Evaluation of InnoTyper® 21 in a sample of Rio de Janeiro population as an alternative forensic panel.

The use of bi-allelic markers such as retrotransposable element insertion polymorphisms or Innuls (for insertion/null) can overcome some limitations of short tandem repeat (STR) loci in typing forensic biological evidence. This study investigated the efficiency of the InnoTyper® 21 Innul markers in an urban admixed population sample in Rio de Janeiro (n = 40) and one highly compromised sample collected as evidence by the Rio de Janeiro police. No significant departures from Hardy-Weinberg equilibrium were detected after the Bonferroni correction (α' ≈ 0.05/20, p < 0.0025), and no significant linkage disequilibrium was observed between markers. Assuming loci independence, the cumulative random match probability (RMP) was 2.3 × 10-8. A lower mean Fis value was obtained for this sample population compared with those of three North American populations (African-American, Southwest Hispanic, US Caucasian). Principal component analysis with the three North American populations and one from 21 East Asian population showed that African Americans segregated as an independent group while US Caucasian, Southwest Hispanic, East Asian, and Rio de Janeiro populations are in a single large heterogeneous group. Also, a full Innuls profile was produced from an evidence sample, despite the DNA being highly degraded. In conclusion, this system is a useful complement to standard STR kits.

A segment of rbcL gene as a potential tool for forensic discrimination of Cannabis sativa seized at Rio de Janeiro, Brazil.

Cannabis sativa, known by the common name marijuana, is the psychoactive drug most widely distributed in the world. Identification of Cannabis cultivars may be useful for association to illegal crops, which may reveal trafficking routes and related criminal groups. This study provides evidence for the performance of a segment of the rbcL gene, through genetic signature, as a tool for identification for C. sativa samples apprehended by the Rio de Janeiro Police, Brazil. The PCR amplified and further sequenced the fragment of approximately 561 bp of 24 samples of C. sativa rbcL gene and showed the same nucleotide sequences, suggesting a possible genetic similarity or identical varieties. Comparing with other Cannabaceae family sequences, we have found 99% of similarity between the Rio de Janeiro sequence and three other C. sativa rbcL genes. These findings suggest that the fragment utilized at this study is efficient in identifying C. sativa samples, therefore, useful in genetic discrimination of samples seized in forensic cases.

Tooth discolouration and internal bleaching after the use of triple antibiotic paste.

AIM: To assess the discolouration of teeth with closed and open apices after placement of triple antibiotic paste (TAP, ciprofloxacin, metronidazole and minocycline) in the pulp chamber and whether discolouration could be reversed by internal bleaching procedures. METHODOLOGY: Twenty extracted human mandibular premolars were divided into 2 groups (n = 10): teeth with closed apices (CA) and teeth with open apices (OA). After conventional access, the TAP was sealed in the pulp chamber for 3 weeks. The paste was removed by a rinse with sodium hypochlorite (NaOCl) and a mixture of sodium perborate and distilled water was sealed in the pulp chamber for 1, 2 and 3 weeks. The shade was measured by a spectrophotometer at six time periods: baseline (T0), after 3 weeks of placement of TAP (T1), after removal of TAP with a NaOCl rinse (T2) and after 1 (T3), 2 (T4) and 3 (T5) weeks of internal bleaching with sodium perborate paste. Data were collected based on the CIELAB-CIE1976 (L*a*b*) system and analysed using t-tests and anova. RESULTS: A significant decrease in the mean values of L* (lightness) was observed after treatment with TAP (T1, P < 0.05). Considerable increases in these values after bleaching with sodium perborate (T3 < T4 < T5) were found in both groups. The only significant difference in the intergroup analysis was between T1 and T2, in which ΔE values in the OA group were higher (P = 0.04). CONCLUSIONS: TAP discoloured the tooth structure, but discolouration could be reversed with sodium perborate paste. In general, teeth with closed and open apices had the same rates of discolouration and bleaching.

Evaluation of a 49 InDel Marker HID panel in two specific populations of South America and one population of Northern Africa.

The majority of STR loci are not ideal for the analysis of forensic samples with degraded and/or low template DNA. One alternative to overcome these limitations is the use of bi-allelic markers, which have low mutation rates and shorter amplicons. Human identification (HID) InDel marker panels have been described in several countries, including Brazil. The commercial kit available is, however, mostly suitable for Europeans, with lower discrimination power for other population groups. Recently, a combination of 49 InDel markers used in four different ethnic groups in the USA has been shown to be more informative than another panel from Portugal, already tested in a Rio de Janeiro sample. However, these 49 InDels have yet to be applied to other admixed or isolated populations. We assessed the efficiency of this panel in two urban admixed populations (Rio de Janeiro, Brazil; Tripoli, Libya) and one isolated Native Brazilian community. All markers are in Hardy-Weinberg equilibrium (HWE) after the Bonferroni correction, and no Linkage disequilibrium was detected. Assuming loci independence and no substructure effect, cumulative RMP was 2.7×10(-18), 1.5×10(-20), and 4.5×10(-20) for Native Brazilian, Rio de Janeiro, and Tripoli populations, respectively. The overall Fst value was 0.05512. Rio de Janeiro and Tripoli showed similar admixture levels, however for Native Brazilians one parental cluster represented over 60 % of the total parental population. We conclude that this panel is suitable for HID on these urban populations, but is less efficient for the isolated group.

