Simultaneous voltammetric detection of ascorbic acid, dopamine and uric acid using a pyrolytic graphite electrode modified into dopamine solution

Pyrolytic graphite electrodes (PGE) were modified into dopamine solutions using phosphate buffer solutions, pH 10 and 6.5, as supporting electrolyte. The modification process involved a previous anodization of the working electrode at +1. 5 V into 0. 1 mol-L-1 NaOH followed by other anodization step, in the same experimental conditions, into dopamine (DA) solutions. pH of the supporting electrolyte performed an important role in the production of a superficial melanin polymeric film, which permitted the simultaneous detection of ascorbic acid (AA), (DA) and uric acid (UA), Delta EAA-DA = 222 mV-, Delta EAA-UA = 360 mV and Delta EDA-UA=138mV, avoiding the superficial poisoning effects. The calculated detection limits were: 1.4 x 10(-6) mol L-1 for uric acid, 1.3x10-(5) molL(-1) for ascorbic acid and 1.1 X 10(-7) mol L-1 for dopamine, with sensitivities of (7.7 +/- 0.5), (0.061 +/- 0.001) and (9.5 +/- 0.05)A mol(-1) cm(-2), respectively, with no mutual interference. Uric acid was determined in urine, blood and serum human samples after dilution in phosphate buffer and no additional sample pre-treatment was necessary. The concentration of uric acid in urine was higher than the values found in blood and serum and the recovery tests (92-102%) indicated that no matrix effects were observed. (C) 2008 Elsevier B.V. All rights reserved.

Prevalence of Human Papillomaviruses in Urine Samples of Male Patients Infected With HIV-1 in Sao Paulo, Brazil

Human papillomavirus is a DNA virus that includes 118 genotypes. HPV16 is responsible for 80% of cervical cancer in women. Men are important reservoirs and major transmitters of HPV to their partners. The aim of this study was to detect HPV DNA and to determine the prevalence of HPV types 6, 11, 16, and 18 in urine samples of men infected with HIV-1. This study included 223 patients infected with HIV-1 from the Center of Reference on HIV/AIDS (CRT-SP) and an outpatient clinic of HIV. Urine samples were collected and after DNA extraction real-time PCR was performed for detection of HPV DNA. Positive samples were then tested by conventional PCR using type-specific primers for the four HPV types. A total of 223 men infected with HIV-1 were tested, 81% of whom were on HAART. Four (5.8%) were positive for HPV6, 18 (26.1%) were positive for HPV11, 22 (31.9%) were positive for HPV16 and five (7.2%) were positive for HPV18 by conventional PCR. Twenty (29%) patients had other HPV types and five patients (1.5%) had multiple types. The mean T CD4+cells count was 517 and 441 cells/mm(3) (P=0.30), in HPV negative and positive men, respectively. The HIV viral load was higher in the HPV negative group than for in the men with HPV (P=0.0002). A 30.9% prevalence of HPV was found in asymptomatic urine samples of men infected with HIV-1. This study suggests that urine may be a useful specimen for HPV screening. J. Med. Virol 81:2007-2011, 2009. (C) 2009 Wiley-Liss, Inc.

Natural Plasmodium infections in Brazilian wild monkeys: Reservoirs for human infections?

Four hundred and forty-eight samples of total blood from wild monkeys living in areas where human autochthonous malaria cases have been reported were screened for the presence of Plasmodium using microscopy and PCR analysis. Samples came from the following distinct ecological areas of Brazil: Atlantic forest (N = 140), semideciduous Atlantic forest (N = 257) and Cerrado (a savannah-like habitat) (N = 51). Thick and thin blood smears of each specimen were examined and Plasmodium infection was screened by multiplex polymerase chain reaction (multiplex PCR). The frequency of Plasmodium infections detected by PCR in Alouatta guariba clamitans in the Sao Paulo Atlantic forest was 11.3% or 8/71 (5.6% for Plasmodium malariae and 5.6% for Plasmodium vivax) and one specimen was positive for Plasmodium falciparum (1.4%); Callithrix sp. (N = 30) and Cebus apella (N = 39) specimens were negative by PCR tests. Microscopy analysis was negative for all specimens from the Atlantic forest. The positivity rate for Alouatta caraya from semideciduous Atlantic forest was 6.8% (16/235) in the PCR tests (5.5, 0.8 and 0.4% for P. malariae, P. falciparum and P. vivax, respectively), while C apella specimens were negative. Parasitological examination of I he samples using thick smears revealed Plasmodium sp. infections in only seven specimens, which had few parasites (3.0%). Monkeys from the Cerrado (a savannah-like habitat) (42 specimens of A. caraya, 5 of Callithrix jacchus and 4 of C. apella) were negative in both tests. The parasitological prevalence of P. vivax and P. malariae in wild monkeys from Atlantic forest and semideciduous Atlantic forest and the finding of a positive result for P.falciparum in Alouatta from both types of forest support the hypothesis that monkeys belonging to this genus could be a potential reservoir. Furthermore, these findings raise the question of the relationship between simian and autochthonous human malaria in extra-Amazonian regions. (C) 2008 Elsevier B.V. All rights reserved.

