Identifying and Characterizing Alternative Molecular Markers for the Symbiotic and Free-Living Dinoflagellate Genus Symbiodinium

Dinoflagellates in the genus Symbiodinium are best known as endosymbionts of corals and other invertebrate as well as protist hosts, but also exist free-living in coastal environments. Despite their importance in marine ecosystems, less than 10 loci have been used to explore phylogenetic relationships in this group, and only the multi-copy nuclear ribosomal Internal Transcribed Spacer (ITS) regions 1 and 2 have been used to characterize fine-scale genetic diversity within the nine clades (A–I) that comprise the genus. Here, we describe a three-step molecular approach focused on 1) identifying new candidate genes for phylogenetic analysis of Symbiodinium spp., 2) characterizing the phylogenetic relationship of these candidate genes from DNA samples spanning eight Symbiodinium clades (A–H), and 3) conducting in-depth phylogenetic analyses of candidate genes displaying genetic divergences equal or higher than those within the ITS-2 of Symbiodinium clade C. To this end, we used bioinformatics tools and reciprocal comparisons to identify homologous genes from 55,551 cDNA sequences representing two Symbiodinium and six additional dinoflagellate EST libraries. Of the 84 candidate genes identified, 7 Symbiodinium genes (elf2, coI, coIII, cob, calmodulin, rad24, and actin) were characterized by sequencing 23 DNA samples spanning eight Symbiodinium clades (A–H). Four genes displaying higher rates of genetic divergences than ITS-2 within clade C were selected for in-depth phylogenetic analyses, which revealed that calmodulin has limited taxonomic utility but that coI, rad24, and actin behave predictably with respect to Symbiodinium lineage C and are potential candidates as new markers for this group. The approach for targeting candidate genes described here can serve as a model for future studies aimed at identifying and testing new phylogenetically informative genes for taxa where transcriptomic and genomics data are available

Novel contrast-enhanced ultrasound imaging in prostate cancer

The purposes of this paper were to present the current status of contrast-enhanced transrectal ultrasound imaging and to discuss the latest achievements and techniques now under preclinical testing. Although grayscale transrectal ultrasound is the standard method for prostate imaging, it lacks accuracy in the detection and localization of prostate cancer. With the introduction of contrast-enhanced ultrasound (CEUS), perfusion imaging of the microvascularization became available. By this, cancer-induced neovascularisation can be visualized with the potential to improve ultrasound imaging for prostate cancer detection and localization significantly. For example, several studies have shown that CEUS-guided biopsies have the same or higher PCa detection rate compared with systematic biopsies with less biopsies needed. This paper describes the current status of CEUS and discusses novel quantification techniques that can improve the accuracy even further. Furthermore, quantification might decrease the user-dependency, opening the door to use in the routine clinical environment. A new generation of targeted microbubbles is now under pre-clinical testing and showed avidly binding to VEGFR-2, a receptor up-regulated in prostate cancer due to angiogenesis. The first publications regarding a targeted microbubble ready for human use will be discussed. Ultrasound-assisted drug delivery gives rise to a whole new set of therapeutic options, also for prostate cancer. A major breakthrough in the future can be expected from the clinical use of targeted microbubbles for drug delivery for prostate cancer diagnosis as well as treatmen

Different Gain/Loss Sensitivity and Social Adaptation Ability in Gifted Adolescents during a Public Goods Game

Gifted adolescents are considered to have high IQs with advanced mathematical and logical performances, but are often thought to suffer from social isolation or emotional mal-adaptation to the social group. The underlying mechanisms that cause stereotypic portrayals of gifted adolescents are not well known. We aimed to investigate behavioral performance of gifted adolescents during social decision-making tasks to assess their affective and social/non-social cognitive abilities. We examined cooperation behaviors of 22 gifted and 26 average adolescents during an iterative binary public goods (PG) game, a multi-player social interaction game, and analyzed strategic decision processes that include cooperation and free-riding. We found that the gifted adolescents were more cooperative than average adolescents. Particularly, comparing the strategies for the PG game between the two groups, gifted adolescents were less sensitive to loss, yet were more sensitive to gain. Additionally, the behavioral characteristics of average adolescents, such as low trust of the group and herding behavior, were not found in gifted adolescents. These results imply that gifted adolescents have a high cognitive ability but a low ability to process affective information or to adapt in social groups compared with average adolescents. We conclude that gain/loss sensitivity and the ability to adapt in social groups develop to different degrees in average and gifted adolescents

Improved Resolution of Reef-Coral Endosymbiont (Symbiodinium) Species Diversity, Ecology, and Evolution through psbA Non-Coding Region Genotyping

