Electrophoretic mobility shift as a molecular beacon-based readout for miRNA detection.

MicroRNAs are short, non-coding RNA sequences involved in gene expression regulation. Quantification of miRNAs in biological fluids involves time consuming and laborious methods such as Northern blotting or PCR-based techniques. Molecular beacons (MB) are an attractive means for rapid detection of miRNAs, although the need for sophisticated readout methods limits their use in research and clinical settings. Here, we introduce a novel method based on delayed electrophoretic mobility, as a quantitative means for detection of miRNAs-MB hybridization. Upon hybridization with the target miRNAs, MB form a fluorescent duplex with reduced electrophoretic mobility, thus bypassing the need for additional staining. In addition to emission of light, the location of the fluorescent band on the gel acts as an orthogonal validation of the target identity, further confirming the specificity of binding. The limit of detection of this approach is approximately 100 pM, depending on the MB sequence. The method is sensitive enough to detect specific red blood cell miRNAs molecules in total RNA, with single nucleotide specificity. Altogether, we describe a rapid and affordable method that offers sensitive detection of single-stranded small DNA and RNA sequences.

Risk factors for mild depression in older women with overactive bladder syndrome-A cross sectional study.

BACKGROUND: Studies demonstrate an association between severe depression and overactive bladder syndrome (OAB). However, mild depression is constantly overlooked. The aim of this study was to evaluate the clinical and sociodemographic factors associated with mild depression in women with OAB. METHODS: Cross-sectional study involving 241 women over 60 years old in Brasilia, Brazil. All patients were subjected to an interview followed by questionnaires and physical examination. The clinical and sociodemographic variables analyzed were age, body mass index, physical activity level, OAB symptoms, presence of gynecological surgery, fecal incontinence, systemic arterial hypertension, Diabetes Mellitus, anxiety (Beck Anxiety Scale). The Geriatric Depression Scale-15 (GDS-15) was used to identify depression. Univariate logistic regression was used to assess the correlation between mild depression and the variables chosen. Variables with a p-value less than 0.2 were included in the multivariate logistic regression analysis. The level of confidence was set at 95%. RESULTS: 121 volunteers suffered from mild depression. The multivariate analysis demonstrated that gynecological surgery (p < .001) and anxiety (p < .001) are factors associated with mild depression. Older women with a history of gynecological surgery and a GDS-15 score of 2.04 were 1.08 times more likely to develop mild depression compared to older women with no history of gynecological surgery. CONCLUSION: Anxiety and a history of gynecological surgery are factors that need to be taken into account and may influence the development of mild depression in older women with OAB. Psychological treatment should be considered an important adjunct in the treatment of women with symptoms of Overactive Bladder Syndrome.

A Survey of the Management of Patients with Temporomandibular Disorders by General Dental Practitioners in Southern Brazil.

PURPOSE: Temporomandibular disorders (TMD) are recognized as one of the most controversial topics in dentistry, despite the fact that both basic science and clinical researchers have currently reached some degree of consensus. This study aimed to conduct a questionnaire-based survey about the management of TMD patients by general dental practitioners (GDPs). MATERIALS AND METHODS: One hundred fifty-one GDPs with a private practice in a city of southern Brazil were included, independent of school of origin, gender, graduation year, and curriculum content. All participants were administered a questionnaire about the management of patients with TMD, and the responses were analyzed by binomial and chi-square tests (α = 0.05). RESULTS: Of the GDPs, 88.7% received TMD patients, who were primarily diagnosed on the basis of medical history (36.6%) or physical examination (30.4%). Of these, 65.4% referred the patients elsewhere, primarily to specialists in occlusion (36.1%) or orthodontics (29.7%). Occlusal splinting was the most commonly used management modality (20.8%), followed by occlusal adjustment (18.1%) and pharmacotherapy (16.6%). Splints were fabricated in maximum habitual intercuspation or centric relation depending on individual patient (54.8%). The hard stabilization form was the most common type of appliance used (35.0%). Moreover, 73.8% of the GDPs did not employ semi-adjustable articulators, and 69.5% adjusted the appliances at the time of fixing. The duration of splint use and the frequency of follow-up were considered patient dependent by 62.1% and 72.8%, respectively. GDPs considered the two major TMD etiologic categories as multifactorial (20.8%) and occlusion (19.9%). Multidisciplinary medical and dental treatment was considered necessary by 97.9%. CONCLUSIONS: The evaluated general dental practitioners manage TMD patients according to international guidelines.

