Structural and functional properties of ryanodine receptor type 3 in zebrafish tail muscle

The ryanodine receptor (RyR)1 isoform of the sarcoplasmic reticulum (SR) Ca²⁺ release channel is an essential component of all skeletal muscle fibers. RyR1s are detectable as “junctional feet” (JF) in the gap between the SR and the plasmalemma or T-tubules, and they are required for excitation–contraction (EC) coupling and differentiation. A second isoform, RyR3, does not sustain EC coupling and differentiation in the absence of RyR1 and is expressed at highly variable levels. Anatomically, RyR3 expression correlates with the presence of parajunctional feet (PJF), which are located on the sides of the SR junctional cisternae in an arrangement found only in fibers expressing RyR3. In frog muscle fibers, the presence of RyR3 and PJF correlates with the occurrence of Ca²⁺ sparks, which are elementary SR Ca²⁺ release events of the EC coupling machinery. Here, we explored the structural and functional roles of RyR3 by injecting zebrafish (Danio rerio) one-cell stage embryos with a morpholino designed to specifically silence RyR3 expression. In zebrafish larvae at 72 h postfertilization, fast-twitch fibers from wild-type (WT) tail muscles had abundant PJF. Silencing resulted in a drop of the PJF/JF ratio, from 0.79 in WT fibers to 0.03 in the morphants. The frequency with which Ca²⁺ sparks were detected dropped correspondingly, from 0.083 to 0.001 sarcomere⁻¹ s⁻¹. The few Ca²⁺ sparks detected in morphant fibers were smaller in amplitude, duration, and spatial extent compared with those in WT fibers. Despite the almost complete disappearance of PJF and Ca²⁺ sparks in morphant fibers, these fibers looked structurally normal and the swimming behavior of the larvae was not affected. This paper provides important evidence that RyR3 is the main constituent of the PJF and is the main contributor to the SR Ca²⁺ flux underlying Ca²⁺ sparks detected in fully differentiated frog and fish fibers.     

Comparative Analysis of Genetic Chemotyping Methods for Fusarium: Tri13 Polymorphism Does not Discriminate between 3‐ and 15‐acetylated Deoxynivalenol Chemotypes in Fusarium graminearum

Genetic chemotyping is an essential tool for characterizing Fusarium populations causing head blight on wheat and other cereals. Three PCR methods, based on tri cluster polymorphism, were optimized and compared on 94 single‐spore isolates obtained from three continents belonging to F. gramineaurm, F. culmorum, F. poae, F. avenaceum and Microdochium nivale. While the methods based on the tri3, tri7 and tri12 polymorphism correctly identified all the tested strains, the method based on tri13 polymorphism was unable to discriminate between the 3‐ and 15‐acetylated DON forms in F. graminearum. It is advised to avoid the use of tri13 polymorphism for genetic chemotyping of the two acetylated chemotypes.     

Continuous feeding of antimicrobial growth promoters to commercial swine during the growing/finishing phase does not modify faecal community erythromycin resistance or community structure

Aims: To investigate the effect of continuous feeding of antimicrobial growth promoters (tylosin or virginiamycin) on the swine faecal community. Methods and Results: The study consisted of two separate on‐farm feeding trials. Swine were fed rations containing tylosin (44 or 88 mg kg^?1 of feed) or virginiamycin (11 or 22 mg kg^?1 of feed) continuously over the growing/finishing phases. The temporal impact of continuous antimicrobial feeding on the faecal community was assessed and compared to nondosed control animals through anaerobic cultivation, the analysis of community 16S rRNA gene libraries and faecal volatile fatty acid content. Feeding either antimicrobial had no detectable effect on the faecal community. Conclusions: Erythromycin methylase genes encoding resistance to the macrolide–lincosamide–streptogramin B (MLS_B) antimicrobials are present at a high level within the faecal community of intensively raised swine. Continuous antimicrobial feeding over the entire growing/finishing phase had no effect on community erm‐methylase gene copy numbers or faecal community structure. Significance and Impact of the Study: Antimicrobial growth promoters are believed to function by altering gut bacterial communities. However, widespread MLS_B resistance within the faecal community of intensively raised swine likely negates any potential effects that these antimicrobials might have on altering the faecal community. These findings suggest that if AGP‐mediated alterations to gut communities are an important mechanism for growth promotion, it is unlikely that these would be associated with the colonic community.     

