The Origin of GPCRs: Identification of Mammalian like Rhodopsin, Adhesion, Glutamate and Frizzled GPCRs in Fungi

G protein-coupled receptors (GPCRs) in humans are classified into the five main families named Glutamate, Rhodopsin, Adhesion, Frizzled and Secretin according to the GRAFS classification. Previous results show that these mammalian GRAFS families are well represented in the Metazoan lineages, but they have not been shown to be present in Fungi. Here, we systematically mined 79 fungal genomes and provide the first evidence that four of the five main mammalian families of GPCRs, namely Rhodopsin, Adhesion, Glutamate and Frizzled, are present in Fungi and found 142 novel sequences between them. Significantly, we provide strong evidence that the Rhodopsin family emerged from the cAMP receptor family in an event close to the split of Opisthokonts and not in Placozoa, as earlier assumed. The Rhodopsin family then expanded greatly in Metazoans while the cAMP receptor family is found in 3 invertebrate species and lost in the vertebrates. We estimate that the Adhesion and Frizzled families evolved before the split of Unikonts from a common ancestor of all major eukaryotic lineages. Also, the study highlights that the fungal Adhesion receptors do not have N-terminal domains whereas the fungal Glutamate receptors have a broad repertoire of mammalian-like N-terminal domains. Further, mining of the close unicellular relatives of the Metazoan lineage, Salpingoeca rosetta and Capsaspora owczarzaki, obtained a rich group of both the Adhesion and Glutamate families, which in particular provided insight to the early emergence of the N-terminal domains of the Adhesion family. We identified 619 Fungi specific GPCRs across 79 genomes and revealed that Blastocladiomycota and Chytridiomycota phylum have Metazoan-like GPCRs rather than the GPCRs specific for Fungi. Overall, this study provides the first evidence of the presence of four of the five main GRAFS families in Fungi and clarifies the early evolutionary history of the GPCR superfamily

A New Method to Predict the Epidemiology of Fungal Keratitis by Monitoring the Sales Distribution of Antifungal Eye Drops in Brazil

Purpose: Fungi are a major cause of keratitis, although few medications are licensed for their treatment. The aim of this study is to observe the variation in commercialisation of antifungal eye drops, and to predict the seasonal distribution of fungal keratitis in Brazil. Methods: Data from a retrospective study of antifungal eye drops sales from the only pharmaceutical ophthalmologic laboratory, authorized to dispense them in Brazil (Opthalmos) were gathered. These data were correlated with geographic and seasonal distribution of fungal keratitis in Brazil between July 2002 and June 2008. Results: A total of 26,087 antifungal eye drop units were sold, with a mean of 2.3 per patient. There was significant variation in antifungal sales during the year (p < 0.01). A linear regression model displayed a significant association between reduced relative humidity and antifungal drug sales (R-2 = 0.17, p < 0.01). Conclusions: Antifungal eye drops sales suggest that there is a seasonal distribution of fungal keratitis. A possible interpretation is that the third quarter of the year (a period when the climate is drier), when agricultural activity is more intense in Brazil, suggests a correlation with a higher incidence of fungal keratitis. A similar model could be applied to other diseases, that are managed with unique, or few, and monitorable medications to predict epidemiological aspects.Conselho Nacional de Desenvolvimento Cientifico e TecnologicoConselho Nacional de Desenvolvimento Cientifico e Tecnologico [302005/2009-9]Fundacao de Apoio ao Ensino, Pesquisa e Assistencia do Hospital das Clinicas da Faculdade de Medicina de Ribeirao Preto da Universidade de Sao PauloFundacao de Apoio ao Ensino, Pesquisa e Assistencia do Hospital das Clinicas da Faculdade de Medicina de Ribeirao Preto da Universidade de Sao Paul

Intracellular Calcium Deficits in Drosophila Cholinergic Neurons Expressing Wild Type or FAD-Mutant Presenilin

Much of our current understanding about neurodegenerative diseases can be attributed to the study of inherited forms of these disorders. For example, mutations in the presenilin 1 and 2 genes have been linked to early onset familial forms of Alzheimer's disease (FAD). Using the Drosophila central nervous system as a model we have investigated the role of presenilin in one of the earliest cellular defects associated with Alzheimer's disease, intracellular calcium deregulation. We show that expression of either wild type or FAD-mutant presenilin in Drosophila CNS neurons has no impact on resting calcium levels but does give rise to deficits in intracellular calcium stores. Furthermore, we show that a loss-of-function mutation in calmodulin, a key regulator of intracellular calcium, can suppress presenilin-induced deficits in calcium stores. Our data support a model whereby presenilin plays a role in regulating intracellular calcium stores and demonstrate that Drosophila can be used to study the link between presenilin and calcium deregulation

