Microsatellite and Wolbachia analysis in Rhagoletis cerasi natural populations: population structuring and multiple infections

Rhagoletis cerasi (Diptera: Tephritidae) is a major pest of sweet and sour cherries in Europe and parts of Asia. Despite its economic significance, there is a lack of studies on the genetic structure of R. cerasi populations. Elucidating the genetic structure of insects of economic importance is crucial for developing phenological‐predictive models and environmental friendly control methods. All natural populations of R. cerasi have been found to harbor the endosymbiont Wolbachia pipientis, which widely affects multiple biological traits contributing to the evolution of its hosts, and has been suggested as a tool for the biological control of insect pests and disease vectors. In the current study, the analysis of 18 R. cerasi populations collected in Greece, Germany, and Russia using 13 microsatellite markers revealed structuring of R. cerasi natural populations, even at close geographic range. We also analyzed the Wolbachia infection status of these populations using 16S rRNA‐, MLST‐ and wsp‐based approaches. All 244 individuals screened were positive for Wolbachia. Our results suggest the fixation of the wCer1 strain in Greece while wCer2, wCer4, wCer5, and probably other uncharacterized strains were also detected in multiply infected individuals. The role of Wolbachia and its potential extended phenotypes needs a thorough investigation in R. cerasi. Our data suggest an involvement of this symbiont in the observed restriction in the gene flow in addition to a number of different ecological factors.     

Autumnal emergence of Anagrus wasps,egg parasitoids of Empoasca vitis,from grapevine leaves and their migration towards brambles

• 1 The Anagrus‘atomus’ parasitoid group (Hymenoptera: Mymaridae), associated with Empoasca vitis (Göthe) (Homoptera: Cicadellidae), overwinters on vegetation surrounding vineyards. The emergence of parasitoid adults from grapevine leaves in autumn was studied in north‐eastern Italy, both in relation to the E. vitis egg‐laying period and to the presence of leafhoppers overwintering as eggs on Rubus bushes.
• 2 Autumnal peaks of Anagrus captured using yellow sticky traps were observed first on grapevines and then on brambles. Parasitoid captures in vineyards were observed for more than 1 month after the last first‐instar nymphs of the grape leafhoppers were noticed. Two species belonging to the A. ‘atomus’ group, Anagrus atomus and Anagrus ustulatus, were captured both on grapevines and brambles.
• 3 Parasitoids of the A.‘atomus’ group can emerge from third‐generation grape leafhopper eggs in accordance with two different development time patterns (i.e. normal or delayed). Individuals with delayed emergence required up to 2.2‐fold more time to develop from an egg to adult than individuals with normal emergence. This meant that some parasitoid adults emerged in autumn from eggs of grape leafhopper laid in August.
• 4 A delayed emergence as a result of a slower development ensures that the A.‘atomus’ parasitoid group is synchronized with the egg‐laying of leafhoppers that overwinter as eggs on Rubus spp.
• 5 Consequently, leafhoppers overwintering as eggs on brambles play a key role in the ecology of the relationship between grape leafhoppers and the A.‘atomus’ parasitoid group.

     

Effects of Radix Astragali and Radix Rehmanniae, the components of an anti-diabetic foot ulcer herbal formula, on metabolism of model CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A4 probe substrates in pooled human liver microsomes and specific CYP isoforms

The present study investigated the effects of Radix Astragali (RA) and Radix Rehmanniae (RR), the major components of an anti-diabetic foot ulcer herbal formula (NF3), on the metabolism of model probe substrates of human CYP isoforms, CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A4, which are important in the metabolism of a variety of xenobiotics. The effects of RA or RR on human CYP1A2 (phenacetin O-deethylase), CYP2C9 (tolbutamide 4-hydroxylase), CYP2D6 (dextromethorphan O-demethylase), CYP2E1 (chlorzoxazone 6-hydroxylase) and CYP3A4 (testosterone 6β-hydroxylase) activities were investigated using pooled human liver microsomes. NF3 competitively inhibited activities of CYP2C9 (IC(50)=0.98mg/ml) and CYP3A4 (IC(50)=0.76mg/ml), with K(i) of 0.67 and 1.0mg/ml, respectively. With specific human CYP2C9 and CYP3A4 isoforms, NF3 competitively inhibited activities of CYP2C9 (IC(50)=0.86mg/ml) and CYP3A4 (IC(50)=0.88mg/ml), with K(i) of 0.57 and 1.6mg/ml, respectively. Studies on RA or RR individually showed that RR was more important in the metabolic interaction with the model CYP probe substrates. RR dose-dependently inhibited the testosterone 6β-hydroxylation (K(i)=0.33mg/ml) while RA showed only minimal metabolic interaction potential with the model CYP probe substrates studied. This study showed that RR and the NF3 formula are metabolized mainly by CYP2C9 and/or CYP3A4, but weakly by CYP1A2, CYP2D6 and CYP2E1. The relatively high K(i) values of NF3 (for CYP2C9 and CYP3A4 metabolism) and RR (for CYP3A4 metabolism) would suggest a low potential for NF3 to cause herb-drug interaction involving these CYP isoforms.     

