Increased Expression and Protein Divergence in Duplicate Genes Is Associated with Morphological Diversification

The differentiation of both gene expression and protein function is thought to be important as a mechanism of the functionalization of duplicate genes. However, it has not been addressed whether expression or protein divergence of duplicate genes is greater in those genes that have undergone functionalization compared with those that have not. We examined a total of 492 paralogous gene pairs associated with morphological diversification in a plant model organism (Arabidopsis thaliana). Classifying these paralogous gene pairs into high, low, and no morphological diversification groups, based on knock-out data, we found that the divergence rate of both gene expression and protein sequences were significantly higher in either high or low morphological diversification groups compared with those in the no morphological diversification group. These results strongly suggest that the divergence of both expression and protein sequence are important sources for morphological diversification of duplicate genes. Although both mechanisms are not mutually exclusive, our analysis suggested that changes of expression pattern play the minor role (33%–41%) and that changes of protein sequence play the major role (59%–67%) in morphological diversification. Finally, we examined to what extent duplicate genes are associated with expression or protein divergence exerting morphological diversification at the whole-genome level. Interestingly, duplicate genes randomly chosen from A. thaliana had not experienced expression or protein divergence that resulted in morphological diversification. These results indicate that most duplicate genes have experienced minor functionalization.     

Identification of Methylated Genes Associated with Aggressive Bladder Cancer

Approximately 500,000 individuals diagnosed with bladder cancer in the U.S. require routine cystoscopic follow-up to monitor for disease recurrences or progression, resulting in over $2 billion in annual expenditures. Identification of new diagnostic and monitoring strategies are clearly needed, and markers related to DNA methylation alterations hold great promise due to their stability, objective measurement, and known associations with the disease and with its clinical features. To identify novel epigenetic markers of aggressive bladder cancer, we utilized a high-throughput DNA methylation bead-array in two distinct population-based series of incident bladder cancer (n = 73 and n = 264, respectively). We then validated the association between methylation of these candidate loci with tumor grade in a third population (n = 245) through bisulfite pyrosequencing of candidate loci. Array based analyses identified 5 loci for further confirmation with bisulfite pyrosequencing. We identified and confirmed that increased promoter methylation of HOXB2 is significantly and independently associated with invasive bladder cancer and methylation of HOXB2, KRT13 and FRZB together significantly predict high-grade non-invasive disease. Methylation of these genes may be useful as clinical markers of the disease and may point to genes and pathways worthy of additional examination as novel targets for therapeutic treatment.     

Identification of Differentially Expressed Genes Associated with Osteochondrosis in Standardbred Horses Using RNA Arbitrarily Primed PCR

The aim of this study was to investigate genes for differential expression in cartilage of foals predisposed to osteochondrosis (OC). Tissue was sampled from the cranial part of the distal intermediate ridge of the tibia in the tarso-crural joint. Foals were considered predisposed to OC when parents had OC at the distal intermediate ridge of the tibia. RNA was isolated and subjected to arbitrarily primed PCR (RAP-PCR) followed by fingerprinting to screen for differentially expressed genes. By verification of results from the RAP-PCR fingerprint screening using real-time RT-PCR, we identified two genes not previously correlated with OC as differentially expressed. The two genes, which were identical to TLK2 and an equine EST, are good targets for future research on OC.     

Identification of Genes Associated with Chlorophyll Accumulation in Flower Petals

Plants have an ability to prevent chlorophyll accumulation, which would mask the bright flower color, in their petals. In contrast, leaves contain substantial amounts of chlorophyll, as it is essential for photosynthesis. The mechanisms of organ-specific chlorophyll accumulation are unknown. To identify factors that determine the chlorophyll content in petals, we compared the expression of genes related to chlorophyll metabolism in different stages of non-green (red and white) petals (very low chlorophyll content), pale-green petals (low chlorophyll content), and leaves (high chlorophyll content) of carnation (Dianthus caryophyllus L.). The expression of many genes encoding chlorophyll biosynthesis enzymes, in particular Mg-chelatase, was lower in non-green petals than in leaves. Non-green petals also showed higher expression of genes involved in chlorophyll degradation, including STAY-GREEN gene and pheophytinase. These data suggest that the absence of chlorophylls in carnation petals may be caused by the low rate of chlorophyll biosynthesis and high rate of degradation. Similar results were obtained by the analysis of Arabidopsis microarray data. In carnation, most genes related to chlorophyll biosynthesis were expressed at similar levels in pale-green petals and leaves, whereas the expression of chlorophyll catabolic genes was higher in pale-green petals than in leaves. Therefore, we hypothesize that the difference in chlorophyll content between non-green and pale-green petals is due to different levels of chlorophyll biosynthesis. Our study provides a basis for future molecular and genetic studies on organ-specific chlorophyll accumulation.     