Preheating with nonminimal kinetic terms.

We present the first (3+1)-dimensional numerical simulations of scalar fields with nonminimal kinetic terms. As an example, we examine the existence and stability of preheating in the presence of a Dirac-Born-Infeld inflaton coupled to a canonical matter field. The simulations represent the full nonlinear theory in the presence of an expanding universe. We show that parametric resonance in the matter field along with self-resonance in the inflaton repopulate the universe with matter particles as efficiently as in traditional preheating.

Targeting the non-structural protein 1 from dengue virus to a dendritic cell population confers protective immunity to lethal virus challenge.

Dengue is the most prevalent arboviral infection, affecting millions of people every year. Attempts to control such infection are being made, and the development of a vaccine is a World Health Organization priority. Among the proteins being tested as vaccine candidates in preclinical settings is the non-structural protein 1 (NS1). In the present study, we tested the immune responses generated by targeting the NS1 protein to two different dendritic cell populations. Dendritic cells (DCs) are important antigen presenting cells, and targeting proteins to maturing DCs has proved to be an efficient means of immunization. Antigen targeting is accomplished by the use of a monoclonal antibody (mAb) directed against a DC cell surface receptor fused to the protein of interest. We used two mAbs (αDEC205 and αDCIR2) to target two distinct DC populations, expressing either DEC205 or DCIR2 endocytic receptors, respectively, in mice. The fusion mAbs were successfully produced, bound to their respective receptors, and were used to immunize BALB/c mice in the presence of polyriboinosinic: polyribocytidylic acid (poly (I:C)), as a DC maturation stimulus. We observed induction of strong anti-NS1 antibody responses and similar antigen binding affinity irrespectively of the DC population targeted. Nevertheless, the IgG1/IgG2a ratios were different between mouse groups immunized with αDEC-NS1 and αDCIR2-NS1 mAbs. When we tested the induction of cellular immune responses, the number of IFN-γ producing cells was higher in αDEC-NS1 immunized animals. In addition, mice immunized with the αDEC-NS1 mAb were significantly protected from a lethal intracranial challenge with the DENV2 NGC strain when compared to mice immunized with αDCIR2-NS1 mAb. Protection was partially mediated by CD4(+) and CD8(+) T cells as depletion of these populations reduced both survival and morbidity signs. We conclude that targeting the NS1 protein to the DEC205(+) DC population with poly (I:C) opens perspectives for dengue vaccine development.

Screening of RB1 alterations in Brazilian patients with retinoblastoma and relatives with retinoma: phenotypic and genotypic associations.

PURPOSE: To identify constitutional alterations of the retinoblastoma 1 gene (RB1) in two cohorts of Brazilian patients with retinoblastoma and to analyze genotype-phenotype associations. METHODS: Molecular screening was carried out by direct sequencing of the 27 RB1 exons and flanking regions in blood DNA of 71 patients with retinoblastoma and 4 relatives with retinoma, and with multiplex ligation-dependent probe amplification (MLPA) in 21 patients. The presumed impact of nucleotide substitutions on the structure of the retinoblastoma protein (pRB) was predicted by Polymorphism Phenotyping-2 (PolyPhen-2). Kaplan-Meier and log-rank test were used for estimating 60-month survival rates. RESULTS: One hundred two nucleotide substitutions were detected, 92 substitutions in 59 patients with retinoblastoma and 10 substitutions in 4 individuals with retinoma. Eight substitutions were novel. The majority of substitutions were intronic (86.2%). More than one substitution was present in 37.3% of patients. Twenty-one duplications and 11 deletions were found in 12 patients; some of which with both types of alterations. Duplications/deletions were found in four patients lacking constitutional alterations when analyzed by sequencing, and in eight patients carrying one or more polymorphic intronic substitutions. The global 60-month survival rate in patients was 91.8% (Confidence Interval95% = 85.0 - 99.1). Significant, lower survival rates were found in extraocular presentation (81.0%) versus intraocular tumors (P = 0.014), first enucleation after 1 month following diagnosis (80.9%) versus earlier first enucleation (P = 0.020), and relapse (100.0%) versus absence of relapse (P = 0.0005). CONCLUSIONS: Fifteen substitutions (4 intronic and 11 exonic) were identified as probably or likely pathogenic. Four of these 11 exonic substitutions were novel. Survival rates, however, were not affected by presence of these probably or likely pathogenic alterations, most of which not found in patients with retinoblastoma from other Latin American countries. These differences might be related to the different ethnic composition of the Latin American cohorts. Portuguese Abstract.

