Preparation of bacterial DNA template by boiling and effect of immunoglobulin G as an inhibitor in real-time PCR for serum samples from patients with brucellosis.

Real-time PCR is a widely used tool for the diagnosis of many infectious diseases. However, little information exists about the influences of the different factors involved in PCR on the amplification efficiency. The aim of this study was to analyze the effect of boiling as the DNA preparation method on the efficiency of the amplification process of real-time PCR for the diagnosis of human brucellosis with serum samples. Serum samples from 10 brucellosis patients were analyzed by a SYBR green I LightCycler-based real-time PCR and by using boiling to obtain the DNA. DNA prepared by boiling lysis of the bacteria isolated from serum did not prevent the presence of inhibitors, such as immunoglobulin G (IgG), which were extracted with the template DNA. To identify and confirm the presence of IgG, serum was precipitated to separate and concentrate the IgG and was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. The use of serum volumes above 0.6 ml completely inhibited the amplification process. The inhibitory effect of IgG in serum samples was not concentration dependent, and it could be eliminated by diluting the samples 1/10 and 1/20 in water. Despite the lack of the complete elimination of the IgG from the template DNA, boiling does not require any special equipment and it provides a rapid, reproducible, and cost-effective method for the preparation of DNA from serum samples for the diagnosis of brucellosis.

Mycobacterium tuberculosis epitope-specific interferon-g production in healthy Brazilians reactive and non-reactive to tuberculin skin test

The interferon (IFN)-γ response to peptides can be a useful diagnostic marker of Mycobacterium tuberculosis (MTB) latent infection. We identified promiscuous and potentially protective CD4+ T-cell epitopes from the most conserved regions of MTB antigenic proteins by scanning the MTB antigenic proteins GroEL2, phosphate-binding protein 1 precursor and 19 kDa antigen with the TEPITOPE algorithm. Seven peptide sequences predicted to bind to multiple human leukocyte antigen (HLA)-DR molecules were synthesised and tested with IFN-γ enzyme-linked immunospot (ELISPOT) assays using peripheral blood mononuclear cells (PBMCs) from 16 Mantoux tuberculin skin test (TST)-positive and 16 TST-negative healthy donors. Eighty-eight percent of TST-positive donors responded to at least one of the peptides, compared to 25% of TST-negative donors. Each individual peptide induced IFN-γ production by PBMCs from at least 31% of the TST-positive donors. The magnitude of the response against all peptides was 182 ± 230 x 106 IFN-γ spot forming cells (SFC) among TST-positive donors and 36 ± 62 x 106 SFC among TST-negative donors (p = 0.007). The response to GroEL2 (463-477) was only observed in the TST-positive group. This combination of novel MTB CD4 T-cell epitopes should be tested in a larger cohort of individuals with latent tuberculosis (TB) to evaluate its potential to diagnose latent TB and it may be included in ELISPOT-based IFN-γ assays to identify individuals with this condition.

Variétés CR polarisées et G-polarisées, partie I

Polarized and G-polarized CR manifolds are smooth manifolds endowed with a double structure: a real foliation &em&F&/em& (given by the action of a Lie group G in the G-polarized case) and a transverse CR distribution. Polarized means that (E,J) is roughly speaking invariant by&em&F&/em&. Both structures are therefore linked up. The interplay between them gives to polarized CR-manifolds a very rich geometry. In this paper, we study the properties of polarized and G-polarized manifolds, putting special emphasis on their deformations.

Electrophoretic characteristics of monoclonal immunoglobulin G of different subclasses.