Genetic analysis of two Portuguese populations of <Emphasis Type="Italic">Ruditapes decussatus</Emphasis> by RAPD profiling

The clam Ruditapes decussatus is commercially important in the south of Portugal. The random amplified polymorphic DNA (RAPD) technique was applied to assess the genetic diversity and population structure of two Portuguese populations occurring in the Ria Formosa (Faro) and the Ria de Alvor, respectively. Twenty-five individuals of each population were investigated by RAPD profiles. Genetic diversity within populations, measured by the percentage of polymorphic loci (%P), varied between 68.57% (Alvor) and 73.88% (Faro). Shannon’s information index (H) and Nei’s gene diversity (h) were 0.281 and 0.176, respectively, for the Alvor population and 0.356 and 0.234 for the Faro population. Overall, genetic variation within R. decussatus populations was high. The total genetic diversity (H _T) was explained by a low variation between populations (G _ST = 0.145), which is consistent with high gene flow (N _m = 2.9). The analysis of molecular variance (AMOVA) showed that 65% of variability is within populations and 35% between populations (Φ_PT = 0.345; P ≥ 0.001). The value of Nei’s genetic distance was 0.0881, showing a low degree of population genetic distance, despite the different geographic origin. This is the first study on the population genetics of R. decussatus by RAPD technique. The results may be useful for restocking programs and aquaculture.     

Removal of pathogen and indicator microorganisms from liquid swine manure in multi-step biological and chemical treatment

Concern has greatly increased about the potential for contamination of water, food, and air by pathogens present in manure. We evaluated pathogen reduction in liquid swine manure in a multi-stage treatment system where first the solids and liquid are separated with polymer, followed by biological nitrogen (N) removal using nitrification and denitrification, and then phosphorus (P) extraction through lime precipitation. Each step of the treatment system was analyzed for Salmonella and microbial indicators of fecal contamination (total coliforms, fecal coliforms, and enterococci). Before treatment, mean concentrations of Salmonella, total coliforms, fecal coliforms, and enterococci were 3.89, 6.79, 6.23 and 5.73 log(10) colony forming units (cfu)/ml, respectively. The flushed manure contained 10,590 mg/l TSS, 8270 mg/l COD, 688 mg/l TKN and 480 mg/l TP, which were reduced >98% by the treatment system. Results showed a consistent trend in reduction of pathogens and microbial indicators as a result of each step in the treatment system. Solid-liquid separation decreased their concentrations by 0.5-1 log(10). Additional biological N removal treatment with alternating anoxic and oxic conditions achieved a higher reduction with average removals of 2.4 log(10) for Salmonella and 4.1-4.5 log(10) for indicator microbes. Subsequent P treatment decreased concentration of Salmonella and pathogen indicators to undetectable level (<0.3 log(10) cfu/ml) due to elevated process pH (10.3). Our results indicate that nitrification/denitrification treatment after solids separation is very effective in reducing pathogens in liquid swine manure and that the phosphorus removal step via alkaline calcium precipitation produces a sanitized effluent which may be important for biosecurity reasons.     

Identification of Key Residues in Virulent Canine Distemper Virus Hemagglutinin That Control CD150/SLAM-Binding Activity

