Detection of the TLR4 1196C > T Polymorphism by Mismatched-Polymerase Chain Reaction Using Plasmid DNA as Internal Control in Restriction Fragment Length Polymorphism Assays

Background: Restriction fragment length polymorphism (RFLP) is a common molecular assay used for genotyping, and it requires validated quality control procedures to prevent mistyping caused by impaired endonuclease activity. We have evaluated the usefulness of a plasmid-based internal control in RFLP assays. Results: Blood samples were collected from 102 individuals with acute myocardial infarction (AMI) and 108 non-AMI individuals (controls) for DNA extraction and laboratory analyses. The 1196C> T polymorphism in the toll-like receptor 4 (TLR4) gene was amplified by mismatched-polymerase chain reaction (PCR). Amplicons and pBluescript II SK-plasmid were simultaneously digested with endonuclease HincII. Fragments were separated on 2% agarose gels. Plasmid was completely digested using up to 55.2 nmL/L DNA solutions and 1 mu L PCR product. Nevertheless, plasmid DNA with 41.4 nM or higher concentrations was incompletely digested in the presence of 7 mL PCR product. In standardized conditions, TLR4 1196C> T variant was accurately genotyped. TLR4 1196T allele frequency was similar between AMI (3.1%) and controls (2.0%, p = 0.948). TLR4 SNP was not associated with AMI in this sample population. In conclusion, the plasmid-based control is a useful approach to prevent mistyping in RFLP assays, and it is validate for genetic association studies such as TLR4 1196C> T.

Multiplex semi-nested RT-PCR with exogenous internal control for simultaneous detection of bovine coronavirus and group A rotavirus

Neonatal calf diarrhea is a multi-etiology syndrome of cattle and direct detection of the two major agents of the syndrome, group A rotavirus and Bovine coronavirus (BCoV) is hampered by their fastidious growth in cell culture. This study aimed at developing a multiplex semi-nested RT-PCR for simultaneous detection of BCoV (N gene) and group A rotavirus (VP1 gene) with the addition of an internal control (mRNA ND5). The assay was tested in 75 bovine feces samples tested previously for rotavirus using PAGE and for BCoV using nested RT-PCR targeted to RdRp gene. Agreement with reference tests was optimal for BCoV (kappa = 0.833) and substantial for rotavirus detection (kappa = 0.648). the internal control, ND5 mRNA, was detected successfully in all reactions. Results demonstrated that this multiplex semi-nested RT-PCR was effective in the detection of BCoV and rotavirus, with high sensitivity and specificity for simultaneous detection of both viruses at a lower cost, providing an important tool for studies on the etiology of diarrhea in cattle. (C) 2010 Elsevier B.V. All rights reserved.

Comparative physical-chemical characterization of encapsulated lipid-based isotretinoin products assessed by particle size distribution and thermal behavior analyses

Isotretinoin is the drug of choice for the management of severe recalcitrant nodular acne. Nevertheless, some of its physical-chemical properties are still poorly known. Hence, the aim of our study consisted to comparatively evaluate the particle size distribution (PSD) and characterize the thermal behavior of the three encapsulated isotretinoin products in oil suspension (one reference and two generics) commercialized in Brazil. Here, we show that the PSD, estimated by laser diffraction and by polarized light microscopy, differed between the generics and the reference product. However, the thermal behavior of the three products, determined by thermogravimetry (TGA), differential thermal (DTA) analyses and differential scanning calorimetry (DSC), displayed no significant changes and were more thermostable than the isotretinoin standard used as internal control. Thus, our study suggests that PSD analyses in isotretinoin lipid-based formulations should be routinely performed in order to improve their quality and bioavailability. (C) 2010 Elsevier B.V. All rights reserved.

Qualitative assessment of stock prices listed on the Sao Paulo stock exchange: An approach from the perspective of homogeneity analysis

The stock market suffers uncertain relations throughout the entire negotiation process, with different variables exerting direct and indirect influence on stock prices. This study focuses on the analysis of certain aspects that may influence these values offered by the capital market, based on the Brazil Index of the Sao Paulo Stock Exchange (Bovespa), which selects 100 stocks among the most traded on Bovespa in terms of number of trades and financial volume. The selected variables are characterized by the companies` activity area and the business volume in the month of data collection, i.e. April/2007. This article proposes an analysis that joins the accounting view of the stock price variables that can be influenced with the use of multivariate qualitative data analysis. Data were explored through Correspondence Analysis (Anacor) and Homogeneity Analysis (Homals). According to the research, the selected variables are associated with the values presented by the stocks, which become an internal control instrument and a decision-making tool when it comes to choosing investments.

The perception of Executives and other Employees on the answer to the Sarbanes-Oxley: case study of a subsidary of the American auto industry established in Brazil

To attend and obtain the systems and. internal controls mechanisms proposed by Sarbanes-Oxley certifications is actually a big challenge,for most of the multinational companies registered in SEC (US Securities and Exchange Commission). This work has the objective of contributing to the analysis of this methodology, not only to attend the law but to reduce cost and generate value through the strengthen of the internal control systems, turning them into animating value generation process mechanisms. So, the idea is to identify the main gaps in the theory through the literature revision and a case study in order to put a question to the main deficiencies, strong points or contributions through the evaluation of the noticed practices. Finally, we can say that a a result of the research and the analyses made in. this case, the vast majority of executives and other employees recognize the benefit that Sarbanes-Oxley Act has brought to the company searched. Also recognize that, although there is still necessity for systemic adequacy and infrastructure, it helps and reinforce reducing and controlling the risks. the system of internal controls in all areas of expertise. They approach and understand that there is the need for a change in the other employees` culture to be inserted in the day-today routine as internal controls, attention to Sarbanes-Oxley and Corporate Governance, making the control cost smaller when compared to the benefits generated.