Monoclonal IgG are commonly observed in various B cell disorders, of which multiple myeloma is the most clinically relevant. In a series of serum samples, we identified by immunofixation 73 monoclonal IgG, including 63 IgG(1), 4 IgG(2), 5 IgG(3), and 1 IgG(4). The light chains were of kappa type in 45 cases, and of lambda type in 28 cases. These monoclonal IgG were further characterized by high resolution two-dimensional polyacrylamide gel electrophoresis (2-DE) in various isoelectric focusing conditions, as well as by 3-DE (2-DE of the proteins extracted from agarose after serum protein agarose electrophoresis). After 2-DE, 38 out of 73 monoclonal gamma chains (52%) were visualized using immobilized pH 3-10 gradients for isoelectric focusing. In 6 cases (8%), gamma chains were only detected using alkaline immobilized pH 6-11 gradients. In 3 cases (4%), 3-DE revealed monoclonal gamma chains hidden by polyclonal gamma chains. Finally, in 26 cases (36%), no monoclonal gamma chains were clearly visualized. Sixty-one monoclonal light chains (84%) were detected using immobilized pH 3-10 gradients, whereas 12 (16%) were not. Monoclonal gamma chains and light chains were highly heterogeneous in terms of pI and M(r). However, a statistically significant correlation (P<0.05) was observed between the position of the monoclonal IgG in agarose gel and the pI of their heavy and light chains (R=0.733, multiple linear regression). Because of the extreme diversity of their heavy and light chains, it appears that a classification of monoclonal IgG based only on their electrophoretic properties is not possible.

Conformations of poly{G}-poly{C} π stacks with high hole mobility

Charge transfer properties of DNA depend strongly on the π stack conformation. In the present paper, we identify conformations of homogeneous poly-{G}-poly-{C} stacks that should exhibit high charge mobility. Two different computational approaches were applied. First, we calculated the electronic coupling squared, V2, between adjacent base pairs for all 1 ps snapshots extracted from 15 ns molecular dynamics trajectory of the duplex G15. The average value of the coupling squared 〈 V2 〉 is found to be 0.0065 eV2. Then we analyze the base-pair and step parameters of the configurations in which V2 is at least an order of magnitude larger than 〈 V2 〉. To obtain more consistent data, ∼65 000 configurations of the (G:C)2 stack were built using systematic screening of the step parameters shift, slide, and twist. We show that undertwisted structures (twist<20°) are of special interest, because the π stack conformations with strong electronic couplings are found for a wide range of slide and shift. Although effective hole transfer can also occur in configurations with twist=30° and 35°, large mutual displacements of neighboring base pairs are required for that. Overtwisted conformation (twist38°) seems to be of limited interest in the context of effective hole transfer. The results may be helpful in the search for DNA based elements for nanoelectronics

Clinical utility of g-cimp and idh1 status as dual prognostic markers in glioblastoma

The glioma CpG island methylator phenotype (G-CIMP) has been shown to be highly correlated with prognosis andwas noted to be highly concordant with IDH1mutation in malignant glioma in the limited number of samples analyzed. To better understand the relationship of G-CIMP with IDH1 mutation status and patient outcome, we examined G-CIMP status in detail in a larger retrospective series of glioblastomas as well as tumor samples from the RTOG 0525 clinical trial. Sampleswere tested for 6 CIMPmarkers andwere correlated with patient outcomes. In the retrospective tumor set (n ¼ 301),we found 3 distinct survival groups based on the number of CIMP markers: 0-1 (CIMP-negative), 2-4 (CIMP-intermediate), and 5 or greater (CIMP-positive) with median survivals 13.8, 20.1, and 90.6 months, respectively. This finding was validated in the RTOG 0525 samples (median survivals 15.0, 20.3, and 37.0 months). Among 787 cases with both IDH and CIMP data, 617 were CIMP-negative, 136 were CIMP-intermediate, and 34 were CIMP-positive. Seven hundred forty-four were wild type for IDH1 mutation, and 43 were mutant. CIMP and IDH status were positively correlated but outliers were found. Among the 610 CIMP-negative tumors, there were 7 IDH-mutant tumors, which showed no difference in outcome. Similarly, among the 34 CIMP-positive tumors, there were 21 IDH-mutant cases, which also showed no difference in outcome. However, among the CIMP-intermediate cases, there were 15 IDH-mutant cases with significantly (p ¼ 0.0003) improved outcome (medians not reached vs. 18.5 months, 2 year survival 87% vs. 32%). Multivariate analysis showed that both IDH1 mutation status and CIMP status were independent predictors of outcome. These findings suggest the clinical utility of refining the CIMP status into negative, intermediate, and positive groups and the finding that both IDH1 and CIMPstatus are important molecular markers in GBM.

