DEBtox theory and matrix population models as helpful tools in understanding the interaction between toxic cyanobacteria and zooplankton

Bioassays were performed to find out how field samples of the toxic cyanobacteria Microcystis aeruginosa affect Moina micrura, a cladoceran found in the tropical Jacarepagua Lagoon (Rio de Janeiro, Brazil). The DEBtox (Dynamic Energy Budget theory applied to toxicity data) approach has been proposed for use in analysing chronic toxicity tests as an alternative to calculating the usual safety parameters (NOEC, ECx). DEBtox theory deals with the energy balance between physiological processes (assimilation, maintenance, growth and reproduction), and it can be used to investigate and compare various hypotheses concerning the mechanism of action of a toxicant. Even though the DEBtox framework was designed for standard toxicity bioassays carried out with standard species (fish, daphnids), we applied the growth and reproduction models to M. micrura, by adapting the data available using a weight-length allometric relationship. Our modelling approach appeared to be very relevant at the individual level, and confirmed previous conclusions about the toxic mechanism. In our study we also wanted to assess the toxic effects at the population level, which is a more relevant endpoint in risk assessment. We therefore incorporated both lethal and sublethal toxic effects in a matrix population model used to calculate the finite rate of population change as a continuous function of the exposure concentration. Alongside this calculation, we constructed a confidence band to predict the critical exposure concentration for population health. Finally, we discuss our findings with regard to the prospects for further refining the analysis of ecotoxicological data.     

<Emphasis Type="Italic">PHEX</Emphasis> analysis in 118 pedigrees reveals new genetic clues in hypophosphatemic rickets

Familial hypophosphatemic rickets is a rare disease, which is mostly transmitted as an X-linked dominant trait, and mutations on the phosphate regulating gene with homologies to endopeptidases on the X-chromosome (PHEX) gene are responsible for the disease in most familial cases. In this study we analyzed PHEX in a large cohort of 118 pedigrees representing 56 familial cases and 62 sporadic cases. The high-resolution melting curves technique was tested as a screening method, along with classical sequencing. PHEX mutations have been found in 87% of familial cases but also in 72% of sporadic cases. Missense mutations were found in 16 probands, two of which being associated with other PHEX mutations resulting into truncated proteins. By plotting missense mutations described so far on a 3D model of PHEX we observed that these mutations focus on two regions located in the inner part of the PHEX protein. Family members of 13 sporadic cases were analyzed and a PHEX mutation was detected in one of the apparently healthy mother. These results highlight the major role of PHEX in X-linked dominant hypophosphatemic rickets, and give new clues regarding the genetic analysis of the disease. A screening of the different family members should be mandatory when a PHEX mutation is assessed in a sporadic case and the search for another PHEX mutation should be systematically proceed when facing a missense mutation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Crosstalk between GABAB and mGlu1a receptors reveals new insight into GPCR signal integration

G protein‐coupled receptors (GPCRs) have critical functions in intercellular communication. Although a wide range of different receptors have been identified in the same cells, the mechanism by which signals are integrated remains elusive. The ability of GPCRs to form dimers or larger hetero‐oligomers is thought to generate such signal integration. We examined the molecular mechanisms responsible for the GABA_B receptor‐mediated potentiation of the mGlu receptor signalling reported in Purkinje neurons. We showed that this effect does not require a physical interaction between both receptors. Instead, it is the result of a more general mechanism in which the βγ subunits produced by the Gi‐coupled GABA_B receptor enhance the mGlu‐mediated Gq response. Most importantly, this mechanism could be generally applied to other pairs of Gi‐ and Gq‐coupled receptors and the signal integration varied depending on the time delay between activation of each receptor. Such a mechanism helps explain specific properties of cells expressing two different Gi‐ and Gq‐coupled receptors activated by a single transmitter, or properties of GPCRs naturally coupled to both types of the G protein.     

