Lsa30, a novel adhesin of Leptospira interrogans binds human plasminogen and the complement regulator C4bp

Pathogenic Leptospira is the etiological agent of leptospirosis, a life-threatening disease that affects populations worldwide. Surface proteins have the potential to promote several activities, including adhesion. This work aimed to study the leptospiral coding sequence (CDS) LIC11087, genome annotated as hypothetical outer membrane protein. The LIC11087 gene was cloned and expressed in Escherichia coil BL21 (DE3) strain by using the expression vector pAE. The recombinant protein tagged with N-terminal 6XHis was purified by metal-charged chromatography and characterized by circular dichroism (CD) spectroscopy. The recombinant protein has the ability to mediate attachment to the extracellular matrix (ECM) components, laminin and plasma fibronectin, and was named Lsa30 (Leptospiral surface adhesin of 30 kDa). Lsa30 binds to laminin and to plasma fibronectin in a dose-dependent and saturable manner, with dissociation equilibrium constants (K-D) of 292 +/- 24 nM and 157 +/- 35 nM, respectively. Moreover, the Lsa30 is a plasminogen (PLC) receptor, capable of generating plasmin, in the presence of activator. This protein may interfere with the complement cascade by interacting with C4bp regulator. The Lsa30 is probably a new surface protein of Leptospira as revealed by immunofluorescence assays with living organisms and the reactivity with antibodies present in serum samples of experimentally infected hamsters. Thus, Lsa30 is a novel versatile protein that may play a role in mediating adhesion and may help pathogenic Leptospira to overcome tissue barriers and to escape the immune system. (C) 2012 Elsevier Ltd. All rights reserved.

The genetic diversity of Plasmodium malariae and Plasmodium brasilianum from human, simian and mosquito hosts in Brazil

Plasmodium malariae is a protozoan parasite that causes malaria in humans and is genetically indistinguishable from Plasmodium brasilianum, a parasite infecting New World monkeys in Central and South America. P. malariae has a wide and patchy global distribution in tropical and subtropical regions, being found in South America, Asia, and Africa. However, little is known regarding the genetics of these parasites and the similarity between them could be because until now there are only a very few genomic sequences available from simian Plasmodium species. This study presents the first molecular epidemiological data for P. malariae and P. brasilianum from Brazil obtained from different hosts and uses them to explore the genetic diversity in relation to geographical origin and hosts. By using microsatellite genotyping, we discovered that of the 14 human samples obtained from areas of the Atlantic forest, 5 different multilocus genotypes were recorded, while in a sample from an infected mosquito from the same region a different haplotype was found. We also analyzed the longitudinal change of circulating plasmodial genetic profile in two untreated non-symptomatic patients during a 12-months interval. The circulating genotypes in the two samples from the same patient presented nearly identical multilocus haplotypes (differing by a single locus). The more frequent haplotype persisted for almost 3 years in the human population. The allele Pm09-299 described previously as a genetic marker for South American P. malariae was not found in our samples. Of the 3 non-human primate samples from the Amazon Region, 3 different multilocus genotypes were recorded indicating a greater diversity among isolates of P. brasilianum compared to P. malariae and thus, P. malariae might in fact derive from P. brasilianum as has been proposed in recent studies. Taken together, our data show that based on the microsatellite data there is a relatively restricted polymorphism of P. malariae parasites as opposed to other geographic locations. (c) 2012 Elsevier B.V. All rights reserved.

Morphological characterization of the nasopalatine region in human fetuses and its association to pathologies

The nasopalatine region is composed of structures such as the vomeronasal organ and nasopalatine duct. The nasopalatine duct may provide the communication of the mouth to the nasal cavity in human fetuses and can be obliterated in an adult human. Knowledge on the development of the nasopalatine region and nasopalatine duct in humans is necessary for understanding the morphology and etiopathogenesis of lesions that occur in this region. Objective: The aim of the present study was to describe the morphological aspects of the nasopalatine region in human fetuses and correlate these aspects with the development of pathologies in this region. Material and Methods: Five human fetuses with no facial or palatine abnormalities were used for the acquisition of specimens from the nasopalatine region. After demineralization, the specimens were histologically processed. Histological cuts were stained with methylene blue to orient the cutting plane and hematoxylin-eosin for the descriptive histological analysis. Results: The age of the fetuses was 8.00, 8.25, 9.00 and 9.25 weeks, and it was not possible to determine the age in the last one. The incisive canal was observed in all specimens as an opening delimited laterally by the periosteum and connecting oral and nasal cavity. The nasopalatine duct is an epithelial structure with the greatest morphological variation, with either unilateral or bilateral occurrence and total patent, partial patent and islet forms. The vomeronasal organ is a bilateral epithelized structure located alongside the nasal septum above the incisive canal in all the fetuses. Conclusions: The incisive canal, nasopalatine duct and vomeronasal organ are distinct anatomic structures. The development of nasopalatine duct cysts may occur in all forms of the nasopalatine duct.