Pseudophakic Retinal Detachment Surgery by 23R01;G Vitrectomy using Slit-Lamp and Non-Contact 90 D Lens.

Background: The purpose of this study is to report the anatomic and functional results of primary 23R01;G vitrectomy using slit-lamp and non-contact 90 D lens for the treatment of pseudophakic rhegmatogenous retinal detachment. Patients and Methods: Pseudophakic eyes were operated by 23R01;G vitrectomy using slit-lamp and non-contact 90 D lens, internal subretinal fluid drainage, cryopexy and internal gas tamponade. The preoperative and postoperative characteristics were analysed. Main outcome measures were anatomic success rates after initial surgical intervention and after reoperation for primary failures, visual outcome at the last follow-up visit, and complications. Results: 46 pseudophakic eyes were included in this retrospective study (October 2013- January 2014). In 40 cases, sulfur hexafluoride 23R02;% gastamponade was used, silicone oil in 6 cases (13R02;%). The retina was reattached successfully after a single surgery in 44 eyes (96R02;%). Recurrence of retinal detachment occurred in 2 eyes. Final anatomic reattachment was obtained in 100R02;% after a second operation. Silicone oil was removed in all eyes. Visual acuity improved significantly from logMAR 0 (IQR 0R01;-R01;0.9) to logMAR 0 (IQR 0R01;-R01;0.2) (pR01;<R01;0.005). Conclusions: Primary 23R01;G vitrectomy using slit-lamp and non contact 90 D lens for the treatment of pseudophakic rhegmatogenous retinal detachment provides a high anatomic and functional success rate and is associated with few complications.

Use of alcohol in the elderly: transcultural validation of the Michigan Alcoholism Screening Test – Geriatric Version (MAST-G)

Objective: To evaluate the internal consistency of the version of the Michigan Alcoholism Screening Test – Geriatric Version (MAST-G) instrument, translated and adapted for Brazil. Method: This was a descriptive, cross-sectional study. Data were collected through a demographic questionnaire, the ICD-10 and the MAST-G, following the steps of translation and cultural adaptation. One hundred eleven elderly in the city of São Carlos, SP, Brazil were interviewed. Results: The mean age of those interviewed was 70 years, with 45% men and 55% women, with the mean education of three years; 92% resided with family; 48% of the subjects consumed alcoholic beverages. The MAST-G presented a good level of reliability, with Cronbach’s α0;=0;0.7873, and good levels of sensitivity and specificity with a cutoff score of five positive responses. Conclusion: The Brazilian version of the MAST-G presented internal consistency values similar to the original English version,showing it to be adequate for use in the national context.





Similarities and convergence in G-7 cycles

This paper examines the properties of G-7 cycles using a multicountry Bayesian panelVAR model with time variations, unit specific dynamics and cross country interdependences.We demonstrate the presence of a significant world cycle and show that country specificindicators play a much smaller role. We detect differences across business cycle phasesbut, apart from an increase in synchronicity in the late 1990s, find little evidence of major structural changes. We also find no evidence of the existence of an Euro area specific cycle or of its emergence in the 1990s.

On the sources of business cycles in the G-7

This paper examines sources of cyclical movements in output, inflation and the term structure of interest rates. It employs a novel identification approach which uses the sign of the cross correlation function in response to shocks to catalog orthogonal disturbances. We find that demand shocks are the dominant source output, inflation and term structure fluctuations in six of the G-7 countries. Within the class of demand disturbances, nominal shocks are dominant, but their importance declined after 1982. Furthermore, there are no significant differences in the proportion of term structure variability explained by different structural sources at different horizons.