Isolation of Prasinoviruses of the Green Unicellular Algae Ostreococcus spp. on a Worldwide Geographical Scale

Ostreococcus spp. are extremely small unicellular eukaryotic green algae found worldwide in marine environments, and they are susceptible to attacks by a diverse group of large DNA viruses. Several biologically distinct species of Ostreococcus are known and differ in the ecological niches that they occupy: while O. tauri (representing clade C strains) is found in marine lagoons and coastal seas, strains belonging to clade A, exemplified by O. lucimarinus, are present in different oceans. We used laboratory cultures of clonal isolates of these two species to assay for the presence of viruses in seawater samples from diverse locations. In keeping with the distributions of their host strains, we found a decline in the abundance of O. tauri viruses from a lagoon in southwest France relative to the Mediterranean Sea, whereas in the ocean, no O. tauri viruses were detected. In contrast, viruses infecting O. lucimarinus were detected from distantly separated oceans. DNA sequencing, phylogenetic analyses using a conserved viral marker gene, and a Mantel test revealed no relationship between geographic and phylogenetic distances in viruses infecting O. lucimarinus.Viruses are the most abundant and genetically diverse biological entities in marine environments (48). The three ways most often used to assess eukaryotic algal virus diversity are (i) using a functional host-virus system to quantify viruses specific to one host strain (i.e., culture-based studies) (4), (ii) using PCR amplification and sequencing a conserved gene (10, 12-14, 28), and (iii) using whole-community genome sequencing (i.e., metagenomics) (6, 8, 31). Recently, the advent of sequencing techniques like shotgun sequencing or pyrosequencing (38) has led to an increase in the number of metagenomics projects. The Global Ocean Sampling (GOS) Expedition has provided a unique opportunity to investigate viral diversity in different environments within the size fraction of 0.1 to 0.8 μm (39). The GOS data revealed highly abundant viral sequences (at least 3% of the predicted proteins had a viral origin) (53). In another study, the analysis of marine viromes from four oceanic regions suggested that the composition of viral assemblages depends on their geographic locations, but these authors conclude that similar viruses are widespread throughout the oceans (2). Despite these new methods and different ways to analyze viral diversity, we still do not really know if “everything is really everywhere” (7).The present study addresses a specific part of this problem: are viruses infecting a single host strain present at geographically distant locations? If several viral strains are identified and characterized, how closely do these viruses resemble one another on a phylogenetic scale? In order to answer these questions, we focused on a microalgal (Prasinophyceae)-virus (Prasinovirus) system. The studied hosts belong to the genus Ostreococcus, a ubiquitous prasinophyte picoeukaryotic alga abundant throughout the oceanic euphotic zone (55, 56). Several strains from this genus were isolated and assigned to four distinct ecotypes according to their growth parameters under different light regimens (22, 36), which correspond to four well-defined phylogenetic clades in an internal transcribed spacer (ITS)-based phylogeny (clades A to D). The complete genome sequences of two Ostreococcus species have been described: O. tauri (19) and O. lucimarinus (35). In the present study, viruses infecting specific host species (Ostreococcus spp.) have been screened from a variety of locations around the world.Among viruses infecting Ostreococcus, the genome of a single strain (OtV5) has been fully sequenced (18), and the phylogenetic relationships among several virus strains have been investigated (4). These viruses belong to the Prasinovirus group, a genus of the Phycodnaviridae family. Many viruses infecting phytoplankton are members of the Phycodnaviridae; they have double-stranded DNA genomes and large polyhedral capsids (20). In the prasinophyte-Prasinovirus system, the hosts and viruses can be grown on solid medium and are easily maintained in the laboratory. Ostreococcus viral strains have been isolated and characterized by phylogenetic analysis based on their B-family DNA polymerase (DNA pol) partial gene sequence (4). This DNA polymerase is a useful marker for phylogenetic analyses because its sequence is well conserved in all known members of nucleocytoplasmic large DNA viruses (NCLDVs) (26), including Phycodnaviridae. Furthermore, several previous studies have examined the abundance and the genetic diversity of marine eukaryotic viruses using environmental sequencing approaches and amplified DNA pol gene fragments (11, 12, 43-46), and Monier et al. (31) used this marker to describe the taxonomic distribution of large DNA viruses from the GOS data.The first stage of this study was to isolate Ostreococcus viruses from different worldwide geographic locations, by culturing on various host strains. In a second stage, these viral strains were characterized via the sequencing of their pol sequence (encoding a part of their DNA polymerase gene), and their specificity toward different host strains was assessed in order to assess the potential host range of the viral strains isolated and to gain a better understanding of their population dynamics and distribution. Finally we compared these new Prasinovirus DNA sequences with metagenomic sequence data (obtained from sampling all around the world) and environmental sequence data identified using BLAST similarity to assess the global distribution of similar Ostreococcus viruses.     