Dynamics of biofilm formation and the interaction between Candida albicans and methicillin-susceptible (MSSA) and -resistant Staphylococcus aureus (MRSA)

Polymicrobial biofilms are an understudied and a clinically relevant problem. This study evaluates the interaction between C. albicans, and methicillin- susceptible (MSSA) and resistant (MRSA) S. aureus growing in single- and dual-species biofilms. Single and dual species adhesion (90 min) and biofilms (12, 24, and 48 h) were evaluated by complementary methods: counting colony-forming units (CFU mL-1), XTT-reduction, and crystal violet staining (CV). The secretion of hydrolytic enzymes by the 48 h biofilms was also evaluated using fluorimetric kits. Scanning electron microscopy (SEM) was used to assess biofilm structure. The results from quantification assays were compared using two-way ANOVAs with Tukey post-hoc tests, while data from enzymatic activities were analyzed by one-way Welch-ANOVA followed by Games-Howell post hoc test ( = 0.05). C. albicans, MSSA and MRSA were able to adhere and to form biofilm in both single or mixed cultures. In general, all microorganisms in both growth conditions showed a gradual increase in the number of cells and metabolic activity over time, reaching peak values between 12 h and 48 h (<0.05). C. albicans single- and dual-biofilms had significantly higher total biomass values (<0.05) than single biofilms of bacteria. Except for single MRSA biofilms, all microorganisms in both growth conditions secreted proteinase and phospholipase-C. SEM images revealed extensive adherence of bacteria to hyphal elements of C. albicans. C. albicans, MSSA, and MRSA can co-exist in biofilms without antagonism and in an apparent synergistic effect, with bacteria cells preferentially associated to C. albicans hyphal forms.CNPq (Council for Technical and Scientific Development) (Grant 400658/2012-7)Fundação para a Ciência e Tecnologia (FCT), Portugal (SFRH/BPD/71076/2010)CAPES(Coordination for the Improvement of Higher Level Personnel

Identification of miRs-143 and -145 that Is Associated with Bone Metastasis of Prostate Cancer and Involved in the Regulation of EMT

The principal problem arising from prostate cancer (PCa) is its propensity to metastasize to bone. MicroRNAs (miRNAs) play a crucial role in many tumor metastases. The importance of miRNAs in bone metastasis of PCa has not been elucidated to date. We investigated whether the expression of certain miRNAs was associated with bone metastasis of PCa. We examined the miRNA expression profiles of 6 primary and 7 bone metastatic PCa samples by miRNA microarray analysis. The expression of 5 miRNAs significantly decreased in bone metastasis compared with primary PCa, including miRs-508-5p, -145, -143, -33a and -100. We further examined other samples of 16 primary PCa and 13 bone metastases using real-time PCR analysis. The expressions of miRs-143 and -145 were verified to down-regulate significantly in metastasis samples. By investigating relationship of the levels of miRs-143 and -145 with clinicopathological features of PCa patients, we found down-regulations of miRs-143 and -145 were negatively correlated to bone metastasis, the Gleason score and level of free PSA in primary PCa. Over-expression miR-143 and -145 by retrovirus transfection reduced the ability of migration and invasion in vitro, and tumor development and bone invasion in vivo of PC-3 cells, a human PCa cell line originated from a bone metastatic PCa specimen. Their upregulation also increased E-cadherin expression and reduced fibronectin expression of PC-3 cells which revealed a less invasive morphologic phenotype. These findings indicate that miRs-143 and -145 are associated with bone metastasis of PCa and suggest that they may play important roles in the bone metastasis and be involved in the regulation of EMT Both of them may also be clinically used as novel biomarkers in discriminating different stages of human PCa and predicting bone metastasis

MicroRNAs and their target gene networks in breast cancer

MicroRNAs (miRNAs) are a major class of small endogenous RNA molecules that post-transcriptionally inhibit gene expression. Many miRNAs have been implicated in several human cancers, including breast cancer. Here we describe the association between altered miRNA signatures and breast cancer tumorigenesis and metastasis. The loss of several tumor suppressor miRNAs (miR-206, miR-17-5p, miR-125a, miR-125b, miR-200, let-7, miR-34 and miR-31) and the overexpression of certain oncogenic miRNAs (miR-21, miR-155, miR-10b, miR-373 and miR-520c) have been observed in many breast cancers. The gene networks orchestrated by these miRNAs are still largely unknown, although key targets have been identified that may contribute to the disease phenotype. Here we report how the observed perturbations in miRNA expression profiles may lead to disruption of key pathways involved in breast cancer

