The Human Paraoxonase 2: An Optimized Procedure for Refolding and Stabilization Facilitates Enzyme Analyses and a Proteomics Approach.

The human paraoxonase 2 (PON2) is the oldest member of a small family of arylesterase and lactonase enzymes, representing the first line of defense against bacterial infections and having a major role in ROS-associated diseases such as cancer, cardiovascular diseases, neurodegeneration, and diabetes. Specific Post-Translational Modifications (PTMs) clustering nearby two residues corresponding to pon2 polymorphic sites and their impact on the catalytic activity are not yet fully understood. Thus, the goal of the present study was to develop an improved PON2 purification protocol to obtain a higher amount of protein suitable for in-depth biochemical studies and biotechnological applications. To this end, we also tested several compounds to stabilize the active monomeric form of the enzyme. Storing the enzyme at 4 °C with 30 mM Threalose had the best impact on the activity, which was preserved for at least 30 days. The catalytic parameters against the substrate 3-Oxo-dodecanoyl-Homoserine Lactone (3oxoC12-HSL) and the enzyme ability to interfere with the biofilm formation of Pseudomonas aeruginosa (PAO1) were determined, showing that the obtained enzyme is well suited for downstream applications. Finally, we used the purified rPON2 to detect, by the direct molecular fishing (DMF) method, new putative PON2 interactors from soluble extracts of HeLa cells.

Serological profiles and evaluation of parasitaemia by PCR and blood culture in individuals chronically infected by Trypanosoma cruzi treated with benzonidazole.

OBJECTIVE: To evaluate the serological and parasitological status of patients with chronic Chagas disease (CD) after chemotherapy with benzonidazole. METHODS: Retrospective study of patients treated with benzonidazole (5 mg/kg/day for 60 days) between 1980 and 2010. Twenty-nine patients who had CD confirmed by two reagent immunological tests and/or one positive xenodiagnosis before treatment were included. Conventional serology (ELISA and IIF) and parasitological tests (haemoculture and N-PCR) were performed. RESULTS: At the time of treatment, the mean age of patients was 36 ± 7.24 years (20-39 years) and the time post-treatment varied from 1 to 29 years. After chemotherapy, all individuals had reagent ELISA and 93.1% had positive results for the IIF test. T. cruzi DNA was detected by N-PCR in 48.3%. Negative results were observed in 41.4% and inconclusive ones in 10.3%. Haemoculture was negative for all individuals. CONCLUSIONS: Our results suggest that N-PCR may be useful in the early identification of therapeutic failure of CD. Although it is difficult to determine parasitological cure in negative N-PCR cases, we can infer that this condition represents a declination of parasitaemia as a favourable consequence of aetiological treatment.

Diffuse Myocardial Fibrosis and Cardiomyocyte Diameter Are Associated With Heart Failure Symptoms in Chagas Cardiomyopathy.

Background: Chronic Chagas cardiomyopathy (CCC) constitutes the most life-threatening consequence of the Trypanosoma cruzi infection. Our goal was to test in CCC the associations of the myocardial tissue phenotype with cardiac dysfunction, and heart failure (HF) severity, using cardiac magnetic resonance (CMR). Methods: We performed a prospective observational cohort of patients with consecutive CCC with a CMR protocol, including ventricular function, myocardial T1, and late gadolinium enhancement (LGE). Extracellular volume (ECV), and intracellular water lifetime, τic, a measure of cardiomyocyte diameter, were compared to CCC disease progression, including Rassi score and New York Heart Association (NYHA) class. An exploratory prognostic analysis was performed to investigate the association of both ECV and τic with CV death. Results: A total of 37 patients with intermediate-to-high-risk CCC were enrolled (Chagas Rassi score ≥7, mean left ventricle (LV) ejection fraction (EF) 32 ± 16%). Myocardial ECV (0.40 ± 0.07) was correlated with Rassi score (r = 0.43; P = 0.009), higher NYHA class, and LV EF (r = -0.51; P = 0.0015). τic decreased linearly with NYHA class (P = 0.007 for non-parametric test of linear trend) and showed a positive association with LV EF (r = 0.47; P = 0.004). Over a median follow-up of 734 days (range: 6-2,943 days), CV death or cardiac transplantation occurred in 10 patients. The Rassi score (heart rate [HR] = 1.3; 95% CI = [1.0, 1.8]; P = 0.028) and ECV (HR = 3.4 for 0.1 change, 95% CI = [1.1, 11.0], P = 0.039) were simultaneously associated with CV death. Conclusion: In patients with intermediate-to-high-risk CCC, an expanded ECV and regression of cardiomyocyte diameter were associated with worsening systolic function and HF severity, respectively. The exploratory analysis indicates that ECV may have a prognostic value to identify patients with CCC at a higher risk for cardiovascular events.