Effective treatment of rat adjuvant-induced arthritis by celastrol.

We have previously reported an increase in interleukin (IL)-1ß and IL-17 levels, and a continuous activation of caspase-1 in early rheumatoid arthritis (RA) patients. These results suggest that drugs targeting IL-1ß regulatory pathways, in addition to tumor necrosis factor (TNF), may constitute promising therapeutic agents in early RA. We have recently used a THP-1 macrophage-like cell line to screen 2320 compounds for those that down-regulate both IL-1ß and TNF secretion. Celastrol was one of the most promising therapeutic candidates identified in that study. Our main goal in the present work was to investigate whether administration of celastrol is able to attenuate inflammation in a rat model of adjuvant-induced arthritis (AIA). Moreover, since IL-1ß is known to play a role in the polarization of Th17 cells, we also investigate whether administration of digoxin, a specific inhibitor of Th17 cells polarization, is able to attenuate inflammation in the same rat model. We found that celastrol administration significantly suppressed joint inflammation. The histological and immunohistochemical evaluation revealed that celastrol-treated rats had a normal joint structure with complete abrogation of the inflammatory infiltrate and cellular proliferation. In contrast, we observed that digoxin administration significantly ameliorated inflammation but only if administrated in the early phase of disease course (after 4days of disease induction), and it was not efficient at inhibiting the infiltration of immune cells within the joint and in preventing damage. Thus, our results suggest that celastrol has significant anti-inflammatory and anti-proliferative properties and can constitute a potential anti-inflammatory drug with therapeutic efficacy in the treatment of immune-mediated inflammatory diseases such as RA. Furthermore, we find that early inhibition of Th17 cells polarization ameliorates arthritis but it is not as effective as celastrol.

Molecular epidemiology of microorganisms isolated from food workers and enteral feeding of public hospitals.

UNLABELLED: This study aimed to compare strains of Staphylococcus aureus and E. coli isolated from food workers and enteral diet samples obtained from 2 public hospitals (H1/H2) in Goiania, Goias, Brazil, by the means of antibiogram and pulsed field gel electrophoresis (PFGE). In the H1, strains of S. aureus were present in 2 enteral diet samples and in 13 food worker swabs. Strains of E. coli were found in an enteral diet sample from H1 and in 2 enteral diet samples from H2 and in 6 food worker swabs in the H1 and in 12 food worker swabs from H2. According to the antibiogram, the 6 susceptibility profiles (A to F) of 15 S. aureus strains colonizing personnel and enteral feeding did not allow the identification of the probable source of diet contamination. All 20 E. coli strains isolated from the H1 and H2 were grouped in 4 phenotypic profiles (A to D). The phenotypes A (H1) and C (H2) showed the same profile for microorganisms isolated from handlers and diets, suggesting more phenotypic similarity among these samples. PFGE genotyping showed that S. aureus isolates from diets were related to a single strain isolated from a food worker suggesting that in this case the reason for the diet contamination may be a result of food handling. The food worker appears to be the most probable source of E. coli contamination for enteral feeding from H2. This fact emphasizes on the food workers as a risk of bacterial transmission for the diets and that the diet chain production must be controlled. PRACTICAL APPLICATION: The study emphasizes the importance of monitoring the enteral diet microbiological quality and the factors associated to its contamination. The study highlights the use of molecular biology as an instrument to correlate strains to determine the origin of the final product contamination.

Analyses of paralytic shellfish toxins and biomarkers in a southern Brazilian reservoir.

The Alagados Reservoir (Brazil) is an important source for the supply of water, recreation and fishery. Since 2002, the occurrence of cyanobacterial blooms (paralytic shellfish toxins - PST producers) have been noted. This study was aimed at the monitoring of PST occurrence in the Reservoir's water and fish. Biomarkers such as ethoxyresorufin-O-deethylase (EROD), glutathione S-transferase (GST), catalase (CAT), and acetylcholinesterase (AchE) activities, lipoperoxidation (LPO), histopathology, and comet assay were analyzed in fish. Water and fish were sampled in spring, summer and autumn. The PST concentrations in water were 5.15, 43.84, and 50.78 ng equiv Saxitoxin/L in the spring, summer and autumn, respectively. The PST muscle concentration was below the limit for shellfish. Gonyautoxins (GTX) were found in water samples and fish muscle, and GTX 5 was the major analogous found in muscle. In the summer samples, the LPO, genetic damage, and the GST and AchE activities increased while in the autumn an increase in EROD activity and genetic damage were observed. In all samplings, histopathological alterations in the fish gills and liver were found. The results showed a seasonal variation in the fishes health, which could be related also to farming activities and to the contaminants bioavailability during the year.

