Prevention, diagnosis and treatment of cervical cancer precursor lesions at the Xingu Indigenous Park, Brazil.

PURPOSE: To describe the prevention, diagnosis, and treatment of cervical cancer precursor lesions at the Xingu Indigenous Park (PIX) from 2005 to 2006. MATERIALS AND METHODS: Observational, transversal study. The research sample consisted of 503 sexually active women aged 12 years and older. The research was performed in three stages: screening, colposcopy, and surgical treatment by large loop excision of the transformation zone. RESULTS: The cytopathological screening coverage was of 99.6%. The rate of cytologic atypia was 11.7%. Together, low-grade squamous intraepithelial lesions (LSILs) and high-grade squamous intraepithelial lesions (HSILs) were observed in 4.6% of the women. The cytological examination returned a sensitivity of 54%, specificity of 97%, a positive predictive value of 88%, and a negative predictive value of 83%. In the anatomopathological examinations of biopsies, the rate of HSILs was 30.2%. The sensitivity of the anatomopathological examination of biopsies was 72.2%, the specificity was 100%, the positive predictive value was 100%, and the negative predictive value was 44.4%. CONCLUSIONS: Viable strategies for preventing, diagnosing, and treating cervical cancer precursor lesions in women from the PIX include increasing annual coverage of cytopathological examinations, early detection of cervical intraepithelial lesions, and treatment and follow-up of detected cases.

Rapid screening and identification of new soluble tannin-salivary protein aggregates in saliva by mass spectrometry (MALDI-TOF-TOF and FIA-ESI-MS).

Astringency is mainly attributed to the interaction between tannins and salivary proteins. Proline-rich proteins, histatins, and statherins are supposed to be the most reactive salivary proteins. This study aims to contribute to the knowledge of the tannin-protein binding process in saliva. It was identified for the first time in several soluble tannin-human salivary protein aggregates. A rapid mass spectrometry analytical method (MALDI-TOF and FIA-ESI-MS) was developed to identify new soluble tannin-human salivary protein aggregates. Three different tannins--procyanidin B3 (B3), procyanidin B2 gallate (B2G), and pentagalloylglucoside (PGG)--were tested to elucidate the tannin selectivity toward histatins, proline-rich proteins, and statherins in human saliva. A greater number of aggregates with a higher molecular weight was found when PGG was tested while no difference in the number and molecular mass range was observed in B3 or B2G salivary protein aggregates. This study confirms for the first time the bilateral selectivity of tannins and protein to yield soluble tannin-human salivary protein complexes. The results confirm that B3 and B2G are more selective than PGG. Furthermore, the families of proteins involved in the majority of B3-salivary protein soluble aggregates were primarly histatins, followed by basic proline-rich proteins and statherins. When B2G was tested, basic proline-rich proteins were involved in a greater number of aggregates, followed by histatines and statherins. Basic proline-rich proteins were also the family of proteins that formed a greater number of PGG-salivary protein aggregates followed by statherins and histatins. Acidic proline-rich proteins and glucosilated proline-rich proteins formed fewer soluble aggregates regardless of the tannin tested. The aggregation process was also found to be influenced by tannin and protein polarity. Indeed, the protein/tannin ratio of soluble aggregates increased with the tannin polarity. On the other hand, the only amphiphilic salivary proteins studied (histatins) formed a greater number of aggregates with the least polar tannin tested (B3).

Disaccharide anthocyanin delphinidin 3-O-sambubioside from Hibiscus sabdariffa L.: Candida antarctica lipase B-catalyzed fatty acid acylation and study of its color properties.

Enzymatic lipophilization is an important process to extend the use of anthocyanins in lipidic media. In this work delphinidin 3-O-sambubioside (Dp3sam) isolated from Hibiscus sabdariffa L. flower was esterified with octanoic acid using Candida antarctica lipase B. The physical-chemical properties of the new lipophilic pigment were studied by UV-vis spectroscopy. Dp3sam with chloride, acetate and formate as counter ions were employed to study the lipophilization reaction. The hydrolysis of the reagent was avoided with a formate counter ion and the expected product was achieved with a noteworthy change of solubility. 1D and 2D NMR characterization of Dp3sam-C8 confirmed that the lipophilization took place at the primary alcohol of the glucoside moiety. Overall, the Dp3sam-C8 ester presents a stabilization of the quinoidal base (blue color) at neutral or moderate alkaline pH, which foresees a potential use of this pigment as a broad kind of industries on lipo-soluble formulations.

