2 (0; 1) provides an asymptotically optimal minimum codegree requirement for the hypergraph to contain a cycle of length @kn

Regular slices for hypergraphs

We present a ‘Regular Slice Lemma’ which, given a k -graph GG, returns a regular (k−1)(k−1)-complex JJ with respect to which GG has useful regularity properties. We believe that many arguments in extremal hypergraph theory are made considerably simpler by using this lemma rather than existing forms of the Strong Hypergraph Regularity Lemma, and advocate its use for this reason

Ku80 Counters Oxidative Stress-Induced DNA Damage and Cataract Formation in the Human Lens

PURPOSE: Oxidative stress in the human lens leads to a wide range of damage including DNA strand breaks, which are likely to contribute to cataract formation. The protein Ku80 is a fundamental component of the nonhomologous end-joining pathway that repairs DNA double strand breaks. This study investigates the putative impact of Ku80 in cataract prevention in the human lens. METHODS: The present study used the human lens epithelial cell line FHL124 and whole human lens organ culture. Targeted siRNA was used to deplete Ku80, with Western blot and immunocytochemistry employed to assess Ku80 expression levels. Oxidative stress was induced with hydrogen peroxide and DNA strand breaks measured by alkaline comet assay and γH2AX foci counts. Visual quality of whole human lenses was measured with image analysis software. RESULTS: Expression of Ku80 was predominately found in the cell nucleus of both FHL124 cells and native human lens epithelium. Treatment of FHL124 cells and whole lens cultures with siRNA targeted against Ku80 resulted in a significant knockdown at the protein level. Application of oxidative stress (30 μM H2O2) created more DNA strand breaks when added to Ku80 knockdown cells than in scrambled siRNA control cells as determined by the alkaline comet assay and the number of γH2AX foci. In whole lens cultures, exposure to 1 mM H2O2 resulted in more lens opacity in Ku80 knockdown lenses than match-paired controls. CONCLUSIONS: Depletion of Ku80 in the lens through acute change or a consequence of aging is likely to increase levels of DNA strand breaks, which could negatively influence physiological function and promote lens opacity. It is therefore feasible that Ku80 plays a role in retarding cataract formation

The Intrinsic Connectome of the Rat Amygdala

The connectomes of nervous systems or parts there of are becoming important subjects of study as the amount of connectivity data increases. Because most tract-tracing studies are performed on the rat, we conducted a comprehensive analysis of the amygdala connectome of this species resulting in a meta-study. The data were imported into the neuroVIISAS system, where regions of the connectome are organized in a controlled ontology and network analysis can be performed. A weighted digraph represents the bilateral intrinsic (connections of regions of the amygdala) and extrinsic (connections of regions of the amygdala to non-amygdaloid regions) connectome of the amygdala. Its structure as well as its local and global network parameters depend on the arrangement of neuronal entities in the ontology. The intrinsic amygdala connectome is a small-world and scale-free network. The anterior cortical nucleus (72 in- and out-going edges), the posterior nucleus (45), and the anterior basomedial nucleus (44) are the nuclear regions that posses most in- and outdegrees. The posterior nucleus turns out to be the most important nucleus of the intrinsic amygdala network since its Shapley rate is minimal. Within the intrinsic amygdala, regions were determined that are essential for network integrity. These regions are important for behavioral (processing of emotions and motivation) and functional (memory) performances of the amygdala as reported in other studies

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 intracellular-associated 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

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

Overall and income specific effect on prevalence of overweight and obesity of 20% sugar sweetened drink tax in UK: econometric and comparative risk assessment modelling study

Objective To model the overall and income specific effect of a 20% tax on sugar sweetened drinks on the prevalence of overweight and obesity in the UK. Design Econometric and comparative risk assessment modelling study. Setting United Kingdom. Population Adults aged 16 and over. Intervention A 20% tax on sugar sweetened drinks. Main outcome measures The primary outcomes were the overall and income specific changes in the number and percentage of overweight (body mass index ≥25) and obese (≥30) adults in the UK following the implementation of the tax. Secondary outcomes were the effect by age group (16-29, 30-49, and ≥50 years) and by UK constituent country. The revenue generated from the tax and the income specific changes in weekly expenditure on drinks were also estimated. Results A 20% tax on sugar sweetened drinks was estimated to reduce the number of obese adults in the UK by 1.3% (95% credible interval 0.8% to 1.7%) or 180 000 (110 000 to 247 000) people and the number who are overweight by 0.9% (0.6% to 1.1%) or 285 000 (201 000 to 364 000) people. The predicted reductions in prevalence of obesity for income thirds 1 (lowest income), 2, and 3 (highest income) were 1.3% (0.3% to 2.0%), 0.9% (0.1% to 1.6%), and 2.1% (1.3% to 2.9%). The effect on obesity declined with age. Predicted annual revenue was £276m (£272m to £279m), with estimated increases in total expenditure on drinks for income thirds 1, 2, and 3 of 2.1% (1.4% to 3.0%), 1.7% (1.2% to 2.2%), and 0.8% (0.4% to 1.2%). Conclusions A 20% tax on sugar sweetened drinks would lead to a reduction in the prevalence of obesity in the UK of 1.3% (around 180 000 people). The greatest effects may occur in young people, with no significant differences between income groups. Both effects warrant further exploration. Taxation of sugar sweetened drinks is a promising population measure to target population obesity, particularly among younger adults