Headspace solid-phase microextraction combined with mass spectrometry as a powerful analytical tool for profiling the terpenoid metabolomic pattern of hop-essential oil derived from Saaz variety

Hop(HumuluslupulusL.,Cannabaceaefamily)isprizedforitsessentialoilcontents,usedin beer production and, more recently, in biological and pharmacological applications. In this work,a methodinvolvingheadspace solid-phase microextractionand gas chromatography– mass spectrometry was developed and optimized to establish the terpenoid (monoterpenes and sesquiterpenes) metabolomic pattern of hop-essential oil derived from Saaz variety as a mean to explore this matrix as a powerful biological source for newer, more selective, biodegradable and naturally produced antimicrobial and antioxidant compounds. Different parameters affecting terpenoid metabolites extraction by headspace solid-phase microextraction were considered and optimized: type of fiber coatings, extraction temperature, extraction time, ionic strength, and sample agitation. In the optimized method, analytes were extracted for 30 min at 40 C in the sample headspace with a 50/30 m divinylbenzene/carboxen/polydimethylsiloxane coating fiber. The methodology allowed the identification of a total of 27 terpenoid metabolites, representing 92.5% of the total Saaz hop-essential oil volatile terpenoid composition. The headspace composition was dominated by monoterpenes (56.1%, 13 compounds), sesquiterpenes (34.9%, 10), oxygenated monoterpenes (1.41%, 3), and hemiterpenes (0.04%, 1) some of which can probably contribute to the hop of Saaz variety aroma. Mass spectrometry analysis revealed that the main metabolites are the monoterpene -myrcene (53.0±1.1% of the total volatile fraction), and the cyclic sesquiterpenes, -humulene (16.6 ± 0.8%), and -caryophyllene (14.7 ± 0.4%), which together represent about 80% of the total volatile fraction from the hop-essential oil. Thesefindingssuggestthatthismatrixcanbeexploredasapowerfulbiosourceofterpenoid metabolites.

Correlação entre a infecção genital pelo vírus do papiloma humano, a resposta imune e os achados colpocitológicos em mulheres grávidas e não grávidas

The genital HPV infection is very common between men and women worldwide, affecting particularly young women, constituting a serious public health problem in less developed regions, favored by the poor living conditions of population. The cytology and colposcopy have notorious importance in the diagnosis of precursor lesions of cervical cancer and therefore its prevention. However, even with such diagnostic tools, the number of women who develop cervical cancer is still high. This study aims to assess the prevalence of genital tract infection by HPV in pregnant and nonpregnant women, evaluating the profile of the immune response presented by the women of these two groups in order to establish correlations among profile of immune response, presence of virus and occurrence of lesions of the uterine cervix. We analyzed specimens obtained from the cervix of 221 patients, 91 pregnant and 130 non-pregnant, aged 14-72 years. The women were subjected to colposcopic and cytologic evaluation detect possible changes in the cervix and then samples were collected in order to perform HPV detection by PCR and real-time PCR for detection of mRNA of pro-inflammatory and anti-inflammatory cytokines. In the present study, the overall prevalence of HPV genital infection was 28.1%; of which 31.9% were pregnant patients and 25.4% in non-pregnant women. Young women under 30 years and those with low educational level education showed a higher risk of HPV infection. Colposcopy showed better correlation with detection of HPV DNA by PCR, when compared to cytology. Generally, HPV infected patients, pregnant or not, exhibited reduced mRNA expression of both pro-inflammatory (IFN-γ, TNF-α) and anti-inflammatory (IL -10) cytokines, when compared to patients not infected by HPV. Nonpregnant patients infected presented increase mRNA expression of IL-17 in patients without injury, whereas those with lesion showed higher mRNA expression of TGF-β. Pregnant women without injury infected exhibited increased mRNA expression of TGF-β. There was no difference in HPV prevalence between pregnant and nonpregnant women. There was a reduction of pro-inflammatory cytokines, except IL-17, in all women infected by HPV. Moreover, we observed an increase of TGF-β in HPV-infected women who are pregnant or not. The results suggest that, in women in this study, HPV infection promoted changes in the profile of cytokines necessary for activation of effective immune response, possibly favoring viral persistence

Associação do receptor toll-like 2 com o estado pró-inflamatório do diabetes tipo 1