Biosynthesis of Silver Nanoparticles from Marine Seaweed Sargassum cinereum and their Antibacterial Activity.

Seaweed extracts of Sargassum cinereum was used as a reducing agent in the eco-friendly extracellular synthesis of silver nanoparticles from an aqueous solution of silver nitrate (AgNO3). High conversion of silver ions to silver nanoparticles was achieved with a reaction temperature of 100(°) and a seaweed extract concentration of 10% with a residential time of 3 h. Formation of silver nanoparticles was characterised by spectrophotometry and the scanning electron microscope. The average particles size was ranging from 45 to 76 nm. Antimicrobial activities indicate the minimum inhibitory concentration of biologically synthesised nanoparticles tested against the pathogen Staphylococcus aureus with 2.5 µl (25 µg/disc). High inhibitions over the growth of Enterobacter aerogenes, Salmonella typhi and Proteus vulgaris were witnessed against the concentrations of 100 µg/disc. Promising potential and the future prospects of S. cinereum nanoparticles in pharmaceutical research are the highlights in this paper.

Antagonistic activity of the fungus Pochonia chlamydosporia on mature and immature Toxocara canis eggs.

In vitro tests were performed to evaluate the ability of 6 isolates of the nematophagous fungus Pochonia chlamydosporia to infect immature and mature Toxocara canis eggs on cellulose dialysis membrane. There was a direct relationship between the number of eggs colonized and the increase in the days of interaction, as well as between the number of eggs colonized and the increase in the concentration of chlamydospores (P<0.05). Immature eggs were more susceptible to infection than mature eggs. The isolate Pc-04 was the most efficient egg parasite until the 7th day, and showed no difference in capacity to infect mature and immature eggs in comparison to Pc-07 at 14 and 21 days of interaction, respectively. Isolate Pc-04 was the most infective on the two evolutionary phases of the eggs at most concentrations, but its ability to infect immature eggs did not differ from that presented by the isolates Pc-07 and Pc-10 at the inoculum level of 5000 chlamydospores. Colonization of infective larvae inside or outside the egg was observed in treatments with the isolates Pc-03, Pc-04, Pc-07 and Pc-10. The isolate Pc-04 of P. chlamydosporia has great biological capacity to destroy immature and mature T. canis eggs in laboratory conditions.

Prevalence of clue cells suggestive for Gardnerella vaginalis in population-based cervical screening in the public versus private health care in Maringá, Paraná, Brazil.

OBJECTIVES: To determine the prevalence of clue cells suggestive for Gardnerella vaginalis in population-based cervical screening in the public (SUS) versus private (RP) health care utilization in Maringá, Paraná, Brazil. METHODS: A retrospective study on the results of 133,966 cytology examinations of patients from the Brazilian Public Health System-SUS (better socio-economic status-SES) and from the Private Network-RP (low SES) done by the Souza Anatomy Laboratory in Maringá, Brazil, from 2004 to 2007. RESULTS: In patients using the SUS, the prevalence of clue cells was 6.75% (n = 3,573), higher (p < 0.05) than for RP users (3.53%, n = 2,813). The clue cells predominated in age range of the 20-49 years from the SUS and RP (p > 0.05). In both the populations, the prevalence and age range did not vary among the 4 years (p > 0.05) and the prevalence declines in age above 45 years (p < 0.05). CONCLUSIONS: The prevalence of clue cells was low and stable in both populations in the 4 years, indicating equilibrium in terms of numbers of bacterial vaginosis (BV) in this localization. The results indicate that SES may influence the epidemiology of BV and that the prevalence declines expressively in older women.

Relation of pretreatment sequence diversity in NS5A region of HCV genotype 1 with immune response between pegylated-INF/ribavirin therapy outcomes.