Ribosomal DNA sequence data abounds from numerous studies on the dinoflagellate endosymbionts of corals, and yet the multi-copy nature and intragenomic variability of rRNA genes and spacers confound interpretations of symbiont diversity and ecology. Making consistent sense of extensive sequence variation in a meaningful ecological and evolutionary context would benefit from the application of additional genetic markers. Sequences of the non-coding region of the plastid psbA minicircle (psbAncr) were used to independently examine symbiont genotypic and species diversity found within and between colonies of Hawaiian reef corals in the genus Montipora. A single psbAncr haplotype was recovered in most samples through direct sequencing (∼80–90%) and members of the same internal transcribed spacer region 2 (ITS2) type were phylogenetically differentiated from other ITS2 types by substantial psbAncr sequence divergence. The repeated sequencing of bacterially-cloned fragments of psbAncr from samples and clonal cultures often recovered a single numerically common haplotype accompanied by rare, highly-similar, sequence variants. When sequence artifacts of cloning and intragenomic variation are factored out, these data indicate that most colonies harbored one dominant Symbiodinium genotype. The cloning and sequencing of ITS2 DNA amplified from these same samples recovered numerically abundant variants (that are diagnostic of distinct Symbiodinium lineages), but also generated a large amount of sequences comprising PCR/cloning artifacts combined with ancestral and/or rare variants that, if incorporated into phylogenetic reconstructions, confound how small sequence differences are interpreted. Finally, psbAncr sequence data from a broad sampling of Symbiodinium diversity obtained from various corals throughout the Indo-Pacific were concordant with ITS lineage membership (defined by denaturing gradient gel electrophoresis screening), yet exhibited substantially greater sequence divergence and revealed strong phylogeographic structure corresponding to major biogeographic provinces. The detailed genetic resolution provided by psbAncr data brings further clarity to the ecology, evolution, and systematics of symbiotic dinoflagellates

Isolation and Characterization of Intestinal Epithelial Cells from Normal and SIV-Infected Rhesus Macaques

Impairment of intestinal epithelial barriers contributes to the progression of HIV/SIV infection and leads to generalized HIV-induced immune-cell activation during chronic infection. Rhesus macaques are the major animal model for studying HIV pathogenesis. However, detailed characterization of isolated rhesus epithelial cells (ECs) from intestinal tissues is not well defined. It is also not well documented whether isolated ECs had any other cell contaminants from intestinal tissues during the time of processing that might hamper interpretation of EC preparations or cultures. In this study, we identify and characterize ECs based on flow cytometry and immunohistochemistry methods using various enzymatic and mechanical isolation techniques to enrich ECs from intestinal tissues. This study shows that normal healthy ECs differentially express HLA-DR, CD23, CD27, CD90, CD95 and IL-10R markers. Early apoptosis and upregulation of ICAM-1 and HLA-DR in intestinal ECs are thought to be the key features in SIV mediated enteropathy. The data suggest that intestinal ECs might be playing an important role in mucosal immune responses by regulating the expression of different important regulatory and adhesion molecules and their function

Heterologous Expression of Membrane Proteins: Choosing the Appropriate Host

International audienceBACKGROUND: Membrane proteins are the targets of 50% of drugs, although they only represent 1% of total cellular proteins. The first major bottleneck on the route to their functional and structural characterisation is their overexpression; and simply choosing the right system can involve many months of trial and error. This work is intended as a guide to where to start when faced with heterologous expression of a membrane protein. METHODOLOGY/PRINCIPAL FINDINGS: The expression of 20 membrane proteins, both peripheral and integral, in three prokaryotic (E. coli, L. lactis, R. sphaeroides) and three eukaryotic (A. thaliana, N. benthamiana, Sf9 insect cells) hosts was tested. The proteins tested were of various origins (bacteria, plants and mammals), functions (transporters, receptors, enzymes) and topologies (between 0 and 13 transmembrane segments). The Gateway system was used to clone all 20 genes into appropriate vectors for the hosts to be tested. Culture conditions were optimised for each host, and specific strategies were tested, such as the use of Mistic fusions in E. coli. 17 of the 20 proteins were produced at adequate yields for functional and, in some cases, structural studies. We have formulated general recommendations to assist with choosing an appropriate system based on our observations of protein behaviour in the different hosts. CONCLUSIONS/SIGNIFICANCE: Most of the methods presented here can be quite easily implemented in other laboratories. The results highlight certain factors that should be considered when selecting an expression host. The decision aide provided should help both newcomers and old-hands to select the best system for their favourite membrane protein