Magnetic nanoparticles hyperthermia in a non-adiabatic and radiating process.

We investigate the magnetic nanoparticles hyperthermia in a non-adiabatic and radiating process through the calorimetric method. Specifically, we propose a theoretical approach to magnetic hyperthermia from a thermodynamic point of view. To test the robustness of the approach, we perform hyperthermia experiments and analyse the thermal behavior of magnetite and magnesium ferrite magnetic nanoparticles dispersed in water submitted to an alternating magnetic field. From our findings, besides estimating the specific loss power value from a non-adiabatic and radiating process, thus enhancing the accuracy in the determination of this quantity, we provide physical meaning to a parameter found in literature that still remained not fully understood, the effective thermal conductance, and bring to light how it can be obtained from experiment. In addition, we show our approach brings a correction to the estimated experimental results for specific loss power and effective thermal conductance, thus demonstrating the importance of the heat loss rate due to the thermal radiation in magnetic hyperthermia.

Violacein antimicrobial activity on Staphylococcus epidermidis and synergistic effect on commercially available antibiotics.

AIMS: The study aimed to assess whether violacein has antimicrobial activity on Staphylococcus epidermidis and synergistically modulates the action of commercially available antimicrobial drugs. METHODS AND RESULTS: Violacein showed excellent antimicrobial activity on biofilm-forming and nonbiofilm-forming S. epidermidis strains (ATCC 35984) (ATCC 12228), with bacteriostatic (MIC = 20 µg ml-1 and 10 µg ml-1 respectively) and bactericidal effects (MBC = 20 µg ml-1 for both strains), observed in short periods of exposure. The violacein bactericidal concentration led to S. epidermidis death after 2-3 h of exposure. Additionally, violacein synergistically modulated the activity of different antimicrobial classes on S. epidermidis ATCC 12228 (81·8%; n = 9) and on S. epidermidis ATCC 35984 (54·5%; n = 6), reducing the MIC of these antibiotics by up to 16-fold. CONCLUSION: Violacein shows excellent antimicrobial activity on S. epidermidis strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Violacein shows the potential for the development of a new drug for the treatment of infections caused by S. epidermidis.

Influence of estrogen therapy on immune markers in postmenopausal women.

OBJECTIVE: To evaluate the impact of estrogen therapy on cellular and humoral immune markers in postmenopausal women. METHODS: This prospective, controlled cohort study included 30 patients who used oral estradiol (1 mg) for 14-17 weeks and 28 patients who served as controls. Total leukocytes and leukocyte subtypes were counted and immunophenotyped by flow cytometry. The concentrations of immunoglobulins and pro- and anti-inflammatory cytokines were also measured in the peripheral blood before and after estrogen therapy. Immunoglobulin E level was measured by electrochemiluminescence, and levels of immunoglobulins A, G, and M were measured by nephelometry. Simultaneous quantification of multiple cytokines was performed by chemiluminescence to measure the serum concentrations of interferon gamma, interleukin (IL)-4, IL-6, IL-10, and IL-17. RESULTS: Hematological cellular components were not significantly different before and after the use of estradiol (p = 0.332-0.984). Serum concentrations of immunoglobulins G, M, E, and A also remained stable (p = 0.248-0.845). Finally, cytokines were not modified throughout the 14-17 weeks of follow-up (p = 0.407-0.873). CONCLUSION: Isolated estrogen therapy with 1 mg of estradiol for 14-17 weeks in postmenopausal women did not modify any of the cellular or humoral immune markers analyzed in this study.

GeLC-MS-based proteomics of Chromobacterium violaceum: comparison of proteome changes elicited by hydrogen peroxide.

Chromobacterium violaceum is a free-living bacillus with several genes that enables it survival under different harsh environments such as oxidative and temperature stresses. Here we performed a label-free quantitative proteomic study to unravel the molecular mechanisms that enable C. violaceum to survive oxidative stress. To achieve this, total proteins extracted from control and C. violaceum cultures exposed during two hours with 8 mM hydrogen peroxide were analyzed using GeLC-MS proteomics. Analysis revealed that under the stress condition, the bacterium expressed proteins that protected it from the damage caused by reactive oxygen condition and decreasing the abundance of proteins responsible for bacterial growth and catabolism. GeLC-MS proteomics analysis provided an overview of the metabolic pathways involved in the response of C. violaceum to oxidative stress ultimately aggregating knowledge of the response of this organism to environmental stress. This study identified approximately 1500 proteins, generating the largest proteomic coverage of C. violaceum so far. We also detected proteins with unknown function that we hypothesize to be part of new mechanisms related to oxidative stress defense. Finally, we identified the mechanism of clustered regularly interspaced short palindromic repeats (CRISPR), which has not yet been reported for this organism.

