Bayesian Coalescent Analysis Reveals a High Rate of Molecular Evolution in GB Virus C

GB virus C/hepatitis G (GBV-C) is an RNA virus of the family Flaviviridae. Despite replicating with an RNA-dependent RNA polymerase, some previous estimates of rates of evolutionary change in GBV-C suggest that it fixes mutations at the anomalously low rate of ∼10⁻⁷ nucleotide substitution per site, per year. However, these estimates were largely based on the assumption that GBV-C and its close relative GBV-A (New World monkey GB viruses) codiverged with their primate hosts over millions of years. Herein, we estimated the substitution rate of GBV-C using the largest set of dated GBV-C isolates compiled to date and a Bayesian coalescent approach that utilizes the year of sampling and so is independent of the assumption of codivergence. This revealed a rate of evolutionary change approximately four orders of magnitude higher than that estimated previously, in the range of 10⁻² to 10⁻³ sub/site/year, and hence in line with those previously determined for RNA viruses in general and the Flaviviridae in particular. In addition, we tested the assumption of host-virus codivergence in GBV-A by performing a reconciliation analysis of host and virus phylogenies. Strikingly, we found no statistical evidence for host-virus codivergence in GBV-A, indicating that substitution rates in the GB viruses should not be estimated from host divergence times.     

Morphologic evaluation and expression of matrix metalloproteinases-2 and 9 and nitric oxide during experimental periodontal disease in rat

The immunopathologic and inflammatory mechanisms involved in periodontal disease (PD) include the participation of host resident, inflammatory cells and chemical mediators. Metalloproteinases (MMPs) and nitric oxide (NO) play essential role in extracellular matrix turnover of periodontal tissue destruction. In this study, by means of RT-PCR through semi-quantitative densitometric scanning methods, the expression of MMPs -2 and -9 and inducible NO synthase (iNOS) was temporally and spatially investigated during the destructive mechanisms of experimentally induced PD in rats. Samples from different periods were microscopically analyzed and compared with the contralateral side (control). Our results showed significant expression of MMP-9 and iNOS in tissues affected by PD, as compared with controls, three days after PD induction, simultaneously with the beginning of alveolar bone loss. At 7 days post induction, only the MMP-9 mRNA presented a significantly higher expression, as compared with the respective controls. Thus, in the rat ligature-induced PD, MMP-9 and iNOS might importantly participate in the early stages of the disease, including inflammatory cell migration, tissue destruction and alveolar bone resorption. Also, we may suggest that the exuberant presence of PMNs may be related to the important expression of iNOS and MMP-9 found at 3 days post induction.     

Low-dose spironolactone reduces reactive oxygen species generation and improves insulin-stimulated glucose transport in skeletal muscle in the TG(mRen2)27 rat

Renin-angiotensin-aldosterone system (RAAS) activation mediates increases in reactive oxygen species (ROS) and impaired insulin signaling. The transgenic Ren2 rat manifests increased tissue renin-angiotensin system activity, elevated serum aldosterone, hypertension, and insulin resistance. To explore the role of aldosterone in the pathogenesis of insulin resistance, we investigated the impact of in vivo treatment with a mineralocorticoid receptor (MR) antagonist on insulin sensitivity in Ren2 and aged-matched Sprague-Dawley (SD) control rats. Both groups (age 6-8 wk) were implanted with subcutaneous time-release pellets containing spironolactone (0.24 mg/day) or placebo over 21 days. Systolic blood pressure (SBP) and intraperitoneal glucose tolerance test were determined. Soleus muscle insulin receptor substrate-1 (IRS-1), tyrosine phosphorylated IRS-1, protein kinase B (Akt) phosphorylation, GLUT4 levels, and insulin-stimulated 2-deoxyglucose uptake were evaluated in relation to NADPH subunit expression/oxidase activity and ROS production (chemiluminescence and 4-hydroxy-2-nonenal immunostaining). Along with increased soleus muscle NADPH oxidase activity and ROS, there was systemic insulin resistance and reduced muscle IRS-1 tyrosine phosphorylation, Akt phosphorylation/activation, and GLUT4 expression in the Ren2 group (each P < 0.05). Despite not decreasing blood pressure, low-dose spironolactone treatment improved soleus muscle insulin signaling parameters and systemic insulin sensitivity in concert with reductions in NADPH oxidase subunit expression/activity and ROS production (each P < 0.05). Our findings suggest that aldosterone contributes to insulin resistance in the transgenic Ren2, in part, by increasing NADPH oxidase activity in skeletal muscle tissue.     

An improved method to obtain antigen-excreting Toxocara canis larvae

Toxocara canis is a dog helminth which causes visceral larva migrans (VLM) when infecting humans as a larva. The infection is demonstrated by detecting IgG antibodies against excretory-secretory larval antigens (ESLA) in serum by ELISA. The production of ESLA involves the collection of adult worms from dog puppy stools, the separation of eggs from dissected uteri, and the in vitro growing of egg-derived larvae, following the time-consuming and laborious protocol described by De Savigny [De Savigny, D.H., 1975. In vitro maintenance of T. canis larvae and a simple method for the production of Toxocara ES antigen for the uses in serodiagnostic tests for visceral larva migrans. Journal of Parasitology 61, 781-782]. In this work, an improved protocol for obtaining T. canis larvae is described. The modifications proposed improved the efficiency of the original De Savigny method in three ways: (i) increasing the parasite yield up to five fold, (ii) improving the larval purity, and (iii) markedly reducing the execution time of the protocol.     

Transesterification of soybean oil catalyzed by sulfated zirconia

Two sulfated zirconias were synthesized and characterized by X-ray diffraction and infrared spectroscopy. They were used as catalysts in the alcoholysis of soybean oil and in the esterification of oleic acid. Using sulfated zirconia prepared by the solvent-free method (S-ZrO(2)) as catalyst, the alcoholysis conversions of soybean oil under optimized conditions (120 degrees C, 1h and 5wt% of catalyst) were 98.6% (methanolysis) and 92% (ethanolysis), respectively. The esterification of oleic acid with methanol was complete after 2h. Zirconia sulfated by standard methods (SZ) had low activity in the methanolysis of soybean oil (conversion of 8.5%) and conventional zirconia (NS) was inactive for methanolysis under the conditions optimized for S-ZrO(2).     

DNA Fingerprinting of Paecilomyces Strains of Potential use for the Biological Control of Pests

Summary Telomeric fingerprinting was found to be highly differentiating for Paecilomyces fumosoroseus and Paecilomyces lilacinus isolates in comparison to intron splice site PCR and is therefore a good method for quality control of future products based on these fungi. Although the telomeric restriction length polymorphisms correctly divided the isolates into their appropriate species, further correlation with host range or geographical origin of the isolates was not found. In this respect, intron splice site PCR was more informative taxonomically. The chromosome numbers inferred from telomeric fingerprints were seven chromosomes for P. lilacinus and between six and nine chromosomes for P. fumosoroseus.