The autophagic pathway is actively modulated by phase II Coxiella burnetii to efficiently replicate in the host cell

The etiologic agent of Q fever Coxiella burnetii, is an intracellular obligate parasite that develops large vacuoles with phagolysosomal characteristics, containing multiple replicating bacteria. We have previously shown that Phase II C. burnetii replicative vacuoles generated after 24-48 h post infection are decorated with the autophagic protein LC3. The aim of the present study was to examine, at earlier stages of infection, the distribution and roles of the small GTPases Rab5 and Rab7, markers of early and late endosomes respectively, as well as of the protein LC3 on C. burnetii trafficking. Our results indicate that: (i) Coxiella phagosomes (Cph) acquire the two Rab proteins sequentially during infection; (ii) overexpression of a dominant negative mutant form of Rab5, but not of Rab7, impaired Coxiella entry, whereas both Rab5 and Rab7 dominant negative mutants inhibited vacuole formation; (iii) Cph colocalized with the protein LC3 as early as 5 min after infection; acquisition of this protein appeared to be a bacterially driven process, because it was inhibited by the bacteriostatic antibiotic chloramphenicol and (iv) C. burnetii delayed the arrival of the typical lysosomal protease cathepsin D to the Cph, which delay is further increased by starvation-induced autophagy. Based on our results we propose that C. burnetii transits through the normal endo/phagocytic pathway but actively interacts with autophagosomes at early times after infection. This intersection with the autophagic pathway delays fusion with the lysosomal compartment possibly favouring the intracellular differentiation and survival of the bacteria.     

Phylogeny of betanodaviruses and molecular evolution of their RNA polymerase and coat proteins

The betanodaviruses are the causative agent of the disease viral nervous necrosis in fishes. Betanodavirus genome consists of two single-stranded positive-sense RNA molecules (RNA1 and RNA2). RNA1 gene encodes the RNA polymerase, named also protein A, while RNA2 encodes the coat protein precursor, the CPp protein. We investigated the evolutionary relationships among betanodaviruses working on partial sequences of both RNA1 and RNA2. Phylogenetic analyses were performed by applying a maximum likelihood approach. The phylogenetic relationships among the major betanodavirus clades SJNNV-IV, TPNNV-III, BFNNV-II and RGNNV-I were resolved differently in the trees obtained, respectively, from RNA1 and RNA2 multiple alignments. The alternative topologies were corroborated by strong bootstrap values. The molecular evolution of proteins A and CPp was also investigated. Protein A appeared to have evolved under strong purifying selection while the CPp protein was subject to both purifying and neutral selection in different amino acid residues. Intragenic recombination in RNA1 and RNA2 genes was investigated by applying several methods and was not detected. Conversely reassortment of RNA1 and RNA2 genes was demonstrated in some isolates. Finally RNA1 and RNA2 genes substitution rates do not follow a clock-like behavior thus impeding estimation of a possible origin time for Betanodavirus genus.     

Detection of Dialister pneumosintes in the subgingival biofilm of subjects with periodontal disease

Dialister pneumosintes has been indicated as a potentially new periodontopathic species. This study evaluated the prevalence of this microorganism in saliva and subgingival biofilm from subjects with different periodontal conditions. Subgingival biofilm and saliva samples from 48 subjects with periodontal health (PH) and 116 patients with chronic periodontitis (CP) were obtained. DNA was extracted from the samples and the presence of D. pneumosintes was determined by PCR. Differences in clinical parameters and frequency of D. pneumosintes between groups were sought by Mann-Whitney, Chi-square and Fisher's exact tests. Overall, D. pneumosintes was detected in 47.8% of the biofilm samples, but only in 3% of saliva samples. CP patients presented a significantly greater mean prevalence of this species in sites with periodontal health and periodontal infection (43.5+/-7.4% and 62.1+/-6.4%, respectively) than PH subjects (29.4+/-7.9%) (Mann-Whitney; p<0.01). Moreover, significant associations between the prevalence of D. pneumosintes and pocket depth (p=0.001), attachment loss (p=0.001) and bleeding on probing (GLM, p=0.014) were observed after adjusting for age and gender. These findings corroborate the association of D. pneumosintes with periodontitis.     