Role of N-Linked Glycans in the Functions of Hepatitis C Virus Envelope Proteins Incorporated into Infectious Virions

Hepatitis C virus (HCV) envelope glycoproteins are highly glycosylated, with generally 4 and 11 N-linked glycans on E1 and E2, respectively. Studies using mutated recombinant HCV envelope glycoproteins incorporated into retroviral pseudoparticles (HCVpp) suggest that some glycans play a role in protein folding, virus entry, and protection against neutralization. The development of a cell culture system producing infectious particles (HCVcc) in hepatoma cells provides an opportunity to characterize the role of these glycans in the context of authentic infectious virions. Here, we used HCVcc in which point mutations were engineered at N-linked glycosylation sites to determine the role of these glycans in the functions of HCV envelope proteins. The mutants were characterized for their effects on virus replication and envelope protein expression as well as on viral particle secretion, infectivity, and sensitivity to neutralizing antibodies. Our results indicate that several glycans play an important role in HCVcc assembly and/or infectivity. Furthermore, our data demonstrate that at least five glycans on E2 (denoted E2N1, E2N2, E2N4, E2N6, and E2N11) strongly reduce the sensitivity of HCVcc to antibody neutralization, with four of them surrounding the CD81 binding site. Altogether, these data indicate that the glycans associated with HCV envelope glycoproteins play roles at different steps of the viral life cycle. They also highlight differences in the effects of glycosylation mutations between the HCVpp and HCVcc systems. Furthermore, these carbohydrates form a “glycan shield” at the surface of the virion, which contributes to the evasion of HCV from the humoral immune response.Hepatitis C virus (HCV) is a single-stranded positive-sense RNA virus that causes serious liver diseases in humans (31). More than 170 million people worldwide are seropositive for HCV and at risk for developing cirrhosis and hepatocellular carcinoma (50). HCV is a small, enveloped virus that belongs to the Hepacivirus genus in the Flaviviridae family (31). Its genome encodes a single polyprotein precursor of about 3,000-amino-acid residues that is cleaved co- and posttranslationally by cellular and viral proteases to yield at least 10 mature products (31). The two envelope glycoproteins, E1 and E2, are released from the polyprotein by signal peptidase cleavages. These two proteins assemble as noncovalent heterodimers, which are retained mainly in the endoplasmic reticulum (ER) (36), and they are found as large disulfide-linked oligomers on the surfaces of HCV particles (46). HCV glycoproteins are involved in the entry process, and since they are present on the surfaces of viral particles, these proteins are the targets of neutralizing antibodies (4, 21).E1 and E2 generally contain 4 and 11 N-glycosylation sites, respectively, all of which have been shown to be modified by glycans (19). Despite variability in HCV envelope glycoprotein sequences, the four glycosylation sites of E1 and nine of E2 are highly conserved, suggesting that the glycans associated with these proteins play an essential role in the HCV life cycle (22). Using retroviral particles pseudotyped with genotype 1a (H strain) HCV envelope glycoproteins (HCVpp), recent studies have determined the potential roles played by these glycans in protein folding, HCV entry, and protection against neutralization (14, 19, 22). Indeed, the lack of glycan E1N1, E1N4, E2N8, or E2N10 strongly affects the incorporation of HCV glycoproteins into HCVpp, suggesting that these glycans are important for correct protein folding (19). Furthermore, mutation of glycosylation sites E2N2 or E2N4 alters HCVpp infectivity despite normal incorporation into pseudotyped particles, suggesting a role for the corresponding glycans in viral entry, at least in this model system (19). Finally, glycans at positions E2N1, E2N6, and E2N11 were shown to reduce the sensitivity of HCVpp to antibody neutralization as well as access of the CD81 coreceptor to its binding site on E2, suggesting that glycans also contribute to HCV evasion of the humoral immune response (14, 22).It has recently been proposed that targeting glycans could be a promising approach to inhibiting viral infection (1). Indeed, HCV, as well as several other viruses with highly glycosylated envelope proteins, can be inhibited by carbohydrate binding agents such as cyanovirin-N and pradimicin A (1, 7, 23). Furthermore, resistance against drugs that target glycans is likely to develop and will probably result in mutations at some glycosylation sites (3, 52). However, since glycans associated with viral envelope proteins play an important role in the viral life cycle, adaptation of viruses to the selective pressure of carbohydrate-binding agents will most likely come at a replicative cost to the virus (2).Although the role of HCV glycans has been studied using mutant recombinant HCV envelope glycoproteins incorporated into HCVpp, these particles do not recapitulate all the functions of HCV envelope proteins. Cell culture-derived virus (HCVcc) (32, 49, 55) assembles in an ER-derived compartment in association with very low density lipoproteins (17, 26), whereas HCVpp are assembled in a post-Golgi compartment and are not associated with lipoproteins (44). Importantly, this leads to differences between HCVpp and HCVcc in the oligomerization of the envelope glycoproteins (46). It is also important to note that the carbohydrate composition of viral glycoproteins can differ when the same virus is grown in different cell lines (13). Thus, HCVpp that are produced in 293T cells are not the most appropriate model for glycosylation studies, since HCV tropism is restricted to the liver. Furthermore, differences in envelope protein glycosylation have been observed between HCVpp and HCVcc particles (46). Differences in some HCV envelope protein functions were also observed when the HCVpp and HCVcc systems were compared (28, 29, 42, 43). The development of the HCVcc system provides, therefore, the opportunity to characterize the role of E1/E2-associated glycans in the context of authentic infectious virions. Here, we analyzed the role of E1/E2 glycans by introducing point mutations at N-linked glycosylation sites in the context of the HCVcc system. The effects of these mutations on virus replication, particle secretion, infectivity, and sensitivity to neutralizing antibodies were investigated. Our results demonstrate that several glycans play an important role in HCVcc assembly and/or infectivity and reduce access of neutralizing antibodies to their epitopes.     

