Bacterial Diversity Assessment In Soil Of An Active Brazilian Copper Mine Using High-throughput Sequencing Of 16s Rdna Amplicons.

Mining activities pose severe environmental risks worldwide, generating extreme pH conditions and high concentrations of heavy metals, which can have major impacts on the survival of organisms. In this work, pyrosequencing of the V3 region of the 16S rDNA was used to analyze the bacterial communities in soil samples from a Brazilian copper mine. For the analysis, soil samples were collected from the slopes (geotechnical structures) and the surrounding drainage of the Sossego mine (comprising the Sossego and Sequeirinho deposits). The results revealed complex bacterial diversity, and there was no influence of deposit geographic location on the composition of the communities. However, the environment type played an important role in bacterial community divergence; the composition and frequency of OTUs in the slope samples were different from those of the surrounding drainage samples, and Acidobacteria, Chloroflexi, Firmicutes, and Gammaproteobacteria were responsible for the observed difference. Chemical analysis indicated that both types of sample presented a high metal content, while the amounts of organic matter and water were higher in the surrounding drainage samples. Non-metric multidimensional scaling (N-MDS) analysis identified organic matter and water as important distinguishing factors between the bacterial communities from the two types of mine environment. Although habitat-specific OTUs were found in both environments, they were more abundant in the surrounding drainage samples (around 50 %), and contributed to the higher bacterial diversity found in this habitat. The slope samples were dominated by a smaller number of phyla, especially Firmicutes. The bacterial communities from the slope and surrounding drainage samples were different in structure and composition, and the organic matter and water present in these environments contributed to the observed differences.

Propylthiouracil Quantification In Human Plasma By High-performance Liquid Chromatography Coupled With Electrospray Tandem Mass Spectrometry: Application In A Bioequivalence Study.

A rapid, sensitive and specific method for quantifying propylthiouracil in human plasma using methylthiouracil as the internal standard (IS) is described. The analyte and the IS were extracted from plasma by liquid-liquid extraction using an organic solvent (ethyl acetate). The extracts were analyzed by high performance liquid chromatography coupled with electrospray tandem mass spectrometry (HPLC-MS/MS) in negative mode (ES-). Chromatography was performed using a Phenomenex Gemini C18 5μm analytical column (4.6mm×150mm i.d.) and a mobile phase consisting of methanol/water/acetonitrile (40/40/20, v/v/v)+0.1% of formic acid. For propylthiouracil and I.S., the optimized parameters of the declustering potential, collision energy and collision exit potential were -60 (V), -26 (eV) and -5 (V), respectively. The method had a chromatographic run time of 2.5min and a linear calibration curve over the range 20-5000ng/mL. The limit of quantification was 20ng/mL. The stability tests indicated no significant degradation. This HPLC-MS/MS procedure was used to assess the bioequivalence of two propylthiouracil 100mg tablet formulations in healthy volunteers of both sexes in fasted and fed state. The geometric mean and 90% confidence interval CI of Test/Reference percent ratios were, without and with food, respectively: 109.28% (103.63-115.25%) and 115.60% (109.03-122.58%) for Cmax, 103.31% (100.74-105.96%) and 103.40% (101.03-105.84) for AUClast. This method offers advantages over those previously reported, in terms of both a simple liquid-liquid extraction without clean-up procedures, as well as a faster run time (2.5min). The LOQ of 20ng/mL is well suited for pharmacokinetic studies. The assay performance results indicate that the method is precise and accurate enough for the routine determination of the propylthiouracil in human plasma. The test formulation with and without food was bioequivalent to reference formulation. Food administration increased the Tmax and decreased the bioavailability (Cmax and AUC).

A Modified Acidic Approach For Dna Extraction From Plant Species Containing High Levels Of Secondary Metabolites.

Purified genomic DNA can be difficult to obtain from some plant species because of the presence of impurities such as polysaccharides, which are often co-extracted with DNA. In this study, we developed a fast, simple, and low-cost protocol for extracting DNA from plants containing high levels of secondary metabolites. This protocol does not require the use of volatile toxic reagents such as mercaptoethanol, chloroform, or phenol and allows the extraction of high-quality DNA from wild and cultivated tropical species.