Trypanosoma cruzi: parasite persistence in tissues in chronic chagasic Brazilian patients.

Chagas disease in the chronic phase may develop into cardiac and/or digestive forms. The pathogenesis of the disease is not yet clear and studies have been carried out to elucidate the role of parasite persistence in affected organs. The aim of this study was to detect and quantify Trypanosoma cruzi in paraffin-embedded tissue samples from chronic patients using NPCR (nested polymerase chain reaction) and QPCR (quantitative polymerase chain reaction) methods. These results were correlated to anatomopathological alterations in the heart and gastrointestinal tract (GIT). Of the 23 patients studied, 18 presented the cardiac form and five presented the cardiodigestive form of Chagas disease. DNA samples were randomly isolated from formalin-fixed paraffin-embedded sections of heart and GIT tissue of 23 necropsies and were analyzed through NPCR amplification. T. cruzi DNA was detected by NPCR in 48/56 (85.7%) heart and 35/42 (83.3%) GIT samples from patients with the cardiac form. For patients with the cardiodigestive form, NPCR was positive in 12/14 (85.7%) heart and in 14/14 (100%) GIT samples. QPCR, with an efficiency of 97.6%, was performed in 13 samples (11 from cardiac and 2 from cardiodigestive form) identified previously as positive by NPCR. The number of T. cruzi copies was compared to heart weight and no statistical significance was observed. Additionally, we compared the number of copies in different tissues (both heart and GIT) in six samples from the cardiac form and two samples from the cardiodigestive form. The parasite load observed was proportionally higher in heart tissues from patients with the cardiac form. These results show that the presence of the parasite in tissues is essential to Chagas disease pathogenesis.

High-Efficiency Biocidal Solution Based on Radiochemically Synthesized Cu-Au Alloy Nanoparticles.

The use of nanotechnologies in the applied biomedical sciences can offer a new way to treat infections and disinfect surfaces, materials, and products contaminated with various types of viruses, bacteria, and fungi. The Cu-Au nanoparticles (NPs) were obtained by an eco-friendly method that allowed the obtaining in a one-step process of size controlled, well dispersed, fully reduced, highly stable NPs at very mild conditions, using high energy ionizing radiations. The gamma irradiation was performed in an aqueous system of Cu2+/Au3+/Sodium Dodecyl Sulfate (SDS)/Ethylene Glycol. After irradiation, the change of color to ruby-red was the first indicator for the formation of NPs. Moreover, the UV-Vis spectra showed a maximum absorption peak between 524 and 540 nm, depending on the copper amount. The Cu-Au NPs presented nearly spherical shapes, sizes between 20 and 90 nm, and a zeta potential of about -44 mV indicating a good electrostatic stability. The biocidal properties performed according to various standards applied in the medical area, in dirty conditions, showed a 5 lg reduction for Staphylococcus aureus, Pseudomonas aeruginosa, and Enterococcus hirae, a 5 lg reduction for both enveloped and non-enveloped viruses such as Adenovirus type 5, Murine Norovirus, and human Coronavirus 229E, and a 4 lg reduction for Candida albicans, respectively. Thus, the radiochemically synthesized Cu-Au alloy NPs proved to have high biocide efficiency against the tested bacteria, fungi, and viruses (both encapsulated and non-encapsulated). Therefore, these nanoparticle solutions are suitable to be used as disinfectants in the decontamination of hospital surfaces or public areas characterized by high levels of microbiological contamination.

