Analysis of the role of COP9 Signalosome (CSN) subunits in K562; the first link between CSN and autophagy

Background  

The COP9/signalosome (CSN) is a highly conserved eight subunit complex that, by deneddylating cullins in cullin-based E3 ubiquitin ligases, regulates protein degradation. Although studied in model human cell lines such as HeLa, very little is known about the role of the CSN in haemopoietic cells.     

Bayesian inference of biochemical kinetic parameters using the linear noise approximation

Background  

Fluorescent and luminescent gene reporters allow us to dynamically quantify changes in molecular species concentration over time on the single cell level. The mathematical modeling of their interaction through multivariate dynamical models requires the deveopment of effective statistical methods to calibrate such models against available data. Given the prevalence of stochasticity and noise in biochemical systems inference for stochastic models is of special interest. In this paper we present a simple and computationally efficient algorithm for the estimation of biochemical kinetic parameters from gene reporter data.     

Design and synthesis of substituted imidazole and triazole N-phenylbenzo[d]oxazolamine inhibitors of retinoic acid metabolizing enzyme CYP26

The design of N-phenylbenzo[d]oxazolamines as CYP26A1 inhibitors involved ligand docking experiments using molecular modeling (FlexX) and analysis of ligand interactions at the binding domain. The synthesis of the benzooxazol-2-yl-[phenyl-imidazol-1-yl-methyl)phenyl]amines was achieved by cyclisation of the corresponding isothiocyanates with subsequent introduction of the haem-binding heterocycle. Triazole and tetrazole derivatives were also prepared for comparison with the lead imidazole derivative. The benzooxazol-2-yl-[phenyl-imidazol-1-yl-methyl)phenyl]amines with small substituents in the phenyl ring were moderately potent CYP26A1 inhibitors (IC(50) 8 and 12 microM) and comparable with liarozole (IC(50) 7 microM).     

Isolation of 11 microsatellite markers in Crepidula convexa (Gastropoda, Calyptraeideae) for parentage analyses

Crepidula convexa, a calyptreid gastropod with direct embryonic development, changes sex from male to female in the course of its lifetime (protandry). Under sex-allocation theory, male reproductive success should be independent from age and size (a proxy used for age). However, this may be counterbalanced by female cryptic choice or gregarious behaviour. Eleven polymorphic microsatellite loci were thus developed to examine paternity of embryos and larvae. This set of loci appears suitable to carry out paternity analyses due to the high exclusion probability of unrelated males given the maternal genotype.     

In Vivo Delta Opioid Receptor Internalization Controls Behavioral Effects of Agonists

Background

GPCRs regulate a remarkable diversity of biological functions, and are thus often targeted for drug therapies. Stimulation of a GPCR by an extracellular ligand triggers receptor signaling via G proteins, and this process is highly regulated. Receptor activation is typically accompanied by desensitization of receptor signaling, a complex feedback regulatory process of which receptor internalization is postulated as a key event. The in vivo significance of GPCR internalization is poorly understood. In fact, the majority of studies have been performed in transfected cell systems, which do not adequately model physiological environments and the complexity of integrated responses observed in the whole animal.

Methods and Findings

In this study, we used knock-in mice expressing functional fluorescent delta opioid receptors (DOR-eGFP) in place of the native receptor to correlate receptor localization in neurons with behavioral responses. We analyzed the pain-relieving effects of two delta receptor agonists with similar signaling potencies and efficacies, but distinct internalizing properties. An initial treatment with the high (SNC80) or low (AR-M100390) internalizing agonist equally reduced CFA-induced inflammatory pain. However, subsequent drug treatment produced highly distinct responses. Animals initially treated with SNC80 showed no analgesic response to a second dose of either delta receptor agonist. Concomitant receptor internalization and G-protein uncoupling were observed throughout the nervous system. This loss of function was temporary, since full DOR-eGFP receptor responses were restored 24 hours after SNC80 administration. In contrast, treatment with AR-M100390 resulted in retained analgesic response to a subsequent agonist injection, and ex vivo analysis showed that DOR-eGFP receptor remained G protein-coupled on the cell surface. Finally SNC80 but not AR-M100390 produced DOR-eGFP phosphorylation, suggesting that the two agonists produce distinct active receptor conformations in vivo which likely lead to differential receptor trafficking.

Conclusions

Together our data show that delta agonists retain full analgesic efficacy when receptors remain on the cell surface. In contrast, delta agonist-induced analgesia is abolished following receptor internalization, and complete behavioral desensitization is observed. Overall these results establish that, in the context of pain control, receptor localization fully controls receptor function in vivo. This finding has both fundamental and therapeutic implications for slow-recycling GPCRs.     

Antigen-Presenting Cells Represent Targets for R5 HIV-1 Infection in the First Trimester Pregnancy Uterine Mucosa

Background

During the first trimester of pregnancy, HIV-1 mother-to-child transmission is relatively rare despite the permissivity of placental cells to cell-to-cell HIV-1 infection. The placenta interacts directly with maternal uterine cells (decidual cells) but the physiological role of the decidua in the control of HIV-1 transmission and whether decidua could be a source of infected cells is unknown.