Protease-activated Receptor-2 (PAR(2)) in Human Periodontitis

No evidence for the role of protease-activated receptor-2 (PAR(2)) in human periodontal disease has been demonstrated so far. Thus, we sought to investigate the expression of PAR(2) mRNA in chronic periodontitis, and to examine whether its expression is related to the presence of PAR(2) potential activators. Microbiological and gingival crevicular fluid samples were collected from individuals with chronic periodontitis and control individuals, and the presence of neutrophil serine proteinase 3 (P3) and Porphyromonas gingivalis was evaluated. PAR(2) mRNA expression was higher (p < 0.001) in those with chronic periodontitis compared with control individuals, and it was statistically decreased (p = 0.0006) after periodontal treatment. Furthermore, those with chronic periodontitis presented higher (p < 0.05) levels of IL-1 alpha, IL-6, IL-8, and TNF-alpha, total proteolytic activity, P. gingivalis prevalence, and P3mRNA expression compared with control individuals. We conclude that PAR(2) mRNA expression and its potential activators are elevated in human chronic periodontitis, therefore suggesting that PAR(2) may play a role in periodontal inflammation.

Paleoamerican Morphology in the Context of European and East Asian Late Pleistocene Variation: Implications for Human Dispersion Into the New World

Early American crania show a different morphological pattern from the one shared by late Native Americans. Although the origin of the diachronic morphological diversity seen on the continents is still debated, the distinct morphology of early Americans is well documented and widely dispersed. This morphology has been described extensively for South America, where larger samples are available. Here we test the hypotheses that the morphology of Early Americans results from retention of the morphological pattern of Late Pleistocene modern humans and that the occupation of the New World precedes the morphological differentiation that gave rise to recent Eurasian and American morphology. We compare Early American samples with European Upper Paleolithic skulls, the East Asian Zhoukoudian Upper Cave specimens and a series of 20 modern human reference crania. Canonical Analysis and Minimum Spanning Tree were used to assess the morphological affinities among the series, while Mantel and Dow-Cheverud tests based on Mahalanobis Squared Distances were used to test different evolutionary scenarios. Our results show strong morphological affinities among the early series irrespective of geographical origin, which together with the matrix analyses results favor the scenario of a late morphological differentiation of modern humans. We conclude that the geographic differentiation of modern human morphology is a late phenomenon that occurred after the initial settlement of the Americas. Am J Phys Anthropol 144:442-453, 2011. (c) 2010 Wiley-Liss, Inc.

Next Generation Sequencing of Pooled Samples Reveals New SNRNP200 Mutations Associated with Retinitis Pigmentosa

The gene SNRNP200 is composed of 45 exons and encodes a protein essential for pre-mRNA splicing, the 200 kDa helicase hBrr2. Two mutations in SNRNP200 have recently been associated with autosomal dominant retinitis pigmentosa (adRP), a retinal degenerative disease, in two families from China. In this work we analyzed the entire 35-Kb SNRNP200 genomic region in a cohort of 96 unrelated North American patients with adRP. To complete this large-scale sequencing project, we performed ultra high-throughput sequencing of pooled, untagged PCR products. We then validated the detected DNA changes by Sanger sequencing of individual samples from this cohort and from an additional one of 95 patients. One of the two previously known mutations (p.S1087L) was identified in 3 patients, while 4 new missense changes (p.R681C, p.R681H, p.V683L, p.Y689C) affecting highly conserved codons were identified in 6 unrelated individuals, indicating that the prevalence of SNRNP200-associated adRP is relatively high. We also took advantage of this research to evaluate the pool-and-sequence method, especially with respect to the generation of false positive and negative results. We conclude that, although this strategy can be adopted for rapid discovery of new disease-associated variants, it still requires extensive validation to be used in routine DNA screenings. (C) 2011 Wiley-Liss, Inc.