Mitofusin 2 in Macrophages Links Mitochondrial ROS Production, Cytokine Release, Phagocytosis, Autophagy, and Bactericidal Activity.

Mitofusin 2 (Mfn2) plays a major role in mitochondrial fusion and in the maintenance of mitochondria-endoplasmic reticulum contact sites. Given that macrophages play a major role in inflammation, we studied the contribution of Mfn2 to the activity of these cells. Pro-inflammatory stimuli such as lipopolysaccharide (LPS) induced Mfn2 expression. The use of the Mfn2 and Mfn1 myeloid-conditional knockout (KO) mouse models reveals that Mfn2 but not Mfn1 is required for the adaptation of mitochondrial respiration to stress conditions and for the production of reactive oxygen species (ROS) upon pro-inflammatory activation. Mfn2 deficiency specifically impairs the production of pro-inflammatory cytokines and nitric oxide. In addition, the lack of Mfn2 but not Mfn1 is associated with dysfunctional autophagy, apoptosis, phagocytosis, and antigen processing. Mfn2floxed;CreLysM mice fail to be protected from Listeria, Mycobacterium tuberculosis, or LPS endotoxemia. These results reveal an unexpected contribution of Mfn2 to ROS production and inflammation in macrophages.

Risk factors for nonunion after nonoperative treatment of displaced midshaft fractures of the clavicle.

BACKGROUND: Identification of patients at higher risk of nonunion after diaphyseal clavicular fractures is desirable to improve patient counseling and enable targeted surgical treatment. METHODS: Seventy-nine percent (941 of 1196) of diaphyseal clavicular fractures were followed to union or nonunion. Demographic, injury, and radiographic characteristics associated with nonunion were determined with use of bivariate and multivariate statistical analyses. RESULTS: In patients who were eighteen years of age or older, 125 (13.3%) of the fractures had clinical and radiographic evidence of nonunion. Factors significantly associated with nonunion on bivariate analysis were sex, smoking status, overall fracture displacement, overlap, translation, and comminution. The factors that maintained significance on multivariate analysis were smoking (odds ratio, 3.76), comminution (odds ratio, 1.75), and fracture displacement (odds ratio, 1.17). If all displaced midshaft fractures were managed operatively, 7.5 procedures would need to be undertaken to prevent a single nonunion. If only fractures with a predicted probability of ≥40% were managed operatively, the number of patients managed operatively to prevent a single nonunion would fall to 1.7. CONCLUSIONS: Thirteen percent of displaced diaphyseal fractures in patients who were at least eighteen years of age did not heal. Smoking was the strongest risk factor, and smoking cessation should be an integral part of treatment. The probability of nonunion in a particular individual can be estimated with use of a statistical model based on known risk factors. This information can be useful when counseling the patient even though nonunion remains difficult to predict accurately in that individual. The number who would need to be treated to prevent a single nonunion can be reduced by identifying those at higher risk.

Evidence of Chagas disease in seronegative Brazilian patients with megaesophagus.

BACKGROUND: After 100 years of research, Chagas disease (CD) remains an important public health problem in Latin America. The symptomatic chronic phase is usually characterized by cardiac or digestive involvement and diagnosis currently relies on the measurement of Trypanosoma cruzi-specific antibodies produced in response to the infection. However, the detection of parasite DNA in seronegative persons has been reported. METHODS: The prevalence of CD in a population with esophageal disorders was assessed by conventional serology. We also detected T. cruzi DNA in blood samples of seronegative and inconclusive patients by nested polymerase chain reaction (N-PCR). RESULTS: The seroprevalence of CD determined by conventional serologic tests (indirect immunofluorescence (IIF) and enzyme-linked immunosorbent assay (ELISA)) was 79% in 513 patients with esophageal disorders. Out of 41 blood samples, N-PCR was positive in 31 (76%) cases for which serology was negative or inconclusive. CONCLUSIONS: As all patients presented with clinical signs suggestive of the digestive form of CD and most of them were born in endemic areas, we highlight the importance of improving diagnosis of the disease and the implications for blood bank screening. Our data suggest that N-PCR is effective in the detection of T. cruzi DNA in patients with inconclusive or negative serology, and it may eventually be useful in the determination of the etiology of megaesophagus.