A 1000-year-old mystery solved: Unlocking the molecular structure for the medieval blue from Chrozophora tinctoria, also known as folium.

The molecular structure of the medieval watercolor known as folium has finally been solved in the 21st century. The interdisciplinary approach taken was the key to producing extracts that had been prepared following medieval instructions, and shows the blue/purple chromophore as the major dye in Chrozophora tinctoria fruits (shell). A multi-analytical characterization of its structure was made using HPLC-DAD-MS, GC-MS, NMR (1H, 13C, COSY, HSQC, HMBC, INADEQUATE), and computational studies. The results demonstrate that the blue compound corresponds to 6'-hydroxy-4,4'-dimethoxy-1,1'-dimethyl-5'-{[3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl]oxy}-[3,3'-bipyridine]-2,2',5,6(1H,1'H)-tetraone, a hermidin derivative, which we named chrozophoridin. Experimental data and computational modeling studies show that this mono-glycosylated dimer is represented by two stable isomers (atropisomers). This is an indispensable piece of knowledge for the characterization of this medieval dye in works of art such as medieval manuscript illuminations and for testing its stability and contributes to the preservation of our cultural heritage.

Uterine cervical neoplasia prevention in Parque Indigena do Xingu.

Results of preventive health measures, diagnosis and treatment applied to Parque Indigena do Xingu native women were studied. Thirty-seven cases of uterine cervical intraepithelial lesions and invasive neoplasias were treated in the local villages without referral to an advanced medical center. LEEPs were carried out in 32 women, three cold knife conizations, one vaginal hysterectomy and one Wertheim Meigs procedure. Results of 53.1% of LEEP surgical procedures did not have margin involvement by the lesions. Bleeding complications were seen in 15.6%. Regular follow-up with two or three cytologic and colposcopic tests in 32 women was carried out. All cases were negative for lesions. Five women were not followed-up due mainly to logistical reasons. Health endeavors adopted in the period 2005-2007 brought about a significant reduction of precursor lesions in this native aboriginal population without screening resources.

Synergistic effect of mixture of two proline-rich-protein salivary families (aPRP and bPRP) on the interaction with wine flavanols.

In this study, we have evaluated by HPLC-DAD, DLS and MALDI-TOF a synergic effect of the coexistence of two salivary-PRP fractions (basic-PRPs and acidic PRPs) on the interaction with flavanols. Results obtained showed noticeable enhancement of the interaction between (epi)catechin and PRPs when both types of proteins are blended. Up to 30 soluble aggregates have been tentatively identified with molecular weight from 4680 to 35,851. (epi)Catechins seem to bind preferentially bPRPs than aPRPs, although the medium size aggregates flavanol-bPRPs formed could favour the interaction with aPRPs giving rise to soluble mixed aggregates.

Identification and characterization of proteolytically resistant gluten-derived peptides.

The lack of digestibility of certain gluten proteins is essential in the development of celiac disease (CD). Gluten proteins are remarkably resistant to luminal and brush-border proteolysis owing to their high proline and glutamine content. Consequently, large fragments remain intact after digestion exerting toxic effects. Intestinal brush-border membrane vesicles (BBMV) have been described as having strong proteolytic activity mainly through prolyl endopeptidase enzymes. The purpose of this work was to monitor the gastrointestinal digestion of specific CD epitopes by means of an in vitro gastrointestinal digestion model that included incubation with brush-border membrane enzymes. Gluten hydrolysates were characterized by mass spectrometry and the immunologic peptides were tracked by searching the main T-cell stimulating epitopes which have been widely described. The immunologic potential of gluten hydrolysates was further analysed by enzyme-linked immunosorbent assay (ELISA). The results showed that the composition of gluten hydrolysates depended on the digestion time and protein structural characteristics. On the other hand, the main T-cell stimulating epitopes formed during hydrolysis depend on the precursor protein. Glutenin oligopeptides were degraded faster whereas gliadin, mainly α-gliadin oligopeptides, remained intact for a long time. MS-based analysis showed that the formation of the epitopes from γ-gliadin and ω-gliadin or glutenin was favoured but they were generally degraded during the gastrointestinal treatment. However, the peptides containing the epitope PFPQPQLPY (α-gliadin) remained intact even after 180 min of digestion time. Overall, from all the epitopes tracked, PFPQPQLPY was the most resistant to in vitro BBMV digestion.

Burkholderia thailandensis as a microbial cell factory for the bioconversion of used cooking oil to polyhydroxyalkanoates and rhamnolipids.

