Differential pathogenesis of lung adenocarcinoma subtypes involving sequence mutations, copy number, chromosomal instability, and methylation

BACKGROUND: Lung adenocarcinoma (LAD) has extreme genetic variation among patients, which is currently not well understood, limiting progress in therapy development and research. LAD intrinsic molecular subtypes are a validated stratification of naturally-occurring gene expression patterns and encompass different functional pathways and patient outcomes. Patients may have incurred different mutations and alterations that led to the different subtypes. We hypothesized that the LAD molecular subtypes co-occur with distinct mutations and alterations in patient tumors. METHODOLOGY/PRINCIPAL FINDINGS: The LAD molecular subtypes (Bronchioid, Magnoid, and Squamoid) were tested for association with gene mutations and DNA copy number alterations using statistical methods and published cohorts (n = 504). A novel validation (n = 116) cohort was assayed and interrogated to confirm subtype-alteration associations. Gene mutation rates (EGFR, KRAS, STK11, TP53), chromosomal instability, regional copy number, and genomewide DNA methylation were significantly different among tumors of the molecular subtypes. Secondary analyses compared subtypes by integrated alterations and patient outcomes. Tumors having integrated alterations in the same gene associated with the subtypes, e.g. mutation, deletion and underexpression of STK11 with Magnoid, and mutation, amplification, and overexpression of EGFR with Bronchioid. The subtypes also associated with tumors having concurrent mutant genes, such as KRAS-STK11 with Magnoid. Patient overall survival, cisplatin plus vinorelbine therapy response and predicted gefitinib sensitivity were significantly different among the subtypes. CONCLUSIONS/ SIGNIFICANCE: The lung adenocarcinoma intrinsic molecular subtypes co-occur with grossly distinct genomic alterations and with patient therapy response. These results advance the understanding of lung adenocarcinoma etiology and nominate patient subgroups for future evaluation of treatment response.     

Insect Innate Immunity Database (IIID): An Annotation Tool for Identifying Immune Genes in Insect Genomes

The innate immune system is an ancient component of host defense. Since innate immunity pathways are well conserved throughout many eukaryotes, immune genes in model animals can be used to putatively identify homologous genes in newly sequenced genomes of non-model organisms. With the initiation of the “i5k” project, which aims to sequence 5,000 insect genomes by 2016, many novel insect genomes will soon become publicly available, yet few annotation resources are currently available for insects. Thus, we developed an online tool called the Insect Innate Immunity Database (IIID) to provide an open access resource for insect immunity and comparative biology research (http://www.vanderbilt.edu/IIID). The database provides users with simple exploratory tools to search the immune repertoires of five insect models (including Nasonia), spanning three orders, for specific immunity genes or genes within a particular immunity pathway. As a proof of principle, we used an initial database with only four insect models to annotate potential immune genes in the parasitoid wasp genus Nasonia. Results specify 306 putative immune genes in the genomes of N. vitripennis and its two sister species N. giraulti and N. longicornis. Of these genes, 146 were not found in previous annotations of Nasonia immunity genes. Combining these newly identified immune genes with those in previous annotations, Nasonia possess 489 putative immunity genes, the largest immune repertoire found in insects to date. While these computational predictions need to be complemented with functional studies, the IIID database can help initiate and augment annotations of the immune system in the plethora of insect genomes that will soon become available.     