Is glycated hemoglobin related to other dysmetabolic variables implicated in the increase of cardiovascular risk in polycystic ovary syndrome? A comparative study.

BACKGROUND: In non-PCOS patients the concentration of glycated hemoglobin (HbA1C) has been employed to identify individuals at higher risk for impaired glucose tolerance (IGT) and diabetes mellitus. A few studies have examined the role of HbA1C in PCOS patients and current results are controversial. AIM: To compare the strength of the association between glycated hemoglobin and other predictors of cardiovascular risk in polycystic ovary syndrome (PCOS). METHODS: This cross-sectional study enrolled 197 PCOS patients and 72 non-PCOS women. Transvaginal ultrasound, biochemical and hormone measurement were performed. Glycated hemoglobin (HbA1C) was correlated with other variables related to dysmetabolic/vascular diseases. RESULTS: The HbA1C levels were 6.0±1.4% and 4.9±0.4% in PCOS patients and non-PCOS controls, respectively (p<0.001). The HbA1C levels were≥5.7% in 46.4% of PCOS and in none of the control subjects (OR=90.8). HbA1C was well-correlated with several anthropometric, metabolic and endocrine parameters. Stepwise multiple regression including HbA1C and other known predictors of cardiovascular risk resulted in a significant model in which body mass index (BMI) and free testosterone exhibited the best correlation with HbA1C (adjusted R(2)=0.530; F=39.8; p<0.001). CONCLUSION: HbA1C was elevated and correlated with anthropometric, biochemical and endocrine variables of metabolic/vascular disease risks in PCOS patients. Combined HbA1C, BMI and free testosterone levels provided a significant model with potential use to evaluate metabolic/vascular disease in PCOS patients.

Identification, characterisation and molecular modelling of two AP endonucleases from base excision repair pathway in sugarcane provide insights on the early evolution of green plants.

Unlike bacteria and mammals, plant DNA repair pathways are not well characterised, especially in monocots. The understanding of these processes in the plant cell is of major importance, since they may be directly involved in plant acclimation and adaptation to stressful environments. Hence, two sugarcane ESTs were identified as homologues of AP endonuclease from the base-excision repair pathway: ScARP1 and ScARP3. In order to understand their probable function and evolutionary origin, structural and phylogenetic studies were performed using bioinformatics approaches. The two predicted proteins present a considerable amino acid sequence similarity, and molecular modelling procedures indicate that both are functional, since the main structural motifs remain conserved. However, inspection of the sort signal regions on the full-length cDNAs indicated that these proteins have a distinct organelle target. Furthermore, variances in their promoter cis-element motifs were also found. Although the mRNA expression pattern was similar, there were significant differences in their expression levels. Taken together, these data raise the hypothesis that the ScARP is an example of a probable gene duplication event that occurred before monocotyledon/dicotyledon segregation, followed by a sub-functionalisation event in the Poaceae, leading to new intracellular targeting and different expression levels.

Cellular and humoral immune responses after short-term oral hormone therapy in postmenopausal women.

OBJECTIVE: To evaluate the cellular and humoral immune responses after oral hormone therapy in postmenopausal women. Study design This was a prospective cohort study, with intervention. The main outcome measures were delayed-type IV cell-mediated hypersensitivity, leukocytes, immunoglobulins, interleukin-6 (IL-6) and interleukin-10 (IL-10). METHODS: The delayed-type cell-mediated hypersensitivity was measured by using five common allergens before and after 3 months of hormone therapy. Each type of leukocyte cell was counted before and after hormone therapy. Different subtypes of lymphocytes were determined by flow cytometry. Immunoglobulins G, A and M were measured by nephelometry; immunoglobulin E was measured by electrochemiluminescence. IL-6 and IL-10 concentrations were determined by chemiluminescence. RESULTS: Hormone therapy increased the response to tuberculin antigen without changing the total number of leukocytes, eosinophils, neutrophils, lymphocytes, and CD4 +, CD8 + B cells. Both monocyte number and CD4 + /CD8 + ratio suffered a slight modification (p = 0.057). Immunoglobulins A, M and E remained unchanged and immunoglobulin G decreased (p = 0.029). IL-6 levels remained stable but IL-10 concentrations increased significantly after hormone therapy. CONCLUSION: Short-term oral hormone treatment has no impact on the cellular immune response but, concerning the humoral immune response, immunoglobulin G decreased and the levels of IL-10 were significantly higher.