How tails guide tail-anchored proteins to their destinations

A large group of diverse, functionally important, and differently localized transmembrane proteins shares a particular membrane topology, consisting of a cytosolic N-terminal region, followed by a transmembrane domain close to the C-terminus. Because of their structure, these C-tail-anchored (TA) proteins must insert into all their target membranes by post-translational pathways. Recent work, based on the development of stringent and sensitive biochemical assays, has demonstrated that novel unexplored mechanisms underlie these post-translational targeting and membrane insertion pathways. Unravelling these pathways will shed light on the biosynthesis and regulation of an important group of membrane proteins and is likely to lead to new concepts in the field of membrane biogenesis.     

TRPM4 Is a Novel Component of the Adhesome Required for Focal Adhesion Disassembly,Migration and Contractility

Cellular migration and contractility are fundamental processes that are regulated by a variety of concerted mechanisms such as cytoskeleton rearrangements, focal adhesion turnover, and Ca²⁺ oscillations. TRPM4 is a Ca²⁺-activated non-selective cationic channel (Ca²⁺-NSCC) that conducts monovalent but not divalent cations. Here, we used a mass spectrometry-based proteomics approach to identify putative TRPM4-associated proteins. Interestingly, the largest group of these proteins has actin cytoskeleton-related functions, and among these nine are specifically annotated as focal adhesion-related proteins. Consistent with these results, we found that TRPM4 localizes to focal adhesions in cells from different cellular lineages. We show that suppression of TRPM4 in MEFs impacts turnover of focal adhesions, serum-induced Ca²⁺ influx, focal adhesion kinase (FAK) and Rac activities, and results in reduced cellular spreading, migration and contractile behavior. Finally, we demonstrate that the inhibition of TRPM4 activity alters cellular contractility in vivo, affecting cutaneous wound healing. Together, these findings provide the first evidence, to our knowledge, for a TRP channel specifically localized to focal adhesions, where it performs a central role in modulating cellular migration and contractility.     

Development and characterization of microsatellite markers for Psychotria tenuinervis (Rubiaceae), a shrub species from the Atlantic forest, and primers transferability from Coffea

The shrub species Psychotria tenuinervis (Rubiaceae) is native to the Brazilian Atlantic forest and is largely found within natural and disturbed forest fragments. Aiming to develop studies on population genetic structure of forest fragment species, eigth microsatellite markers were developed for P. tenuinervis. Also, 15 loci already developed for Coffea (Rubiaceae) were tested for transferability to this species. We utilized 45 individuals from natural populations of three different fragments-anthropic edge, interior fragment and natural edge, within the Brazilian Atlantic forest. The average number of alleles per locus was 2.5 (two–four alleles/locus). These loci will be useful for future population genetic studies aiming to the conservation and management of this species.     

Biochemical composition of a dominant detritivorous fish Prochilodus lineatus along pollution gradients in the Paraná-Río de la Plata Basin

The biochemical composition of muscle, liver and stomach contents of a detritivorous fish Prochilodus lineatus was analysed and compared to settling particles and sediments along pollution gradients over 1500 km of the Río de la Plata Basin to evaluate the effects of anthropogenic discharges in a detritus food chain. The stomach contents of P. lineatus collected in the polluted Metropolitan Buenos Aires coast were enriched in proteins, carbohydrates and lipids, similar to settling particulates collected in the sewer area, and two to five times higher than underlying sediments, supporting the interpretation that P. lineatus feeds on unconsolidated organic flocs freshly decanted from mixed industrial and sewage outfalls. Fish from Buenos Aires had consistently higher standard length (L_S) and mass (M_T) slopes (b = 3·5), condition indexes (K = 3·01 ± 0·47, mean ±s.d .) and muscle fat content (fat = 23·8 ± 13·8% wet mass, mean ±s.d .) relative to northern fish (b = 2·7, K = 2·22 ± 0·39, fat = 3·4 ± 3·2% wet mass, respectively), suggesting that sewage‐derived organic matter was an enriched diet, which allowed an enhanced body mass gain and fat accumulation compared to organic‐poor vegetal detritus in the north Paraná area. Buenos Aires fish also showed higher hepato‐somatic indices (mean ±s.d . I_H 1·41 ± 0·49 v. 0·70 ± 0·32, respectively), which correlated with their two to three orders of magnitude higher hydrocarbon and polychlorinated biphenyl (PCB) loads, suggesting an enhanced detoxifying metabolism. The northward migration of fatty P. lineatus was evidenced by the presence of clear outliers in the L_S and M_T relationship, K and fat content along the Paraná River.