MyD88 Is Required for Protection from Lethal Infection with a Mouse-Adapted SARS-CoV

A novel human coronavirus, SARS-CoV, emerged suddenly in 2003, causing approximately 8000 human cases and more than 700 deaths worldwide. Since most animal models fail to faithfully recapitulate the clinical course of SARS-CoV in humans, the virus and host factors that mediate disease pathogenesis remain unclear. Recently, our laboratory and others developed a recombinant mouse-adapted SARS-CoV (rMA15) that was lethal in BALB/c mice. In contrast, intranasal infection of young 10-week-old C57BL/6 mice with rMA15 results in a nonlethal infection characterized by high titer replication within the lungs, lung inflammation, destruction of lung tissue, and loss of body weight, thus providing a useful model to identify host mediators of protection. Here, we report that mice deficient in MyD88 (MyD88^−/−), an adapter protein that mediates Toll-like receptor (TLR), IL-1R, and IL-18R signaling, are far more susceptible to rMA15 infection. The genetic absence of MyD88 resulted in enhanced pulmonary pathology and greater than 90% mortality by day 6 post-infection. MyD88^−/− mice had significantly higher viral loads in lung tissue throughout the course of infection. Despite increased viral loads, the expression of multiple proinflammatory cytokines and chemokines within lung tissue and recruitment of inflammatory monocytes/macrophages to the lung was severely impaired in MyD88^−/− mice compared to wild-type mice. Furthermore, mice deficient in chemokine receptors that contribute to monocyte recruitment to the lung were more susceptible to rMA15-induced disease and exhibited severe lung pathology similar to that seen in MyD88^−/−mice. These data suggest that MyD88-mediated innate immune signaling and inflammatory cell recruitment to the lung are required for protection from lethal rMA15 infection.     

Boundary Questionnaire Results in the Mentally Healthy Elderly

The Hartmann Boundary Questionnaire was administered twice, with six months in between, to 61 Swiss subjects over 60 years of age taking part in an investigation into the effects of dream-telling on five variables: well-being, sleep quality, sleep duration, dream recall and dream tone. In addition, dream epoch, i.e., the age of life of the dreamer as perceived in the dream, was recorded for those who told dreams. In addition to this study group in which the members told dreams there were two control groups. Those in the first control group were asked about well-being and sleep quality but not about dreams or dreaming, while those in the second control group were additionally asked how many dreams they had retained, how frequently they had occurred and about the dream tone (pleasant/unpleasant). All study participants were given the Hartmann Boundary Questionnaire at the beginning (pre-test) and again at the end of the six month study period (post-test). The retest reliability was high (r = 0.872 for the whole sample). We report here the relationships obtained between the questionnaire scores and age, group membership, gender and the number of dreams that were retained over a 26 week testing period. No significant correlations were found for age, group membership or dream recall. There was, however, a small, significant boundary score difference between women and men for the pre-test, indicating thinner boundaries for women, but this difference was no longer significant in the post-test.     

Progress toward the development of a genetically engineered attenuated hepatitis A virus vaccine.

Mutations which positively affect growth of hepatitis A virus in cell culture may negatively affect growth in vivo. Therefore, development of an attenuated vaccine for hepatitis A may require a careful balancing of mutations to produce a virus that will grow efficiently in cells suitable for vaccine production and still maintain a satisfactory level of attenuation in vivo. Since such a balance could be achieved most directly by genetic engineering, we are analyzing mutations that accumulated during serial passage of the HM-175 strain of hepatitis A virus in MRC-5 cell cultures in order to determine the relative importance of the mutations for growth in MRC-5 cells and for attenuation in susceptible primates. Chimeric viral genomes of the HM-175 strain were constructed from cDNA clones derived from a virulent virus and from two attenuated viruses adapted to growth in African green monkey kidney (AGMK) and MRC-5 cells, respectively. Viruses encoded by these chimeric genomes were recovered by in vitro or in vivo transfection and assessed for their ability to grow in cultured MRC-5 cells or to cause hepatitis in primates (tamarins). The only MRC-5-specific mutations that substantially increased the efficiency of growth in MRC-5 cells were a group of four mutations in the 5' noncoding (NC) region. These 5' NC mutations and a separate group of 5' NC mutations that accumulated during earlier passages of the HM-175 virus in primary AGMK cells appeared, independently and additively, to result in decreased biochemical evidence of hepatitis in tamarins. However, neither group of 5' NC mutations had a demonstrable effect on the extent of virus excretion or liver pathology in these animals.     