Genotoxicity assessment in aquatic environment impacted by the presence of heavy metals.

The aim of this study was to access the genotoxic potential of Extremoz Lake waters in Northeastern Brazilian coast, using the Allium cepa system, piscine micronucleus test and comet assay. In addition, heavy metal levels were quantified by atomic absorption flame spectrometry. The results of the A. cepa system showed significant changes in the frequency of chromosome aberrations and in the mitotic index compared to negative control. No significant changes were observed in micronuclei frequency in the erythrocytes of Oreochromis niloticus. The comet assay showed a statistically significant alteration in the level of DNA breaks of O. niloticus. Chemical analysis detected an increase in heavy metal levels in different sampling periods. These results point out a state of deterioration of water quality at Extremoz Lake, caused by heavy metal contamination and genotoxic activity. It is recommended to establish a monitoring program for the presence of genotoxic agents in this water lake.

Human choriogonadotrophin protein core and sugar branches heterogeneity: basic and clinical insights.

BACKGROUND: Human chorionic gonadotrophin (hCG) is measured in serum and urine for the early detection of ectopic pregnancy, patients with higher risk of miscarriage, embryos or fetuses with chromosome abnormalities, prediction of pre-eclampsia or fetal growth restriction and identification or follow-up of trophoblast neoplasia. This review examines basic knowledge on the heterogeneity of hCG protein core and sugar branches and its relevance to assays used in a clinical setting. METHODS: The databases Scielo and Medline/Pubmed were consulted for identification of the most relevant published papers. Search terms were gonadotrophin, glycoprotein structure, hCG structure and molecular forms of hCG. RESULTS: The synthesis of alpha (hCGalpha) and beta (hCGbeta) peptide chains and their further glycosylation involve the complex action of different enzymes. After assembly, hCG reaches the cell surface and is secreted as a bioactive heterodimer. The complex cascade of enzymes acting in hCG secretion results in heterogeneous molecular forms. The hCG molecules are differently metabolized by the liver, ovary and kidney, but the majority of hCG forms are excreted in the urine. Intact hCG, hCGalpha, hCGbeta, hyperglycosylated (hCGh), nicked (hCGn) and core fragment of hCGbeta (hCGbetacf) forms have relevant clinical use. The immunogenicity of each hCG variant, their epitopes distribution and the available antibodies are important for the development of specific assays. Depending on the prevalent form or proportion in relation to the intact hCG, the choice of assay for measurement of a specific molecule in a particular clinical setting is paramount. CONCLUSIONS: Measurement of hCG and/or its related molecules is useful in clinical practice, but greater awareness is needed worldwide regarding the use of new sensitive and specific assays tailored for different clinical applications.

Gonadotrophin dynamics during reproductive life.

OBJECTIVE: To evaluate the follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels in early follicular phase throughout the reproductive years. METHOD: FSH and LH concentrations were determined by radioimmunoassay (RIA). Linear and polynomial regressions were carried out considering basal FSH as the dependent and age as the independent variable. RESULTS: FSH levels increased throughout the reproductive years (P<0.025). A positive correlation between age and basal FSH levels was detected (P<0.05). The Pearson squared coefficient of r(2)=0.889 was obtained. Using polynomial regression, the inclination of the parabole (Y=7.97-0.009x+0.057x(2)) was 0.359 and the generalized correlation coefficient was r=0.795. The goodness of fit analysis showed that the parabole may better represent the phenomenon (F=4.7; P<0.05). The LH levels remained constant, increasing only beyond 40 years of age. CONCLUSION: The FSH levels rose in a nonlinear way during the reproductive life and the LH concentrations increased discreetly only in patients over 40 years of age.