Peptidomic Analysis of Human Cell Lines

Peptides have been proposed to function in intracellular signaling within the cytosol. Although cytosolic peptides are considered to be highly unstable, a large number of peptides have been detected in mouse brain and other biological samples. In the present study, we evaluated the peptidome of three diverse cell lines: SH-SY5Y, MCF7, and HEIC293 cells. A comparison of the peptidomes revealed considerable overlap in the identity of the peptides found in each cell line. The majority of the observed peptides are not derived from the most abundant or least stable proteins in the cell, and approximately half of the cellular peptides correspond to the N- or C- termini of the precursor proteins. Cleavage site analysis revealed a preference for hydrophobic residues in the PI position. Quantitative peptidomic analysis indicated that the levels of most cellular peptides are not altered in response to elevated intracellular calcium, suggesting that calpain is not responsible for their production. The similarity of the peptidomes of the three cell lines and the lack of correlation with the predicted cellular degradome implies the selective formation or retention of these peptides, consistent with the hypothesis that they are functional in the cells.

Patterns of Antibodies Against Latent and Lytic Antigens of Human Herpesvirus 8 in an Endemic Population and Patients With Kaposi`s Sarcoma in Mozambique

The patterns of antibodies against latent and lytic antigens of human herpesvirus 8 (HHV-8) were assessed using immunofluorescence assays of samples from 155 persons seropositive for HHV-8 seen at public health centers and 24 patients with Kaposi`s sarcoma (KS) from Mozambique. Of the 155 persons without KS, 48(31%) had antibodies against latent antigens only, 29 (18.7%) had antibodies against lytic antigens only, and 78 (50.3%) had antibodies against both types of antigen. The HHV-8 antibody titer tended to increase with age until age 40, after which it began to decrease. High titers of antibodies against latent and lytic antigens of HHV-8 were detected mostly in persons co-infected with HIV, and these increased titers could have a predictive value. All patients with KS except four patients who were seronegative for HHV-8 had elevated titers of HHV-8 antibodies, predominantly against latent antigens. The data suggest the potential for an increase in the development of KS in this endemic area for HHV-8. J. Med. Virol. 82:1576-1581, 2010. (C) 2010 Wiley-Liss, Inc.

A Comparative Study of the Frequency of Antibody and Titers Against Human Herpesvirus 8 Latent and Lytic Antigens in ""At-Risk"" Individuals and Among Patients With Kaposi`s Sarcoma

Differences in the prevalence of human herpesvirus 8 (HHV-8) and Kaposi`s sarcoma (KS) have been described, depending on the study population and their geographic origin. A cross-sectional study aimed at detecting the frequency and titers of antibodies against HHV-8 latent and lytic antigens in serum samples from individuals with different risk-factors for HHV-8 infection, as well as predictive marker identification in patients with KS, was conducted. Serum samples were collected from seven groups of individuals: 75 patients with AIDS-KS, 5 with classic KS, 16 with African KS, 495 with HIV/AIDS, 805 patients with chronic kidney disease, 683 handicapped individuals, and 757 health care workers. Samples were evaluated for the presence and titers of HHV-8-specific antibodies to latent and lytic antigens using ""in house"" immunofluorescence assays. The results were analyzed by the Chi-square, Fisher`s exact test, Kruskal-Wallis and/or Mann-Whitney U-tests. The frequencies of HHV-8 antibodies were as follows: 87.5-100% in patients with KS, 20.4% in patients with HIV/AIDS, 18% in patients with chronic kidney disease, 1.6% in handicapped individuals, and 1.1% in health care workers. A greater number of samples were antibody positive to lytic antigens. Elevated titers of antibodies to latent and lytic antigens, mostly among patients with KS, were detected. Using established serological assays, different ""at-risk"" populations for HHV-8 infection/disease were detected in this geographic area, confirming HIV/AIDS and identifying patients with chronic kidney disease as high-risk groups. It is suggested that a longitudinal evaluation of antibody titers in patients with chronic kidney disease be undertaken to confirm their predictive value in the development of KS. J. Med. Virol. 81: 1292-1297, 2009. (C) 2009 Wiley-Liss, Inc.