Overwintering Is Associated with Reduced Expression of Immune Genes and Higher Susceptibility to Virus Infection in Honey Bees

The eusocial honey bee, Apis mellifera, has evolved remarkable abilities to survive extreme seasonal differences in temperature and availability of resources by dividing the worker caste into two groups that differ in physiology and lifespan: summer and winter bees. Most of the recent major losses of managed honey bee colonies occur during the winter, suggesting that winter bees may have compromised immune function and higher susceptibility to diseases. We tested this hypothesis by comparing the expression of eight immune genes and naturally occurring infection levels of deformed wing virus (DWV), one of the most widespread viruses in A. mellifera populations, between summer and winter bees. Possible interactions between immune response and physiological activity were tested by measuring the expression of vitellogenin and methyl farnesoate epoxidase, a gene coding for the last enzyme involved in juvenile hormone biosynthesis. Our data show that high DWV loads in winter bees correlate with reduced expression of genes involved in the cellular immune response and physiological activity and high expression of humoral immune genes involved in antibacterial defense compared with summer bees. This expression pattern could reflect evolutionary adaptations to resist bacterial pathogens and economize energy during the winter under a pathogen landscape with reduced risk of pathogenic viral infections. The outbreak of Varroa destructor infestation could have overcome these adaptations by promoting the transmission of viruses. Our results suggest that reduced cellular immune function during the winter may have increased honey bee’s susceptibility to DWV. These results contribute to our understanding of honey bee colony losses in temperate regions.     

Identification of Novel Genes Associated with Alginate Production in Pseudomonas aeruginosa Using Mini-himar1 Mariner Transposon-mediated Mutagenesis

Pseudomonas aeruginosa is a Gram-negative, environmental bacterium with versatile metabolic capabilities. P. aeruginosa is an opportunistic bacterial pathogen which establishes chronic pulmonary infections in patients with cystic fibrosis (CF). The overproduction of a capsular polysaccharide called alginate, also known as mucoidy, promotes the formation of mucoid biofilms which are more resistant than planktonic cells to antibiotic chemotherapy and host defenses. Additionally, the conversion from the nonmucoid to mucoid phenotype is a clinical marker for the onset of chronic infection in CF. Alginate overproduction by P. aeruginosa is an endergonic process which heavily taxes cellular energy. Therefore, alginate production is highly regulated in P. aeruginosa. To better understand alginate regulation, we describe a protocol using the mini-himar1 transposon mutagenesis for the identification of novel alginate regulators in a prototypic strain PAO1. The procedure consists of two basic steps. First, we transferred the mini-himar1 transposon (pFAC) from host E. coli SM10/λpir into recipient P. aeruginosa PAO1 via biparental conjugation to create a high-density insertion mutant library, which were selected on Pseudomonas isolation agar plates supplemented with gentamycin. Secondly, we screened and isolated the mucoid colonies to map the insertion site through inverse PCR using DNA primers pointing outward from the gentamycin cassette and DNA sequencing. Using this protocol, we have identified two novel alginate regulators, mucE (PA4033) and kinB (PA5484), in strain PAO1 with a wild-type mucA encoding the anti-sigma factor MucA for the master alginate regulator AlgU (AlgT, σ²²). This high-throughput mutagenesis protocol can be modified for the identification of other virulence-related genes causing change in colony morphology.     