Functional interactions between the estrogen receptor and the transcription activator Sp1 regulate the estrogen-dependent transcriptional activity of the vitellogenin A1 io promoter.

Two distinct, TATA box-containing promoters regulate the transcriptional activity of the Xenopus vitellogenin A1 gene. These two promoters are of different strength and are separated by 1.8 kilobase pairs of untranslated sequence. Estrogen receptor (ER) and its ligand, 17beta-estradiol, induce the activity of both promoters. The estrogen response elements (EREs) are located proximal to the downstream i promoter while no ERE-like sequences have been identified in the vicinity of the upstream io promoter. We show here, that transcriptional activity of the upstream io promoter is Sp1-dependent. Moreover, we demonstrate that estrogen inducibility of the io promoter results from functional interactions between the io bound Sp1 and the ER bound at the proximity of i. Functional interactions between Sp1 and ER do not require the presence of a TATA box for transcriptional activation, as is demonstrated using the acyl-CoA oxidase promoter. The relative positions that ER and Sp1 occupy with respect to the initiation site determines whether these two transcription activators can synergize for transcription initiation.

Molecular heterogeneity of the beta-core fragment of human chorionic gonadotrophin.

We have analysed the structure and composition of the beta-core fragment of human chorionic gonadotrophin (beta C-hCG) from fresh urine specimens obtained from pregnant women and compared our findings with those previously proposed by other groups using different protocols. SDS-PAGE separation of reduced beta C-hCG demonstrated two major bands with apparent molecular weights of M(r) 8900 and M(r) 7500. The molecular weight of the agalacto beta C-hCG was estimated to be M(r) 10,218 from the amino acid analysis after high-performance liquid chromatography (HPLC) separation. Moreover, HPLC separation of its reduced and S-carboxymethylated peptides resulted in three peaks, but only two of them could be sequenced and demonstrated to be the previously reported beta 6-40 (M(r) 5000) and beta 55-92 (M(r) 5300) peptides of the beta hCG subunit. The results showed that 56-78% of beta C-hCG molecules of molecular weight M(r) 12,800 were able to bind Concanavalin A (Con A). While most were lacking all the peripheral monosaccharides and terminated in mannose, some retained other sugar residues on their antennae. Direct carbohydrate analysis showed the following molar content normalized to six mannose molecules: galactose 2.8, glucosamine 5.3, galactosamine 0.3, fucose 1.7 and sialic acid 3.0. Approximately 22-44% of the beta C-hCG molecules did not bind Con A (Con A non-reactive forms), of which 88% were totally deprived of sugar units and had an apparent molecular weight of approximately M(r) 10,000, and 12% were weakly reactive to Con A and reactive to anion exchange (negatively charged forms), being incompletely trimmed of their oligosaccharide chains. Comparison of our results with those of two other groups have indicated that the differences noted among preparations are due to either the source or the methods used to purify and characterize this fragment. In addition, our results showed significant microheterogeneity on the N-linked oligosaccharide moieties with some molecules apparently having no sugar molecules. These results have implications for the origins of beta C-hCG, suggesting secretion of some molecules without sugar chains and in other cases possible metabolism of hCG in the peripheral tissues.

Beta-core fragment of human chorionic gonadotropin in cervical intraepithelial neoplasia (CIN).

A low molecular weight glycoprotein immunologically identical with the beta-core fragment of hCG (beta C-hCG) has been described in invasive tumors of the genital tract, particularly carcinoma of the cervix. A previous report has also suggested increased urinary concentrations in subjects with cervical intraepithelial neoplasia (CIN). This prospective study of 107 patients with CIN was conducted to determine concentrations of beta C-hCG in the urine compared to a reference population without CIN. All subjects underwent a cervical smear and colposcopy, with biopsy when indicated. Between 11 and 18% of patients had urinary concentrations of beta C-hCG greater than the upper limit of the reference group and these results were confirmed when corrected for urinary creatinine concentration. A substantial number of subjects (19%) also had a positive result in a C-terminal immunoassay. It is concluded that preinvasive carcinoma of the cervix may secrete hCG or beta C-hCG in the earliest stages although measurement of beta C-hCG is unlikely to prove to be a valuable diagnostic marker in CIN.