Dipstick-type biosensor for visual detection of DNA with oligonucleotide-decorated colored polystyrene microspheres as reporters

In recent years, there is a continuously growing interest in the development of biosensors for rapid, simple and inexpensive DNA tests suitable for the small laboratory or for on-site testing. Detection is accomplished through electrochemical, optical or gravimetric transduction. We report on the development of disposable dipstick-type DNA biosensors that employ oligonucleotide-decorated colored polystyrene microspheres as reporters and enable visual detection of DNA sequences without the use of instrumentation. The biosensors have been designed to detect DNA molecules that contain both, a biotin moiety and a segment that is complementary to the oligonucleotide attached on the surface of blue or red microspheres. Capture of the hybrids by immobilized streptavidin at the test zone results in the formation of a colored line. The biosensors were applied to: (a) detection of single-stranded DNA, (b) detection of PCR-amplified double-stranded DNA and (c) genotyping of single nucleotide polymorphisms (SNP). The results were compared with sensors based on gold nanoparticle reporters. It is also demonstrated that the microspheres offer the potential for multicolor detection of specific DNA sequences.     

Inactivation of endotoxin by human plasma gelsolin

Septic shock from bacterial endotoxin, triggered by the release of lipopolysaccharide (LPS) molecules from the outer wall of Gram-negative bacteria, is a major cause of human death for which there is no effective treatment once the complex inflammatory pathways stimulated by these small amphipathic molecules are activated. Here we report that plasma gelsolin, a highly conserved human protein, binds LPS from various bacteria with high affinity. Solid-phase binding assays, fluorescence measurements, and functional assays of actin depolymerizing effects show that gelsolin binds more tightly to LPS than it does to its other known lipid ligands, phosphatidylinositol 4,5-bisphosphate and lysophosphatidic acid. Gelsolin also competes with LPS-binding protein (LBP), a high-affinity carrier for LPS. One result of gelsolin-LPS binding is inhibition of the actin binding activity of gelsolin as well as the actin depolymerizing activity of blood serum. Simultaneously, effects of LPS on cellular functions, including cytoskeletal actin remodeling, and collagen-induced platelet activation by pathways independent of toll-like receptors (TLRs) are neutralized by gelsolin and by a peptide based on gelsolin residues 160-169 (GSN160-169) which comprise part of gelsolin's phosphoinositide binding site. Additionally, TLR-dependent NF-kappaB translocation in astrocytes appears to be blocked by gelsolin. These results show a strong effect of LPS on plasma gelsolin function and suggest that some effects of endotoxin in vivo may be mediated or inhibited by plasma gelsolin.     

The human papillomavirus 16 E6 protein binds to Fas-associated death domain and protects cells from Fas-triggered apoptosis