Determining the C-reactive protein level in patients with different clinical forms of chagas disease.

Chagas disease is caused by Trypanosoma cruzi and remains a health problem in the developing countries of South America. The condition leads to cardiac conduction disturbances and chronic heart failure. In this study, 136 individuals were evaluated by the Chagas Disease Study Group of the Hospital de la Universidad Estatal de Campinas in Brazil to determine the relationship between chronic heart failure and the serum C-reactive protein (CRP) level. When patients were stratified according to the different clinical presentations of Chagas disease, it was found that the CRP levels in those with severe heart disease and non-Chagasic cardiopathy were significantly higher than in controls or those with mild heart disease (P< .05), even when participants were stratified by age (i.e. <40 and > or =40 years). There was a direct linear correlation between age and CRP level, such that the older the individual, the higher the CRP level. These data provide further evidence for an association between chronic inflammation and the development of heart failure. Although CRP elevations are not exclusively related to Chagas disease, the CRP level may be a useful marker for the progression of Chagas disease to a more advanced phase.

Chagas' disease and HIV co-infection in patients without effective antiretroviral therapy: prevalence, clinical presentation and natural history.

The objectives of this study were to establish the prevalence of Chagas' disease among HIV seropositive patients and to define the clinical profile of co-infected cases. Cross-sectional study: the prevalence of co-infected subjects was 1.3% and there was no significant difference between co-infected and non co-infected patients relative to race, birthplace, home address and CD4 T cells. The co-infected group comprised predominantly women and mean age and median viral load were higher. Longitudinal study: included 20 patients (12 women) and described the clinical presentation and natural history of concomitant infections. The mean follow-up time was 35.8 months, mean age was 43+/-8.7 years and 60% of patients were white. During the follow-up, a total of 113 serological tests for Chagas' disease were performed: 89 (78.8%) were reactive/positive, 21 (18.6%) were doubtful and three (2.6%) were non-reactive/negative. Positive results for xenodiagnosis were high (81%). At the baseline evaluation, thirteen patients had the indeterminate form of Chagas' disease and seven cardiopathy. One patient developed from indeterminate to digestive form, three had a reactivation of Chagas' disease in the central nervous system, all had parasitological confirmation and received specific treatment. There were 11 deaths. Thus, HIV-infected patients should be tested for Chagas' disease when epidemiologically relevant.

Trypanosoma cruzi: parasite persistence in tissues in chronic chagasic Brazilian patients

Chagas disease in the chronic phase may develop into cardiac and/or digestive forms. The pathogenesis of the disease is not yet clear and studies have been carried out to elucidate the role of parasite persistence in affected organs. The aim of this study was to detect and quantify Trypanosoma cruzi in paraffin-embedded tissue samples from chronic patients using NPCR (nested polymerase chain reaction) and QPCR (quantitative polymerase chain reaction) methods. These results were correlated to anatomopathological alterations in the heart and gastrointestinal tract (GIT). Of the 23 patients studied, 18 presented the cardiac form and five presented the cardiodigestive form of Chagas disease. DNA samples were randomly isolated from formalin-fixed paraffin-embedded sections of heart and GIT tissue of 23 necropsies and were analyzed through NPCR amplification. T. cruzi DNA was detected by NPCR in 48/56 (85.7 percent) heart and 35/42 (83.3 percent) GIT samples from patients with the cardiac form. For patients with the cardiodigestive form, NPCR was positive in 12/14 (85.7 percent) heart and in 14/14 (100 percent) GIT samples. QPCR, with an efficiency of 97.6 percent, was performed in 13 samples (11 from cardiac and 2 from cardiodigestive form) identified previously as positive by NPCR. The number of T. cruzi copies was compared to heart weight and no statistical significance was observed. Additionally, we compared the number of copies in different tissues (both heart and GIT) in six samples from the cardiac form and two samples from the cardiodigestive form. The parasite load observed was proportionally higher in heart tissues from patients with the cardiac form. These results show that the presence of the parasite in tissues is essential to Chagas disease pathogenesis.

