Identification of ribosomal proteins specific to higher eukaryotic organisms

This report describes the identification of a novel protein named PS1D (Genbank accession number ), which is composed of an S1-like RNA-binding domain, a (cysteine)x3-(histidine) CCCH-zinc finger, and a very basic carboxyl domain. PS1D is expressed as two isoforms, probably resulting from the alternative splicing of mRNA. The long PS1D isoform differs from the short one by the presence of 48 additional amino acids at its amino-terminal extremity. Analysis of PS1D subcellular distribution by cell fractionation reveals that this protein belongs to the core of the eukaryotic 60S ribosomal subunit. Interestingly, PS1D protein is a highly conserved protein among mammalians as murine, human, and simian PS1D homologues share more than 95% identity. In contrast, no homologous protein is found in lower eukaryotes such as yeast and Caenorhabditis elegans. These observations indicate that PS1D is the first eukaryotic ribosomal protein that is specific to higher eukaryotes.

Identification of microRNAs expressed in two mosquito vectors, Aedes albopictus and Culex quinquefasciatus.

BACKGROUND: MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression in a variety of organisms, including insects, vertebrates, and plants. miRNAs play important roles in cell development and differentiation as well as in the cellular response to stress and infection. To date, there are limited reports of miRNA identification in mosquitoes, insects that act as essential vectors for the transmission of many human pathogens, including flaviviruses. West Nile virus (WNV) and dengue virus, members of the Flaviviridae family, are primarily transmitted by Aedes and Culex mosquitoes. Using high-throughput deep sequencing, we examined the miRNA repertoire in Ae. albopictus cells and Cx. quinquefasciatus mosquitoes. RESULTS: We identified a total of 65 miRNAs in the Ae. albopictus C7/10 cell line and 77 miRNAs in Cx. quinquefasciatus mosquitoes, the majority of which are conserved in other insects such as Drosophila melanogaster and Anopheles gambiae. The most highly expressed miRNA in both mosquito species was miR-184, a miRNA conserved from insects to vertebrates. Several previously reported Anopheles miRNAs, including miR-1890 and miR-1891, were also found in Culex and Aedes, and appear to be restricted to mosquitoes. We identified seven novel miRNAs, arising from nine different precursors, in C7/10 cells and Cx. quinquefasciatus mosquitoes, two of which have predicted orthologs in An. gambiae. Several of these novel miRNAs reside within a ~350 nt long cluster present in both Aedes and Culex. miRNA expression was confirmed by primer extension analysis. To determine whether flavivirus infection affects miRNA expression, we infected female Culex mosquitoes with WNV. Two miRNAs, miR-92 and miR-989, showed significant changes in expression levels following WNV infection. CONCLUSIONS: Aedes and Culex mosquitoes are important flavivirus vectors. Recent advances in both mosquito genomics and high-throughput sequencing technologies enabled us to interrogate the miRNA profile in these two species. Here, we provide evidence for over 60 conserved and seven novel mosquito miRNAs, expanding upon our current understanding of insect miRNAs. Undoubtedly, some of the miRNAs identified will have roles not only in mosquito development, but also in mediating viral infection in the mosquito host.

The characterization of twenty sequenced human genomes.

We present the analysis of twenty human genomes to evaluate the prospects for identifying rare functional variants that contribute to a phenotype of interest. We sequenced at high coverage ten "case" genomes from individuals with severe hemophilia A and ten "control" genomes. We summarize the number of genetic variants emerging from a study of this magnitude, and provide a proof of concept for the identification of rare and highly-penetrant functional variants by confirming that the cause of hemophilia A is easily recognizable in this data set. We also show that the number of novel single nucleotide variants (SNVs) discovered per genome seems to stabilize at about 144,000 new variants per genome, after the first 15 individuals have been sequenced. Finally, we find that, on average, each genome carries 165 homozygous protein-truncating or stop loss variants in genes representing a diverse set of pathways.

Identification of the endophilins (SH3p4/p8/p13) as novel binding partners for the beta1-adrenergic receptor.

Several G-protein coupled receptors, such as the beta1-adrenergic receptor (beta1-AR), contain polyproline motifs within their intracellular domains. Such motifs in other proteins are known to mediate protein-protein interactions such as with Src homology (SH)3 domains. Accordingly, we used the proline-rich third intracellular loop of the beta1-AR either as a glutathione S-transferase fusion protein in biochemical "pull-down" assays or as bait in the yeast two-hybrid system to search for interacting proteins. Both approaches identified SH3p4/p8/p13 (also referred to as endophilin 1/2/3), a SH3 domain-containing protein family, as binding partners for the beta1-AR. In vitro and in human embryonic kidney (HEK) 293 cells, SH3p4 specifically binds to the third intracellular loop of the beta1-AR but not to that of the beta2-AR. Moreover, this interaction is mediated by the C-terminal SH3 domain of SH3p4. Functionally, overexpression of SH3p4 promotes agonist-induced internalization and modestly decreases the Gs coupling efficacy of beta1-ARs in HEK293 cells while having no effect on beta2-ARs. Thus, our studies demonstrate a role of the SH3p4/p8/p13 protein family in beta1-AR signaling and suggest that interaction between proline-rich motifs and SH3-containing proteins may represent a previously underappreciated aspect of G-protein coupled receptor signaling.

