A novel surface protein of Trichomonas vaginalis is regulated independently by low iron and contact with vaginal epithelial cells

Background  

Trichomonosis caused by Trichomonas vaginalis is the number one, non-viral sexually transmitted disease (STD) that affects more than 250 million people worldwide. Immunoglobulin A (IgA) has been implicated in resistance to mucosal infections by pathogens. No reports are available of IgA-reactive proteins and the role, if any, of this class of antibody in the control of this STD. The availability of an IgA monoclonal antibody (mAb) immunoreactive to trichomonads by whole cell (WC)-ELISA prompted us to characterize the IgA-reactive protein of T. vaginalis.     

Cryo X-ray nano-tomography of vaccinia virus infected cells

We have performed full-field cryo X-ray microscopy in the water window photon energy range on vaccinia virus (VACV) infected cells to produce tomographic reconstructions. PtK2 cells were infected with a GFP-expressing VACV strain and frozen by plunge fast freezing. The infected cells were selected by light fluorescence microscopy of the GFP marker and subsequently imaged in the X-ray microscope under cryogenic conditions. Tomographic tilt series of X-ray images were used to yield three-dimensional reconstructions showing different cell organelles (nuclei, mitochondria, filaments), together with other structures derived from the virus infection. Among them, it was possible to detect viral factories and two types of viral particles related to different maturation steps of VACV (immature and mature particles), which were compared to images obtained by standard electron microscopy of the same type of cells. In addition, the effect of radiation damage during X-ray tomographic acquisition was analyzed. Thin sections studied by electron microscopy revealed that the morphological features of the cells do not present noticeable changes after irradiation. Our findings show that cryo X-ray nano-tomography is a powerful tool for collecting three-dimensional structural information from frozen, unfixed, unstained whole cells with sufficient resolution to detect different virus particles exhibiting distinct maturation levels.     

Role of helix 0 of the N-BAR domain in membrane curvature generation

A group of proteins with cell membrane remodeling properties is also able to change dramatically the morphology of liposomes in vitro, frequently inducing tubulation. For a number of these proteins, the mechanism by which this effect is exerted has been proposed to be the embedding of amphipathic helices into the lipid bilayer. For proteins presenting BAR domains, removal of an N-terminal amphipathic α-helix (H0-NBAR) results in much lower membrane tubulation efficiency, pointing to a fundamental role of this protein segment. Here, we studied the interaction of a peptide corresponding to H0-NBAR with model lipid membranes. H0-NBAR bound avidly to anionic liposomes but partitioned weakly to zwitterionic bilayers, suggesting an essentially electrostatic interaction with the lipid bilayer. Interestingly, it is shown that after membrane incorporation, the peptide oligomerizes as an antiparallel dimer, suggesting a potential role of H0-NBAR in the mediation of BAR domain oligomerization. Through monitoring the effect of H0-NBAR on liposome shape by cryoelectron microscopy, it is clear that membrane morphology is not radically changed. We conclude that H0-NBAR alone is not able to induce vesicle curvature, and its function must be related to the promotion of the scaffold effect provided by the concave surface of the BAR domain.     

New Insights into Rotavirus Entry Machinery: Stabilization of Rotavirus Spike Conformation Is Independent of Trypsin Cleavage

The infectivity of rotavirus, the main causative agent of childhood diarrhea, is dependent on activation of the extracellular viral particles by trypsin-like proteases in the host intestinal lumen. This step entails proteolytic cleavage of the VP4 spike protein into its mature products, VP8* and VP5*. Previous cryo-electron microscopy (cryo-EM) analysis of trypsin-activated particles showed well-resolved spikes, although no density was identified for the spikes in uncleaved particles; these data suggested that trypsin activation triggers important conformational changes that give rise to the rigid, entry-competent spike. The nature of these structural changes is not well understood, due to lack of data relative to the uncleaved spike structure. Here we used cryo-EM and cryo-electron tomography (cryo-ET) to characterize the structure of the uncleaved virion in two model rotavirus strains. Cryo-EM three-dimensional reconstruction of uncleaved virions showed spikes with a structure compatible with the atomic model of the cleaved spike, and indistinguishable from that of digested particles. Cryo-ET and subvolume average, combined with classification methods, resolved the presence of non-icosahedral structures, providing a model for the complete structure of the uncleaved spike. Despite the similar rigid structure observed for uncleaved and cleaved particles, trypsin activation is necessary for successful infection. These observations suggest that the spike precursor protein must be proteolytically processed, not to achieve a rigid conformation, but to allow the conformational changes that drive virus entry.     

