Evolution of substrate-specific gene expression and RNA editing in brown rot wood-decaying fungi.

Fungi that decay wood have characteristic associations with certain tree species, but the mechanistic bases for these associations are poorly understood. We studied substrate-specific gene expression and RNA editing in six species of wood-decaying fungi from the 'Antrodia clade' (Polyporales, Agaricomycetes) on three different wood substrates (pine, spruce, and aspen) in submerged cultures. We identified dozens to hundreds of substrate-biased genes (i.e., genes that are significantly upregulated in one substrate relative to the other two substrates) in each species, and these biased genes are correlated with their host ranges. Evolution of substrate-biased genes is associated with gene family expansion, gain and loss of genes, and variation in cis- and trans- regulatory elements, rather than changes in protein coding sequences. We also demonstrated widespread RNA editing events in the Antrodia clade, which differ from those observed in the Ascomycota in their distribution, substitution types, and the genomic environment. Moreover, we found that substrates could affect editing positions and frequency, including editing events occurring in mRNA transcribed from wood-decay-related genes. This work shows the extent to which gene expression and RNA editing differ among species and substrates, and provides clues into mechanisms by which wood-decaying fungi may adapt to different hosts

Integrative Discovery of Epigenetically Derepressed Cancer Testis Antigens in NSCLC

Background: Cancer/testis antigens (CTAs) were first discovered as immunogenic targets normally expressed in germline cells, but differentially expressed in a variety of human cancers. In this study, we used an integrative epigenetic screening approach to identify coordinately expressed genes in human non-small cell lung cancer (NSCLC) whose transcription is driven by promoter demethylation. Methodology/Principal Findings: Our screening approach found 290 significant genes from the over 47,000 transcripts incorporated in the Affymetrix Human Genome U133 Plus 2.0 expression array. Of the top 55 candidates, 10 showed both differential overexpression and promoter region hypomethylation in NSCLC. Surprisingly, 6 of the 10 genes discovered by this approach were CTAs. Using a separate cohort of primary tumor and normal tissue, we validated NSCLC promoter hypomethylation and increased expression by quantitative RT-PCR for all 10 genes. We noted significant, coordinated coexpression of multiple target genes, as well as coordinated promoter demethylation, in a large set of individual tumors that was associated with the SCC subtype of NSCLC. In addition, we identified 2 novel target genes that exhibited growth- promoting effects in multiple cell lines. Conclusions/Significance: Coordinated promoter demethylation in NSCLC is associated with aberrant expression of CTAs and potential, novel candidate protooncogenes that can be identified using integrative discovery techniques. These findings have significant implications for discovery of novel CTAs and CT antigen directed immunotherapy. © 2009 Glazer et al

Global Analysis of Predicted G Protein-Coupled Receptor Genes in the Filamentous Fungus, Neurospora crassa.

G protein-coupled receptors (GPCRs) regulate facets of growth, development, and environmental sensing in eukaryotes, including filamentous fungi. The largest predicted GPCR class in these organisms is the Pth11-related, with members similar to a protein required for disease in the plant pathogen Magnaporthe oryzae. However, the Pth11-related class has not been functionally studied in any filamentous fungal species. Here, we analyze phenotypes in available mutants for 36 GPCR genes, including 20 Pth11-related, in the model filamentous fungus Neurospora crassa. We also investigate patterns of gene expression for all 43 predicted GPCR genes in available datasets. A total of 17 mutants (47%) possessed at least one growth or developmental phenotype. We identified 18 mutants (56%) with chemical sensitivity or nutritional phenotypes (11 uniquely), bringing the total number of mutants with at least one defect to 28 (78%), including 15 mutants (75%) in the Pth11-related class. Gene expression trends for GPCR genes correlated with the phenotypes observed for many mutants and also suggested overlapping functions for several groups of co-transcribed genes. Several members of the Pth11-related class have phenotypes and/or are differentially expressed on cellulose, suggesting a possible role for this gene family in plant cell wall sensing or utilization