Morbillivirus cell entry is controlled by hemagglutinin (H), an envelope-anchored viral glycoprotein determining interaction with multiple host cell surface receptors. Subsequent to virus-receptor attachment, H is thought to transduce a signal triggering the viral fusion glycoprotein, which in turn drives virus-cell fusion activity. Cell entry through the universal morbillivirus receptor CD150/SLAM was reported to depend on two nearby microdomains located within the hemagglutinin. Here, we provide evidence that three key residues in the virulent canine distemper virus A75/17 H protein (Y525, D526, and R529), clustering at the rim of a large recessed groove created by β-propeller blades 4 and 5, control SLAM-binding activity without drastically modulating protein surface expression or SLAM-independent F triggering.Paramyxoviruses are enveloped nonsegmented negative-strand RNA viruses that inject their genetic information into target cells by fusing their lipid envelope with the plasma membrane of the host cell at a neutral pH. Plasma membrane fusion activity is achieved by the concerted action of two viral membrane-bound glycoproteins. The attachment protein (hemagglutinin [H], hemagglutinin-neuraminidase [HN], or attachment [G], depending on the viral genus) is thought to bind a host cell surface receptor, in turn activating the fusion (F) protein, which will then undergo large-scale structural rearrangements, leading to plasma membrane fusion activity (9, 10, 19). In addition, both viral surface glycoproteins may mediate fusion activity between two contacting neighboring cells (22, 27). Virus-induced cell-cell fusion activity eventually leads to multinucleated cell formation (also termed syncytium formation) and, ultimately, to cell lysis.The crystal structure of the measles virus hemagglutinin (MeV-H) has recently become available (3, 7, 8). Interestingly, the overall β-propeller structure consisting of six β-sheets was well conserved compared to already determined paramyxovirus HN structures (4, 12, 29). The canine distemper virus H (CDV-H) protein has a short N-terminal cytoplasmic tail followed by a transmembrane domain and a large C-terminal ectodomain (1). It is suggested that the ectodomain consists of a stalk region with an α-helical coiled-coil configuration (13, 28) that supports a globular head domain containing the receptor recognition site and antigenic regions of the protein (11).Recently, site-directed mutagenesis aimed at identifying residues throughout the MeV-H ectodomain that might selectively control membrane fusion activity in a receptor-dependent manner (CD150/SLAM, CD46, or a yet-unidentified putative epithelial cell receptor [EpR]) was conducted. Indeed, four key residues, located in two connected microdomains (site 1 and site 2) on MeV-H globular head β-propeller blade 5, were necessary to uphold SLAM-dependent fusogenicity. Mutations in each one of the four amino acids resulted in a selective inhibition of SLAM-dependent fusion activity (H-SLAM-blind; HSB [25]). Interestingly, the latter quartet of residues were subsequently demonstrated not to be involved in SLAM-binding activity but presumably were involved in controlling SLAM-dependent F triggering (14). An additional residue (isoleucine 194), located within MeV-H β-propeller blade 6 but in contact with site 2, was next shown to govern interaction with the universal morbillivirus SLAM receptor (14). Consequently, the corresponding residues of both microdomains were mutated in the H protein of the virulent CDV strain 5804P and were also demonstrated to control SLAM-dependent fusion activity (24), although for CDV, full ablation of fusion activity required the substitutions in both microdomains and in two additional neighboring amino acids (CDV-H residues in site 1, D526, I527, S528, and R529; in site 2, Y547 and T548). Moreover, using a CDV-H 3D homology model, the two microdomains were demonstrated to be in very close proximity to one another (compared to those of MeV-H) but not in direct contact (24). Subsequently, a recombinant CDV bearing a SLAM-blind H protein was reported to be completely attenuated in ferrets, a phenotype associated with reduced immunosuppression and lack of neurovirulence (26). However, the precise molecular mechanisms sustaining HSB-dependent lack of fusion support activity was not elucidated and remains to be determined.     

Respiratory responses of three Antarctic ascidians and a sea pen to increased sediment concentrations

Glacial retreat and subglacial bedrock erosion are consequences of rapid regional warming on the West Antarctic Peninsula. Sedimentation of fine-grained eroded particles can impact the physiology of filter-feeding benthic organisms. We investigated the effect of increasing concentrations of sediment on the oxygen consumption of suspension feeding species, the ascidians Molgula pedunculata, Cnemidocarpa verrucosa, Ascidia challengeri, and the pennatulid Malacobelemnon daytoni in Potter Cove (South Shetland Islands, Antarctica). In A. challengeri and C. verrucosa, oxygen consumption increased gradually up to a critical sediment concentration (C _crit) where species oxygen consumption was maximal (O _max in mg O₂?g^?1dm?day^?1) and further addition of sediments decreased respiration. C _crit was 200?mg?L^?1 for A. challengeri (O _max of 0.651?±?0.238) and between 100 and 200?mg?L^?1 for C. verrucosa (O _max of 0.898?±?0.582). Oxygen consumption of M. pedunculata increased significantly even at low sediment concentrations (15–50?mg sediment?L^?1). Contrary to the ascidians, sediment exposure did not affect oxygen consumption of the sea pen. The tiered response to sedimentation in the four species corroborates recent field observations that detected a reduction in the abundance of the sensitive ascidian M. pedunculata from areas strongly affected by glacial sediment discharge, whereas sea pens are increasing in abundance. Our investigation relates consequences (population shifts in filter-feeder communities) to causes (glacial retreat) and is of importance for modelling of climate change effects in Antarctic shallow coastal areas.