Hepatitis C virus (HCV) infection frequently persists despite substantial virus-specific immune responses and the combination of pegylated interferon (INF)-α and ribavirin therapy. Major histocompatibility complex class I restricted CD8(+) T cells are responsible for the control of viraemia in HCV infection, and several studies suggest protection against viral infection associated with specific HLAs. The reason for low rates of sustained viral response (SVR) in HCV patients remains unknown. Escape mutations in response to cytotoxic T lymphocyte are widely described; however, its influence in the treatment outcome is ill understood. Here, we investigate the differences in CD8 epitopes frequencies from the Los Alamos database between groups of patients that showed distinct response to pegylated α-INF with ribavirin therapy and test evidence of natural selection on the virus in those who failed treatment, using five maximum likelihood evolutionary models from PAML package. The group of sustained virological responders showed three epitopes with frequencies higher than Non-responders group, all had statistical support, and we observed evidence of selection pressure in the last group. No escape mutation was observed. Interestingly, the epitope VLSDFKTWL was 100% conserved in SVR group. These results suggest that the response to treatment can be explained by the increase in immune pressure, induced by interferon therapy, and the presence of those epitopes may represent an important factor in determining the outcome of therapy.

Relation of pretreatment sequence diversity in NS5A region ofHCV genotype 1 with immune response between pegylated- INF/ribavirin therapy outcomes

Hepatitis C virus (HCV) infection frequently persists despite substantial virus-specific immune responsesand the combination of pegylated interferon (INF)-a and ribavirin therapy. Major histocompatibility complex class Irestricted CD8+ T cells are responsible for the control of viraemia in HCV infection, and several studies suggestprotection against viral infection associated with specific HLAs. The reason for low rates of sustained viral response (SVR) in HCV patients remains unknown. Escape mutations in response to cytotoxic T lymphocyte are widely described; however, its influence in the treatment outcome is ill understood. Here, we investigate the differences in CD8 epitopes frequencies from the Los Alamos database between groups of patients that showed distinct response to pegylated a-INF with ribavirin therapy and test evidence of natural.

Hepatitis B virus and hepatitis delta virus genotypes in outbreaks of fulminant hepatitis (Labrea black fever) in the western Brazilian Amazon region.

The genotypes of hepatitis B (HBV) and delta (HDV) viruses circulating among fulminant hepatitis cases from the western Amazon Basin of Brazil were characterized in this study. HBV and HDV isolates were obtained from liver samples from 14 patients who developed fulminant hepatitis and died during 1978-1989. HBV DNA and HDV RNA were detected in all samples. Phylogenetic analyses of HDV sequences showed that they all clustered with previously characterized sequences of HDV genotype 3 (HDV-3). HBV genotypes F, A and D were found in 50.0, 28.6 and 21.4 % of cases, respectively. These results confirm the predominance of HDV-3 in South America and its association with the severe form of hepatitis, and the finding of the co-infection of HDV-3 with different genotypes of HBV suggests that the association between HDV-3 and HBV-F is not necessarily causally related to a more severe clinical course of infection.

Conservation of hepatitis C virus nonstructural protein 3 amino acid sequence in viral isolates during liver transplantation.

As a consequence of selective pressure exerted by the immune response during hepatitis C virus (HCV) infection, a high rate of nucleotide mutations in the viral genome is observed which leads to the emergence of viral escape mutants. The aim of this study was to evaluate the evolution of the amino acid (aa) sequence of the HCV nonstructural protein 3 (NS3) in viral isolates after liver transplantation. Six patients with HCV-induced liver disease undergoing liver transplantation (LT) were followed up for sequence analysis. Hepatitis C recurrence was observed in all patients after LT. The rate of synonymous (dS) nucleotide substitutions was much higher than that of nonsynonymous (dN) ones in the NS3 encoding region. The high values of the dS/dN ratios suggest no sustained adaptive evolution selection pressure and, therefore, absence of specific NS3 viral populations. Clinical genotype assignments were supported by phylogenetic analysis. Serial samples from each patient showed lower mean nucleotide genetic distance when compared with samples of the same HCV genotype and subtype. The NS3 samples studied had an N-terminal aa sequence with several differences as compared with reference ones, mainly in genotype 1b-infected patients. After LT, as compared with the sequences before, a few reverted aa substitutions and several established aa substitutions were observed at the N-terminal of NS3. Sites described to be involved in important functions of NS3, notably those of the catalytic triad and zinc binding, remained unaltered in terms of aa sequence. Rare or frequent aa substitutions occurred indiscriminately in different positions. Several cytotoxic T lymphocyte epitopes described for HCV were present in our 1b samples. Nevertheless, the deduced secondary structure of the NS3 protease showed a few alterations in samples from genotype 3a patients, but none were seen in 1b cases. Our data, obtained from patients under important selective pressure during LT, show that the NS3 protease remains well conserved, mainly in HCV 3a patients. It reinforces its potential use as an antigenic candidate for further studies aiming at the development of a protective immune response.