Serum microrna biomarkers for detection of non-small cell lung cancer

Non small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality world-wide and the majority of cases are diagnosed at late stages of disease. There is currently no cost-effective screening test for NSCLC, and the development of such a test is a public health imperative. Recent studies have suggested that chest computed tomography screening of patients at high risk of lung cancer can increase survival from disease, however, the cost effectiveness of such screening has not been established. In this Phase I/II biomarker study we examined the feasibility of using serum miRNA as biomarkers of NSCLC using RT-qPCR to examine the expression of 180 miRNAs in sera from 30 treatment naive NSCLC patients and 20 healthy controls. Receiver operating characteristic curves (ROC) and area under the curve were used to identify differentially expressed miRNA pairs that could distinguish NSCLC from healthy controls. Selected miRNA candidates were further validated in sera from an additional 55 NSCLC patients and 75 healthy controls. Examination of miRNA expression levels in serum from a multi-institutional cohort of 50 subjects (30 NSCLC patients and 20 healthy controls) identified differentially expressed miRNAs. A combination of two differentially expressed miRNAs miR-15b and miR-27b, was able to discriminate NSCLC from healthy controls with sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of 100% in the training set. Upon further testing on additional 130 subjects (55 NSCLC and 75 healthy controls), this miRNA pair predicted NSCLC with a specificity of 84% (95% CI 0.73-0.91), sensitivity of 100% (95% CI; 0.93-1.0), NPV of 100%, and PPV of 82%. These data provide evidence that serum miRNAs have the potential to be sensitive, cost-effective biomarkers for the early detection of NSCLC. Further testing in a Phase III biomarker study in is necessary for validation of these results. © 2012 Hennessey et al

Conserved white-rot enzymatic mechanism for wood decay in the Basidiomycota genus Pycnoporus

White-rot (WR) fungi are pivotal decomposers of dead organic matter in forest ecosystems and typically use a large array of hydrolytic and oxidative enzymes to deconstruct lignocellulose. However, the extent of lignin and cellulose degradation may vary between species and wood type. Here, we combined comparative genomics, transcriptomics and secretome proteomics to identify conserved enzymatic signatures at the onset of wood-decaying activity within the Basidiomycota genus Pycnoporus. We observed a strong conservation in the genome structures and the repertoires of protein-coding genes across the four Pycnoporus species described to date, despite the species having distinct geographic distributions. We further analysed the early response of P. cinnabarinus, P. coccineus and P. sanguineus to diverse (ligno)-cellulosic substrates. We identified a conserved set of enzymes mobilized by the three species for breaking down cellulose, hemicellulose and pectin. The co-occurrence in the exo-proteomes of H2O2-producing enzymes with H2O2-consuming enzymes was a common feature of the three species, although each enzymatic partner displayed independent transcriptional regulation. Finally, cellobiose dehydrogenase-coding genes were systematically co-regulated with at least one AA9 lytic polysaccharide monooxygenase gene, indicative of enzymatic synergy in vivo. This study highlights a conserved core white-rot fungal enzymatic mechanism behind the wood-decaying process.Peer reviewe

Somatic mutations affect key pathways in lung adenocarcinoma

Determining the genetic basis of cancer requires comprehensive analyses of large collections of histopathologically well- classified primary tumours. Here we report the results of a collaborative study to discover somatic mutations in 188 human lung adenocarcinomas. DNA sequencing of 623 genes with known or potential relationships to cancer revealed more than 1,000 somatic mutations across the samples. Our analysis identified 26 genes that are mutated at significantly high frequencies and thus are probably involved in carcinogenesis. The frequently mutated genes include tyrosine kinases, among them the EGFR homologue ERBB4; multiple ephrin receptor genes, notably EPHA3; vascular endothelial growth factor receptor KDR; and NTRK genes. These data provide evidence of somatic mutations in primary lung adenocarcinoma for several tumour suppressor genes involved in other cancers - including NF1, APC, RB1 and ATM - and for sequence changes in PTPRD as well as the frequently deleted gene LRP1B. The observed mutational profiles correlate with clinical features, smoking status and DNA repair defects. These results are reinforced by data integration including single nucleotide polymorphism array and gene expression array. Our findings shed further light on several important signalling pathways involved in lung adenocarcinoma, and suggest new molecular targets for treatment.National Human Genome Research InstituteWe thank A. Lash, M.F. Zakowski, M.G. Kris and V. Rusch for intellectual contributions, and many members of the Baylor Human Genome Sequencing Center, the Broad Institute of Harvard and MIT, and the Genome Center at Washington University for support. This work was funded by grants from the National Human Genome Research Institute to E.S.L., R.A.G. and R.K.W.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62885/1/nature07423.pd

