Exploring gene expression biomarker candidates for neurobehavioral impairment from total sleep deprivation.

BACKGROUND: Although sleep deprivation is associated with neurobehavioral impairment that may underlie significant risks to performance and safety, there is no reliable biomarker test to detect dangerous levels of impairment from sleep loss in humans. This study employs microarrays and bioinformatics analyses to explore candidate gene expression biomarkers associated with total sleep deprivation (TSD), and more specifically, the phenotype of neurobehavioral impairment from TSD. Healthy adult volunteers were recruited to a sleep laboratory for seven consecutive days (six nights). After two Baseline nights of 10 h time in bed, 11 subjects underwent an Experimental phase of 62 h of continuous wakefulness, followed by two Recovery nights of 10 h time in bed. Another six subjects underwent a well-rested Control condition of 10 h time in bed for all six nights. Blood was drawn for measuring gene expression on days two, four, and six at 4 h intervals from 08:00 to 20:00 h, corresponding to 12 timepoints across one Baseline, one Experimental, and one Recovery day. RESULTS: Altogether 212 genes changed expression in response to the TSD Treatment, with most genes exhibiting down-regulation during TSD. Also, 28 genes were associated with neurobehavioral impairment as measured by the Psychomotor Vigilance Test. The results support previous findings associating TSD with the immune response and ion signaling, and reveal novel candidate biomarkers such as the Speedy/RINGO family of cell cycle regulators. CONCLUSIONS: This study serves as an important step toward understanding gene expression changes during sleep deprivation. In addition to exploring potential biomarkers for TSD, this report presents novel candidate biomarkers associated with lapses of attention during TSD. Although further work is required for biomarker validation, analysis of these genes may aid fundamental understanding of the impact of TSD on neurobehavioral performance.

Proceedings of the 2013 MidSouth Computational Biology and Bioinformatics Society (MCBIOS) Conference.

The tenth annual conference of the MidSouth Computational Biology and Bioinformatics Society (MCBIOS 2013), "The 10th Anniversary in a Decade of Change: Discovery in a Sea of Data", took place at the Stoney Creek Inn & Conference Center in Columbia, Missouri on April 5-6, 2013. This year's Conference Chairs were Gordon Springer and Chi-Ren Shyu from the University of Missouri and Edward Perkins from the US Army Corps of Engineers Engineering Research and Development Center, who is also the current MCBIOS President (2012-3). There were 151 registrants and a total of 111 abstracts (51 oral presentations and 60 poster session abstracts).

Insights into evolution of multicellular fungi from the assembled chromosomes of the mushroom Coprinopsis cinerea (Coprinus cinereus).

The mushroom Coprinopsis cinerea is a classic experimental model for multicellular development in fungi because it grows on defined media, completes its life cycle in 2 weeks, produces some 10(8) synchronized meiocytes, and can be manipulated at all stages in development by mutation and transformation. The 37-megabase genome of C. cinerea was sequenced and assembled into 13 chromosomes. Meiotic recombination rates vary greatly along the chromosomes, and retrotransposons are absent in large regions of the genome with low levels of meiotic recombination. Single-copy genes with identifiable orthologs in other basidiomycetes are predominant in low-recombination regions of the chromosome. In contrast, paralogous multicopy genes are found in the highly recombining regions, including a large family of protein kinases (FunK1) unique to multicellular fungi. Analyses of P450 and hydrophobin gene families confirmed that local gene duplications drive the expansions of paralogous copies and the expansions occur in independent lineages of Agaricomycotina fungi. Gene-expression patterns from microarrays were used to dissect the transcriptional program of dikaryon formation (mating). Several members of the FunK1 kinase family are differentially regulated during sexual morphogenesis, and coordinate regulation of adjacent duplications is rare. The genomes of C. cinerea and Laccaria bicolor, a symbiotic basidiomycete, share extensive regions of synteny. The largest syntenic blocks occur in regions with low meiotic recombination rates, no transposable elements, and tight gene spacing, where orthologous single-copy genes are overrepresented. The chromosome assembly of C. cinerea is an essential resource in understanding the evolution of multicellularity in the fungi.