The present work assessed the feasibility of used cooking oil as a low cost carbon source for rhamnolipid biosurfactant production employing the strain Burkholderia thailandensis. According to the results, B. thailandensis was able to produce rhamnolipids up to 2.2 g/L, with the dominant congener being the di-rhamnolipid Rha-Rha-C14-C14. Rhamnolipids had the ability to reduce the surface tension to 37.7 mN/m and the interfacial tension against benzene and oleic acid to 4.2 and 1.5 mN/m, while emulsification index against kerosene reached up to 64%. The ability of B. thailandensis to accumulate intracellular biopolymers, in the form of polyhydroxyalkanoates (PHA), was also monitored. Polyhydroxybutyrate (PHB) was accumulated simultaneously and consisted of up to 60% of the cell dry weight. PHB was further characterized in terms of its molecular weight and thermal properties. This is the first study reporting the simultaneous production of polyhydroxyalkanoates and rhamnolipids by the non-pathogen rhamnolipid producer B. thailandensis.

Red wine antioxidants protect hippocampal neurons against ethanol-induced damage: a biochemical, morphological and behavioral study.

Chronic ethanol consumption increases oxidative stress, which accounts for the striking neurological changes seen in this condition. Notwithstanding, there is well-documented evidence that polyphenols, present in grape skin and seeds, exhibit a strong antioxidant activity. As red wine is rich in polyphenols, the aim of the present work was to evaluate their putative protective effects on the hippocampal formation by applying biochemical, morphological and behavioral approaches. Six-month old male Wistar rats were fed with red wine (ethanol content adjusted to 20%) and the results were compared with those from ethanol-treated (20%) rats and pair-fed controls. Biochemical markers of oxidative stress (lipid peroxidation, glutathione levels and antioxidant enzyme activities) were assessed on hippocampal homogenates. Lipofuscin pigment, an end product of lipid peroxidation, was quantified in hippocampal cornu ammonis 1 and 3 (CA1 and CA3) pyramidal neurons using stereological methods. All animals were behaviorally tested on the Morris water maze in order to assess their spatial learning and memory skills. In red wine-treated rats, lipid peroxidation was the lowest while presenting the highest levels of reduced glutathione and an induction of antioxidant enzyme activities. Morphological findings revealed that, contrary to ethanol, red wine did not increase lipofuscin deposition in CA1 and CA3 pyramidal neurons. Besides, red wine-treated animals learned the water maze task at a higher rate than ethanol group and had better performance scores by the end of the training period and on a probe trial. Actually, no significant differences were found between pair-fed controls and red wine-treated rats in morphological and behavioral data. Thus, our findings demonstrate that chronic consumption of red wine, unlike the ethanol solution alone, does not lead to a decline in hippocampal-dependent spatial memory. This may be due to the ability of red wine polyphenols to improve the antioxidant status in the brain and to prevent free radical-induced neuronal damage.

Chromatographic and mass spectrometry analysis of wheat flour prolamins, the causative compounds of celiac disease.

Immunogenic gluten peptides trigger Celiac Disease (CD), an adaptive immune response in genetically predisposed individuals. Given the structural similarity between all gluten proteins their individual CD influence is not clear. Hence, the extraction, separation and characterization of wheat gluten proteins have become relevant to measure their individual potential immunoreactivity. Wheat proteins were extracted from commercial wheat flour and further isolated by preparative HPLC. The resulting richest gliadin sub-fractions were characterized by nano-LC-MS/MS following a shotgun proteomic approach in order to identify the prolamins in the original commercial wheat flour. It was found that the gliadin extract was additionally composed of glutenins and avenin-like proteins. Accurate prolamin identification has emerged as a need to delve deep into the influence of each fraction on the onset of celiac disease. After protein characterization, the immunoreactivity towards the main epitope related to CD was verified by ELISA and western blotting for several different gluten fractions.

Pharmacokinetics of table and Port red wine anthocyanins: a crossover trial in healthy men.

This study was designed to evaluate the pharmacokinetics of Port and table red wine anthocyanins in healthy men. Volunteers were recruited to drink 250 mL of a table red wine (221 mg of anthocyanins) and 150 mL of young Port red wine (49 mg of anthocyanins). Venous blood was collected from participants at 0, 15, 30, 60 and 120 min after wine ingestion. Urine samples were collected at baseline and at 120 min. Anthocyanins and anthocyanin metabolites in plasma and urine samples were quantified by HPLC-DAD and tentatively identified by LC-MS. Red wine anthocyanins were detected in their intact forms in both plasma and urine samples, but the glucuronylated metabolites of peonidin and malvidin (PnGlucr and MvGlucr) were the two main derivatives detected after both red wine consumptions. For the first time, and supported by the synthesis of Mv3Glucr, the main pathway followed by Mv3glc after absorption was described and involves anthocyanidin conjugation with glucuronic acid after glucose removal. Despite the lower total content of anthocyanins ingested when volunteers drank Port wine, no differences were observed in the plasma Cmax of MvGlucr and PnGlucr after table and Port red wine consumption. The relative bioavailability of anthocyanins in Port wine was 96.58 ± 5.74%, compared to the anthocyanins present in red wine. In conclusion, both Port and table red wines are good sources of bioavailable anthocyanins.