Inflammation has been pointed out as an important factor in development of chronic diseases, as diabetes. Hyperglycemia condition would be responsible by toll-like receptors, TLR2 and TLR4, and, consequently by local and systemic inflammation induction. Thus, the objective of present study was to evaluate type 1 Diabetes mellitus (T1DM) pro-inflammatory state through mRNA expression of TLRs 2 and 4 and proinflammatory cytokines IL-1β, IL-6 and TNF-α correlating to diabetic nephropathy. In order to achieve this objective, 76 T1DM patients and 100 normoglycemic (NG) subjects aged between 6 and 20 years were evaluated. T1DM subjects were evaluated as a total group DM1, and considering glycemic control (good glycemic control DM1G, and poor glycemic control DM1P) and considering time of diagnosis (before achieving 5 years of diagnosis DM1< 5yrs, and after achieving 5 years of diagnosis DM1 <5yrs). Metabolic control was evaluated by glucose and glycated hemoglobin concentrations; to assess renal function serum urea, creatinine, albumin, total protein and urinary albumin-to-creatinine ratio were determined and to evaluate hepatic function, AST and ALT serum activities were measured. Pro-inflammatory status was assessed by mRNA expression of TLRs 2 and 4 and the inflammatory cytokines IL-1β, IL-6 and TNF-α. Except for DM1G group (18.4%), DM1NC patients (81.6%) showed a poor glycemic control, with glycated hemoglobin (11,2%) and serum glucose (225,5 md/dL) concentrations significantly increased in relation to NG group (glucose: 76,5mg/dL and glycated hemoglobin: 6,9%). Significantly enhanced values of urea (20%) and ACR (20,8%) and diminished concentrations of albumin (5,7%) and total protein (13,6%) were found in T1DM patients, mainly associated to a poor glycemic control (DM1P increased values of urea: 20% and ACR:49%, and diminished of albumin: 13,6% and total protein:13,6%) and longer disease duration (DM1 <5yrs - increased values of urea: 20% and ACR:20,8%, and diminished of albumin: 14,3% and total protein:13,6%). As regarding pro-inflammatory status evaluation, significantly increased mRNA expressions were presented for TLR2 (37,5%), IL-1β (43%), IL-6 (44,4%) and TNF-α (15,6%) in T1DM patients in comparison to NG, mainly associated to DM1P (poor glycemic control TLR2: 82%, IL-1β: 36,8% increase) and DM1 <5yrs (longer time of diagnosis TLR2: 85,4%, IL-1β: 46,5% increased) groups. Results support the existence of an inflammatory state mediated by an increased expression of TLR2 and pro-inflammatory cytokines IL-1β, IL-6 and TNF-α in T1DM

Virulência de Aphanocladium album (Preuss) Gams e Verticillium lecanii (Zimm.) Viégas (Deuteromycotina: Hyphomycetes) para o Percevejo-de-renda da seringueira, Leptopharsa heveae (Drake & Poor) (Hemiptera: Tingidae)

The lace bug, Leptopharsa heveae is an insect that causes serious damage on rubber trees. In laboratory condition, the strains of Verticillium lecanii (ARSEF 6430, 6431 e 6432) and Aphanocladium album (ARSEF 6433) were tested on third-and fifth-instar nymphs and adults of L. heveae to evaluate their virulence using 2.4 x 10(5) and 2.4 x 10(7) conidia/mL. The bioassays were carried out using Petri dishes whose inner bottoms were covered with damp filter papers. Each Petri dish contained five insects and one rubber tree leaflet. The plates were covered with PVC film to provide high relative humidity, maintained at 26 +/- 0.5 degrees C and a photophase of 14 hours. The Probit analysis was calculated from mortality date of insects killed by fungi. In the highest concentrations, ARSEF 6430 was more virulent for third instar nymphs, and the LT50 was 1.9 days. For the fifth instar, the strains ARSEF 6430, 6433 and 6432 showed similar virulence with LT50 of 2.6, 2.6 and 3.2 days, respectively. For adults, ARSEF 6431 was the most virulent strain with the LT50 recorded at 2.0 days. The smallest concentration did not always cause more than 50% mortality.

Biologia de Leptopharsa heveae Drake & Poor (Heteroptera: Tingidae) e a relação de suas exigências térmicas com a flutuação populacional em seringueira

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Parasitismo de Erythmelus tingitiphagus (Soares) (Hymenoptera: Mymaridae) em ovos de Leptopharsa heveae Drake & Poor (Hemiptera: Tingidae), em plantios de seringueira (Hevea brasiliensis Müell. Arg.)

