Expression of recombinant human mutant granulocyte colony stimulating factor (Nartograstim) in Escherichia coli

The human granulocyte colony stimulating factor (hG-CSF) plays an important role in hematopoietic cell proliferation/differentiation and has been widely used as a therapeutic agent for treating neutropenias. Nartograstim is a commercial G-CSF that presents amino acid changes in specific positions when compared to the wildtype form, which potentially increase its activity and stability. The aim of this work was to develop an expression system in Escherichia coli that leads to the production of large amounts of a recombinant hG-CSF (rhG-CSF) biosimilar to Nartograstim. The nucleotide sequence of hg-csf was codon-optimized for expression in E. coli. As a result, high yields of the recombinant protein were obtained with adequate purity, structural integrity and biological activity. This protein has also been successfully used for the production of specific polyclonal antibodies in mice, which could be used in the control of the expression and purification in an industrial production process of this recombinant protein. These results will allow the planning of large-scale production of this mutant version of hG-CSF (Nartograstim), as a potential new biosimilar in the market.

Effects of the combination of low-level laser irradiation and recombinant human bone morphogenetic protein-2 in bone repair

Low-level laser irradiation (LLLI) and recombinant human bone morphogenetic protein type 2 (rhBMP-2) have been used to stimulate bone formation. LLLI stimulates proliferation of osteoblast precursor cells and cell differentiation and rhBMP-2 recruits osteoprogenitor cells to the bone healing area. This in vivo study evaluated the effects of LLLI and rhBMP-2 on the bone healing process in rats. Critical bone defects were created in the parietal bone in 42 animals, and the animals were divided into six treatment groups: (1) laser, (2) 7 mu g of rhBMP-2, (3) laser and 7 mu g of rhBMP-2, (4) 7 mu g of rhBMP-2/monoolein gel, (5) laser and 7 mu g rhBMP-2/monoolein gel, and (6) critical bone defect controls. A gallium-aluminum-arsenide diode laser was used (wavelength 780 nm, output power 60 mW, beam area 0.04 cm(2), irradiation time 80 s, energy density 120 J/cm(2), irradiance 1.5 W/cm(2)). After 15 days, the calvarial tissues were removed for histomorphometric analysis. Group 3 defects showed higher amounts of newly formed bone (37.89%) than the defects of all the other groups (P < 0.05). The amounts of new bone in defects of groups 1 and 4 were not significantly different from each other (24.00% and 24.75%, respectively), but were significantly different from the amounts in the other groups (P < 0.05). The amounts of new bone in the defects of groups 2 and 5 were not significantly different from each other (31.42% and 31.96%, respectively), but were significantly different from the amounts in the other groups (P < 0.05). Group 6 defects had 14.10% new bone formation, and this was significantly different from the amounts in the other groups (P < 0.05). It can be concluded that LLLI administered during surgery effectively accelerated healing of critical bone defects filled with pure rhBMP-2, achieving a better result than LLLI alone or the use of rhBMP-2 alone.

Influence of low-level laser associated with osteogenic proteins recombinant human BMP-2 and Hevea brasiliensis on bone repair in Wistar rats

This study analyzed the newly formed bone tissue after application of recombinant human BMP-2 (rhBMP-2) and P-1 (extracted from Hevea brasiliensis) proteins, 2 weeks after the creation of a critical bone defect in male Wistar rats treated or not with a low-intensity laser (GaAlAs 780 nm, 60 mW of power, and energy density dose of 30 J/cm2). The animals were divided into two major groups: (1) bone defect plus low-intensity laser treatment and (2) bone defect without laser irradiation. The following subgroups were also analyzed: (a) 5 mu g of pure rhBMP-2; (b) 5 mu g of pure P-1 fraction; (c) 5 mu g of rhBMP-2/monoolein gel; (d) 5 mu g of P-1 fraction/monoolein gel; (e) pure monoolein gel. Comparisons of the groups receiving laser treatment with those that did not receive laser irradiation show differences in the areas of new bone tissue. The group treated with 5 mu g of rhBMP-2 and laser irradiation was not significantly different (P >0.05) than the nonirradiated group that received the same treatment. The irradiated, rhBMP-2/monoolein gel treatment group showed a lower area of bone formation than the nonirradiated, rhBMP-2/gel monoolein treatment group (P < 0.001). The area of new bone tissue in the other nonirradiated and irradiated groups was not significantly different (P > 0.05). Furthermore, the group that received the 5 mu g of rhBMP-2 application showed the greatest bone formation. We conclude that the laser treatment did not interfere with the area of new bone tissue growth and that the greatest stimulus for bone formation involved application of the rhBMP-2 protein. Microsc. Res. Tech. 2011. (c) 2011 Wiley Periodicals, Inc.

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.