Grade 3 vulvar and anal intraepithelial neoplasia in a HIV seropositive child--therapeutic result: case report.

A case report of a HIV seropositive 8-year-old child with vulvar and anal border neoplasia, both grade 3, and the adopted therapeutic management are presented. The mother reported the history of a progressively growing verrucous lesion in the vulva since the age of three and a half years. On physical examination a pigmented and elevated lesion was observed in the whole vulvar region extending to the anal region and intergluteal sulcus. After biopsies and anatomic pathological examination, antiretroviral therapy, adequate for age, and topical application of podophyllotoxin associated with Thuya officinalis extract was started. Three months afterwards vaporization and CO2 laser excision were performed in five sequential sessions, thereafter associated with topical imiquimod application. After the first two sessions of laser therapy early relapses occurred. After four weeks of imiquimod use, already a significant improvement of the lesions was observed, making the following laser therapy sessions easier. We conclude that antiretroviral therapy associated with podophyllotoxin and Thuya was not effective regarding regression of the lesions. Laser therapy alone led to early relapses. The local use of imiquimod associated with laser was effective in decreasing and controling the lesions.

Anatomical observations on the ending of the human thoracic duct.

Fifty-one cadavers of human adults of both sexes were examined for the ramifications and site of opening of the cervical segment of the thoracic duct. It was found that the duct opened into veins of the neck through either one (84.31%) or two branches (15.69%). The most frequent site of opening was in the junction of the left internal jugular vein with the subclavian vein (18 individuals), but another frequent site (15 individuals) was in the internal jugular vein. The duct opened at the angle formed by the internal jugular vein and the internal posterior jugular vein in five individuals, and at a variety of the other locations in the remaining cadavers. These results are compared with the findings of previous investigators.

Structural features of procyanidin interactions with salivary proteins.

Procyanidin dimers and trimer C1 were synthesized, whereas (-)-epicatechin O-gallate and B2-3"-O-gallate were isolated from grape seeds. Human saliva was separated into two fractions. One of these was mainly alpha-amylase and the other mainly proline-rich proteins (PRPs). The procyanidin compounds were combined with each of the saliva protein fractions and with bovine serum albumin. The protein-polyphenol interactions were observed using nephelometry. (+)-Catechin had a higher tannin specific activity (TSA) for PRPs than (-)-epicatechin (1.45 versus 0.65 nephelos turbidity units/mg of polyphenol). This indicated the effect of the stereochemistry of flavan-3-ols on their interaction with proteins. Procyanidin dimers linked through a C(4)-C(8) interflavanoid bond had consistently greater TSA than their counterparts with a C(4)-C(6) linkage. Esterification of a galloyl group to the C(3) hydroxyl function of (-)-epicatechin or to the epicatechin moiety of procyanidin dimer B2 increased TSA. This was not as strong an effect for the dimer, probably as a result of the expected "closed" structure of B2-3"-O-gallate.

Evolution and stability of anthocyanin-derived pigments during Port wine aging.

For three years, the evolution of the three major anthocyanidin monoglucosides (malvidin 3-glucoside, malvidin 3-acetylglucoside, and malvidin 3-coumaroylglucoside) and their anthocyanin-pyruvic acid adducts was monitored in Port wines stored in oak barrels. The degradation reactions of all pigments followed first-order kinetics in all the wines studied. The degradation rate constants of the anthocyanin-pyruvic acid adducts were much lower than those of the anthocyanidin monoglucosides. The results of both anthocyanins and pyruvic acid adducts show that acylation on the sugar moiety of all the pigments decreased their stability in wine. The levels of malvidin 3-glucoside-pyruvic acid adduct and its acylated forms increased right after wine fortification with wine spirit before starting to decrease around 100 days. The initial formation of anthocyanin-pyruvic acid adducts was concurrent with the degradation of anthocyanidin monoglucosides.

Role of polyphenols in copper complexation in red wines.