DEFENSE EVOLUTION IN THE GRACILARIACEAE (RHODOPHYTA): SUBSTRATE‐REGULATED OXIDATION OF AGAR OLIGOSACCHARIDES IS MORE ANCIENT THAN THE OLIGOAGAR‐ACTIVATED OXIDATIVE BURST1

Combined phylogenetic, physiological, and biochemical approaches revealed that differences in defense‐related responses among 17 species belonging to the Gracilariaceae were consistent with their evolutionary history. An oxidative burst response resulting from activation of NADPH oxidase was always observed in two of the subgenera of Gracilaria sensu lato (Gracilaria, Hydropuntia), but not in Gracilariopsis and in species related to Gracilaria chilensis (“chilensis” clade). On the other hand, all species examined except Gracilaria tenuistipitata var. liui and Gracilariopsis longissima responded with up‐regulation of agar oligosaccharide oxidase to an challenge with agar oligosaccharides. As indicated by pharmacological experiments conducted with Gracilaria chilensis and Gracilaria sp. “dura,” the up‐regulation of agar oligosaccharide oxidase involved an NAD(P)H‐dependent signaling pathway, but not kinase activity. By contrast, the activation of NADPH oxidase requires protein phosphorylation. Both responses are therefore independent, and the agar oligosaccharide‐activated oxidative burst evolved after the capacity to oxidize agar oligosaccharide, probably providing additional defensive capacity to the most recently differentiated clades of Gracilariaceae. As demonstrated with Gracilaria gracilis, Gracilaria dura, and Gracilariopsis longissima, the different responses to agar oligosaccharides allow for a fast and nondestructive distinction among different clades of gracilarioids that are morphologically convergent. Based upon sequences of the chloroplast‐encoded rbcL gene, this study suggests that at least some of the samples from NW America recorded as Gs. lemanaeiformis are probably Gs. chorda. Moreover, previous records of Gracilaria conferta from Israel are shown to be based upon misidentification of Gracilaria sp. “dura,” a species that belongs to the Hydropuntia subgenus.