De novo assembly and characterization of a maternal and developmental transcriptome for the emerging model crustacean Parhyale hawaiensis

<p>Abstract</p> <p>Background</p> <p>Arthropods are the most diverse animal phylum, but their genomic resources are relatively few. While the genome of the branchiopod <it>Daphnia pulex </it>is now available, no other large-scale crustacean genomic resources are available for comparison. In particular, genomic resources are lacking for the most tractable laboratory model of crustacean development, the amphipod <it>Parhyale hawaiensis</it>. Insight into shared and divergent characters of crustacean genomes will facilitate interpretation of future developmental, biomedical, and ecological research using crustacean models.</p> <p>Results</p> <p>To generate a transcriptome enriched for maternally provided and zygotically transcribed developmental genes, we created cDNA from ovaries and embryos of <it>P. hawaiensis</it>. Using 454 pyrosequencing, we sequenced over 1.1 billion bases of this cDNA, and assembled them <it>de novo </it>to create, to our knowledge, the second largest crustacean genomic resource to date. We found an unusually high proportion of C2H2 zinc finger-containing transcripts, as has also been reported for the genome of the pea aphid <it>Acyrthosiphon pisum</it>. Consistent with previous reports, we detected trans-spliced transcripts, but found that they did not noticeably impact transcriptome assembly. Our assembly products yielded 19,067 unique BLAST hits against <b>nr </b>(E-value cutoff e-10). These included over 400 predicted transcripts with significant similarity to <it>D. pulex </it>sequences but not to sequences of any other animal. Annotation of several hundred genes revealed <it>P. hawaiensis </it>homologues of genes involved in development, gametogenesis, and a majority of the members of six major conserved metazoan signaling pathways.</p> <p>Conclusions</p> <p>The amphipod <it>P. hawaiensis </it>has higher transcript complexity than known insect transcriptomes, and trans-splicing does not appear to be a major contributor to this complexity. We discuss the importance of a reliable comparative genomic framework within which to consider findings from new crustacean models such as <it>D. pulex </it>and <it>P. hawaiensis</it>, as well as the need for development of further substantial crustacean genomic resources.</p

A prospective phase II trial exploring the association between tumor microenvironment biomarkers and clinical activity of ipilimumab in advanced melanoma

<p>Abstract</p> <p>Background</p> <p>Ipilimumab, a fully human monoclonal antibody that blocks cytotoxic T-lymphocyte antigen-4, has demonstrated an improvement in overall survival in two phase III trials of patients with advanced melanoma. The primary objective of the current trial was to prospectively explore candidate biomarkers from the tumor microenvironment for associations with clinical response to ipilimumab.</p> <p>Methods</p> <p>In this randomized, double-blind, phase II biomarker study (ClinicalTrials.gov NCT00261365), 82 pretreated or treatment-naïve patients with unresectable stage III/IV melanoma were induced with 3 or 10 mg/kg ipilimumab every 3 weeks for 4 doses; at Week 24, patients could receive maintenance doses every 12 weeks. Efficacy was evaluated per modified World Health Organization response criteria and safety was assessed continuously. Candidate biomarkers were evaluated in tumor biopsies collected pretreatment and 24 to 72 hours after the second ipilimumab dose. Polymorphisms in immune-related genes were also evaluated.</p> <p>Results</p> <p>Objective response rate, response patterns, and safety were consistent with previous trials of ipilimumab in melanoma. No associations between genetic polymorphisms and clinical activity were observed. Immunohistochemistry and histology on tumor biopsies revealed significant associations between clinical activity and high baseline expression of FoxP3 (p = 0.014) and indoleamine 2,3-dioxygenase (p = 0.012), and between clinical activity and increase in tumor-infiltrating lymphocytes (TILs) between baseline and 3 weeks after start of treatment (p = 0.005). Microarray analysis of mRNA from tumor samples taken pretreatment and post-treatment demonstrated significant increases in expression of several immune-related genes, and decreases in expression of genes implicated in cancer and melanoma.</p> <p>Conclusions</p> <p>Baseline expression of immune-related tumor biomarkers and a post-treatment increase in TILs may be positively associated with ipilimumab clinical activity. The observed pharmacodynamic changes in gene expression warrant further analysis to determine whether treatment-emergent changes in gene expression may be associated with clinical efficacy. Further studies are required to determine the predictive value of these and other potential biomarkers associated with clinical response to ipilimumab.</p