Constitutive RB1 mutation in a child conceived by in vitro fertilization: implications for genetic counseling.

BACKGROUND: The purpose of this study was to identify mutations associated with bilateral retinoblastoma in a quadruplet conceived by in vitro fertilization, and to trace the parental origin of mutations in the four quadruplets and their father. METHODS: Mutational screening was carried out by sequencing. Genotyping was carried out for determining quadruplet zygosity. RESULTS: The proband was a carrier of a novel RB1 constitutive mutation (g.2056C>G) which was not detected in her father or her unaffected sisters, and of two other mutations (g.39606 C>T and g.174351T>A) also present in two monozygotic sisters. The novel mutation probably occurred de novo while the others were of likely maternal origin. The novel mutation, affecting the Kozak consensus at the 5'UTR of RB1 and g.174351T>A were likely associated to retinoblastoma in the proband. CONCLUSION: Molecular diagnosis of retinoblastoma requires genotypic data of the family for determining hereditary transmission. In the case of children generated by IVF with oocytes from an anonymous donor which had been stored in a cell repository, this might not be successfully accomplished, making precise diagnosis impracticable for genetic counseling.

Hereditary retinoblastoma transmitted by maternal germline mosaicism.

BACKGROUND: Investigating transmission of a constitutive, g78238C > T (R552X), RB1 mutation in four affected children descended from three different unaffected fathers and an unaffected mother. PROCEDURES: Sequence data analyses and allele-specific PCR assays were used to investigate the presence of the mutation in four affected children, five unaffected sibs (or half-sibs), and the unaffected mother. Haplotyping was carried out for confirming that the children descended from different fathers. RESULTS: Haplotyping excluded the possibility of paternal transmission of a de novo mutation and provided evidence of maternal germline mosaicism. The mutation was apparently absent in blood- and buccal cell-DNA of the mother who also showed a normal fundoscopy. CONCLUSIONS: Our findings indicated that mosaicism was restricted to the maternal germline. The mutational event must have occurred at least 4 weeks post-conception, unlike the early mutational events of most mosaics, occurring between fertilization and the 8th day of conception. The implications of these findings are discussed in view that genetic counselling should discriminate between germline mosaicism and de novo events in pseudo-low-penetrant hereditary retinoblastoma.

Effect of beta-lactoglobulin polymorphism and seasonality on bovine milk composition.

The objective was to evaluate the effect of beta-lactoglobulin (beta-lg) polymorphism and seasonality on milk composition (fat, lactose, total solids, milk urea nitrogen, total protein, true protein, casein and somatic cell counts) of Holstein and Girolando cows. Milk and blood samples from 278 Holsteins cows and 156 Girolando cows were taken during two dry seasons and two rainy seasons, for milk composition analysis and to determine beta-lg genotypes, respectively. BB genotype was the most frequent for both breeds, followed by AA genotype for Holstein (BB>AA>AB) and by AB for Girolando cows (BB>AB>AA). No differences were found in milk compositional characteristics among genetic variants of beta-lg (AA, AB and BB) either between Holstein or Girolando cows. No association between milk composition and beta-lg genetic polymorphism was observed. During the dry season, independently of the breed considered, higher contents of lactose, true protein, casein and casein:true protein ratio were found.

Changes in dental arch measurements of young adults with normal occlusion--a longitudinal study.

This study evaluates the longitudinal changes in occlusal dimensions in young adults. The sample was composed of 27 individuals (13 male and 14 female individuals) who presented dental Class I and normal occlusion, according to the inclusion protocol. All teeth were present except for the third molars. The mean age of the subjects was 21 years and two months initially and 28 years and four months at the end of the observation period. The measurements collected in both arches were overbite, overjet, intercanine and intermolar distances, irregularity index, and perimeter. All measurements were taken from study dental casts by a calibrated operator, using a digital caliper. The paired t-test was used to evaluate the changes, using the difference between the initial and final mean measurements. Data indicated that the overbite increased 0.39 mm, the incisor irregularity increased 0.38 mm in the upper arch and 0.54 mm in the lower arch, and the arch perimeter decreased 0.67 mm in the upper arch and 0.71 mm in the lower arch (P > .05). The other measurements did not show significant changes. No sexual dimorphism was observed. It could be concluded that occlusal dimensions change throughout adult life. In the sample studied, changes were statistically significant for overbite, incisors irregularity, and arch perimeter after a mean period of seven years and two months. These changes can be observed in both sexes.