The genome of the versatile nitrogen fixer Azorhizobium caulinodans ORS571.

BACKGROUND: Biological nitrogen fixation is a prokaryotic process that plays an essential role in the global nitrogen cycle. Azorhizobium caulinodans ORS571 has the dual capacity to fix nitrogen both as free-living organism and in a symbiotic interaction with Sesbania rostrata. The host is a fast-growing, submergence-tolerant tropical legume on which A. caulinodans can efficiently induce nodule formation on the root system and on adventitious rootlets located on the stem. RESULTS: The 5.37-Mb genome consists of a single circular chromosome with an overall average GC of 67% and numerous islands with varying GC contents. Most nodulation functions as well as a putative type-IV secretion system are found in a distinct symbiosis region. The genome contains a plethora of regulatory and transporter genes and many functions possibly involved in contacting a host. It potentially encodes 4717 proteins of which 96.3% have homologs and 3.7% are unique for A. caulinodans. Phylogenetic analyses show that the diazotroph Xanthobacter autotrophicus is the closest relative among the sequenced genomes, but the synteny between both genomes is very poor. CONCLUSION: The genome analysis reveals that A. caulinodans is a diazotroph that acquired the capacity to nodulate most probably through horizontal gene transfer of a complex symbiosis island. The genome contains numerous genes that reflect a strong adaptive and metabolic potential. These combined features and the availability of the annotated genome make A. caulinodans an attractive organism to explore symbiotic biological nitrogen fixation beyond leguminous plants.

FELINES: a utility for extracting and examining EST-defined introns and exons.

FELINES (Finding and Examining Lots of Intron 'N' Exon Sequences) is a utility written to automate construction and analysis of high quality intron and exon sequence databases produced from EST (expressed sequence tag) to genomic sequence alignments. We demonstrated the various programs of the FELINES utility by creating intron and exon sequence databases for the fungal organism Schizosaccharomyces pombe from alignments of EST to genomic sequences. In addition, we analyzed our constructed S.pombe sequence databases and the well-established Saccharomyces cerevisiae intron database from Manuel Ares' Laboratory for conserved sequence motifs. FELINES was shown to be useful for characterizing branchsites, polypyrimidine tracts and 5' and 3' splice sites in the intron databases and exonic splicing enhancers (ESEs) in S.pombe exons. FELINES is available at http://www.genome.ou.edu/informatics.html.

Microarray characterization of gene expression changes in blood during acute ethanol exposure.

BACKGROUND: As part of the civil aviation safety program to define the adverse effects of ethanol on flying performance, we performed a DNA microarray analysis of human whole blood samples from a five-time point study of subjects administered ethanol orally, followed by breathalyzer analysis, to monitor blood alcohol concentration (BAC) to discover significant gene expression changes in response to the ethanol exposure. METHODS: Subjects were administered either orange juice or orange juice with ethanol. Blood samples were taken based on BAC and total RNA was isolated from PaxGene™ blood tubes. The amplified cDNA was used in microarray and quantitative real-time polymerase chain reaction (RT-qPCR) analyses to evaluate differential gene expression. Microarray data was analyzed in a pipeline fashion to summarize and normalize and the results evaluated for relative expression across time points with multiple methods. Candidate genes showing distinctive expression patterns in response to ethanol were clustered by pattern and further analyzed for related function, pathway membership and common transcription factor binding within and across clusters. RT-qPCR was used with representative genes to confirm relative transcript levels across time to those detected in microarrays. RESULTS: Microarray analysis of samples representing 0%, 0.04%, 0.08%, return to 0.04%, and 0.02% wt/vol BAC showed that changes in gene expression could be detected across the time course. The expression changes were verified by qRT-PCR.The candidate genes of interest (GOI) identified from the microarray analysis and clustered by expression pattern across the five BAC points showed seven coordinately expressed groups. Analysis showed function-based networks, shared transcription factor binding sites and signaling pathways for members of the clusters. These include hematological functions, innate immunity and inflammation functions, metabolic functions expected of ethanol metabolism, and pancreatic and hepatic function. Five of the seven clusters showed links to the p38 MAPK pathway. CONCLUSIONS: The results of this study provide a first look at changing gene expression patterns in human blood during an acute rise in blood ethanol concentration and its depletion because of metabolism and excretion, and demonstrate that it is possible to detect changes in gene expression using total RNA isolated from whole blood. The analysis approach for this study serves as a workflow to investigate the biology linked to expression changes across a time course and from these changes, to identify target genes that could serve as biomarkers linked to pilot performance.