Comparison of specific immunoassays for detection of the beta-core human chorionic gonadotrophin fragment in body fluids.

We have validated two new methods, one radioimmunoassay (RIA) and one immunoradiometric assay (IRMA), for the detection of beta-core hCG fragment (beta C-hCG) in body fluids. In addition, we have compared their performance with two other assays designed for beta C-hCG quantification. The RIA uses a rabbit polyclonal antibody raised against pure beta C-hCG which has a high affinity constant, is sensitive to 5 pmol/l, and has significant cross-reaction only with the free beta LH subunit. The IRMA, designed in a liquid phase, uses the same polyclonal antibody associated with a 125I-labelled mouse monoclonal antibody (32H2) raised against beta hCG, is sensitive to 1.5 pmol/l, and does not cross-react significantly with any related glycoprotein. Comparison between these two assays and two others previously published was made by measuring beta C-hCG in urine from healthy pregnant women (n = 47) and gave correlation coefficients higher than r = 0.960 with any combination. Analysis of beta C-hCG in urine of non-pregnant subjects (n = 238) showed measurable beta C-hCG in 8.8% (levels ranged from 5 to 34 pmol/l) with the IRMA and 88.3% with the RIA (n = 30; ranging from 28.4 to 228 pmol/l) (P = 0.05). We concluded that, despite different affinities of the antibody involved and different cross-reactivities with related glycoproteins, the four assays we examined may be equally employed to detect beta C-hCG in pregnancy urine. However, the IRMA appears to be more appropriate for beta C-hCG analysis in non-pregnant individuals, specifically in postmenopausal women because of the high cross-reactivity of the RIA with free beta LH or beta fragments of other glycoproteins. These studies have significance for our understanding of the physiology of beta C-hCG in cancer, pregnancy and after the menopause.

Distribution of the beta-core human chorionic gonadotrophin fragment in human body fluids.

The origins of a fragment of the human chorionic gonadotrophin (hCG) molecule, beta-core (beta C-hCG) were studied by analysis of beta C-hCG concentrations in biological fluids. In addition, the ability of the placenta to produce the fragment and the metabolism of hCG to beta C-hCG by human granulosa cells was determined in tissue culture. Finally the conversion of exogenous hCG to beta C-hCG was studied in vivo. The fragment was present in pregnancy urine as well as that from premenopausal and postmenopausal subjects. The highest concentrations were found in pregnant women. Ratios of beta C-hCG to intact hCG were higher in pregnancy urine when radioimmunoassay (RIA) was used compared with immunoradiometric assay (IRMA) (0.67 and 0.37 respectively). Concentrations of beta C-hCG were higher in postmenopausal urine than in premenopausal specimens. A significant amount of a high molecular weight beta C-hCG immunoreactive material was found in serum samples after size separation, and the molar ratio of beta C-hCG/hCG was estimated as 0.019. Amniotic fluid also contained small quantities of two forms of immunoreactive beta C-hCG and the ratio of 0.01 for authentic beta C-hCG/hCG increased to 0.026 when the high molecular weight form was considered. Cultured trophoblastic tissue released material with beta C-hCG immunoreactivity in the medium and chromatographic separation revealed that the majority of this material was of higher molecular weight compared with the authentic beta C-hCG form. beta C-hCG was the principal glycoprotein found in follicular fluid after hyperstimulated folliculogenesis and intramuscular injection of 5000 IU hCG. We also demonstrated that 26% of follicular fluid samples (n = 50) were positive for beta C-hCG; levels ranged from 5.2 to 23.0 pmol/l (13.1 +/- 5.7); S.D.) when a specific IRMA was used. The RIA could detect beta C-hCG in 48 samples (96%), levels ranging from 7.0 to 28.5 pmol/l (19.4 +/- 5.2). Moreover, granulosa cells cultured in the presence of hCG were able to degrade the intact molecule to both high molecular weight and authentic immunoreactive forms of beta C-hCG. After gel filtration, material of molecular weight over a wide range and immunoreactive for beta C-hCG was present in human seminal plasma. Assaying 74 samples of this fluid by IRMA, beta C-hCG was detected in 42 (56.7%), levels ranging between 5.5 and 59.5 pmol/l (24.9 +/- 15.2).(ABSTRACT TRUNCATED AT 400 WORDS)

Urinary concentrations of beta core fragment of hCG throughout pregnancy.