High risk strains of human papillomavirus (HPV), such as HPV 16, cause human cervical carcinoma. The E6 protein of HPV 16 mediates the rapid degradation of the tumor suppressor p53, although this is not the only function of E6 and cannot completely explain its transforming potential. Previous work in our laboratory has demonstrated that E6 can protect cells from tumor necrosis factor-induced apoptosis by binding to the C-terminal end of tumor necrosis factor R1, thus blocking apoptotic signal transduction. In this study, E6 was shown to also protect cells from apoptosis induced via the Fas pathway. Furthermore, use of an inducible E6 expression system demonstrated that this protection is dose-dependent, with higher levels of E6 leading to greater protection. Although E6 suppresses activation of both caspase 3 and caspase 8, it does not affect apoptotic signaling through the mitochondrial pathway. Mammalian two-hybrid and in vitro pull-down assays were then used to demonstrate that E6 binds directly to the death effector domain of Fas-associated death domain (FADD), with deletion and site-directed mutants enabling the localization of the E6-binding site to the N-terminal end of the FADD death effector domain. E6 is produced in two forms as follows: a full-length version of approximately 16 kDa and a smaller version of about half that size corresponding to the N-terminal half of the full-length protein. Pull-down and functional assays demonstrated that the full-length version, but not the small version of E6, was able to bind to FADD and to protect cells from Fas-induced apoptosis. In addition, binding to E6 leads to degradation of FADD, with the loss of cellular FADD proportional to the amount of E6 expressed. These results support a model in which E6-mediated degradation of FADD prevents transmission of apoptotic signals via the Fas pathway.     

Community Case Management of Fever Due to Malaria and Pneumonia in Children Under Five in Zambia: A Cluster Randomized Controlled Trial

Background

Pneumonia and malaria, two of the leading causes of morbidity and mortality among children under five in Zambia, often have overlapping clinical manifestations. Zambia is piloting the use of artemether-lumefantrine (AL) by community health workers (CHWs) to treat uncomplicated malaria. Valid concerns about potential overuse of AL could be addressed by the use of malaria rapid diagnostics employed at the community level. Currently, CHWs in Zambia evaluate and treat children with suspected malaria in rural areas, but they refer children with suspected pneumonia to the nearest health facility. This study was designed to assess the effectiveness and feasibility of using CHWs to manage nonsevere pneumonia and uncomplicated malaria with the aid of rapid diagnostic tests (RDTs).

Methods and Findings

Community health posts staffed by CHWs were matched and randomly allocated to intervention and control arms. Children between the ages of 6 months and 5 years were managed according to the study protocol, as follows. Intervention CHWs performed RDTs, treated test-positive children with AL, and treated those with nonsevere pneumonia (increased respiratory rate) with amoxicillin. Control CHWs did not perform RDTs, treated all febrile children with AL, and referred those with signs of pneumonia to the health facility, as per Ministry of Health policy. The primary outcomes were the use of AL in children with fever and early and appropriate treatment with antibiotics for nonsevere pneumonia. A total of 3,125 children with fever and/or difficult/fast breathing were managed over a 12-month period. In the intervention arm, 27.5% (265/963) of children with fever received AL compared to 99.1% (2066/2084) of control children (risk ratio 0.23, 95% confidence interval 0.14–0.38). For children classified with nonsevere pneumonia, 68.2% (247/362) in the intervention arm and 13.3% (22/203) in the control arm received early and appropriate treatment (risk ratio 5.32, 95% confidence interval 2.19–8.94). There were two deaths in the intervention and one in the control arm.

Conclusions

The potential for CHWs to use RDTs, AL, and amoxicillin to manage both malaria and pneumonia at the community level is promising and might reduce overuse of AL, as well as provide early and appropriate treatment to children with nonsevere pneumonia.

Trial registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00513500","term_id":"NCT00513500"}}NCT00513500 Please see later in the article for the Editors'' Summary     

Moderate Exercise and Fibrinolytic Potential in Obese Sedentary Men with Metabolic Syndrome

Objective: To investigate the impact of 30‐minute walking exercise at 70% Vo _2max on tissue plasminogen activator (t‐PA) Ag and plasminogen activator inhibitor type 1 (PAI‐1) Ag in obese sedentary males. Research Methods and Procedures: A controlled observational study of the effect of a 30‐minute acute exercise bout at 70% Vo _2max on plasma t‐PA antigen and PAI‐1 antigen in 10 obese sedentary males matched for age, ethnic origin, and smoking status with 10 nonobese sedentary male controls. Results: The obese group remained hypofibrinolytic compared with the nonobese group at all time‐points before, during, and after exercise. t‐PA increased in both groups with exercise before returning to baseline values 30 minutes after exercise. PAI‐1 did not significantly change in either group with exercise but rose significantly 30 minutes after exercise in the obese group. Discussion: The reduction in fibrinolytic potential in the obese group represents an increase in acute thrombotic risk and could account for the increased incidence of exercise‐associated myocardial infarction observed in sedentary obese groups.