Percutaneous aspiration biopsy in perineal recurrences after abdominoperineal extirpations.

The authors recommend the introduction of percutaneous aspiration biopsy--a new method used by them for demonstrating perineal recurrences after abdominoperineal extirpations. The method is fast and easy to perform, simple and can be used for screening. It may be an addition to the therapeutic and diagnostic arsenal.

[Evaluation of operative mortality among patients aged over 80 between 1970 and 1980]. / Auswertung zur Operationsmortalität bei über 80jährigen Patienten von 1970-1980.

The second surgical department of the Postgraduate Medical School conducted a 10-year analysis covering patients aged over 80. The results suggest that elective surgery is advisable even at this advanced age. A sound pre-operative diagnosis can help ensure that aged patients undergo an operation at the earliest possible date, the death rate from surgery carried out in a late acute stage being much higher. The authors take issue with the view -- still regarded as a dogma in the surgical world -- that the operation should be "minimal". They note that good results in "geriatric" surgery depend on good management and adequate pre-operative and post-operative observation.

Determinación de la concentración plasmática de proteína C reactiva en pacientes con diferentes formas clínicas de la enfermedad de Chagas / Determining the C-reactive protein level in patients with different clinical forms of Chagas disease

La enfermedad de Chagas (EC) es una infección causada por Trypanosoma cruzi que aún hoy constituye un problema de salud en los países en desarrollo de Sudamérica y causa trastornos de la conducción e insuficiencia cardiaca crónica. En este trabajo, el Grupo de Estudios de Enfermedad de Chagas del Hospital de la Universidad Estatal de Campinas ha analizado a 136 individuos para investigar la relación entre insuficiencia cardiaca crónica y concentración sérica de proteína C reactiva (PCR). La PCR estratificada según diferentes apariciones clínicas de la EC reveló que los pacientes con cardiopatía grave y cardiopatía no chagásica tenían estadísticamente concentraciones de PCR mayores que los otros grupos de control y cardiopatía leve (p < 0,05), incluso estratificando por edad ( < 40 y ≥ 40 años). Hubo una correlación lineal positiva entre edad y PCR, de manera que cuanto mayores los individuos, más altos los valores de PCR. Estos datos refuerzan la asociación entre inflamación crónica y aparición de insuficiencia cardiaca. Aunque la elevaciones de PCR no están exclusivamente relacionadas con la EC, serían un marcador asequible de la evolución de la EC hacia fases avanzadas (AU)

Chagas disease is caused by Trypanosoma cruzi and remains a health problem in the developing countries of South America. The condition leads to cardiac conduction disturbances and chronic heart failure. In this study, 136 individuals were evaluated by the Chagas Disease Study Group of the Hospital de la Universidad Estatal de Campinas in Brazil to determine the relationship between chronic heart failure and the serum C-reactive protein (CRP) level. When patients were stratified according to the different clinical presentations of Chagas disease, it was found that the CRP levels in those with severe heart disease and non-Chagasic cardiopathy were significantly higher than in controls or those with mild heart disease (P < .05), even when participants were stratified by age (i.e. <40 and 8805 40 years there was a direct linear correlation between age crp level such that the older individual higher these data provide further evidence for an association chronic inflammation development of heart failure although elevations are not exclusively related to chagas disease may be useful marker progression more advanced phase (AU)