A comparative study of discriminating human heart failure etiology using gene expression profiles

BACKGROUND: Human heart failure is a complex disease that manifests from multiple genetic and environmental factors. Although ischemic and non-ischemic heart disease present clinically with many similar decreases in ventricular function, emerging work suggests that they are distinct diseases with different responses to therapy. The ability to distinguish between ischemic and non-ischemic heart failure may be essential to guide appropriate therapy and determine prognosis for successful treatment. In this paper we consider discriminating the etiologies of heart failure using gene expression libraries from two separate institutions. RESULTS: We apply five new statistical methods, including partial least squares, penalized partial least squares, LASSO, nearest shrunken centroids and random forest, to two real datasets and compare their performance for multiclass classification. It is found that the five statistical methods perform similarly on each of the two datasets: it is difficult to correctly distinguish the etiologies of heart failure in one dataset whereas it is easy for the other one. In a simulation study, it is confirmed that the five methods tend to have close performance, though the random forest seems to have a slight edge. CONCLUSIONS: For some gene expression data, several recently developed discriminant methods may perform similarly. More importantly, one must remain cautious when assessing the discriminating performance using gene expression profiles based on a small dataset; our analysis suggests the importance of utilizing multiple or larger datasets

Assembly of the Candida albicans genome into sixteen supercontigs aligned on the eight chromosomes

For Assembly 20 of the Candida albicans genome, the sequence of each of the eight chromosomes was determined, revealing new insights into gene family creation and dispersion, subtelomere organization, and chromosome evolution

Functional Analysis of Human Hematopoietic Stem Cell Gene Expression Using Zebrafish

Although several reports have characterized the hematopoietic stem cell (HSC) transcriptome, the roles of HSC-specific genes in hematopoiesis remain elusive. To identify candidate regulators of HSC fate decisions, we compared the transcriptome of human umbilical cord blood and bone marrow CD34(+)CD33(−)CD38(−)Rho(lo)c-kit(+) cells, enriched for hematopoietic stem/progenitor cells with CD34(+)CD33(−)CD38(−)Rho(hi) cells, enriched in committed progenitors. We identified 277 differentially expressed transcripts conserved in these ontogenically distinct cell sources. We next performed a morpholino antisense oligonucleotide (MO)-based functional screen in zebrafish to determine the hematopoietic function of 61 genes that had no previously known function in HSC biology and for which a likely zebrafish ortholog could be identified. MO knock down of 14/61 (23%) of the differentially expressed transcripts resulted in hematopoietic defects in developing zebrafish embryos, as demonstrated by altered levels of circulating blood cells at 30 and 48 h postfertilization and subsequently confirmed by quantitative RT-PCR for erythroid-specific hbae1 and myeloid-specific lcp1 transcripts. Recapitulating the knockdown phenotype using a second MO of independent sequence, absence of the phenotype using a mismatched MO sequence, and rescue of the phenotype by cDNA-based overexpression of the targeted transcript for zebrafish spry4 confirmed the specificity of MO targeting in this system. Further characterization of the spry4-deficient zebrafish embryos demonstrated that hematopoietic defects were not due to more widespread defects in the mesodermal development, and therefore represented primary defects in HSC specification, proliferation, and/or differentiation. Overall, this high-throughput screen for the functional validation of differentially expressed genes using a zebrafish model of hematopoiesis represents a major step toward obtaining meaningful information from global gene profiling of HSCs

Development and Use of an Efficient System for Random mariner Transposon Mutagenesis To Identify Novel Genetic Determinants of Biofilm Formation in the Core Enterococcus faecalis Genome▿ †

Enterococcus faecalis is a gram-positive commensal bacterium of the gastrointestinal tract and an important opportunistic pathogen. Despite the increasing clinical significance of the enterococci, most of the genetic analysis of these organisms has focused on mobile genetic elements, and existing tools for manipulation and analysis of the core E. faecalis chromosome are limited. We are interested in a comprehensive analysis of the genetic determinants for biofilm formation encoded within the core E. faecalis genome. To identify such determinants, we developed a substantially improved system for transposon mutagenesis in E. faecalis based on a mini-mariner transposable element. Mutagenesis of wild-type E. faecalis with this element yielded predominantly mutants carrying a single copy of the transposable element, and insertions were distributed around the entire chromosome in an apparently random fashion. We constructed a library of E. faecalis transposon insertion mutants and screened this library to identify mutants exhibiting a defect in biofilm formation. Biofilm-defective mutants were found to carry transposon insertions both in genes that were previously known to play a role in biofilm formation and in new genes lacking any known function; for several genes identified in the screen, complementation analysis confirmed a direct role in biofilm formation. These results provide significant new information about the genetics of enterococcal biofilm formation and demonstrate the general utility of our transposon system for functional genomic analysis of E. faecalis

Functional Genomics of Enterococcus faecalis: Multiple Novel Genetic Determinants for Biofilm Formation in the Core Genome▿ †

