Molecular quantification of symbiotic dinoflagellate algae of the genus Symbiodinium

The dinoflagellate microalga Symbiodinium is the dominant algal symbiont in corals and related marine animals. To explore the incidence of mixed infections, methods employing real-time quantitative polymerase chain reaction (QPCR) and fluorescence in situ hybridization (FISH) were developed. In experiments focusing on Symbiodinium clades A and B, QPCR and FISH results were well correlated and generally more precise and sensitive than those from the endpoint PCR-restriction fragment length polymorphism analysis (PCR-RFLP) traditionally used for this application, thus increasing the detected incidence of mixed infections. For example, the prevalence of mixed infections in the sea anemone Condylactis gigantea was 40% by PCR-RFLP and 80%-90% by QPCR and FISH. However, the use of QPCR and FISH was limited by inter-host variation in the rRNA gene copy number per Symbiodinium cell, precluding any single conversion factor between QPCR signal and Symbiodinium cell number; and one FISH probe that gave excellent hybridization efficiency with cultured Symbiodinium yielded variable results with Symbiodinium from symbioses. After controlling for these caveats, QPCR studies revealed that field-collected hosts previously described as universally unialgal bore up to 1.6% of the alternative clade. Further research is required to establish the contribution that algal cells at low density in symbiosis and external to the symbiosis make to the minor clade.     

CFTR mutations in Turkish and North African cystic fibrosis patients in Europe: implications for screening

AIMS: To obtain more insight into the variability of the CFTR mutations found in immigrant cystic fibrosis (CF) patients who are living in Europe now, and to estimate the test sensitivity of different frequently used methods of DNA analysis to detect CF carriers or patients among these Turkish or North African immigrants. METHODS: A survey among 373 European CF centers asking which CFTR mutations had been found in Turkish and North African CF patients. RESULTS: 31 and 26 different mutations were reported in Turkish and North African patients, identifying 64.2% (113/176) and 87.4% (118/135) alleles, respectively (p < 0.001). The mean sensitivity (detection rate) of three most common CFTR mutation panels to detect these mutations differed between Turkish and North African people, 44.9% (79/176) versus 69.6% (94/135) (p < 0.001), and can be increased to 57.4% (101/176) and 79.3% (107/135) (p < 0.001), respectively, by expanding these panels with 13 mutations which have been found on two or more alleles. CONCLUSION: 35.8% and 12.6%, respectively, of CF alleles in Turkish and North African patients living in Europe now had not been identified. Among these populations, the test sensitivity of common CFTR mutation panels is insufficient for use in screening programs in Europe, even after expansion with frequent Turkish and North African mutations. This raises questions about whether and how to implement CF carrier and neonatal screening in a multiethnic society.     

Distribution of the invasive New Zealand mudsnail (Potamopyrgus antipodarum) in the Columbia River Estuary and its first recorded occurrence in the diet of juvenile Chinook salmon (Oncorhynchus tshawytscha)

Estuaries play an important role as nurseries and migration corridors for Chinook salmon and other fishes. The invasive New Zealand mudsnail, Potamopyrgus antipodarum (Gray, 1843), has been noted in the Columbia River Estuary and other estuaries in the western USA, yet no studies have addressed the estuarine impacts of this invader. Our data show P. antipodarum is currently found in five peripheral bays and many tributaries of the Columbia River Estuary, where it can constitute a major portion of the benthic invertebrate biomass and where it co-occurs with native amphipod species. We review the history of the P. antipodarum invasion in the Columbia River Estuary and discuss potential impacts on estuarine food webs. We also report the first occurrence of P. antipodarum in the diet of juvenile Chinook salmon from the Columbia River Estuary. Although present in Chinook diets at very low frequencies, our observations of P. antipodarum in Chinook gut contents may represent early stages of food web change due to the establishment of dense estuarine snail populations. Additional research is needed to determine the effects of P. antipodarum on benthic resources, native benthic invertebrates, and benthic predators. We encourage biologists working in western USA estuaries to be alert to the possibility of encountering P. antipodarum in benthic habitats and predator diets.

Anion channel activation and proton pumping inhibition involved in the plasma membrane depolarization induced by ABA in Arabidopsis thaliana suspension cells are both ROS dependent

In Arabidopsis thaliana suspension cells, ABA was previously shown to promote the activation of anion channels and the reduction of proton pumping that both contribute to the plasma membrane depolarization. These two ABA responses were shown to induce two successive [Ca(2+)](cyt) spikes. As reactive oxygen species (ROS) have emerged as components of ABA signaling pathways especially by promoting [Ca(2+)](cyt) variations, we studied whether ROS were involved in the regulation of anion channels and proton pumps activities. Here we demonstrated that ABA induced ROS production which triggered the second of the two [Ca(2+)](cyt) increases observed in response to ABA. Blocking ROS generation using diphenyleneiodonium (DPI) impaired the proton pumping reduction, the anion channel activation and the RD29A gene expression in response to ABA. Furthermore, H(2)O(2) was shown to activate anion channels and to inhibit plasma membrane proton pumping, as did ABA. However, ROS partially mimicked ABA's effects since H(2)O(2) treatment elicited anion channel activation but not the subsequent expression of the RD29A gene as did ABA. This suggests that expression of the RD29A gene in response to ABA results from the activation of multiple concomitant signaling pathways: blocking of one of them would impair gene expression whereas stimulating only one would not. We conclude that ROS are a central messenger of ABA in the signaling pathways leading to the plasma membrane depolarization induced by ABA.     

8-[2-(4-Aryl-1-piperazinyl)ethyl]-2H-1,4-benzoxazin-3(4H)-ones: Dual-acting 5-HT1 receptor antagonists and serotonin reuptake inhibitors—Part II

8-[2-(4-Aryl-1-piperazinyl)ethyl]-2H-1,4-benzoxazin-3(4H)-ones have been identified as highly potent 5-HT_1A/B/D receptor antagonists with and without additional SerT activity and a high degree of selectivity over hERG potassium channels. Modulation of the different target activities gave compounds with a range of profiles suitable for further in vivo characterization.     

Consensus-degenerate hybrid oligonucleotide primers (CODEHOPs) for the detection of novel viruses in non-human primates

Consensus-degenerate hybrid oligonucleotide primers (CODEHOPs) have proven to be a powerful tool for the identification of novel genes. CODEHOPs are designed from highly-conserved regions of multiply-aligned protein sequences from members of a gene family and are used in PCR amplification to identify distantly-related genes. The CODEHOP approach has been used to identify novel pathogens by targeting amino acid motifs conserved in specific pathogen families. We initiated a program utilizing the CODEHOP approach to develop PCR-based assays targeting a variety of viral families that are pathogens in non-human primates. We have also developed and further improved a computer program and website to facilitate the design of CODEHOP PCR primers. Here, we detail the method for the development of pathogen-specific CODEHOP PCR assays using the papillomavirus family as a target. Papillomaviruses constitute a diverse virus family infecting a wide variety of mammalian species, including humans and non-human primates. We demonstrate that our pan-papillomavirus CODEHOP assay is broadly reactive with all major branches of the virus family and show its utility in identifying a novel non-human primate papillomavirus in cynomolgus macaques.