Tissue-engineered cardiac patch for advanced functional maturation of human ESC-derived cardiomyocytes.

Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) provide a promising source for cell therapy and drug screening. Several high-yield protocols exist for hESC-CM production; however, methods to significantly advance hESC-CM maturation are still lacking. Building on our previous experience with mouse ESC-CMs, we investigated the effects of 3-dimensional (3D) tissue-engineered culture environment and cardiomyocyte purity on structural and functional maturation of hESC-CMs. 2D monolayer and 3D fibrin-based cardiac patch cultures were generated using dissociated cells from differentiated Hes2 embryoid bodies containing varying percentage (48-90%) of CD172a (SIRPA)-positive cardiomyocytes. hESC-CMs within the patch were aligned uniformly by locally controlling the direction of passive tension. Compared to hESC-CMs in age (2 weeks) and purity (48-65%) matched 2D monolayers, hESC-CMs in 3D patches exhibited significantly higher conduction velocities (CVs), longer sarcomeres (2.09 ± 0.02 vs. 1.77 ± 0.01 μm), and enhanced expression of genes involved in cardiac contractile function, including cTnT, αMHC, CASQ2 and SERCA2. The CVs in cardiac patches increased with cardiomyocyte purity, reaching 25.1 cm/s in patches constructed with 90% hESC-CMs. Maximum contractile force amplitudes and active stresses of cardiac patches averaged to 3.0 ± 1.1 mN and 11.8 ± 4.5 mN/mm(2), respectively. Moreover, contractile force per input cardiomyocyte averaged to 5.7 ± 1.1 nN/cell and showed a negative correlation with hESC-CM purity. Finally, patches exhibited significant positive inotropy with isoproterenol administration (1.7 ± 0.3-fold force increase, EC50 = 95.1 nm). These results demonstrate highly advanced levels of hESC-CM maturation after 2 weeks of 3D cardiac patch culture and carry important implications for future drug development and cell therapy studies.

Cryptococcus gattii VGIII isolates causing infections in HIV/AIDS patients in Southern California: identification of the local environmental source as arboreal.

Ongoing Cryptococcus gattii outbreaks in the Western United States and Canada illustrate the impact of environmental reservoirs and both clonal and recombining propagation in driving emergence and expansion of microbial pathogens. C. gattii comprises four distinct molecular types: VGI, VGII, VGIII, and VGIV, with no evidence of nuclear genetic exchange, indicating these represent distinct species. C. gattii VGII isolates are causing the Pacific Northwest outbreak, whereas VGIII isolates frequently infect HIV/AIDS patients in Southern California. VGI, VGII, and VGIII have been isolated from patients and animals in the Western US, suggesting these molecular types occur in the environment. However, only two environmental isolates of C. gattii have ever been reported from California: CBS7750 (VGII) and WM161 (VGIII). The incongruence of frequent clinical presence and uncommon environmental isolation suggests an unknown C. gattii reservoir in California. Here we report frequent isolation of C. gattii VGIII MATα and MATa isolates and infrequent isolation of VGI MATα from environmental sources in Southern California. VGIII isolates were obtained from soil debris associated with tree species not previously reported as hosts from sites near residences of infected patients. These isolates are fertile under laboratory conditions, produce abundant spores, and are part of both locally and more distantly recombining populations. MLST and whole genome sequence analysis provide compelling evidence that these environmental isolates are the source of human infections. Isolates displayed wide-ranging virulence in macrophage and animal models. When clinical and environmental isolates with indistinguishable MLST profiles were compared, environmental isolates were less virulent. Taken together, our studies reveal an environmental source and risk of C. gattii to HIV/AIDS patients with implications for the >1,000,000 cryptococcal infections occurring annually for which the causative isolate is rarely assigned species status. Thus, the C. gattii global health burden could be more substantial than currently appreciated.

Simultaneous transcranial magnetic stimulation and single-neuron recording in alert non-human primates.

