Bacterial endosymbiont localization in Hyalesthes obsoletus, the insect vector of Bois noir in Vitis vinifera

One emerging disease of grapevine in Europe is Bois noir (BN), a phytoplasmosis caused by "Candidatus Phytoplasma solani" and spread in vineyards by the planthopper Hyalesthes obsoletus (Hemiptera: Cixiidae). Here we present the first full characterization of the bacterial community of this important disease vector collected from BN-contaminated areas in Piedmont, Italy. Length heterogeneity PCR and denaturing gradient gel electrophoresis analysis targeting the 16S rRNA gene revealed the presence of a number of bacteria stably associated with the insect vector. In particular, symbiotic bacteria detected by PCR with high infection rates in adult individuals fell within the "Candidatus Sulcia muelleri" cluster in the Bacteroidetes and in the "Candidatus Purcelliella pentastirinorum" group in the Gammaproteobacteria, both previously identified in different leafhoppers and planthoppers. A high infection rate (81%) was also shown for another symbiont belonging to the Betaproteobacteria, designated the HO1-V symbiont. Because of the low level of 16S rRNA gene identity (80%) with the closest relative, an uncharacterized symbiont of the tick Haemaphysalis longicornis, we propose the new name "Candidatus Vidania fulgoroideae." Other bacterial endosymbionts identified in H. obsoletus were related to the intracellular bacteria Wolbachia pipientis, Rickettsia sp., and "Candidatus Cardinium hertigii." Fluorescent in situ hybridization coupled with confocal laser scanning microscopy and transmission electron microscopy showed that these bacteria are localized in the gut, testicles, and oocytes. As "Ca. Sulcia" is usually reported in association with other symbiotic bacteria, we propose that in H. obsoletus, it may occur in a bipartite or even tripartite relationship between "Ca. Sulcia" and "Ca. Purcelliella," "Ca. Vidania," or both.     

Interaction of fosfomycin with the Glycerol 3-phosphate Transporter of Escherichia coli

Background

Fosfomycin is widely used to treat urinary tract and pediatric gastrointestinal infections of bacteria. It is supposed that this antibiotic enters cells via two transport systems, including the bacterial Glycerol-3-phosphate Transporter (GlpT). Impaired function of GlpT is one mechanism for fosfomycin resistance.

Methods

The interaction of fosfomycin with the recombinant and purified GlpT of Escherichia coli reconstituted in liposomes has been studied. IC₅₀ and the half-saturation constant of the transporter for external fosfomycin (K_i) were determined by transport assay of [¹⁴C]glycerol-3-phosphate catalyzed by recombinant GlpT. Efficacy of fosfomycin on growth rates of GlpT defective bacteria strains transformed with recombinant GlpT was measured.

Results

Fosfomycin, externally added to the proteoliposomes, poorly inhibited the glycerol-3-phosphate/glycerol-3-phosphate antiport catalyzed by the reconstituted transporter with an IC₅₀ of 6.4 mM. A kinetic analysis revealed that the inhibition was completely competitive, that is, fosfomycin interacted with the substrate-binding site and the K_i measured was 1.65 mM. Transport assays performed with proteoliposomes containing internal fosfomycin indicate that it was not very well transported by GlpT. Complementation study, performed with GlpT defective bacteria strains, indicated that the fosfomycin resistance, beside deficiency in antibiotic transporter, could be due to other gene defects.

Conclusions

The poor transport observed in a reconstituted system together with the high value of K_i and the results of complementation study well explain the usual high dosage of this drug for the treatment of the urinary tract infections.

General significance

This is the first report regarding functional analysis of interaction between fosfomycin and GlpT.     