A Circulating MicroRNA Profile Is Associated with Late-Stage Neovascular Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is the leading cause of severe vision impairment in Western populations over 55 years. A growing number of gene variants have been identified which are strongly associated with an altered risk to develop AMD. Nevertheless, gene-based biomarkers which could be dysregulated at defined stages of AMD may point toward key processes in disease mechanism and thus may support efforts to design novel treatment regimens for this blinding disorder. Circulating microRNAs (cmiRNAs) which are carried by nanosized exosomes or microvesicles in blood plasma or serum, have been recognized as valuable indicators for various age-related diseases. We therefore aimed to elucidate the role of cmiRNAs in AMD by genome-wide miRNA expression profiling and replication analyses in 147 controls and 129 neovascular AMD patients. We identified three microRNAs differentially secreted in neovascular (NV) AMD (hsa-mir-301-3p, p_corrected = 5.6*10⁻⁵, hsa-mir-361-5p, p_corrected = 8.0*10⁻⁴ and hsa-mir-424-5p, p_corrected = 9.6*10⁻³). A combined profile of the three miRNAs revealed an area under the curve (AUC) value of 0.727 and was highly associated with NV AMD (p = 1.2*10⁻⁸). To evaluate subtype-specificity, an additional 59 AMD cases with pure unilateral or bilateral geographic atrophy (GA) were analyzed for microRNAs hsa-mir-301-3p, hsa-mir-361-5p, and hsa-mir-424-5p. While we found no significant differences between GA AMD and controls neither individually nor for a combined microRNAs profile, hsa-mir-424-5p levels remained significantly higher in GA AMD when compared to NV (p_corrected<0.005). Pathway enrichment analysis on genes predicted to be regulated by microRNAs hsa-mir-301-3p, hsa-mir-361-5p, and hsa-mir-424-5p, suggests canonical TGFβ, mTOR and related pathways to be involved in NV AMD. In addition, knockdown of hsa-mir-361-5p resulted in increased neovascularization in an in vitro angiogenesis assay.     

An Expression Profile of Active Genes in Human Lung

An expression profile ofgenes active in the human lung was obtainedby collecting 797 partial sequences from a 3'-directed cDNAlibrary. Three genes were found to produce mRNA each of whichcomprised more than 1% of total mRNA. These three have beenidentified as genes for pulmonary surfactant apoprotein (PSP-A),Clara cells 10-kDa secretory protein, and HLA-E heavy chain.In the remaining 745 clones, 221 were composed of89 speciesthat occurred recurrently, and 524 clones appeared only once.Because the 3'-directed cDNA library faithfully represents themRNA population in the source tissue, these numbers representthe relative activities ofthe gene expression. Altogether 437gene species were novel, and 179 gene species were identifiedin GenBank. A significant portion ofthese genes encode proteinsfound in secretory proteins, cell surface proteins, and componentsin the protein synthesis machinery, representing the functionof the lung.     

Cancer Evolution Is Associated with Pervasive Positive Selection on Globally Expressed Genes

Cancer is an evolutionary process in which cells acquire new transformative, proliferative and metastatic capabilities. A full understanding of cancer requires learning the dynamics of the cancer evolutionary process. We present here a large-scale analysis of the dynamics of this evolutionary process within tumors, with a focus on breast cancer. We show that the cancer evolutionary process differs greatly from organismal (germline) evolution. Organismal evolution is dominated by purifying selection (that removes mutations that are harmful to fitness). In contrast, in the cancer evolutionary process the dominance of purifying selection is much reduced, allowing for a much easier detection of the signals of positive selection (adaptation). We further show that, as a group, genes that are globally expressed across human tissues show a very strong signal of positive selection within tumors. Indeed, known cancer genes are enriched for global expression patterns. Yet, positive selection is prevalent even on globally expressed genes that have not yet been associated with cancer, suggesting that globally expressed genes are enriched for yet undiscovered cancer related functions. We find that the increased positive selection on globally expressed genes within tumors is not due to their expression in the tissue relevant to the cancer. Rather, such increased adaptation is likely due to globally expressed genes being enriched in important housekeeping and essential functions. Thus, our results suggest that tumor adaptation is most often mediated through somatic changes to those genes that are important for the most basic cellular functions. Together, our analysis reveals the uniqueness of the cancer evolutionary process and the particular importance of globally expressed genes in driving cancer initiation and progression.     