Resolution of aviation forensic toxicology findings with the aid of DNA profiling.

Body components of aviation accident fatalities are often scattered, disintegrated, commingled, contaminated, and/or putrefied at accident scenes. These situations may impose difficulties in victim identification/tissue matching. The prevalence of misidentification in relation to aviation accident forensic toxicology has not been well established. Therefore, the Civil Aerospace Medical Institute (CAMI) toxicology database was searched for the 1998-2008 period for those cases wherein DNA profiling was performed to resolve identity issue of the samples submitted to CAMI for toxicological evaluation. During this period, biological samples from the casualties of a total of 3523 accidents were submitted to CAMI. The submitted samples were primarily from pilots. Out of the 3523 accidents, at least, one fatality had occurred in 3366 (≈ 96%) accidents; thus, these accidents were considered fatal accidents. Accordingly, biological samples from 3319 pilots (3319 of the 3366 accidents) were received at CAMI for toxicological testing. Of these 3319 pilots, 3275 (≈ 99%) were fatally injured. DNA profiling was performed in 15 (≈ 0.5%) of the 3319 accidents. The profiling was conducted upon the requests of families in two accidents, accident investigators in three, and pathologists in four. In six accidents, contradictory toxicological findings led CAMI to initiate DNA profiling. The requests made by families and investigators were primarily triggered by inconsistency between the toxicological results and the history of drug use of the victims, while by pathologists because of commingling of samples. In three (20%) of the 15 accidents, at least one submitted sample was misidentified or mislabeled. The present study demonstrated that the number of aviation accident cases requiring DNA profiling was small and this DNA approach was effectively applied in resolving aviation toxicology findings associated with those accidents.

Examining smoking-induced differential gene expression changes in buccal mucosa.

BACKGROUND: Gene expression changes resulting from conditions such as disease, environmental stimuli, and drug use, can be monitored in the blood. However, a less invasive method of sample collection is of interest because of the discomfort and specialized personnel necessary for blood sampling especially if multiple samples are being collected. Buccal mucosa cells are easily collected and may be an alternative sample material for biomarker testing. A limited number of studies, primarily in the smoker/oral cancer literature, address this tissue's efficacy as an RNA source for expression analysis. The current study was undertaken to determine if total RNA isolated from buccal mucosa could be used as an alternative tissue source to assay relative gene expression. METHODS: Total RNA was isolated from swabs, reverse transcribed and amplified. The amplified cDNA was used in RT-qPCR and microarray analyses to evaluate gene expression in buccal cells. Initially, RT-qPCR was used to assess relative transcript levels of four genes from whole blood and buccal cells collected from the same seven individuals, concurrently. Second, buccal cell RNA was used for microarray-based differential gene expression studies by comparing gene expression between a group of female smokers and nonsmokers. RESULTS: An amplification protocol allowed use of less buccal cell total RNA (50 ng) than had been reported previously with human microarrays. Total RNA isolated from buccal cells was degraded but was of sufficient quality to be used with RT-qPCR to detect expression of specific genes. We report here the finding of a small number of statistically significant differentially expressed genes between smokers and nonsmokers, using buccal cells as starting material. Gene Set Enrichment Analysis confirmed that these genes had a similar expression pattern to results from another study. CONCLUSIONS: Our results suggest that despite a high degree of degradation, RNA from buccal cells from cheek mucosa could be used to detect differential gene expression between smokers and nonsmokers. However the RNA degradation, increase in sample variability and microarray failure rate show that buccal samples should be used with caution as source material in expression studies.