Clinical correlations of human cytomegalovirus strains and viral load in kidney transplant recipients

Little is known about clinical differences associated with cytomegalovirus (CMV) infection by distinct strains in renal transplant patients. Different clinical pictures may be associated with specific viral genotypes. viral load, as well as host factors. The objective of this study was to identify CMV strains to determine viral load (antigenemia), and their correlation with clinical data in renal transplant recipients. Seventy-one patients were enrolled, comprising 91 samples. After selection, polymorphonuclear cells were used to amplify and sequence the gB region of CMV DNA. The sequences were analyzed to ascertain the frequency of different genotypes. Additionally, the results of this Study showed that the gB coding gene presents a great variability, revealing a variety of patterns: classical gB (1.4%), gB1V (46.4%), classical gB2 (35.2%), gB2V (2.8%), gB3 (1.4%), classical gB4 (4.9%) and gB4V (4.9%). The mean viral load in kidney transplant patient was 75.1 positive cells (1-1000). A higher viral load was observed in patients with genotype 4 infection. Statistically significant differences were detected between gB1 and gB4 (p=0.010), and between gB2 and gB4 (p=0.021). The average numbers of positive cells in relation to clinical presentation were: 34.5 in asymptomatic, 49.5 in CMV associated syndrome and 120.7 in patients with invasive disease (p=0.048). As a group, gB1 was the most frequent strain and revealed a potential risk for developing invasive disease. Viral load also seemed to be important as a marker associated with clinical presentation of the disease. (C) 2008 Elsevier B.V. All rights reserved.

Molecular characterization of human erythrovirus B19 strains obtained from patients with several clinical presentations in the Amazon region of Brazil

Background: Human erythrovirus B 19, endemic in the Amazon region since 1990, is associated with a wide spectrum of clinical presentations. Objectives: To assess the prevalence of erythrovirus B 19 infection and the relative frequency of erythrovirus B 19 genotypes in patients in the Amazon region with various clinical presentations. Study design: A total of 487 clinical samples obtained from patients with symptoms suggestive of erythrovirus infection were tested using specific IgM and IgG antibody assays (ELISA) and PCR for viral DNA detection. Partial VP1 and VP2 regions were sequenced and genotyped by phylogenetic reconstruction. Results: B 19 DNA was detected in 117 (24%) of 487 samples. Of these, 106 (91%) isolates were genotype I and II (9%) were genotype 3. No genotype 2 was found. Genotype I had three clusters (A1, A2 and 13) and all genotype 3 sequences were subtype 3b. All patients with hernatological disorders within cluster B of genotype I were infected by the sarne B 19 lineage, suggesting that this lineage of B 19 may have been transmitted via transfusion of blood products. Conclusion: We reported two genotypes, I and 3b, with three genotype I clusters co-circulating in the Amazon region during the past 10 years. (C) 2008 Elsevier B.V. All rights reserved.

Hospital-acquired human bocavirus in infants

Human bocavirus (HBoV) is a respiratory pathogen that affects young children. We screened 511 nasopharyngeal aspirates for hospital-acquired HBoV from infants hospitalised with respiratory infection from January to December 2008. Among 55 children with HBoV infection, 10 cases were hospital-acquired. Compared with the community-acquired cases, coinfection with other respiratory viruses in these patients was uncommon. HBoV should be considered for inclusion in screening protocols for nosocomial childhood respiratory infections, especially in intensive care units. (C) 2010 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved.