Ecological differentiation and cladogenesis of Baldellia (L.) Parl. (Alismataceae)

The Iberian Peninsula is home to a large number of endemic species that often show strong genetic subdivisions which indicate possible isolation of populations in the past. Numerous phylogeographic and phylogenetic studies on Iberian flora and fauna have revealed this territory to be the origin of genetic differentiation during the Pleistocene Ice Age. To better understand the influence of Plio-Pleistocene climatic oscillations on the cladogenesis and population structuring of refugial taxa restricted to freshwaters of the Iberian Peninsula, we studied the evolutionary history of the small European genus Baldellia, both from a phylogenetic/phylogeographic perspective and regarding its ecological characteristics. Molecular analyses were based on four plastid regions and nuclear ITS2 ribosomal spacer from individuals belonging to geographically isolated Baldellia populations, while the relationships between climate and Baldellia species distribution was investigated by redundancy analysis applied to the climatic data with the occurrence of Baldellia species as external variables. The absence of a clear phylogenetic signal most likely reflects a common and recent cladogenesis for Baldellia. In contrast, the more rapidly evolving plastid sequences indicate a recent phylogeographic history of refugial repartition and ecological segregation probably occurring during the last glaciation, a scenario well supported by marked differences in species?? ecological preferences and climate analysis.     

Comparative role of acetylation along c-SRC/ETS1 signaling pathway in bone metastatic and invasive mammary cell phenotypes

Metastatic cells switch between different modes of migration through supramolecular plasticity mechanism(s) still largely unknown. The aim of the present paper was to clarify some molecular aspects of the epigenetic control of migration of 1833-bone metastatic cells compared to MDA-MB231-parental mammary carcinoma cells. Active c-Src overexpression enhanced 1833-cell spontaneous migration and CXCR4-mediated chemoinvasion toward CXCL12 ligand. Only in metastatic cells, in fact, c-Src seemed to stabilize nuclear CXCR4-protein receptor possibly due to tyrosine phosphorylation, by impairing protein-degradative smear and causing instead an electrophoretic-mobility shift; the cytosolic steady-state level of CXCR4 was enhanced, and the protein appeared also phosphorylated. These findings suggested the triggering of unique signaling pathways in metastasis for homing of breast-cancer cells to congenial environment of specific organs. Microenvironmental stimuli activating c-Src might influence Ets1 binding to CXCR4 promoter and consequent transactivation, as well as CXCR4 post-translational regulatory mechanisms such as phosphorylation. Enhancement of Ets1 activity and CXCR4 induction by c-Src overexpression were prevented by histone deacetylase (HDAC) blockade. In contrast, HDAC inhibition with trichostatin A increased cytosolic phosphorylated CXCR4 expression in MDA-MB231 cells, but Ets1 involvement was practically unneeded. c-Src might be suggested as a bio-marker predicting metastasis sensitivity patterns to HDAC inhibitors. Rationally designed and individualized therapy may become possible as more is learned about the target molecules of HDAC's inhibitory agents and their roles, as undertaken for CXCR4 that is likely to be crucial for homing, angiogenesis and survival in a c-Src-dependent manner in bone-metastatic mammary cells.     

Analyses of recombinant stereotypic IGHV3-21-encoded antibodies expressed in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) cells that use IgH encoded by IGHV3-21 and that have a particular stereotypic third CDR (HCDR3), DANGMDV (motif-1), almost invariably express Ig L chains (IgL) encoded by IGLV3-21, whereas CLL that use IGHV3-21-encoded IgH with another stereotypic HCDR3, DPSFYSSSWTLFDY (motif-2), invariably express κ-IgL encoded by IGKV3-20. This nonstochastic pairing could reflect steric factors that preclude these IgH from pairing with other IgL or selection for an Ig with a particular Ag-binding activity. We generated rIg with IGHV3-21-encoded IgH with HCDR3 motif-1 or -2 and IgL encoded by IGKV3-20 or IGLV3-21. Each IgH paired equally well with matched or mismatched κ- or λ-IgL to form functional Ig, which we screened for binding to an array of different Ags. Ig with IGLV3-21-encoded λ-IgL could bind with an affinity of ～ 2 × 10(-6) M to protein L, a cell-wall protein of Peptostreptococcus magnus, independent of the IgH, indicating that protein L is a superantigen for IGLV3-21-encoded λ-IgL. We also detected Ig binding to cofilin, a highly conserved actin-binding protein. However, cofilin binding was independent of native pairing of IgH and IgL and was not specific for Ig with IgH encoded by IGHV3-21. We conclude that steric factors or the binding activity for protein L or cofilin cannot account for the nonstochastic pairing of IgH and IgL observed for the stereotypic Ig made by CLL cells that express IGHV3-21.     