EST library sequencing of genes expressed during early limb regeneration in the fiddler crab and transcriptional responses to ecdysteroid exposure in limb bud explants.

We have constructed directional and randomly primed cDNA libraries from mRNAs isolated during progressive stages of fiddler crab (Uca pugilator) limb regeneration. Data from these libraries are being assembled into an on-line database (http://www.genome.ou.edu/crab.html) that is both BLAST and keyword searchable; the data set is also available through GenBank. The first characterized library was made from mRNA isolated 4 days post-autotomy, when the first sign of morphological differentiation, cuticle secretion, is observed. Analysis of 1698 cDNA clones led to assignment of 473 contigs and 417 singlets, for a total of 890 sequences. Of these, ∼86% showed no assignments to characterized genes on database searching, while 14% could be assigned to a known ortholog in the COG (Clusters of Orthologous Groups) database. BLAST searches to specific protein domains in the Gene Ontology database led to assignments for ∼40% of the assembled sequences. Sequence similarity searches of other crustacean EST databases produced hits to 13-30% of the Uca query sequences. The ESTs include several genes that may be potentially ecdysteroid-responsive, such as homologs to chaperone proteins and cuticle protein genes, as well as homologs to arthropod proteins involved in retinoid/terpenoid metabolism. We have tested 3 potential candidate genes for their ability to be induced by ecdysteroid in limb bud explants; an arthropodial cuticle protein gene, and the nuclear receptor genes EcR and RXR. A subset of early blastemal limb buds (8 days post autotomy) show a positive response to ecdysteroid by 1-1.5 h, followed by a decrease in transcript abundance at longer periods of sustained incubation. Later stage buds (12 days post autotomy-late premolt) show decreases in steady-state mRNA levels by 1.5 h, or are completely refractory to ecdysteroid exposure.

Mating factor linkage and genome evolution in basidiomycetous pathogens of cereals.

Sex in basidiomycete fungi is controlled by tetrapolar mating systems in which two unlinked gene complexes determine up to thousands of mating specificities, or by bipolar systems in which a single locus (MAT) specifies different sexes. The genus Ustilago contains bipolar (Ustilago hordei) and tetrapolar (Ustilago maydis) species and sexual development is associated with infection of cereal hosts. The U. hordei MAT-1 locus is unusually large (approximately 500 kb) and recombination is suppressed in this region. We mapped the genome of U. hordei and sequenced the MAT-1 region to allow a comparison with mating-type regions in U. maydis. Additionally the rDNA cluster in the U. hordei genome was identified and characterized. At MAT-1, we found 47 genes along with a striking accumulation of retrotransposons and repetitive DNA; the latter features were notably absent from the corresponding U. maydis regions. The tetrapolar mating system may be ancestral and differences in pathogenic life style and potential for inbreeding may have contributed to genome evolution.

Differential gene expression in Festuca under heat stress conditions.

Fescues (Festuca sp.) are major cool-season forage and turf grass species around the world. Heat stress is one of the limiting factors in the production of fescues as forage in the southern Great Plains of the US. Heat responsive gene transcripts were cloned by using suppression subtractive hybridization between a heat-tolerant and a heat-sensitive fescue genotype subjected to a slowly increased temperature mimicking the natural conditions. The temperature in the growth chamber containing the plants was gradually increased from 24 degrees C to 44 degrees C over a period of 2 weeks. Three subtractions were conducted between samples of the two genotypes collected after 12 h of exposure to 39, 42, and 44 degrees C. A total of 2495 ESTs were generated, of which 1800 clustered into 434 contigs and 656 were singlets. The putative functions of ESTs were predicted by BLASTX. Nearly 30% of the contigs and 39% of the singlets had no similarity to GenBank sequences. Differentially expressed genes selected by subtractions were classified into 10 broad categories according to their putative functions generated by BLAST analysis. Under heat-stress conditions, cell maintenance, chloroplast associated and photosynthesis-, protein synthesis-, signalling-, and transcription factor-related genes had higher expression levels in the heat-tolerant genotype. Genes related to metabolism and stress had higher expression in the heat-sensitive genotype. The expression of 17 selected gene transcripts were examined by RT-PCR using plant tissues of the two genotypes grown under heat stress and under optimal temperature conditions (24 degrees C) for fescue. Results from RT-PCR confirmed the differential expressions of these transcripts. The differential expressions of at least 11 of these genes were attributable to heat stress rather than to differences in the genetic backgrounds of the genotypes.