Islet Neogenesis-associated Protein (ingap): The Role Of Its Endogenous Production As A Positive Modulator Of Insulin Secretion.

Islet neogenesis-associated protein (INGAP) is a peptide found in pancreatic exocrine-, duct- and islet- non-β-cells from normal hamsters. Its increase induced by either its exogenous administration or by the overexpression of its gene enhances β-cell secretory function and increases β-cell mass by a combination of stimulation of cell replication and islet neogenesis and reduction of β-cell apoptosis. We studied the potential modulatory role of endogenous INGAP in insulin secretion using two different experimental approaches. Hamster islets transfected with INGAP-small interfering RNA (INGAP-siRNA) were used to study glucose-stimulated insulin secretion (GSIS). In parallel, freshly isolated islets were incubated with high glucose and the same concentration of either a specific anti-INGAP rabbit serum or normal rabbit serum. INGAP-siRNA transfected islets reduced their INGAP mRNA and protein content by 35.1% and 47.2%, respectively whereas GSIS decreased by 25.8%. GSIS by transfected islets attained levels comparable to those recorded in control islets when INGAP pentadecapeptide (INGAP-PP) was added to the culture medium. INGAP antibody in the medium decreased significantly GSIS in a dose-dependent manner. These results indicate that endogenous INGAP plays a physiological positive modulatory role in insulin secretion, supporting its possible use in the treatment of prediabetes and Type 2 diabetes.

Screening The Life Cycle Of Schistosoma Mansoni Using High-resolution Mass Spectrometry.

Schistosomiasis is a common tropical disease caused by Schistosoma species Schistosomiasis' pathogenesis is known to vary according to the worms' strain. Moreover, high parasitical virulence is directly related to eggs release and granulomatous inflammation in the host's organs. This virulence might be influenced by different classes of molecules, such as lipids. Therefore, better understanding of the metabolic profile of these organisms is necessary, especially for an increased potential of unraveling strain virulence mechanisms and resistance to existing treatments. In this report, direct-infusion electrospray high-resolution mass spectrometry (ESI(+)-HRMS) along with the lipidomic platform were employed to rapidly characterize and differentiate two Brazilian S. mansoni strains (BH and SE) in three stages of their life cycle: eggs, miracidia and cercariae, with samples from experimental animals (Swiss/SPF mice). Furthermore, urine samples of the infected and uninfected mice were analyzed to assess the possibility of direct diagnosis. All samples were differentiated using multivariate data analysis, PCA, which helped electing markers from distinct lipid classes; phospholipids, diacylglycerols and triacylglycerols, for example, clearly presented different intensities in some stages and strains, as well as in urine samples. This indicates that biochemical characterization of S. mansoni may help narrowing-down the investigation of new therapeutic targets according to strain composition and aggressiveness of disease. Interestingly, lipid profile of infected mice urine varies when compared to control samples, indicating that direct diagnosis of schistosomiasis from urine may be feasible.

The Real-life Experience With Cardiovascular Complications In The First Dose Of Fingolimod For Multiple Sclerosis.

Fingolimod is a new and efficient treatment for multiple sclerosis (MS). The drug administration requires special attention to the first dose, since cardiovascular adverse events can be observed during the initial six hours of fingolimod ingestion. The present study consisted of a review of cardiovascular data on 180 patients with MS receiving the first dose of fingolimod. The rate of bradycardia in these patients was higher than that observed in clinical trials with very strict inclusion criteria for patients. There were less than 10% of cases requiring special attention, but no fatal cases. All but one patient continued the treatment after this initial dose. This is the first report on real-life administration of fingolimod to Brazilian patients with MS, and one of the few studies with these characteristics in the world.

Inactivation Of Ampk Mediates High Phosphate-induced Extracellular Matrix Accumulation Via Nox4/tgfß-1 Signaling In Human Mesangial Cells.