Evaluation of laccases and melanization in clinical and environmental Cryptococcus neoformans samples by non-denaturing PAGE

The increased incidence of infections caused by the opportunistic pathogen Cryptococcus neoformans, which mainly affects immunocompromised patients but can also infect immunocompetent individuals, has needed additional studies on this micro-organism`s pathogenicity and factors related to virulence, such as enzyme production, for a better understanding of the aetiology of cryptococcosis. The aim of this study was to verify the applicability of non-denaturing PAGE for analysis of laccases by quantification of the amount of melanin pigment produced by clinical and environmental strains of C. neoformans. After incubation of the gel with the substrate L-dopa, strains produced melanin spots of a bright brown to black colour. Quantification of these spots was performed by densitometry analysis and the amount of melanin produced was calculated and compared among the strains. All strains showed laccase activity. Serotype B strains showed a higher melanin intensity than serotype A strains. Over half of the clinical strains (56.2%) showed the lowest melanin intensities, suggesting that melanin production may not be the main virulence factor against host defence. The clinical strain ICB 88 revealed two melanin spots on the gel, indicating the presence of two laccase isoforms. The environmental strains showed the highest values of melanin intensity, which may be related to previous exposure to environmental stress conditions.

Molecular characterization of human polyomavirus JC in Brazilian AIDS patients with and without progressive multifocal leukoencephalopathy

Background: JC virus (JCV), the causative agent of progressive multifocal leukoencephalopathy (PML), is classified in 8 different genotypes. Previous reports have suggested a positive association between specific genotypes and PML. Objective: To compare genotypes and adaptive mutations of JCV strains from Brazilian AIDS patients with and without PML. Study design: The VP1 region of JCV was amplified by polymerase chain reaction from cerebrospinal fluid samples from 51 patients with PML and from urine samples of 47 patients with AIDS without central nervous system disease. Genotyping was done by phylogenetic analysis. Amino acid replacement and selection pressures were also investigated. Results: JCV genotype frequency distributions showed that genotypes 2 (32.7%), 1 (26.5%) and 3 (23.5%) were the most prevalent. Genotype 1 had a positive association (p < 0.0001) and genotype 3 showed an inverse association (p < 0.001) with PML. A previously undescribed point mutation at residue 91 (L/I or L/V) and (L/P), non-genotype-associated, was found in 5/49 (10.2%) and 2/47 (4.3%) JCV sequences from PML and non-PML patients, respectively. This mutation was under positive selection only in PML patients. A previously described substitution of T-A in position 128 showed a significant difference between PML and non-PML cases (70% versus 16%, respectively, p < 0.0005). Conclusion: In Brazilian patients with AIDS, JCV genotype 1 showed a strong association with PML (p < 0.0001) and JCV genotype 3 showed an inverse association with PML. The possible association of aminoacids substitution in residues 91 and 128 with PML in patients with AIDS must be further investigated. (C) 2010 Elsevier B.V. All rights reserved.

Factors associated with increased prevalence of human papillomavirus infection in a cohort of HIV-Infected Brazilian women

Objectives: Human papillomavirus (HPV) infection is a major risk factor for cervical disease. Using baseline data from the HIV-infected cohort of Evandro Chagas Clinical Research Institute at Fiocruz, Rio de Janeiro, Brazil, factors associated with an increased prevalence of HPV were assessed. Methods: Samples from 634 HIV-infected women were tested for the presence of HPV infection using hybrid capture 11 and polymerase chain reaction. Prevalence ratios (PR) were estimated using Poisson regression analysis with robust variance. Results: The overall prevalence of HPV infection was 48%, of which 94% were infected with a high-risk HPV. In multivariate analysis, factors independently associated with infection with high-risk HPV type were: younger age (<30 years of age; PR 1.5, 95% confidence interval (CI) 1.1-2.1), current or prior drug use (PR 1.3, 95% CI 1.0-1.6), self-reported history of HPV infection (PR 1.2, 95% CI 0.96-1.6), condom use in the last sexual intercourse (PR 1.3, 95% CI 1.1-1.7), and nadir CD4+ T-cell count <100 cells/mm(3) (PR 1.6, 95% CI 1.2-2.1). Conclusions: The estimated prevalence of high-risk HPV-infection among HIV-infected women from Rio de Janeiro, Brazil, was high. Close monitoring of HPV-related effects is warranted in all HIV-infected women, in particular those of younger age and advanced immunosuppression. (C) 2008 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.