Classification of Genes and Putative Biomarker Identification Using Distribution Metrics on Expression Profiles

Background

Identification of genes with switch-like properties will facilitate discovery of regulatory mechanisms that underlie these properties, and will provide knowledge for the appropriate application of Boolean networks in gene regulatory models. As switch-like behavior is likely associated with tissue-specific expression, these gene products are expected to be plausible candidates as tissue-specific biomarkers.

Methodology/Principal Findings

In a systematic classification of genes and search for biomarkers, gene expression profiles (GEPs) of more than 16,000 genes from 2,145 mouse array samples were analyzed. Four distribution metrics (mean, standard deviation, kurtosis and skewness) were used to classify GEPs into four categories: predominantly-off, predominantly-on, graded (rheostatic), and switch-like genes. The arrays under study were also grouped and examined by tissue type. For example, arrays were categorized as ‘brain group’ and ‘non-brain group’; the Kolmogorov-Smirnov distance and Pearson correlation coefficient were then used to compare GEPs between brain and non-brain for each gene. We were thus able to identify tissue-specific biomarker candidate genes.

Conclusions/Significance

The methodology employed here may be used to facilitate disease-specific biomarker discovery.     

Identification of miRNAs Expression Profile in Gastric Cancer Using Self-Organizing Maps (SOM)

In this paper, an unsupervised artificial neural network was implemented to identify the patters of specific signatures. The networkwas based on the differential expression of miRNAs (under or over expression) found in healthy or cancerous gastric tissues.Among the tissues analyzes, the neural network evaluated 514 miRNAs of gastric tissue that exhibited significant differentialexpression. The result suggested a specific expression signature nine miRNAs (hsa-mir-21, hsa-mir-29a, hsa-mir-29c, hsa-mir-148a,hsa-mir-141, hsa-let-7b, hsa-mir-31, hsa-mir-451, and hsa-mir-192), all with significant values (p-value < 0.01 and fold change > 5) thatclustered the samples into two groups: healthy tissue and gastric cancer tissue. The results obtained “in silico” must be validated ina molecular biology laboratory; if confirmed, this method may be used in the future as a risk marker for gastric cancerdevelopment.     

OAS Gene Family Expression Is Associated with HIV-Related Neurocognitive Disorders

HIV-associated neurocognitive disorders are common in HIV-infected individuals, even in the combination antiretroviral therapy (c-ART) era. Several mechanisms are involved in neuronal damage, including chronic inflammation immune activation. Mammalian 2′-5′-oligoadenylate synthetase (OAS) genes are produced in response to interferon (IFN), mainly by monocytes, and exert their antiviral functions by activation of RNase L that degrades viral and cellular RNAs. In this study, we aimed at exploring OAS gene family RNA expression in simian immunodeficiency virus encephalitis (SIVE), in HIV-associated neurocognitive disorders (HAND), and in HIV-associate dementia (HAD). We analyzed three microarray datasets obtained from the NCBI in order to assess the expression levels of OAS gene family network in brain biopsies of macaques with SIVE vs uninfected animals, as well as post-mortem brain of individuals with HAND (on or off ART) vs uninfected controls and three brain regions of HIV-infected individuals with both neurocognitive impairment (HAD) and encephalitis (HIVE). All OAS genes were upregulated both in SIVE and in HAND. OAS expression was significantly higher in high-viremic individuals; increased expression levels persisted in cART subjects when compared to healthy controls. OAS gene network analysis showed that several genes belonging to the type I IFN pathway, especially CXCL10 and IFIT3, were similarly upregulated in SIVE/HAND. Furthermore, we identified a significant upregulation of OAS gene family RNA expression in basal ganglia, white matter, and frontal cortex of HIV-1, HAD, and HAD/HIVE patients compared to healthy subjects. OAS gene family expression is increased in brain sections from individuals with HAND, HAD, and HIVE as well as macaques with SIVE. OAS family expression is likely to be induced by IFN as a consequence of viral replication in the CNS. Its long-term upregulation may contribute to the chronic inflammatory status and neurocognitive impairment we still observe in virologically suppressed individuals on c-ART.