High phosphate (Pi) levels and extracellular matrix (ECM) accumulation are associated with chronic kidney disease progression. However, how high Pi levels contribute to ECM accumulation in mesangial cells is unknown. The present study investigated the role and mechanism of high Pi levels in ECM accumulation in immortalized human mesangial cells (iHMCs). iHMCs were exposed to normal (0.9 mM) or increasing Pi concentrations (2.5 and 5 mM) with or without diferent blockers or activators. NOX4, phosphorylated AMPK (p-AMPK), phosphorylated SMAD3 (p-SMAD3), fibronectin (F/N), collagen IV (C-IV) and alpha-smooth muscle actin (α-SMA) expression was assessed via western blot and immunofluorescence. Lucigenin-enhanced chemiluminescence, and dihydroethidium (DHE) assessed NADPH oxidase activity and superoxide (SO), respectively. In iHMCs, a Pi transporter blocker (PFA) abrogated high Pi-induced AMPK inactivation, increase in NADPH oxidase-induced reactive oxygen species (ROS) levels, NOX4, p-SMAD3, α-SMA and C-IV expression. AMPK activation by AICAR, NOX4 silencing or NADPH oxidase blocker prevented high Pi-induced DHE levels, p-SMAD3, F/N, C-IV and α-SMA expression. AMPK inactivation with NOX4-induced ROS formation and transforming growth factor ß-1 (TGFß-1) signaling activation mediates high Pi-induced ECM accumulation in iHMCs. Maneuvers increasing AMPK or reducing NOX4 activity may contribute to renal protection under hyperphosphatemia.

Gonadal Hormones Modulate The Responsiveness To Local β-blocker-induced Antinociception In The Temporomandibular Joint Of Male And Female Rats.

We have previously demonstrated that blockade of β-adrenoreceptors (β-AR) located in the temporomandibular joint (TMJ) of rats suppresses formalin-induced TMJ nociceptive behaviour in both male and female rats, but female rats are more responsive. In this study, we investigated whether gonadal hormones modulate the responsiveness to local β-blocker-induced antinociception in the TMJ of rats. Co-administration of each of the selective β1 (atenolol), β2 (ICI 118.551) and β3 (SR59230A)-AR antagonists with equi-nociceptive concentrations of formalin in the TMJ of intact, gonadectomized and hormone-treated gonadectomized male and female rats. Atenolol, ICI 118.551 and SR59230A significantly reduced formalin-induced TMJ nociception in a dose response fashion in all groups tested. However, a lower dose of each β-AR antagonist was sufficient to significantly reduce nociceptive responses in gonadectomized but not in intact and testosterone-treated gonadectomized male rats. In the female groups, a lower dose of β1 -AR antagonist was sufficient to significantly reduce nociceptive responses in gonadectomized but not in intact or gonadectomized rats treated with progesterone or a high dose of oestradiol; a lower dose of β2 -AR antagonist was sufficient to significantly reduce nociceptive responses in gonadectomized but not in intact and gonadectomized rats treated with low or high dose of oestradiol. Gonadal hormones may reduce the responsiveness to local β-blocker-induced antinociception in the TMJ of male and female rats. However, their effect depends upon their plasma level, the subtype of β-AR and the dose of β-blockers used.

High-resolution Transcript Profiling Of The Atypical Biotrophic Interaction Between Theobroma Cacao And The Fungal Pathogen Moniliophthora Perniciosa.

Witches' broom disease (WBD), caused by the hemibiotrophic fungus Moniliophthora perniciosa, is one of the most devastating diseases of Theobroma cacao, the chocolate tree. In contrast to other hemibiotrophic interactions, the WBD biotrophic stage lasts for months and is responsible for the most distinctive symptoms of the disease, which comprise drastic morphological changes in the infected shoots. Here, we used the dual RNA-seq approach to simultaneously assess the transcriptomes of cacao and M. perniciosa during their peculiar biotrophic interaction. Infection with M. perniciosa triggers massive metabolic reprogramming in the diseased tissues. Although apparently vigorous, the infected shoots are energetically expensive structures characterized by the induction of ineffective defense responses and by a clear carbon deprivation signature. Remarkably, the infection culminates in the establishment of a senescence process in the host, which signals the end of the WBD biotrophic stage. We analyzed the pathogen's transcriptome in unprecedented detail and thereby characterized the fungal nutritional and infection strategies during WBD and identified putative virulence effectors. Interestingly, M. perniciosa biotrophic mycelia develop as long-term parasites that orchestrate changes in plant metabolism to increase the availability of soluble nutrients before plant death. Collectively, our results provide unique insight into an intriguing tropical disease and advance our understanding of the development of (hemi)biotrophic plant-pathogen interactions.