Dual activity of aminoarylthiazoles on the trafficking and gating defects of the cystic fibrosis transmembrane conductance regulator chloride channel caused by cystic fibrosis mutations

A large fraction of mutations causing cystic fibrosis impair the function of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel by causing reduced channel activity (gating defect) and/or impaired exit from the endoplasmic reticulum (trafficking defect). Such defects need to be treated with separate pharmacological compounds termed potentiators and correctors, respectively. Here, we report the characterization of aminoarylthiazoles (AATs) as compounds having dual activity. Cells expressing mutant CFTR were studied with functional assays (fluorescence-based halide transport and short circuit current measurements) to assess the effect of acute and chronic treatment with compounds. We found that AATs are effective on F508del, the most frequent cystic fibrosis mutation, which is associated with both a gating and a trafficking defect. AATs are also effective on mutations like G1349D and G551D, which cause only a gating defect. Evaluation of a panel of AAT analogs identified EN277I as the most effective compound. Incubation of cells expressing mutant CFTR with EN277I caused a strong stimulation of channel activity as demonstrated by single channel recordings. Compounds with dual activity such as AATs may be useful for the development of effective drugs for the treatment of cystic fibrosis.     

Protective effects of cyanidin-3-O-glucoside from blackberry extract against peroxynitrite-induced endothelial dysfunction and vascular failure

Anthocyanins are a group of naturally occurring phenolic compounds as colorants in several plants, flowers and fruits. These pigments have a great importance as quality indicators, as chemotaxonomic markers and antioxidants.The content of blackberry (Rubus species) juice was investigated by HPLC/ESI/MS using narrow bore HPLC columns. Using this method we demonstrated that cyanidin-3-O-glucoside represents about 80% of the total anthocyanin contents in blackberry extract. Here we investigated antioxidant activity of the blackberry juice and cyanidin-3-O-glucoside on the endothelial dysfunction in cells and in vascular rings exposed to peroxynitrite. In human umbilical vein endothelial cells (HUVEC) in vitro, peroxynitrite caused a significant suppression of mitochondrial respiration (38 +/- 2.1% of control cells), as measured by the mitochondrial-dependent conversion of the dye MTT to formazan. Peroxynitrite caused DNA strand breakage (63 +/- 1.9% single strand vs 3 +/- 0.9% single strand in control cells), as measured by the alkaline unwinding assay, and caused an activation of PARS, as measured by the incorporation of radiolabeled NAD(+) to nuclear proteins. Blackberry juice (different dilutions that contained 80 ppm;40 ppm;14.5 ppm of cyanidin-3-O-glucoside) and cyanidin-3-O-glucoside (as chloride) (0.085 microM; 0.028 microM; 0.0085 microM) reduced the peroxynitrite-induced suppression of mitochondrial respiration, DNA damage and PARS activation in HUVECs. Vascular rings exposed to peroxynitrite exhibited reduced endothelium-dependent relaxant responses in response to acetylcholine as well as a vascular contractility dysfunction in response to norepinephrine. The development of this peroxynitrite-induced vascular dysfunction was ameliorated by the blackberry juice (different dilutions that contained 80 ppm;40 ppm;14.5 ppm of cyanidin-3-O-glucoside) and cyanidin-3-O-glucoside (as chloride) (0.085 microM;0.028 microM;0.0085 microM).In conclusion our findings clearly demonstrate that blackberry juice containing cyanidin-3-O-glucoside is a scavenger of peroxynitrite and that exert a protective effect against endothelial dysfunction and vascular failure induced by peroxynitrite.     