Introns and splicing elements of five diverse fungi.

Genomic sequences and expressed sequence tag data for a diverse group of fungi (Saccharomyces cerevisiae, Schizosaccharomyces pombe, Aspergillus nidulans, Neurospora crassa, and Cryptococcus neoformans) provided the opportunity to accurately characterize conserved intronic elements. An examination of large intron data sets revealed that fungal introns in general are short, that 98% or more of them belong to the canonical splice site (ss) class (5'GU...AG3'), and that they have polypyrimidine tracts predominantly in the region between the 5' ss and the branch point. Information content is high in the 5' ss, branch site, and 3' ss regions of the introns but low in the exon regions adjacent to the introns in the fungi examined. The two yeasts have broader intron length ranges and correspondingly higher intron information content than the other fungi. Generally, as intron length increases in the fungi, so does intron information content. Homologs of U2AF spliceosomal proteins were found in all species except for S. cerevisiae, suggesting a nonconventional role for U2AF in the absence of canonical polypyrimidine tracts in the majority of introns. Our observations imply that splicing in fungi may be different from that in vertebrates and may require additional proteins that interact with polypyrimidine tracts upstream of the branch point. Theoretical protein homologs for Nam8p and TIA-1, two proteins that require U-rich regions upstream of the branch point to function, were found. There appear to be sufficient differences between S. cerevisiae and S. pombe introns and the introns of two filamentous members of the Ascomycota and one member of the Basidiomycota to warrant the development of new model organisms for studying the splicing mechanisms of fungi.

EGF receptor gene mutations are common in lung cancers from "never smokers" and are associated with sensitivity of tumors to gefitinib and erlotinib.

Somatic mutations in the tyrosine kinase (TK) domain of the epidermal growth factor receptor (EGFR) gene are reportedly associated with sensitivity of lung cancers to gefitinib (Iressa), kinase inhibitor. In-frame deletions occur in exon 19, whereas point mutations occur frequently in codon 858 (exon 21). We found from sequencing the EGFR TK domain that 7 of 10 gefitinib-sensitive tumors had similar types of alterations; no mutations were found in eight gefitinib-refractory tumors (P = 0.004). Five of seven tumors sensitive to erlotinib (Tarceva), a related kinase inhibitor for which the clinically relevant target is undocumented, had analogous somatic mutations, as opposed to none of 10 erlotinib-refractory tumors (P = 0.003). Because most mutation-positive tumors were adenocarcinomas from patients who smoked <100 cigarettes in a lifetime ("never smokers"), we screened EGFR exons 2-28 in 15 adenocarcinomas resected from untreated never smokers. Seven tumors had TK domain mutations, in contrast to 4 of 81 non-small cell lung cancers resected from untreated former or current smokers (P = 0.0001). Immunoblotting of lysates from cells transiently transfected with various EGFR constructs demonstrated that, compared to wild-type protein, an exon 19 deletion mutant induced diminished levels of phosphotyrosine, whereas the phosphorylation at tyrosine 1092 of an exon 21 point mutant was inhibited at 10-fold lower concentrations of drug. Collectively, these data show that adenocarcinomas from never smokers comprise a distinct subset of lung cancers, frequently containing mutations within the TK domain of EGFR that are associated with gefitinib and erlotinib sensitivity.