Accumulation of Demolybdo Nitrate Reductase during Induction and Its Separation from Active Nitrate Reductase in Chlorella

During induction of nitrate reductase in Chlorella vulgaris,synthesis of the precursor, demolybdo cytochrome c reductase,exceeds the synthesis of active enzyme. Evidence is also presentedwhich shows that the purification procedure of Funkhouser etal. [(1980) Plant Physiol. 65: 939] separates demolybdo cytochromec reductase from active nitrate reductase. ¹Supported in part by a grant to B. V. from the Deutsche Forschungsgemeinschaftand a contribution of the Texas Agricultural Experiment Station. (Received July 27, 1983; Accepted September 13, 1983)     

Additional effects of monovalent cations on the lysine-sensitive aspartokinase of E. coli B

The apparent specificity of activation of lysine-sensitive aspartokinase (E.C.2.7.2.4) from E. coli by monovalent cations differs depending on the assay used and on the Mg2+ concentration. Activity is nearly absolutely dependent on and is highly specific for a monovalent cation in the aspartate semialdehyde dehydrogenase coupled assay or the adenosine triphosphate-adenosine diphosphate exchange assay. Little specificity for monovalent cations is observed using the aspartyl hydroxamate assay. Activation and specificity are also altered by Mg2+ concentrations at a constant 5 mM nucleotide concentration. At a low (1.25 or 1.6 mM)Mg2+ concentration, monovalent cation activation and specificity are nearly absolute. Less dependence on monovalent cations and less specificity are observed at a higher Mg2+ concentration (6 mM). Li+ inhibits aspartokinase competitively with respect to either K+ or NH4+. Monovalent cations are also thermoprotective and differential thermal inactivation experiments at 56 degrees C reveal that NH4+ and K+, either of which will produce maximum catalytic activity, interact differently with aspartokinase. K+ interacts with positive cooperativity, whereas NH4+ does not. K+, NH4+, and Na+ are about equally effective in enhancing the dissociation of the aspartokinase-aspartylphosphate complex. Li+ is less effective.     

Purification of myo-Inositol 1-Phosphate Synthase from Rice Cell Culture by Affinity Chromatography

myo-Inositol 1-phosphate synthase (EC 5.5.1.4) is the enzyme which catalyzes the synthesis of the precursor for the myo-inositol oxidation pathway. Rice callus grown in suspension culture provides a good source of plant enzyme. Use has been made of a noncompetitive inhibitor to prepare an affinity column for this enzyme. With this column, the enzyme from rice callus has been purified 1500-fold in a single step, about 9000-fold over-all, to a specific activity of 0.078 units per milligram of protein. This is an order of magnitude greater than previous purifications of the plant enzyme.     

Monoclonal antibody isolation of type II pneumocytes

A monoclonal antibody that identifies a membrane molecule unique in rat lung for type II alveolar epithelial cells was used to isolate these cells from enzymatically dispersed lung cells by fluorescence-activated cell sorting. Although multistep physical separation techniques have permitted the isolation of large quantities of these cells and flow cytometry has been used by others to isolate lamellar body-containing cells, the application of this antibody-directed sorting has distinct advantages. Because the marker molecule is expressed on immature type II cells prior to the development of lamellar bodies, the antibody will also permit their isolation and study.     

Estimating time since infection in early homogeneous HIV-1 samples using a poisson model

Background  

The occurrence of a genetic bottleneck in HIV sexual or mother-to-infant transmission has been well documented. This results in a majority of new infections being homogeneous, i.e., initiated by a single genetic strain. Early after infection, prior to the onset of the host immune response, the viral population grows exponentially. In this simple setting, an approach for estimating evolutionary and demographic parameters based on comparison of diversity measures is a feasible alternative to the existing Bayesian methods (e.g., BEAST), which are instead based on the simulation of genealogies.