Four monovarietal red wines, Tinta Roriz, Tinta Barroca, Touriga Nacional, and Touriga Francesa, were studied for their complexometric properties toward copper. For the purpose, ion selective electrode potentiometric titrations were carried out, and the data were treated by two methods suitable for heterogeneous ligands: the Scatchard plot, which provided the complexation capacity (CC(total) = maximum number of moles of sites per liter), and the differential equilibrium function (DEF), which provided the conditional stability constants of the sites effectively involved in the metal complexation (K(DEF)) at a given titration point. The different wines displayed similar properties: CC(total) ranged from 4.5x10(-3) (Tinta Barroca) to 4.9x10(-3) M (Touriga Francesa), and log K(DEF) values were between 4.2 and 6.5. The range of log(|Cu|(bound)/CC(total)) = log theta embraced in the titration was between -0.2 and -1.9. With regard to the natural levels of copper, from 4.4x10(-7) (Tinta Roriz) to 1.3x10(-6) M (Touriga Nacional), and ligands (CC(total)) in the wines, it can be expected that copper will be strongly complexed in the studied wines (log K(DEF) > 6). Studies carried out, in parallel, for solutions of isolated seed tannins and skin polyphenols (mainly anthocyans) showed that their contribution to the CC(total) of the wines was <40% for tannins and <2% for skin polyphenols. However, skin polyphenols were shown to be much stronger copper ligands (log K(DEF) = 6.7, log theta = -1.5) than tannins (log K(DEF) = 4.5, log theta = -1.5).

Identification of some volatile descriptors of the rock-rose-like aroma of fortified red wines from douro demarcated region.

2,2,6-Trimethylcyclohexanone and ethyl dihydrocinnamate, known sensorial descriptors of rock-rose (Cistus ladaniferus) aroma, are reported as minor constituents of young fortified wines from the Douro Demarcated region (vintage 1997) for the first time. These compounds were respectively identified by GC-MS in two different fractions (A and B) obtained by preparative GC. Quantitative analysis by GC-MS was performed on 14 fortified wines originating from the different Douro subregions. Ethyl dihydrocinnamate was found to be present at concentrations between 2.3 and 6.7 microgram/L, while 2,2,6-trimethylcyclohexanone ranged from 0.05 to 0.4 microgram/L.

Occurrence of anthocyanin-derived pigments in red wines.

Several anthocyanin-derived pigments that showed UV-visible spectra different from those of the original grape anthocyanins were detected by HPLC-DAD analysis in 1-year-old bottled Port wines from the Douro region. Among these, three malvidin 3-glucoside derived pigments were detected in large amounts, representing approximately 60% of the total anthocyanidin monoglucosides content. These pigments were isolated, purified, and identified by LSI-MS and NMR ((1)H, DQF-COSY, ROESY, HSQC, and HMBC) techniques. The major pigment is malvidin 3-glucoside pyruvic adduct, previously characterized, and the other two corresponded to its respective acetyl and coumaroyl glucoside derivatives. The latter is reported for the first time in red wines.

Chemicals with sweet aroma descriptors found in Portuguese wines from the Douro region: 2,6,6-trimethylcyclohex-2-ene-1,4-dione and diacetyl.

2,6,6-Trimethylcyclohex-2-ene-1,4-dione (TMCHD), a norisoprenoid with a sweet honey aroma descriptor, is reported for the first time as a minor constituent of single-varietal table and fortified wines from the demarcated Douro region. Olfactory gas chromatography (GC-O) of a volatile wine extract, previously isolated by preparative gas chromatography, indicated the presence of a zone containing an intense honey descriptor. The targeted odor compound was identified by GC-MS, GC-O, and Kovats index. Quantitative analysis using a selected characteristic ion (m/z 96) indicated that young Douro fortified wines from the 1997 vintage contained up to 4 microg/L TMCHD. The sweet honey sensory threshold limit for TMCHD in a model Port wine solution was found to be 25 microg/L. TMCHD is therefore only likely to contribute as a collective element to Port wine aroma. The wine volatile diacetyl was identified as a strong contributor to the sweet caramel aroma descriptor often associated with Port.

Direction of the mental canal in human mandibles.

The direction of the mental canal was calculated in the horizontal and vertical planes with a view to aiding anaesthetic techniques. Based on our data, it is advisable to give the following approximate inclinations to the needle when penetrating the mental canal in anaesthetic procedures: 55 degrees postero-anteriorly in relation to a horizontal plane and 40 degrees latero-medially in relation to a horizontal line tangent to the body of the mandible at the level of the mental foramen.