Effects of water flow on ablation rate and morphological changes in human enamel and dentin after Er:YAG laser irradiation

Purpose: To investigate the laboratory effect of Er:YAG laser on ablation rate and morphological changes in human enamel and dentin with varying water flow. Methods: 23 human third molars were sectioned in mesio-distal and buccal-lingual directions. The slabs were flattened and weighted on an analytical laboratory balance (control). A 4-mm(2) area was demarcated and the samples were randomly assigned into three groups according to water flow employed during the laser irradiation (1.0, 1.5, and 2.0 mL/minute). An Er:YAG laser was used to ablate enamel (80.22-J/cm(2), 300 mJ/4Hz) and dentin (96.26-J/cm(2), 250 mJ/4Hz). After irradiation, the samples were immersed in distilled water for 1 hour and then weighted again. The final mass was obtained and laser-irradiated substrate mass loss was calculated by the difference between the initial and final mass. Afterwards, specimens were prepared for SEM. Results: Data were submitted to ANOVA and Tukey's test (P< 0.05). It was observed that the 2.0 mL/minute resulted in a higher mass loss, 1.0 mL/minute showed a lower mass loss, and 1.5 mL/minute demonstrated intermediate results (P< 0.05). The increase of water flow promoted less melting areas and cracks. Furthermore, dentin was more ablated than enamel. It may be concluded that the water flow of Er:YAG laser and the substrates affected the ablation rate. Among the tested parameters, 2.0 mL/minute improved the ability of ablation in enamel and dentin, with less morphologic surface alteration. (Am J Dent 20 12;25:332-336).

Expression of Clock Genes in Human Subcutaneous and Visceral Adipose Tissues

Disrupted circadian rhythms are associated with obesity and metabolic alterations, but little is known about the participation of peripheral circadian clock machinery in these processes. The aim of the present study was to analyze RNA expression of clock genes in subcutaneous (SAT) and visceral (VAT) adipose tissues of male and female subjects in AM (morning) and PM (afternoon) periods, and its interactions with body mass index (BMI). Ninety-one subjects (41 +/- 11 yrs of age) presenting a wide range of BMI (21.4 to 48.6 kg/m(2)) were included. SAT and VAT biopsies were obtained from patients undergoing abdominal surgeries. Clock genes expressions were evaluated by qRT-PCR. The only clock gene that showed higher expression (p < .0001) in SAT in comparison to VAT was PER1 of female (372%) and male (326%) subjects. Different patterns of expression between the AM and PM periods were observed, in particular REV-ERBa, which was reduced (p < .05) at the PM period in SAT and VAT of both women and men (women: similar to 53% lower; men: similar to 78% lower), whereas CLOCK expression was not altered. Relationships between clock genes were different in SAT vs. VAT. BMI was negatively correlated with SATPER1 (r = -.549; p = .001) and SATPER2 (r = -.613; p = .0001) and positively with VATCLOCK (r = .541; p = .001) and VATBMAL1 (r = .468; p = .007) only in women. These data suggest that the circadian clock machinery of adipose tissue depots differs between female and male subjects, with a sex-specific effect observed for some genes. BMI correlated with clock genes, but at this moment it is not possible to establish the cause-effect relationship. (Author correspondence: mzanquetta@gmail.com)

AN EFFICIENT HPLC-UV METHOD FOR THE QUANTITATIVE DETERMINATION OF CEFADROXIL IN HUMAN PLASMA AND ITS APPLICATION IN PHARMACOKINETIC STUDIES

Cefadroxil is a semi-synthetic first-generation oral cephalosporin used in the treatment of mild to moderate infections of the respiratory and urinary tracts, skin and soft tissue infections. In this work a simple, rapid, economic and sensitive HPLC-UV method is described for the quantitative determination of cefadroxil in human plasma samples using lamivudine as internal standard. Sample pre-treatment was accomplished through protein precipitation with acetonitrile and chromatographic separation was performed with a mobile phase consisting of a mixture of sodium dihydrogen phosphate monohydrate solution, methanol and acetonitrile in the ratio of 90:8:2 (v/v/v) at a flow rate of 1.0mL/min. The proposed method is linear between 0.4 to 40.0 mu g/mL and its average recovery is 102.21% for cefadroxil and 97.94% for lamivudine. The method is simple, sensitive, reproducible, less time consuming for determination of cefadroxil in human plasma. The method can therefore be recommended for pharmacokinetics studies, including bioavailability and bioequivalence studies.