Generation of a Chinese Hamster Ovary Cell Line Producing Recombinant Human Glucocerebrosidase

Impaired activity of the lysosomal enzyme glucocerebrosidase (GCR) results in the inherited metabolic disorder known as Gaucher disease. Current treatment consists of enzyme replacement therapy by administration of exogenous GCR. Although effective, it is exceptionally expensive, and patients worldwide have a limited access to this medicine. In Brazil, the public healthcare system provides the drug free of charge for all Gaucher's patients, which reaches the order of $ 84million per year. However, the production of GCR by public institutions in Brazil would reduce significantly the therapy costs. Here, we describe a robust protocol for the generation of a cell line producing recombinant human GCR. The protein was expressed in CHO-DXB11 (dhfr(-)) cells after stable transfection and gene amplification with methotrexate. As expected, glycosylated GCR was detected by immunoblotting assay both as cell-associated (similar to 64 and 59 kDa) and secreted (63-69 kDa) form. Analysis of subclones allowed the selection of stable CHO cells producing a secreted functional enzyme, with a calculated productivity of 5.14 pg/cell/day for the highest producer. Although being laborious, traditionalmethods of screening high-producing recombinant cellsmay represent a valuable alternative to generate expensive biopharmaceuticals in countries with limited resources.

Recent benthic foraminiferal distribution and related environmental factors in Ezcurra Inlet, King George Island, Antarctica

This investigation attempts to determine which environmental parameters of the bottom water and sediment control recent foraminifera fauna at Ezcurra Inlet (King George Island, Antarctica), using data collected during four summers (2002/03, 2003/04, 2004/05 and 2006/07). The study revealed that Ezcurra Inlet contain typical Antarctic foraminifera fauna with three distinct assemblages and few differences in environmental parameters. The species Bolivina pseudopunctata, Fursenkoina fusiformis, Portatrochammina antarctica, and Adercotryma glomerata were abundant in the samples. An elevated abundance, richness and diversity were common at the entrance of the inlet at depths greater than 55 m, where the inlet was characterized by low temperatures and muddy sand. In the inner part of the inlet (depth 30-55 m), richness and diversity were low and the most significant species were Cassidulinoides parkerianus, C. porrectus, and Psammosphaera fusca. Shallow waters showed low values of richness and abundance and high temperatures coupled with coarser sediment. In areas with high suspended matter concentrations and pH values associated with low salinity the most representative species were Hippocrepinella hirudinea and Hemisphaerammina bradyi.

Perinatal and early adulthood factors associated with adiposity

We used body mass index (BMI) and waist circumference (WC) as fat indicators to assess whether perinatal and early adulthood factors are associated with adiposity in early adulthood. We hypothesized that risk factors differ between men and women and are also different when WC is used for measuring adiposity as opposed to BMI. We conducted a longitudinal study based on a sample of 2,063 adults from the 1978/1979 Ribeirao Preto birth cohort. Adjustment was performed using four sequential multiple linear regression models stratified by sex. Both perinatal and early adulthood variables influenced adulthood BMI and WC. The associations differed between men and women and depending on the measure of abdominal adiposity (BMI or WC). Living with a partner, for both men and women, and high fat and alcohol intake in men were factors that were consistently associated with higher adulthood BMI and WC levels. The differences observed between sexes may point to different lifestyles of men and women, suggesting that prevention policies should consider gender specific strategies.

Relationship Between Oral Health-Related Quality of Life, Oral Health, Socioeconomic, and General Health Factors in Elderly Brazilians

Objectives To assess the impact of oral health on quality of life in elderly Brazilians and to evaluate its association with clinical oral health measures and socioeconomic and general health factors. Design Cross-sectional study. Setting Population-based cohort study on health, well-being, and aging. Participants Eight hundred fifty-seven participants representing 588,384 community-dwelling elderly adults from the city of Sao Paulo, Brazil. MeasurementS Self-perceived impact of oral health on quality of life was measured using the Geriatric Oral Health Assessment Index (GOHAI), with scores categorized as good, moderate, or poor, indicating low, moderate, and high degrees of negative impact on quality of life, respectively. Results Nearly half of the individuals had good GOHAI scores (44.7% of overall sample, 45.9% of dentate participants, and 43.4% of edentulous participants). In the overall sample, those with poor self-rated general health and a need for dental prostheses were more likely to have poor and moderate GOHAI scores. Individuals with depression were significantly more likely to have poor GOHAI scores. No socioeconomic variables were related to the outcome, except self-perception of sufficient income, which was a protective factor against a poor GOHAI score in dentate participants. Conclusion Moderate and high degrees of negative impact of oral health on quality of life were associated with general health and clinical oral health measures, independent of socioeconomic factors.

Physical and psychosocial risk factors for musculoskeletal disorders in Brazilian and Italian nurses

As part of the international CUPID investigation, we compared physical and psychosocial risk factors for musculoskeletal disorders among nurses in Brazil and Italy. Using questionnaires, we collected information on musculoskeletal disorders and potential risk factors from 751 nurses employed in public hospitals. By fitting country-specific multiple logistic regression models, we investigated the association of stressful physical activities and psychosocial characteristics with site-specific and multisite pain, and associated sickness absence. We found no clear relationship between low back pain and occupational lifting, but neck and shoulder pain were more common among nurses who reported prolonged work with the arms in an elevated position. After adjustment for potential confounding variables, pain in the low back, neck and shoulder, multisite pain, and sickness absence were all associated with somatizing tendency in both countries. Our findings support a role of somatizing tendency in predisposition to musculoskeletal disorders, acting as an important mediator of the individual response to triggering exposures, such as work-load.