Methodology/Principal Findings

To answer to this question, decidual mononuclear cells were exposed to HIV-1 in vitro. Decidual cells were shown to be more susceptible to infection by an R5 HIV-1, as compared to an X4 HIV-1. Infected cells were identified by flow cytometry analysis. The results showed that CD14⁺ cells were the main targets of HIV-1 infection in the decidua. These infected CD14⁺ cells expressed DC-SIGN, CD11b, CD11c, the Fc gamma receptor CD16, CD32 and CD64, classical MHC class-I and class-II and maturation and activation molecules CD83, CD80 and CD86. The permissivity of decidual tissue was also evaluated by histoculture. Decidual tissue was not infected by X4 HIV-1 but was permissive to R5 HIV-1. Different profiles of infection were observed depending on tissue localization.

Conclusions/Significance

The presence of HIV-1 target cells in the decidua in vitro and the low rate of in utero mother-to-child transmission during the first trimester of pregnancy suggest that a natural control occurs in vivo limiting cell-to-cell infection of the placenta and consequently infection of the fetus.     

Development of a PCR-RFLP marker to genetically distinguish Prosorhynchus crucibulum and Prosorhynchus aculeatus

The cercariae stages of Prosorhynchus crucibulum and Prosorhynchus aculeatus are morphologically indistinguishable. However, the differentiation of these two species is crucial to understand the transmission dynamics between these primary hosts (mussels) and the secondary hosts (fish). In this way, the objective of this study is to develop an accurate molecular identification tool to differentiate the cercariae stage of P. crucibulum and P. aculeatus. We targeted the 18S nuclear ribosomal DNA region by PCR amplification and sequenced this amplicon. By generating these sequences, we developed a RFLP tool with the use of the enzymes HincII and FokI that produced different restriction profiles between P. crucibulum and P. aculeatus. Each enzyme generated different-sized fragments specific to the species examined and no cross-reaction between the species was detected in their restriction pattern. By sequencing, no intraspecific-polymorphism was detected since there is 100% homology among P. aculeatus or P. crucibulum. These results indicate that PCR-linked restriction analysis of the 18S rDNA region provided us with rapid and reliable molecular tools for distinction of the cercariae of these species. The sequences generated were deposited in GenBank accession numbers for P. crucibulum cercariae (FJ463407, FJ463408 and FJ463409FJ463408FJ463409) and adult worm (FJ429096, FJ429097), and for P. aculeatus adult (FJ429094 and FJ429095).     

Anti-microfouling Activity of Lipidic Metabolites from the Invasive Brown Alga Sargassum muticum (Yendo) Fensholt

The purification of the chloroform extract from the brown invasive macroalga Sargassum muticum, through a series of chromatographic separations, yielded 12 fractions that were tested against strains of bacteria, microalgae, and fungi involved in marine biofilm formation. The chemical composition of four (a, c, g, and k) out of the six fractions that exhibited anti-microfouling activity was investigated. Fraction a contained saturated and unsaturated linear hydrocarbons (C₁₂–C₂₇). Arachidonic acid was identified as the major metabolite in fraction c whereas fraction g contained mainly palmitic, linolenic, and palmitoleic acids. Fraction k was submitted to further purification yielding the fraction kAcaF1e that was composed of galactoglycerolipids, active against the growth of two of the four bacterial strains (Shewanella putrefaciens and Polaribacter irgensii) and all tested fungi. These promising results, in particular the isolation and the activity of galactoglycerolipids, attest the potential of the huge biomass of S. muticum as a source of new environmentally friendly antifouling compounds.     

Microbial community dynamics in a seasonally anoxic fjord: Saanich Inlet, British Columbia

Dissolved oxygen concentration plays a major role in shaping biotic interactions and nutrient flows within marine ecosystems. Throughout the global ocean, regions of low dissolved oxygen concentration (hypoxia) are a common and expanding feature of the water column, with major feedback on productivity and greenhouse gas cycling. To better understand microbial diversity underlying biogeochemical transformations within oxygen-deficient oceanic waters, we monitored and quantified bacterial and archaeal community dynamics in relation to dissolved gases and nutrients during a seasonal stratification and deep water renewal cycle in Saanich Inlet, British Columbia, a seasonally anoxic fjord. A number of microbial groups partitioned within oxygen-deficient waters including Nitrospina and SAR324 affiliated with the δ- proteobacteria , SAR406 and γ- proteobacteria related to thiotrophic gill symbionts of deep-sea clams and mussels. Microbial diversity was highest within the hypoxic transition zone decreasing dramatically within anoxic basin waters and temporal patterns of niche partitioning were observed along defined gradients of oxygen and phosphate. These results provide a robust comparative phylogenetic framework for inferring systems metabolism of nitrogen, carbon and sulfur cycling within oxygen-deficient oceanic waters and establish Saanich Inlet as a tractable model for studying the response of microbial communities to changing levels of water column hypoxia.