Yeasts associated with Manteca

Manteca is a traditional milk product of southern Italy produced from whey deriving from Caciocavallo Podolico cheese-making. This study was undertaken to obtain more information about the microbiological properties of this product and particularly about the presence, metabolic activities, and technological significance of the different yeast species naturally occurring in Manteca. High numbers of yeasts were counted after 7 days ripening (10(4)-10(5) cfu g(-1)) and then decreased to 10(2) at the end. A total of 179 isolates were identified and studied for their phenotypic and genotypic characteristics. The most frequently encountered species were Trichosporon asahii (45), Candida parapsilosis (33), Rhodotorula mucilaginosa (32), Candida inconspicua (29). Some of these yeasts showed lipolytic activity (32 strains) and proteolytic activity (29 strains), NaCl resistance up to 10% and growth up to 45 degrees C (42 strains). Biogenic amines were formed by proteolytic strains, in particular phenylethylamine, putrescine and spermidine. Spermidine was produced by all the yeasts tested in this work, but only Trichosporon produced a great quantity of this compound. Histamine was not detectable. Caseinolytic activity was common to almost all strains, corresponding to the ability to efficiently split off amino-terminal amino acids. The highest and most constant activity expressed by all species was X-prolyl-dipeptidyl aminopeptidase. The findings suggest that the presence of yeasts may play a significant role in justifying interactions with lactic acid bacteria, and consequently with their metabolic activity in the definition of the peculiar characteristics of Manteca cheese.     

Treasure from garden: chemical profiling,pharmacology and biotechnology of mulleins

The genus Verbascum (mulleins), belonging to the family Scrophulariaceae, comprises about 360 species of flowering plants. The leaves, flowers and whole aerial parts of Verbascum spp. have been widely used in traditional medicine for the treatment of respiratory and inflammatory disorders and also display powerful wound healing activity. Verbascum species are found to accumulate several groups of bioactive molecules, therefore they might be utilized as attractive sources of new (drug) leads. The present review attempts to provide an up-to-date comprehensive overview on phytochemical and pharmacological aspects of Verbacum spp. research along with some successful examples of growing (and transforming) mulleins in vitro.     

A Prospective Study of Romanian Agriculture Workers for Zoonotic Influenza Infections

Background

In this prospective study we sought to examine seroepidemiological evidence for acute zoonotic influenza virus infection among Romanian agricultural workers.

Methods

Sera were drawn upon enrollment (2009) and again at 12 and 24 months from 312 adult agriculture workers and 51 age-group matched controls. Participants were contacted monthly for 24 months and queried regarding episodes of acute influenza-like illnesses (ILI). Cohort members meeting ILI criteria permitted respiratory swab collections as well as acute and convalescent serum collection. Serologic assays were performed against 9 avian, 3 swine, and 3 human influenza viruses.

Results

During the two-year follow-up, a total of 23 ILI events were reported. Two subjects'' specimens were identified as influenza A by rRT-PCR. During the follow-up period, three individuals experienced elevated microneutralization antibody titers ≥1∶80 against three (one each) avian influenza viruses: A/Teal/Hong Kong/w312/97(H6N1), A/Hong Kong/1073/1999(H9N2), or A/Duck/Alberta/60/1976(H12N5). However, none of these participants met the criteria for poultry exposure. A number of subjects demonstrated four-fold increases over time in hemagglutination inhibition (HI) assay titers for at least one of the three swine influenza viruses (SIVs); however, it seems likely that two of these three responses were due to cross-reacting antibody against human influenza. Only elevated antibody titers against A/Swine/Flanders/1/1998(H3N2) lacked evidence for such confounding. In examining risk factors for elevated antibody against this SIV with multiple logistic regression, swine exposure (adjusted OR = 1.8, 95% CI 1.1–2.8) and tobacco use (adjusted OR = 1.8; 95% CI 1.1–2.9) were important predictors.

Conclusions

While Romania has recently experienced multiple incursions of highly pathogenic avian influenza among domestic poultry, this cohort of Romanian agriculture workers had sparse evidence of avian influenza virus infections. In contrast, there was evidence, especially among the swine exposed participants, of infections with human and one swine H3N2 influenza virus.