Preserved Fertility In A Patient With Gynecomastia Associated With The P.pro695ser Mutation In The Androgen Receptor.

The androgen insensitivity syndrome (AIS) is described as a dysfunction of the androgen receptor (AR) in 46,XY individuals, which can be associated with mutations in the AR gene or can be due to unknown mechanisms. Different mutations in AIS generally cause variable phenotypes that range from a complete hormone resistance to a mild form usually associated with male infertility. The purpose of this study was to search for mutations in the AR gene in a fertile man with gynecomastia and to evaluate the influence of the mutation on the AR transactivation ability. Sequencing of the AR gene revealed the p.Pro695Ser mutation. It is located within the AR ligand-binding domain. Bioinformatics analysis indicated a deleterious role, which was verified after testing transactivation activity and N-/C-terminal (N/C) interaction by in vitro expression of a reporter gene and 2-hybrid assays. p.Pro695Ser showed low levels of both transactivation activity and N/C interaction at low dihydrotestosterone (DHT) conditions. As the ligand concentration increased, both transactivation activity and N/C interaction also increased and reached normal levels. Therefore, this study provides functional insights for the p.Pro695Ser mutation described here for the first time in a patient with mild AIS. The expression profile of p.Pro695Ser not only correlates to the patient's phenotype, but also suggests that a high-dose DHT therapy may overcome the functional deficit of the mutant AR.

Low Hdl Cholesterol But Not High Ldl Cholesterol Is Independently Associated With Subclinical Coronary Atherosclerosis In Healthy Octogenarians.

Although low-density lipoprotein cholesterol (LDL-C) has been consistently demonstrated a predictor of atherosclerotic disease in a large spectrum of clinical settings, among individuals aged of 80 years or older this concept is uncertain. This study was evaluated in a carefully selected population if the association between LDL-C and coronary atherosclerotic burden remains significant in the very elderly. Individuals aged of 80 years or older (n = 208) who spontaneously sought primary prevention care and have never manifested cardiovascular disease, malnutrition, neoplastic or consumptive disease were enrolled for a cross-sectional analysis. Medical evaluation, anthropometric measurements, blood tests and cardiac computed tomography were obtained. In analyses adjusted for age, gender, diabetes, systolic and diastolic blood pressure, smoking and statin therapy, no association was found between coronary calcium score (CCS) and LDL-C [1.79 (0.75-4.29)]. There was no association between triglycerides and CCS. The association between high-density lipoprotein cholesterol (HDL-C) and CCS was significant and robust in unadjusted [0.32 (0.15-0.67)] as well as in the fully adjusted analysis [0.34 (0.15-0.75)]. The present study confirms in a healthy cohort of individuals aged of 80 years or more that while the association between LDL-C and coronary atherosclerosis weakens with aging, the opposite occurs with the levels of HDL-C.

Post Hoc Analysis Of The Patricia Randomized Trial Of The Efficacy Of Human Papillomavirus Type 16 (hpv-16)/hpv-18 As04-adjuvanted Vaccine Against Incident And Persistent Infection With Nonvaccine Oncogenic Hpv Types Using An Alternative Multiplex Type-specific Pcr Assay For Hpv Dna.