Transcranial magnetic stimulation (TMS) is a widely used, noninvasive method for stimulating nervous tissue, yet its mechanisms of effect are poorly understood. Here we report new methods for studying the influence of TMS on single neurons in the brain of alert non-human primates. We designed a TMS coil that focuses its effect near the tip of a recording electrode and recording electronics that enable direct acquisition of neuronal signals at the site of peak stimulus strength minimally perturbed by stimulation artifact in awake monkeys (Macaca mulatta). We recorded action potentials within ∼1 ms after 0.4-ms TMS pulses and observed changes in activity that differed significantly for active stimulation as compared with sham stimulation. This methodology is compatible with standard equipment in primate laboratories, allowing easy implementation. Application of these tools will facilitate the refinement of next generation TMS devices, experiments and treatment protocols.

Evidence-ranked motif identification.

cERMIT is a computationally efficient motif discovery tool based on analyzing genome-wide quantitative regulatory evidence. Instead of pre-selecting promising candidate sequences, it utilizes information across all sequence regions to search for high-scoring motifs. We apply cERMIT on a range of direct binding and overexpression datasets; it substantially outperforms state-of-the-art approaches on curated ChIP-chip datasets, and easily scales to current mammalian ChIP-seq experiments with data on thousands of non-coding regions.

The NRF-1/α-PAL transcription factor regulates human E2F6 promoter activity

E2F6 is widely expressed in human tissues and cell lines. Recent studies have demonstrated its involvement in developmental patterning and in the regulation of various genes implicated in chromatin remodelling. Despite a growing number of studies, nothing is really known concerning the E2F6 expression regulation. To understand how cells control E2F6 expression, we analysed the activity of the previously cloned promoter region of the human E2F6 gene. DNase I footprinting, gel electrophoretic-mobility shift, transient transfection and site-directed mutagenesis experiments allowed the identification of two functional NRF-1/α-PAL (nuclear respiratory factor-1/α-palindrome-binding protein)-binding sites within the human E2F6 core promoter region, which are conserved in the mouse and rat E2F6 promoter region. Moreover, ChIP (chromatin immunoprecipitation) analysis demonstrated that overexpressed NRF-1/α-PAL is associated in vivo with the E2F6 promoter. Furthermore, overexpression of full-length NRF-1/α-PAL enhanced E2F6 promoter activity, whereas expression of its dominant-negative form reduced the promoter activity. Our results indicate that NRF-1/α-PAL is implicated in the regulation of basal E2F6 gene expression.

Identification of CDH1 germline missense mutations associated with functional inactivation of the E-cadherin protein in young gastric cancer probands

E-cadherin is involved in the formation of cell-junctions and the maintenance of epithelial integrity. Direct evidence of E-cadherin mutations triggering tumorigenesis has come from the finding of inactivating germline mutations of the gene (CDH1) in hereditary diffuse gastric cancer (HDGC). We screened a series of 66 young gastric cancer probands for germline CDH1 mutations, and two novel missense alterations together with an intronic variant were identified. We then analysed the functional significance of the exonic missense variants found here as well as a third germline missense variant that we previously identified in a HGDC family. cDNAs encoding either the wild-type protein or mutant forms of E-cadherin were stably transfected into CHO (Chinese hamster ovary) E-cadherin-negative cells. Transfected cell-lines were characterized in terms of aggregation, motility and invasion. We show that a proportion of apparently sporadic early-onset diffuse gastric carcinomas are associated with germline alterations of the E-cadherin gene. We also demonstrate that a proportion of missense variants are associated with significant functional consequences, suggesting that our cell model can be used as an adjunct in deciding on the potential pathogenic role of identified E-cadherin germline alterations.

A systematic methodology to assess the impact of human factors in ship design

Evaluating ship layout for human factors (HF) issues using simulation software such as maritimeEXODUS can be a long and complex process. The analysis requires the identification of relevant evaluation scenarios; encompassing evacuation and normal operations; the development of appropriate measures which can be used to gauge the performance of crew and vessel and finally; the interpretation of considerable simulation data. Currently, the only agreed guidelines for evaluating HFs performance of ship design relate to evacuation and so conclusions drawn concerning the overall suitability of a ship design by one naval architect can be quite different from those of another. The complexity of the task grows as the size and complexity of the vessel increases and as the number and type of evaluation scenarios considered increases. Equally, it can be extremely difficult for fleet operators to set HFs design objectives for new vessel concepts. The challenge for naval architects is to develop a procedure that allows both accurate and rapid assessment of HFs issues associated with vessel layout and crew operating procedures. In this paper we present a systematic and transparent methodology for assessing the HF performance of ship design which is both discriminating and diagnostic. The methodology is demonstrated using two variants of a hypothetical naval ship.

Assessing the suitability of ship design for human factors issues associated with evacuation and normal operations

Evaluating ship layout for human factors (HF) issues using simulation software such as maritimeEXODUS can be a long and complex process. The analysis requires the identification of relevant evaluation scenarios; encompassing evacuation and normal operations; the development of appropriate measures which can be used to gauge the performance of crew and vessel and finally; the interpretation of considerable simulation data. In this paper we present a systematic and transparent methodology for assessing the HF performance of ship design which is both discriminating and diagnostic. The methodology is demonstrated using two variants of a hypothetical naval ship.