The rubber tree lace bug, Leptopharsa heveae Drake & Poor occurs in high populations in rubber tree plantations and it is a limiting factor in rubber production due to the loss of photosynthetic tissue. The control of the pest has been made mainly with chemical products, which cause environmental contamination. The alternative would be the use of biological control agents, however, information about L. heveae natural enemies are scarce. The parasitoid Erythmelus tingitiphagus (Soares) parasitize eggs of the rubber tree lace bug. The aim of this study was to verify the occurrence of E. tingitiphagus in plantations of several rubber tree clones, located in Itiquira town, Mato Grosso State. The plant leaflets of the clones RRIM 600, PR 255, GT 1, PB 235 and PB 217 were collected weekly from October 2005 to February 2006. Parasitism was recorded during the entire study period. The parasitism rate of L. heveae eggs in the different clones ranged from 16.8 to 20.6%.

Electromyographic analysis of the masseter and buccinator muscles with the Pro-Fono Facial Exerciser use in bruxers

The aim of this study was to evaluate the efficiency of the Pro-Fono Facial Exerciser (Pro-Fono Productos Especializaclos para Fonoaudiologia Ltda.., Barueri/SP, Brazil) to decrease bruxism, as well as the correlation between the masseter and the buccinator muscles using electromyography (EMG). In this study, 39 individuals ranging from 23 to 48 years of age were selected from a dental school and then underwent surface EMG at three different periods of time: 0, 10, and 70 days. They were divided into a normal control group, a bruxer control group (without device), and an experimental bruxer group who used the device. The bruxer group showed a greater masseter EMG amplitude when compared to the normal group, while the experimental group had deceased activity with a reduction in symptoms. The buccinator EMG spectral analysis of the experimental bruxist group showed asynchronous contractions of the masseter muscle (during jaw opening) after using the Pr6-Fono Facial Exerciser. The normal group also showed asynchronous contractions. Upon correlation of the data between these muscles, the inference is that there is a reduction in bruxism when activating the buccinator muscle.

Oxidation of melatonin by AAPH-derived peroxyl radicals: Evidence of a pro-oxidant effect of melatonin

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Mudanças climáticas globais e o compromisso pró-ecológico de adolescentes natalenses

In recent years, much has been discussed about global climate changes (GCCs), popularly known as global warming. The scientific evidences point out to the influence of human actions for its drastic intensification. Therefore, studies of the psychological aspects involved become relevant. This study aimed at the investigation of the views of adolescents concerning GCCs, and the possible relations between those views and their pro-ecological commitment. Such commitment is measured by willingness for engagement in pro-environmental behaviors; environmentalism attitudes, like ecocentric and anthropocentric; consideration of future consequences; and ecological worldviews. Participants were 348 adolescents who answered a questionnaire containing questions about socio-demographic data, open questions about the practice of environmental care, and about GCCs, and the scales of Ecocentric and Anthropocentric Environmentalism, the Consideration of Future Consequences and the Ecological Worldviews assessment scale. From the inter-relationships between variables, procedures carried out by means of descriptive and correlacional statistics, it was observed that 55% of teenagers said that they did not engage in actions of environmental care, which was associated with apathyanthropocentric, immediatism, and individualism. The consideration of future consequences joined the practice of environmental care, corroborating evidence from the literature. It was evident that views concerning GCCs were superficial; adolescents perceive it as a generic environmental problem, and are confused with other problems such as pollution. This study found no association between views about GCCs and the indicators of pro-ecological commitment, perhaps due to the conceptual confusion about the subject. However, the lack of environmental care actions and other indicators of non-commitment (apathy-anthropocentric, individualism and immediatism) were associated with conceptually poor or incomplete responses (with no indication of cause, consequence or responsibility for the problem), demonstrating diminished knowledge and the failure to consider these issues

Silibinin modulates the NF-kappa b pathway and pro-inflammatory cytokine production by mononuclear cells from preeclamptic women

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Decreased fertility in poor responder women is not related to oocyte morphological status

Introduction: In women showing impaired fertility, a decreased response to ovarian stimulation is a major problem, limiting the number of oocytes to be used for assisted reproduction techniques (ART). Despite the several definitions of poor response, it is still a matter of debate whether young poor responder patients also show a decrease in oocyte quality. The objective in this study was to investigate whether poor ovarian response to the superstimulation protocol is accompanied by impaired oocyte quality.Material and methods: This study included 313 patients younger than 35 years old, undergoing intracytoplasmic sperm injection. Patients with four or fewer MII oocytes (poor-responder group, PR, n = 57) were age-matched with normoresponder patients (NR, n = 256).Results: A higher rate of oocyte retrieval and a trend towards an increase in MII oocyte rate were observed in the NR group when compared to the PR group (71.6 +/- 1.1% and 74.1 +/- 1.0% vs. 56.3 +/- 2.9% and 66.5 +/- 3.7%; p < 0.0001 and p = 0.056, respectively). A trend toward increased implantation rates was observed in the NR group when compared to the PR group (44 and 24.5 +/- 2.0% vs. 28.8 and 16.4 +/- 3.9%; p = 0.0305 and p = 0.0651, respectively).Conclusions: Low response to ovarian stimulation is apparently not related to impaired oocyte quality. However, embryos produced from poor responder oocytes show impaired capacity to implant and to carry a pregnancy to term.