Activity, movement and secretive behavior of a threatened arboreal folivore, the thin-spined porcupine, in the Atlantic forest of southern Bahia, Brazil

Activity and behavior patterns are important components of a given species ecological strategy, as they have profound implications for its survival and reproduction. Here, we studied the activities, movements and secretive behavior of the thin-spined porcupine Chaetomys subspinosus (Rodentia: Erethizontidae), a threatened arboreal folivore in the Brazilian Atlantic rainforest. We aimed to ascertain the behavioral strategies used by this species as well as its responses to seasonal and daily climatic changes. Four radio-collared individuals were followed continuously for 72-h in the summer and winter, as well as during 146 half-night sessions conducted from April 2005 to September 2006 in forest remnants in southern Bahia. The thin-spined porcupines were nocturnally active (17:30-05:40 h), with peaks in activity and movement from 19:00 to 20:00 h and 03:00 to 04:00 h. Animals followed a circadian rhythm of activity during both the summer and winter. During the diel cycle, porcupines spent 74% of their time resting, 14% feeding, 11% traveling and 2% performing other activities. Distance traveled during the diel cycle averaged 277.5 +/- 117.9 m sd. The mean movement rate during the night was 21.6 +/- 30.1 m/h sd. No significant changes in activity budget or daily distance traveled were observed between seasons, most likely in response to the low fluctuations in climatic conditions and food availability throughout the year in the study region. However, rainfall reduced the time that the animals spent on feeding activities and explained day-to-day differences in activity budgets. We also provide details about intraspecific interactions and defecation behavior. Our observations confirmed that thin-spined porcupines, similar to other folivorous species, present low activity levels and short daily movements, and have adopted various cryptic habits, such as nocturnality, a solitary lifestyle, the tendency to leave offspring alone most of the time and defecation in concealed latrines.

Judiciary as the last resort to protect children and adolescents: intersectoral actions, investment in human resources, and structuring of services

This study aimed to identify the work developed by the Judiciary to prevent sexual violence against children and adolescents within the family. The approach to social representations in a cultural perspective was used. The field study consisted in the 1st and 2nd Court of Crimes against Children and Adolescents, at the State Supreme Court of Pernambuco, Brazil. Participant observation, semi-structured interviews, and focus group with 17 subjects were the techniques for data collection, analyzed through the interpretation of meanings, allowing the identification of the category "The Judiciary as the ultimate level" and the following subcategories: "The public policies to prevent violence" and "The structure and dynamics of Courts". This study allows the visualization of the Judiciary's limitations with regard to the full protection and absolute priority, and that the work along with the victims demands investments in structure and human resources.

A DNA vaccine against tuberculosis based on the 65 kDa heat-shock protein differentially activates human macrophages and dendritic cells

Abstract Background A number of reports have demonstrated that rodents immunized with DNA vaccines can produce antibodies and cellular immune responses presenting a long-lasting protective immunity. These findings have attracted considerable interest in the field of DNA vaccination. We have previously described the prophylactic and therapeutic effects of a DNA vaccine encoding the Mycobacterium leprae 65 kDa heat shock protein (DNA-HSP65) in a murine model of tuberculosis. As DNA vaccines are often less effective in humans, we aimed to find out how the DNA-HSP65 stimulates human immune responses. Methods To address this question, we analysed the activation of both human macrophages and dendritic cells (DCs) cultured with DNA-HSP65. Then, these cells stimulated with the DNA vaccine were evaluated regarding the expression of surface markers, cytokine production and microbicidal activity. Results It was observed that DCs and macrophages presented different ability to uptake DNA vaccine. Under DNA stimulation, macrophages, characterized as CD11b+/CD86+/HLA-DR+, produced high levels of TNF-alpha, IL-6 (pro-inflammatory cytokines), and IL-10 (anti-inflammatory cytokine). Besides, they also presented a microbicidal activity higher than that observed in DCs after infection with M. tuberculosis. On the other hand, DCs, characterized as CD11c+/CD86+/CD123-/BDCA-4+/IFN-alpha-, produced high levels of IL-12 and low levels of TNF-alpha, IL-6 and IL-10. Finally, the DNA-HSP65 vaccine was able to induce proliferation of peripheral blood lymphocytes. Conclusion Our data suggest that the immune response is differently activated by the DNA-HSP65 vaccine in humans. These findings provide important clues to the design of new strategies for using DNA vaccines in human immunotherapy.