The ability of Enterococcus faecalis to form robust biofilms on host tissues and on abiotic surfaces such as catheters likely plays a major role in the pathogenesis of opportunistic antibiotic-resistant E. faecalis infections and in the transfer of antibiotic resistance genes. We have carried out a comprehensive analysis of genetic determinants of biofilm formation in the core genome of E. faecalis. Here we describe 68 genetic loci predicted to be involved in biofilm formation that were identified by recombinase in vivo expression technology (RIVET); most of these genes have not been studied previously. Differential expression of a number of these determinants during biofilm growth was confirmed by quantitative reverse transcription-PCR, and genetic complementation studies verified a role in biofilm formation for several candidate genes. Of particular interest was genetic locus EF1809, predicted to encode a regulatory protein of the GntR family. We isolated 14 independent nonsibling clones containing the putative promoter region for this gene in the RIVET screen; EF1809 also showed the largest increase in expression during biofilm growth of any of the genes tested. Since an in-frame deletion of EF1809 resulted in a severe biofilm defect that could be complemented by the cloned wild-type gene, we have designated EF1809 ebrA (enterococcal biofilm regulator). Most of the novel genetic loci identified in our studies are highly conserved in gram-positive bacterial pathogens and may thus constitute a pool of uncharacterized genes involved in biofilm formation that may be useful targets for drug discovery

Whole genome transcription profiling of <it>Anaplasma phagocytophilum </it>in human and tick host cells by tiling array analysis

<p>Abstract</p> <p>Background</p> <p><it>Anaplasma phagocytophilum </it>(<it>Ap</it>) is an obligate intracellular bacterium and the agent of human granulocytic anaplasmosis, an emerging tick-borne disease. <it>Ap </it>alternately infects ticks and mammals and a variety of cell types within each. Understanding the biology behind such versatile cellular parasitism may be derived through the use of tiling microarrays to establish high resolution, genome-wide transcription profiles of the organism as it infects cell lines representative of its life cycle (tick; ISE6) and pathogenesis (human; HL-60 and HMEC-1).</p> <p>Results</p> <p>Detailed, host cell specific transcriptional behavior was revealed. There was extensive differential <it>Ap </it>gene transcription between the tick (ISE6) and the human (HL-60 and HMEC-1) cell lines, with far fewer differentially transcribed genes between the human cell lines, and all disproportionately represented by membrane or surface proteins. There were <it>Ap </it>genes exclusively transcribed in each cell line, apparent human- and tick-specific operons and paralogs, and anti-sense transcripts that suggest novel expression regulation processes. Seven <it>virB2 </it>paralogs (of the bacterial type IV secretion system) showed human or tick cell dependent transcription. Previously unrecognized genes and coding sequences were identified, as were the expressed <it>p44/msp2 </it>(major surface proteins) paralogs (of 114 total), through elevated signal produced to the unique hypervariable region of each – 2/114 in HL-60, 3/114 in HMEC-1, and none in ISE6.</p> <p>Conclusion</p> <p>Using these methods, whole genome transcription profiles can likely be generated for <it>Ap</it>, as well as other obligate intracellular organisms, in any host cells and for all stages of the cell infection process. Visual representation of comprehensive transcription data alongside an annotated map of the genome renders complex transcription into discernable patterns.</p

Growth and survival of <i>E. faecalis</i> OG1RF, Δ<i>rsh</i>, Δ<i>relQ</i>, and Δ<i>rsh</i>Δ<i>relQ</i> in human (A) serum and (B) whole blood <i>ex vivo</i>.

<p>Overnight cultures of the four strains were diluted 1∶20 in serum or whole blood and incubated at 37°C. Dilutions of aliquots collected at 0, 3 (panel B), 6 (panel A), 9, 24, 48, and 72 hours were plated to assess bacterial survival. Results are reported as percent survival with the 0 hour time point set to 100%. Values and error bars represent the mean ± SEM of n = 3 independent replicates. Data were analyzed for statistical significance with two-way ANOVA followed by Dunnett’s multiple comparisons test. *, p<0.05; **, p<0.01; ***, p<0.001.</p

Survival of <i>E. faecalis</i> OG1RF, Δ<i>rsh</i>, Δ<i>relQ</i>, and Δ<i>rsh</i>Δ<i>relQ</i> in subdermal abscesses at (A) early and (B) late time points post-inoculation.

<p>Subdermal abscess infections with the four strains were carried out as described in the text and in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115839#pone-0115839-g001" target="_blank">Fig. 1</a>. Results are reported as percent survival with the 0 hour time point set to 100%. Values and error bars represent the mean ± SEM of n = 2 rabbits. Data from the same rabbits were separated into panels (A) and (B) for clarity. (A) Student’s t-test comparing Δ<i>rsh</i> and Δ<i>rsh</i>Δ<i>relQ</i>, α = 0.1: red #, p = 0.08; red ∧, p = 0.06. (B) One-way ANOVA followed by Tukey’s Multiple Comparison post-hoc test: black *, p<0.05 for OG1RF versus Δ<i>relQ</i>; black **, p<0.01 for OG1RF versus Δ<i>rsh</i>Δ<i>relQ</i>; blue *, p<0.05 for Δ<i>rsh</i> versus Δ<i>rsh</i>Δ<i>relQ</i>.</p

Functional categories of genes found by microarray to be differentially expressed in <i>E. faecalis</i> OG1RF <i>in</i><i>vivo</i> during subdermal abscess infection.

<p>The numbers of up- (right) and down-regulated (left) genes at two (top) or eight (bottom) hours post-inoculation are indicated on the horizontal axis.</p