Are poor responders patients at higher risk for producing aneuploid embryos in vitro?

Purpose To test the hypothesis that aged women with poor ovarian response express an increase on embryo chromosomal alterations when compared to aged women who presented normal response.Methods Couples undergoing intracytoplasmic sperm injection cycles with preimplantation genetic screening, were subdivided into two groups: Poor Responder group (n = 34), patients who produced a parts per thousand currency sign4 oocytes; and Normoresponder group (n = 50), patients who produced a parts per thousand yen5 oocytes. Groups were compared regarding cycles' outcomes and aneuploidy frequency.Results There were no significant differences between and groups regarding the fertilization rate (p = 0.6861), clinical pregnancy (p = 0.9208), implantation (p = 0.6863), miscarriage (p = 0.6788) and the percentage of aneuploid embryos (p = 0.270). Embryo transfer rate was significantly lower on poor responder group (p = 0.0128) and logistic regression confirmed the influence of poor response on the chance of embryo transfer (p = 0.016).Conclusions Aged females responding poorly to gonadotrophins are not at a higher risk for producing aneuploid embryos in vitro.

Enhanced natural killer activity and production of pro-inflammatory cytokines in mice selected for high acute inflammatory response (AIRmax)

Strains of mice with maximal and minimal acute inflammatory responsiveness (AIRmax and AIRmin, respectively) were developed through selective breeding based on their high- or low-acute inflammatory responsiveness. Previous reports have shown that AIRmax mice are more resistant to the development of a variety of tumours than AIRmin mice, including spontaneous metastasis of murine melanoma. Natural killer activity is involved in immunosurveillance against tumour development, so we analysed the number and activity of natural killer cells (CD49b(+)), T-lymphocyte subsets and in vitro cytokine production by spleen cells of normal AIRmax and AIRmin mice. Analysis of lymphocyte subsets by flow cytometry showed that AIRmax mice had a higher relative number of CD49b(+) cells than AIRmin mice, as well as cytolytic activity against Yac.1 target cells. The number of CD3(+) CD8(+) cells was also higher in AIRmax mice. These findings were associated with the ability of spleen cells from AIRmax mice in vitro to produce higher levels of the pro-inflammatory cytokines tumour necrosis factor-alpha, interleukin-12p40 and interferon-gamma but not the anti-inflammatory interleukin-10. Taken together, our data suggest that the selective breeding to achieve the AIRmax and AIRmin strains was able to polarize the genes associated with cytotoxic activity, which can be responsible for the antitumour resistance observed in AIRmax mice.

DNA methylation in the CTCF-binding site I and the expression pattern of the H19 gene: Does positive expression predict poor prognosis in early stage head and neck carcinomas?

Loss of allele-specific expression by the imprinted genes IGF2 and H19 has been correlated with a differentially methylated region (DMR) upstream to the H19 gene. The H19-DMR contains seven potential CCCTC-binding factor (CTCF) binding sites. CTCF is a chromatin insulator and a multifunctional transcription factor whose binding to the H19-DMR is suppressed by DNA methylation. Our study included a group of 41 head and neck squamous cell carcinoma (HNSCC) samples. The imprinting status of the H19 gene was analyzed in 11 out of 35 positive cases for H19 gene expression, and only 1 of them showed loss of imprinting. We detected a significant correlation (P=0.041, Fisher's exact test) between H19 expression and tumor recurrence. Among H19 positive cases, six were T2, in which five developed recurrence and/or metastasis. Inversely, in the group of tumors that showed no H19 gene expression, 5 out of 24 were T2 and only I presented regional recurrence. These data support the hypothesis that H19 expression could be used as a prognostic marker to indicate recurrence in early stage tumors. We also examined the methylation of the CTCF binding site 1 in a subgroup of these samples. The H19 gene silencing and loss of imprinting were not correlated with the methylation pattern of the CTCF binding site 1. However, the significant correlation between H19 expression and tumor recurrence suggest that this transcript could be a marker for the progression of HNSCC. (c) 2005 Wiley-Liss, Inc.