OBJECTIVE: We sought to determine a reference range for urinary immunoreactive beta core fragment of hCG (beta C-hCG) in pregnancy, the ratio between beta C-hCG and intact hCG, and the earliest detectable rise of beta C-hCG in urine. METHODS: Urine was obtained from 741 pregnant women between 6-41 weeks' gestation, as well as from women undergoing donor insemination with timed ovulation peaks. RESULTS: The beta core fragment of hCG reached a maximum between 8-15 weeks, with a decrease between 20-29 weeks. The molar ratio of beta C-hCG to intact hCG was always greater than 1. CONCLUSION: In pregnancy, beta C-hCG concentrations increase in the urine in parallel to intact hCG but at a higher molar ratio, suggesting either placental production of beta C-hCG or enhanced metabolism of hCG to beta C-hCG in peripheral organs.

Stability of immunoreactive beta-core fragment of hCG.

The beta core fragment of hCG (beta C-hCG) accounts for a large proportion of hCG immunoreactivity in the urine of pregnant women. It is often increased in the urine of patients with gynecologic tumors and may become an important diagnostic tool in early pregnancy and cancers. Despite the importance of beta C-hCG, little is known about its stability in urine under conditions of differing pH and temperature. This study examined the effect of repeated freeze-thaw cycles; storage at room temperature, 4C, and -20 C over several months; and the effect of alteration of urine pH. The two specific immunoassays for beta C-hCG used do not significantly cross-react with intact hCG or the free alpha subunit. Despite different cross-reactivities of the antibodies to the free beta subunit and higher immunoactivity when the radioimmunoassay was used, there was excellent correlation between the two assays in pregnancy urine. This suggests that there is little free beta subunit in urine from pregnant women. In addition, this study evaluated the stability of intact hCG and beta-hCG under identical conditions. No alterations in their immunoactivities were found under most conditions of storage. It is concluded that, for clinical purposes, beta C-hCG as well as intact hCG and free beta subunit are very stable molecules.

Measurement of human chorionic gonadotrophin (hCG): indications and techniques for the clinical laboratory.

The structure of human chorionic gonadotrophin (hCG) is so similar to that of luteinizing hormone (LH) that a variety of assay techniques have been devised to differentiate between these two hormones. The principal indications for measurement of hCG using these methods have not changed greatly over the past decade but the improvements in the sensitivity, specificity and the development of assays for free subunits and metabolic fragments have expanded the use of hCG assays. The review discusses the use of hCG measurement in a routine clinical immunoassay laboratory and emphasizes different requirements for clinical situations.

Identification of two steroid-responsive promoters of different strength controlled by the same estrogen-responsive element in the 5'-end region of the Xenopus laevis vitellogenin gene A1.

A structural and functional analysis of the 5'-end region of the Xenopus laevis vitellogenin gene A1 revealed two transcription initiation sites located 1.8 kilobases apart. A RNA polymerase II binding assay indicates that both promoters form initiation complexes efficiently. In vitro, using a transcription assay derived from a HeLa whole-cell extract, the upstream promoter is more than 10-fold stronger than the downstream one. In contrast, both promoters have a similar strength in a HeLa nuclear extract. In vivo, that is in estrogen-stimulated hepatocytes, it is the downstream promoter homologous to the one used by the other members of the vitellogenin gene family, which is 50-fold stronger than the upstream promoter. Thus, if functional vitellogenin mRNA results from this latter activity, it would contribute less than 1% to the synthesis of vitellogenin by fully induced Xenopus hepatocytes expressing the four vitellogenin genes. In contrast, both gene A1 promoters are silent in uninduced hepatocytes. Transfection experiments using the Xenopus cell line B3.2 in which estrogen-responsiveness has been introduced reveal that the strong downstream promoter is controlled by an estrogen responsive element (ERE) located 330 bp upstream of it. The upstream promoter can also be controlled by the same ERE. Since the region comprising the upstream promoter is flanked by a 200 base pair long inverted repeat with stretches of homology to other regions of the X. laevis genome, we speculate that it might have been inserted upstream of the vitellogenin gene A1 by a recombination event and consequently brought under control of the ERE lying 1.5 kilobases downstream.