[Microbiological quality of human milk from a Brazilian milk bank]. / Qualidade microbiológica de leite humano obtido em banco de leite.

OBJECTIVE: The objectives of the present study were to determine the prevalence of potentially pathogenic microorganisms that indicate the hygienic and sanitary conditions of human milk samples collected at a Human Milk Bank. METHODS: Three hundred and thirty eight (338) samples of human milk collected from a milk bank in a maternity in the municipality of Goiânia, in the state of Goias, Brazil were submitted to microbiological analysis. The latter were plated on McConkey agar according to the type of bacteria. Among the total number of samples collected, 194 consisted of raw milk and the remaining 144 were pasteurized milk. RESULTS: The presence of Staphylococcus spp., Streptococcus spp., yeasts and molds, and Enterobacteriaceae was verified in the raw milk samples. Staphylococcus aureus were isolated in 10 (5.2%) samples, Staphylococcus epidermidis in 28 (14.4%) samples, Streptococcus spp. in three (1.6%) samples, yeasts and molds in 43 (22.2%) and Enterobacteriaceae in 49 (25.3%) samples. In a hundred and forty four (144) samples which underwent thermal treatment Staphylococcus aureus was detected in five (3.5%) samples, Staphylococcus epidermidis in 15 (10.4%), Staphylococcus lugdenensis in two (1.4%), Streptococcus spp. in four (2.8%), yeasts and molds in 37 (25.7%), and Enterobacteriaceae in nine (6.3%). CONCLUSIONS: Analysis indicated a high degree of contamination in raw human milk, and as for the pasteurized milk, despite elimination of the great majority of potentially pathogenic microorganisms, the percentage of yeasts and molds was higher than in raw milk, demonstrating that a lower degree of initial contamination would be necessary for pasteurization to be an efficient means of microbiological control.

Qualidade microbiológica de leite humano obtido em banco de leite / Microbiological quality of human milk from a Brazilian milk bank

Determinar a prevalência de microorganismos indicadores e potencialmente patogênicos que indicam as condições higiênico-sanitárias das amostras de leite humano ordenhado coletadas em banco de leite. Foram realizadas análises microbiológicas de 338 amostras de leite humano ordenhado, sendo 194 de leite cru e 144, pasteurizado, coletadas em banco de leite humano de um hospital materno infantil de Goiânia, GO. As análises microbiológicas foram realizadas com semeadura em ágar Mc Conkey, de acordo com o tipo de bactéria. No leite cru, verificou-se a presença de Staphylococcus spp. Streptococcus spp., bolores e leveduras e Enterobacteriaceae. Observou-se que Staphylococcus aureus esteve presente em 10 (5,2porcento) amostras. Staphylococcus epidermidis em 28 (14,4porcento), Streptococcus spp. em três (1,6porcento), bolores e leveduras em 43 (22, 2porcento) e Enterobacteriaceae em 49 (25,3porcento). Das 144 amostras de leite humano ordenhado pasteurizado, detectaram-se Staphylococcus aureus em cinco (3,5porcento) , Staphylococcus epidermidis em 15 (10,4porcento), Staphylococcus lugdenesis em duas (1,4porcento), Streptococcus spp. em quatro (2,8porcento), bolores e leveduras em 37 (25,7porcento) e Enterobacteriaceae em nove (6,3porcento). Os resultados mostraram um alto grau de contaminaçäo no leite cru. No leite pasteurizado, apesar da eliminaçäo da grande maioria de microorganismos potencialmente patogênicos, a percentagem de bolores e leveduras excedeu a de leite cru, mostrando a necessidade de obtençäo de um leite com carga microbiana inicial mais baixa para que a pasteurizaçäo seja eficiente no controle microbiológico.

Uso de implantes osseointegrados na movimentaçäo dentária: relato de caso / The use of osseointegrated implants in tooth movement. A case report

O presente trabalho relata um caso clínico onde foi utilizado um implante osseointegrado, agindo como ancoragem para movimentaçäo ortodôntica. O caso exemplifica a importância de um diagóstico e plano de tratamento multidisciplinar, visando aprimorar o tratamento reabilitador de pacientes adultos com perdas múltiplas. É dado destaque para a necessidade de determinaçäo do local exato de colocaçäo do implante, para que sejam atendidos os objetivos ortodônticos e protéticos