Optimising vessel layout using human factors simulation

Evaluating ship layout for human factors (HF) issues using simulation software such as maritimeEXODUS can be a long and complex process. The analysis requires the identification of relevant evaluation scenarios; encompassing evacuation and normal operations; the development of appropriate measures which can be used to gauge the performance of crew and vessel and finally; the interpretation of considerable simulation data. In this paper we present a systematic and transparent methodology for assessing the HF performance of ship design which is both discriminating and diagnostic.

The relationship between internet identification, internet anxiety and internet use

This paper reports a study investigating the relationship between Internet identification, Internet anxiety and Internet use. The participants were 446 students (319 females and 127 males) from two universities in the UK and one university in Australia. Measures of Internet identification and Internet anxiety were developed. The majority of participants were NOT anxious about using the Internet, although there were approximately 8% who showed evidence of Internet anxiety. There was a significant and negative relationship between Internet anxiety and Internet use. Those who were more anxious about using the Internet used the Internet less, although the magnitude of effect was small. There was a positive and significant relationship between Internet use and Internet identification. Those who scored high on the measure of Internet identification used the Internet more than those who did not. There was also a significant and negative relationship between Internet anxiety and Internet identification. Finally, males had a significantly higher Internet identification score than females. Implications of these findings are discussed.

Impact of human activities on benthic biotopes and species

Executive Summary 1. The Marine Life Information Network (MarLIN) has been developed since 1998. Defra funding has supported a core part of its work, the Biology and Sensitivity Key Information Sub-programme. This report relates to Biology and Sensitivity work for the period 2001-2004. 2. MarLIN Biology and Sensitivity research takes information on the biology of species to identify the likely effects of changing environmental conditions linked to human activities on those species. In turn, species that are key functional, key structural, dominant, or characteristic in a biotope (the habitat and its associated species) are used to identify biotope sensitivity. Results are displayed over the World Wide Web and can be accessed via a range of search tools that make the information of relevance to environmental management. 3. The first Defra contract enabled the development of criteria and methods of research, database storage methods and the research of a wide range of species. A contract from English Nature and Scottish Natural Heritage enabled biotopes relevant to marine SACs to be researched. 4. Defra funding in 2001-2004 has especially enabled recent developments to be targeted for research. Those developments included the identification of threatened and declining species by the OSPAR Biodiversity Committee, the development of a new approach to defining sensitivity (part of the Review of Marine Nature Conservation), and the opportunity to use Geographical Information Systems (GIS) more effectively to link survey data to MarLIN assessments of sensitivity. 5. The MarLIN database has been developed to provide a resource to 'pick-and-mix' information depending on the questions being asked. Using GIS, survey data that provides locations for species and biotopes has been linked to information researched by MarLIN to map the likely sensitivity of an area to a specified factor. Projects undertaken for the Irish Sea pilot (marine landscapes), in collaboration with CEFAS (fishing impacts) and with the Countryside Council for Wales (oil spill response) have demonstrated the application of MarLIN information linked to survey data in answering, through maps, questions about likely impacts of human activities on seabed ecosystems. 6. GIS applications that use MarLIN sensitivity information give meaningful results when linked to localized and detailed survey information (lists of species and biotopes as point source or mapped extents). However, broad landscape units require further interpretation. 7. A new mapping tool (SEABED map) has been developed to display data on species distributions and survey data according to search terms that might be used by an environmental manager. 8. MarLIN outputs are best viewed on the Web site where the most up-to-date information from live databases is available. The MarLIN Web site receives about 1600 visits a day. 9. The MarLIN approach to assessing sensitivity and its application to environmental management were presented in papers at three international conferences during the current contract and a 'touchstone' paper is to be published in the peer-reviewed journal Hydrobiologia. The utility of MarLIN information for environmental managers, amongst other sorts of information, has been described in an article in Marine Pollution Bulletin. 10. MarLIN information is being used to inform the identification of potential indicator species for implementation of the Water Framework Directive including initiatives by ICES. 11. Non-Defra funding streams are supporting the updating of reviews and increasing the amount of peer review undertaken; both of which are important to the maintenance of the resource. However, whilst MarLIN information is sufficiently wide ranging to be used in an 'operational' way for marine environmental protection and management, new initiatives and the new biotopes classification have introduced additional species and biotopes that will need to be researched in the future. 12. By the end of the contract, the Biology and Sensitivity Key Information database contained full Key Information reviews on 152 priority species and 117 priority biotopes, together with basic information on 412 species; a total of 564 marine benthic species.