The efficacy of the human papillomavirus type 16 (HPV-16)/HPV-18 AS04-adjuvanted vaccine against cervical infections with HPV in the Papilloma Trial against Cancer in Young Adults (PATRICIA) was evaluated using a combination of the broad-spectrum L1-based SPF10 PCR-DNA enzyme immunoassay (DEIA)/line probe assay (LiPA25) system with type-specific PCRs for HPV-16 and -18. Broad-spectrum PCR assays may underestimate the presence of HPV genotypes present at relatively low concentrations in multiple infections, due to competition between genotypes. Therefore, samples were retrospectively reanalyzed using a testing algorithm incorporating the SPF10 PCR-DEIA/LiPA25 plus a novel E6-based multiplex type-specific PCR and reverse hybridization assay (MPTS12 RHA), which permits detection of a panel of nine oncogenic HPV genotypes (types 16, 18, 31, 33, 35, 45, 52, 58, and 59). For the vaccine against HPV types 16 and 18, there was no major impact on estimates of vaccine efficacy (VE) for incident or 6-month or 12-month persistent infections when the MPTS12 RHA was included in the testing algorithm versus estimates with the protocol-specified algorithm. However, the alternative testing algorithm showed greater sensitivity than the protocol-specified algorithm for detection of some nonvaccine oncogenic HPV types. More cases were gained in the control group than in the vaccine group, leading to higher point estimates of VE for 6-month and 12-month persistent infections for the nonvaccine oncogenic types included in the MPTS12 RHA assay (types 31, 33, 35, 45, 52, 58, and 59). This post hoc analysis indicates that the per-protocol testing algorithm used in PATRICIA underestimated the VE against some nonvaccine oncogenic HPV types and that the choice of the HPV DNA testing methodology is important for the evaluation of VE in clinical trials. (This study has been registered at ClinicalTrials.gov under registration no. NCT00122681.).

Reference Values For High-density Lipoprotein Particle Size And Volume By Dynamic Light Scattering In A Brazilian Population Sample And Their Relationships With Metabolic Parameters.

Current data indicate that the size of high-density lipoprotein (HDL) may be considered an important marker for cardiovascular disease risk. We established reference values of mean HDL size and volume in an asymptomatic representative Brazilian population sample (n=590) and their associations with metabolic parameters by gender. Size and volume were determined in HDL isolated from plasma by polyethyleneglycol precipitation of apoB-containing lipoproteins and measured using the dynamic light scattering (DLS) technique. Although the gender and age distributions agreed with other studies, the mean HDL size reference value was slightly lower than in some other populations. Both HDL size and volume were influenced by gender and varied according to age. HDL size was associated with age and HDL-C (total population); non- white ethnicity and CETP inversely (females); HDL-C and PLTP mass (males). On the other hand, HDL volume was determined only by HDL-C (total population and in both genders) and by PLTP mass (males). The reference values for mean HDL size and volume using the DLS technique were established in an asymptomatic and representative Brazilian population sample, as well as their related metabolic factors. HDL-C was a major determinant of HDL size and volume, which were differently modulated in females and in males.

Histopathological Alterations In The Prostates Of Mongolian Gerbils Exposed To A High-fat Diet And Di-n-butyl Phthalate Individually Or In Combination.

Both high-fat diet and exposure to endocrine-disrupting chemicals have been implicated in susceptibility to pathological prostate lesions, but the consequences of combining the two have not yet been examined. We evaluated the effects of gestational and postnatal exposure to a high-fat diet (20% fat) and low doses of di-n-butyl phthalate (DBP; 5mg/kg/day), individually or in combination, on the tissue response and incidence of pathological lesions in the ventral prostate of adult gerbils. Continuous intake of a high-fat diet caused dyslipidemia, hypertrophy, and promoted the development of inflammatory, premalignant and malignant prostate lesions, even in the absence of obesity. Life-time DBP exposure was obesogenic and dyslipidemic and increased the incidence of premalignant prostate lesions. Combined exposure to DBP and a high-fat diet also caused prostate hypertrophy, but the effects were less severe than those of individual treatments; combined exposure neither induced an inflammatory response nor altered serum lipid content.

Characterization Of Anfo Explosive By High Accuracy Esi(±)-ftms With Forensic Identification On Real Samples By Easi(-)-ms.

Ammonium nitrate fuel oil (ANFO) is an explosive used in many civil applications. In Brazil, ANFO has unfortunately also been used in criminal attacks, mainly in automated teller machine (ATM) explosions. In this paper, we describe a detailed characterization of the ANFO composition and its two main constituents (diesel and a nitrate explosive) using high resolution and accuracy mass spectrometry performed on an FT-ICR-mass spectrometer with electrospray ionization (ESI(±)-FTMS) in both the positive and negative ion modes. Via ESI(-)-MS, an ion marker for ANFO was characterized. Using a direct and simple ambient desorption/ionization technique, i.e., easy ambient sonic-spray ionization mass spectrometry (EASI-MS), in a simpler, lower accuracy but robust single quadrupole mass spectrometer, the ANFO ion marker was directly detected from the surface of banknotes collected from ATM explosion theft.