Extreme differences in rates of molecular evolution of foraminifera revealed by comparison of ribosomal DNA sequences and the fossil record

Foraminifera have one of the best known fossil records among the unicellular eukaryotes. However, the origin and phylogenetic relationships of the extant foraminiferal lineages are poorly understood. To test the current paleontological hypotheses on evolution of foraminifera, we sequenced about 1,000 base pairs from the 3' end of the small subunit rRNA gene (SSU rDNA) in 22 species representing all major taxonomic groups. Phylogenies were derived using neighbor- joining, maximum-parsimony, and maximum-likelihood methods. All analyses confirm the monophyletic origin of foraminifera. Evolutionary relationships within foraminifera inferred from rDNA sequences, however, depend on the method of tree building and on the choice of analyzed sites. In particular, the position of planktonic foraminifera shows important variations. We have shown that these changes result from the extremely high rate of rDNA evolution in this group. By comparing the number of substitutions with the divergence times inferred from the fossil record, we have estimated that the rate of rDNA evolution in planktonic foraminifera is 50 to 100 times faster than in some benthic foraminifera. The use of the maximum-likelihood method and limitation of analyzed sites to the most conserved parts of the SSU rRNA molecule render molecular and paleontological data generally congruent.      

Global transcriptional response of pig brain and lung to natural infection by Pseudorabies virus

Background  

Pseudorabies virus (PRV) is an alphaherpesviruses whose native host is pig. PRV infection mainly causes signs of central nervous system disorder in young pigs, and respiratory system diseases in the adult.     

Defining the healthy "core microbiome" of oral microbial communities

Background  

Most studies examining the commensal human oral microbiome are focused on disease or are limited in methodology. In order to diagnose and treat diseases at an early and reversible stage an in-depth definition of health is indispensible. The aim of this study therefore was to define the healthy oral microbiome using recent advances in sequencing technology (454 pyrosequencing).     

Mitochondrial heteroplasmy and DNA barcoding in Hawaiian <Emphasis Type="Italic">Hylaeus</Emphasis> (<Emphasis Type="Italic">Nesoprosopis</Emphasis>) bees (Hymenoptera: Colletidae)

Background  

The past several years have seen a flurry of papers seeking to clarify the utility and limits of DNA barcoding, particularly in areas such as species discovery and paralogy due to nuclear pseudogenes. Heteroplasmy, the coexistence of multiple mitochondrial haplotypes in a single organism, has been cited as a potentially serious problem for DNA barcoding but its effect on identification accuracy has not been tested. In addition, few studies of barcoding have tested a large group of closely-related species with a well-established morphological taxonomy. In this study we examine both of these issues, by densely sampling the Hawaiian Hylaeus bee radiation.     

Identification of a Vitis vinifera endo‐β‐1,3‐glucanase with antimicrobial activity against Plasmopara viticola

Inducible plant defences against pathogens are stimulated by infections and comprise several classes of pathogenesis‐related (PR) proteins. Endo‐β‐1,3‐glucanases (EGases) belong to the PR‐2 class and their expression is induced by many pathogenic fungi and oomycetes, suggesting that EGases play a role in the hydrolysis of pathogen cell walls. However, reports of a direct effect of EGases on cell walls of plant pathogens are scarce. Here, we characterized three EGases from Vitis vinifera whose expression is induced during infection by Plasmopara viticola, the causal agent of downy mildew. Recombinant proteins were expressed in Escherichia coli. The enzymatic characteristics of these three enzymes were measured in vitro and in planta. A functional assay performed in vitro on germinated P. viticola spores revealed a strong anti‐P. viticola activity for EGase3, which strikingly was that with the lowest in vitro catalytic efficiency. To our knowledge, this work shows, for the first time, the direct effect against downy mildew of EGases of the PR‐2 family from Vitis.     

alpha-Tocopherol and protein kinase C inhibition enhance platelet-derived nitric oxide release.

Platelet activation is tightly regulated by products of the endothelium and platelets including nitric oxide (NO). Excess vascular oxidative stress has been associated with impaired NO release, and antioxidant status has been shown to alter endothelium-derived NO bioactivity. Although physiological levels of a-tocopherol are known to inhibit platelet function, the effect of a-tocopherol on platelet NO release is unknown. Loading platelets with physiologic levels of a-tocopherol increased platelet NO production approximately 1.5-fold (Pa-tocopherol, platelet NO release increased 50% (Pa-Tocopherol-loaded platelets also produced 74% less superoxide as compared with control (Pa-tocopherol inhibited PKC-dependent eNOS phosphorylation as determined by immunoprecipitation. Lastly, platelets isolated from NOS3-deficient mice released 80% less superoxide as compared with control animals (P=0.011), and incubation of NOS III-deficient platelets with 500 mM a-tocopherol only caused a modest additional decrease in platelet superoxide release (NS). Thus, a-tocopherol appears to enhance platelet NO release both in vitro and in vivo through antioxidant- and PKC-dependent mechanisms.