Exploring the binding properties and structural stability of an opsin in the chytrid Spizellomyces punctatus using comparative and molecular modeling

Background Opsin proteins are seven transmembrane receptor proteins which detect light. Opsins can be classified into two types and share little sequence identity: type 1, typically found in bacteria, and type 2, primarily characterized in metazoa. The type 2 opsins (Rhodopsins) are a subfamily of G-protein coupled receptors (GPCRs), a large and diverse class of seven transmembrane proteins and are generally restricted to metazoan lineages. Fungi use light receptors including opsins to sense the environment and transduce signals for developmental or metabolic changes. Opsins characterized in the Dikarya (Ascomycetes and Basidiomycetes) are of the type 1 bacteriorhodopsin family but the early diverging fungal lineages have not been as well surveyed. We identified by sequence similarity a rhodopsin-like GPCR in genomes of early diverging chytrids and examined the structural characteristics of this protein to assess its likelihood to be homologous to animal rhodopsins and bind similar chromophores. Methods We used template-based structure modeling, automated ligand docking, and molecular modeling to assess the structural and binding properties of an identified opsin-like protein found in Spizellomyces punctatus, a unicellular, flagellated species belonging to Chytridiomycota, one of the earliest diverging fungal lineages. We tested if the sequence and inferred structure were consistent with a solved crystal structure of a type 2 rhodopsin from the squid Todarodes pacificus. Results Our results indicate that the Spizellomyces opsin has structural characteristics consistent with functional animal type 2 rhodopsins and is capable of maintaining a stable structure when associated with the retinaldehyde chromophore, specifically the 9-cis-retinal isomer. Together, these results support further the homology of Spizellomyces opsins to animal type 2 rhodopsins. Discussion This represents the first test of structure/function relationship of a type 2 rhodopsin identified in early branching fungal lineages, and provides a foundation for future work exploring pathways and components of photoreception in early fungi

Exploring the binding properties and structural stability of an opsin in the chytrid Spizellomyces punctatus using comparative and molecular modeling.

BackgroundOpsin proteins are seven transmembrane receptor proteins which detect light. Opsins can be classified into two types and share little sequence identity: type 1, typically found in bacteria, and type 2, primarily characterized in metazoa. The type 2 opsins (Rhodopsins) are a subfamily of G-protein coupled receptors (GPCRs), a large and diverse class of seven transmembrane proteins and are generally restricted to metazoan lineages. Fungi use light receptors including opsins to sense the environment and transduce signals for developmental or metabolic changes. Opsins characterized in the Dikarya (Ascomycetes and Basidiomycetes) are of the type 1 bacteriorhodopsin family but the early diverging fungal lineages have not been as well surveyed. We identified by sequence similarity a rhodopsin-like GPCR in genomes of early diverging chytrids and examined the structural characteristics of this protein to assess its likelihood to be homologous to animal rhodopsins and bind similar chromophores.MethodsWe used template-based structure modeling, automated ligand docking, and molecular modeling to assess the structural and binding properties of an identified opsin-like protein found in Spizellomyces punctatus, a unicellular, flagellated species belonging to Chytridiomycota, one of the earliest diverging fungal lineages. We tested if the sequence and inferred structure were consistent with a solved crystal structure of a type 2 rhodopsin from the squid Todarodes pacificus.ResultsOur results indicate that the Spizellomyces opsin has structural characteristics consistent with functional animal type 2 rhodopsins and is capable of maintaining a stable structure when associated with the retinaldehyde chromophore, specifically the 9-cis-retinal isomer. Together, these results support further the homology of Spizellomyces opsins to animal type 2 rhodopsins.DiscussionThis represents the first test of structure/function relationship of a type 2 rhodopsin identified in early branching fungal lineages, and provides a foundation for future work exploring pathways and components of photoreception in early fungi