Rab27a controls HIV-1 assembly by regulating plasma membrane levels of phosphatidylinositol 4,5-bisphosphate

During the late stages of the HIV-1 replication cycle, the viral polyprotein Pr55^Gag is recruited to the plasma membrane (PM), where it binds phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) and directs HIV-1 assembly. We show that Rab27a controls the trafficking of late endosomes carrying phosphatidylinositol 4-kinase type 2 α (PI4KIIα) toward the PM of CD4⁺ T cells. Hence, Rab27a promotes high levels of PM phosphatidylinositol 4-phosphate and the localized production of PI(4,5)P₂, therefore controlling Pr55^Gag membrane association. Rab27a also controls PI(4,5)P₂ levels at the virus-containing compartments of macrophages. By screening Rab27a effectors, we identified that Slp2a, Slp3, and Slac2b are required for the association of Pr55^Gag with the PM and that Slp2a cooperates with Rab27a in the recruitment of PI4KIIα to the PM. We conclude that by directing the trafficking of PI4KIIα-positive endosomes toward the PM, Rab27a controls PI(4,5)P₂ production and, consequently, HIV-1 replication.     

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.     

Phylogeography, genetic structure and population divergence time of cheetahs in Africa and Asia: evidence for long-term geographic isolates

The cheetah (Acinonyx jubatus) has been described as a species with low levels of genetic variation. This has been suggested to be the consequence of a demographic bottleneck 10 000–12 000 years ago (ya) and also led to the assumption that only small genetic differences exist between the described subspecies. However, analysing mitochondrial DNA and microsatellites in cheetah samples from most of the historic range of the species we found relatively deep phylogeographic breaks between some of the investigated populations, and most of the methods assessed divergence time estimates predating the postulated bottleneck. Mitochondrial DNA monophyly and overall levels of genetic differentiation support the distinctiveness of Northern‐East African cheetahs (Acinonyx jubatus soemmeringii). Moreover, combining archaeozoological and contemporary samples, we show that Asiatic cheetahs (Acinonyx jubatus venaticus) are unambiguously separated from African subspecies. Divergence time estimates from mitochondrial and nuclear data place the split between Asiatic and Southern African cheetahs (Acinonyx jubatus jubatus) at 32 000–67 000 ya using an average mammalian microsatellite mutation rate and at 4700–44 000 ya employing human microsatellite mutation rates. Cheetahs are vulnerable to extinction globally and critically endangered in their Asiatic range, where the last 70–110 individuals survive only in Iran. We demonstrate that these extant Iranian cheetahs are an autochthonous monophyletic population and the last representatives of the Asiatic subspecies A. j. venaticus. We advocate that conservation strategies should consider the uncovered independent evolutionary histories of Asiatic and African cheetahs, as well as among some African subspecies. This would facilitate the dual conservation priorities of maintaining locally adapted ecotypes and genetic diversity.     

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.     

Serologic surveillance for selected viral agents in captive and free-ranging populations of Arabian oryx (Oryx leucoryx) from Saudi Arabia and the United Arab Emirates

A total of 294 sera collected between 1999 and 2001 from eight captive and one free-ranging herds of Arabian oryx (Oryx leucoryx) distributed in Saudi Arabia (SA) and the United Arab Emirates (UAE) were assayed for antibodies against 13 selected viral agents. Arabian oryx have been exposed to bluetongue virus (BTV), epizootic hemorrhagic disease virus (EHDV), rinderpest virus (RPV), bovine respiratory syncytial virus (BRSV), bovine adenovirus 3 (BAV-3), cervid herpesvirus-1, foot-and-mouth disease virus, equine herpesvirus 9, and bovine viral diarrhea virus. The high seroprevalence to BTV and EHDV in the UAE and SA indicates that Arabian oryx are likely to be susceptible to infection by these viruses and therefore could act as a source of virus to vectors during the infective stage of infection. Moreover, antibodies were detected against RPV and BRSV in sera from SA and against BAV-3 in sera from the UAE. No antibodies were found against bovine herpesvirus-1, caprine herpesvirus-1, enzootic bovine leucosis virus, and peste des petits ruminants virus. On the basis of these results, caution should be applied when considering translocation of Arabian oryx, and only those proven to be free of infectious agents that might present a risk to other species should be moved.