Improved HIV-1 RNA quantitation by COBAS AmpliPrep/COBAS TaqMan HIV-1 Test, v2.0 using a novel dual-target approach.

BACKGROUND: HIV-1 RNA viral load is a key parameter for reliable treatment monitoring of HIV-1 infection. Accurate HIV-1 RNA quantitation can be impaired by primer and probe sequence polymorphisms as a result of tremendous genetic diversity and ongoing evolution of HIV-1. A novel dual HIV-1 target amplification approach was realized in the quantitative COBAS AmpliPrep/COBAS TaqMan HIV-1 Test, v2.0 (HIV-1 TaqMan test v2.0) to cope with the high genetic diversity of the virus. OBJECTIVES AND STUDY DESIGN: The performance of the new assay was evaluated for sensitivity, dynamic range, precision, subtype inclusivity, diagnostic and analytical specificity, interfering substances, and correlation with the COBAS AmpliPrep/COBAS TaqMan HIV-1 (HIV-1 TaqMan test v1.0) predecessor test in patients specimens. RESULTS: The new assay demonstrated a sensitivity of 20 copies/mL, a linear measuring range of 20-10,000,000 copies/mL, with a lower limit of quantitation of 20 copies/mL. HIV-1 Group M subtypes and HIV-1 Group O were quantified within +/-0.3 log(10) of the assigned titers. Specificity was 100% in 660 tested specimens, no cross reactivity was found for 15 pathogens nor any interference for endogenous substances or 29 drugs. Good comparability with the predecessor assay was demonstrated in 82 positive patient samples. In selected clinical samples 35/66 specimens were found underquantitated in the predecessor assay; all were quantitated correctly in the new assay. CONCLUSIONS: The dual-target approach for the HIV-1 TaqMan test v2.0 enables superior HIV-1 Group M subtype coverage including HIV-1 Group O detection. Correct quantitation of specimens underquantitated in the HIV-1 TaqMan test v1.0 test was demonstrated.

The IFNL3/4 ΔG variant increases susceptibility to cytomegalovirus retinitis among HIV-infected patients.

BACKGROUND: Cytomegalovirus (CMV) retinitis is a major cause of visual impairment and blindness among patients with uncontrolled HIV infections. Whereas polymorphisms in interferon-lambda 3 (IFNL3, previously named IL28B) strongly influence the clinical course of hepatitis C, few studies examined the role of such polymorphisms in infections due to viruses other than hepatitis C virus. OBJECTIVES: To analyze the association of newly identified IFNL3/4 variant rs368234815 with susceptibility to CMV-associated retinitis in a cohort of HIV-infected patients. DESIGN AND METHODS: This retrospective longitudinal study included 4884 white patients from the Swiss HIV Cohort Study, among whom 1134 were at risk to develop CMV retinitis (CD4 nadir <100R02;/μl and positive CMV serology). The association of CMV-associated retinitis with rs368234815 was assessed by cumulative incidence curves and multivariate Cox regression models, using the estimated date of HIV infection as a starting point, with censoring at death and/or lost follow-up. RESULTS: A total of 40 individuals among 1134 patients at risk developed CMV retinitis. The minor allele of rs368234815 was associated with a higher risk of CMV retinitis (log-rank test PR02;=R02;0.007, recessive mode of inheritance). The association was still significant in a multivariate Cox regression model (hazard ratio 2.31, 95% confidence interval 1.09-4.92, PR02;=R02;0.03), after adjustment for CD4 nadir and slope, HAART and HIV-risk groups. CONCLUSION: We reported for the first time an association between an IFNL3/4 polymorphism and susceptibility to AIDS-related CMV retinitis. IFNL3/4 may influence immunity against viruses other than HCV.