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).

Perinatal and early life factors associated with symptoms of depression in Brazilian children

Background: Few studies have been conducted on the association between perinatal and early life factors with childhood depression and results are conflicting. Our aim was to estimate the prevalence and perinatal and early life factors associated with symptoms of depression in children aged 7 to 11 years from two Brazilian birth cohorts. Methods: The study was conducted on 1444 children whose data were collected at birth and at school age, in 1994 and 2004/2005 in Ribeirao Preto, where they were aged 10-11 years and in 1997/98 and 2005/06 in Sao Luis, where children were aged 7-9 years. Depressive symptoms were investigated with the Child Depression Inventory (CDI), categorized as yes (score >= 20) and no (score < 20). Adjusted and non-adjusted prevalence ratios (PR) were estimated by Poisson regression with robust estimation of the standard errors. Results: The prevalence of depressive symptoms was 3.9% (95% CI = 2.5-5.4) in Ribeirao Preto and 13.7% (95% CI = 11.0-16.4) in Sao Luis. In the adjusted analysis, in Ribeirao Preto, low birth weight (PR = 3.98; 95% CI = 1.72-9.23), skilled and semi-skilled manual occupation (PR = 5.30; 95% CI = 1.14-24.76) and unskilled manual occupation and unemployment (PR = 6.65; 95% CI = 1.16-38.03) of the household head were risk factors for depressive symptoms. In Sao Luis, maternal schooling of 0-4 years (PR = 2.39; 95% CI = 1.31-4.34) and of 5 to 8 years (PR = 1.80; 95% CI = 1.08-3.01), and paternal age < 20 years (PR = 1.92; 95% CI = 1.02-3.61), were independent risk factors for depressive symptoms. Conclusions: The prevalence of depressive symptoms was much higher in the less developed city, Sao Luis, than in the more developed city, Ribeirao Preto, and than those reported in several international studies. Low socioeconomic level was associated with depressive symptoms in both cohorts. Low paternal age was a risk factor for depressive symptoms in the less developed city, Sao Luis, whereas low birth weight was a risk factor for depressive symptoms in the more developed city, Ribeirao Preto.

Genetic and environmental risk factors in congenital heart disease functionally converge in protein networks driving heart development

Congenital heart disease (CHD) occurs in similar to 1% of newborns. CHD arises from many distinct etiologies, ranging from genetic or genomic variation to exposure to teratogens, which elicit diverse cell and molecular responses during cardiac development. To systematically explore the relationships between CHD risk factors and responses, we compiled and integrated comprehensive datasets from studies of CHD in humans and model organisms. We examined two alternative models of potential functional relationships between genes in these datasets: direct convergence, in which CHD risk factors significantly and directly impact the same genes and molecules and functional convergence, in which risk factors significantly impact different molecules that participate in a discrete heart development network. We observed no evidence for direct convergence. In contrast, we show that CHD risk factors functionally converge in protein networks driving the development of specific anatomical structures (e.g., outflow tract, ventricular septum, and atrial septum) that are malformed by CHD. This integrative analysis of CHD risk factors and responses suggests a complex pattern of functional interactions between genomic variation and environmental exposures that modulate critical biological systems during heart development.

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.

Spatial analysis of leprosy incidence and associated socioeconomic factors

OBJECTIVE: To identify clusters of the major occurrences of leprosy and their associated socioeconomic and demographic factors. METHODS: Cases of leprosy that occurred between 1998 and 2007 in Sao Jose do Rio Preto (southeastern Brazil) were geocodified and the incidence rates were calculated by census tract. A socioeconomic classification score was obtained using principal component analysis of socioeconomic variables. Thematic maps to visualize the spatial distribution of the incidence of leprosy with respect to socioeconomic levels and demographic density were constructed using geostatistics. RESULTS: While the incidence rate for the entire city was 10.4 cases per 100,000 inhabitants annually between 1998 and 2007, the incidence rates of individual census tracts were heterogeneous, with values that ranged from 0 to 26.9 cases per 100,000 inhabitants per year. Areas with a high leprosy incidence were associated with lower socioeconomic levels. There were identified clusters of leprosy cases, however there was no association between disease incidence and demographic density. There was a disparity between the places where the majority of ill people lived and the location of healthcare services. CONCLUSIONS: The spatial analysis techniques utilized identified the poorer neighborhoods of the city as the areas with the highest risk for the disease. These data show that health departments must prioritize politico-administrative policies to minimize the effects of social inequality and improve the standards of living, hygiene, and education of the population in order to reduce the incidence of leprosy.