Microvascular Lymphatic Density Analysis in Cutaneous Regressive and Nonregressive Superficial Spreading Melanomas Using the Lymphatic Marker D2-40

Background: The prognostic significance of spontaneous regression in melanoma, especially thin lesions, has been a controversial issue for the past 20 years, although recent studies suggest that extensive and late regression may be related to worse prognosis. Many data suggest that lymphangiogenesis predicts metastatic spread in melanoma. Methods: We have quantified lymphatic microvascular density (LMVD) in thin (<= 1.0 mm) superficial spreading melanomas comparing regressive and nonregressive melanomas, regressive and nonregressive areas from the same tumor, and early and late histological stages of regression in the same tumor. In addition, we tried to correlate lymphangiogenesis and tumor growth phase. We conducted histological examinations and immunohistochemical analyses using monoclonal antibody D2-40 with subsequent quantification by image analysis of 37 melanomas, 16 regressive and 21 nonregressive (controls). Results: We found higher LMVD in the late stage of regression compared with nonregressive area (internal control) of regressive melanomas. Conclusions: Our study suggest that the late stage of spontaneous regression in thin melanomas may be related to worse prognosis as it showed higher LMVD, and evidence shows that this is related with increased risk of metastatic spread. But this supposition must be confirmed by a longer follow-up for detection of lymph node metastases.

Galectin-3 Overexpression in Invasive Laryngeal Carcinoma, Assessed by Computer-assisted Analysis

The larynx is the most common site of malignancy in the upper aerodigestive tract. In Brazil, malignant laryngeal lesions represent 2% of all cancers, with similar to 3000 annual deaths. The association between human papillomavirus (HPV) and laryngeal cancer is still controversial. The aim of the present retrospective study was to determine the expression of galectin-3 immunoperoxidase in laryngeal carcinoma by examining paraffin-em bedded larynx biopsies from 65 patients, 10 in situ laryngeal carcinomas, 27 laryngeal carcinomas without metastases, and 28 with metastases. Twenty-eight cervical lymph nodes from patients with metastatic lesions were also evaluated. Nested PCR was performed to detect and type HPV DNA. Galectin-3 expression was assessed by immunohistochemistry using a computer-assisted system. Among 65 patients, 55 (84.6%)were positive to beta-globin (internal control); 10 (15.4%) patients were beta-globin negative and were excluded from the HPV evaluation. Thus, 7 (12.7%) out of 55 patients were HPV positive and 48 (87.3%) out of 55 patients were HPV negative. High expression of galectin-3 was observed in invasive laryngeal tumors, suggesting that galectin-3 could be associated with the invasiveness and aggressiveness of laryngeal carcinomas. (J Histochem Cytochem 57:665-673, 2009)

Control of interceptive actions is based on expectancy of time to target arrival

In this study the hypothesis that interceptive movements are controlled on the basis of expectancy of time to target arrival was tested. The study was conducted through assessment of temporal errors and kinematics of interceptive movements to a moving virtual target. Initial target velocity was kept unchanged in part of the trials, and in the others it was decreased 300 ms before the due time of target arrival at the interception position, increasing in 100 ms time to target arrival. Different probabilities of velocity decrease ranging from 25 to 100% were compared. The results revealed that while there were increasing errors between probabilities of 25 and 75% for unchanged target velocity, the opposite relationship was observed for target velocity decrease. Kinematic analysis indicated that movement timing adjustments to target velocity decrease were made online. These results support the conception that visuomotor integration in the interception of moving targets is mediated by an internal forward model whose weights can be flexibly adjusted according to expectancy of time to target arrival.

Macro-Fiber Composite Actuators for Flow Control of a Variable Camber Airfoil

This research employs solid-state actuators for delay of flow separation seen in airfoils at low Reynolds numbers. The flow control technique investigated here is aimed for a variable camber airfoil that employs two active surfaces and a single four-bar (box) mechanism as the internal structure. To reduce separation, periodic excitation to the flow around the leading edge of the airfoil is induced by a total of nine piezocomposite actuated clamped-free unimorph benders distributed in the spanwise direction. An electromechanical model is employed to design an actuator capable of high deformations at the desired frequency for lift improvement at post-stall angles. The optimum spanwise distribution of excitation for increasing lift coefficient is identified experimentally in the wind tunnel. A 3D (non-uniform) excitation distribution achieved higher lift enhancement in the post-stall region with lower power consumption when compared to the 2D (uniform) excitation distribution. A lift coefficient increase of 18.4% is achieved with the identified non-uniform excitation mode at the bender resonance frequency of 125 Hz, the flow velocity of 5 m/s and at the reduced frequency of 3.78. The maximum lift (Clmax) is increased 5.2% from the baseline. The total power consumption of the flow control technique is 639 mW(RMS).

Comparison Between Voltage Control Structures of Synchronous Machines

This paper compares the behaviour of two different control structures of automatic voltage regulators of synchronous machines equipped with static excitation systems. These systems have a fully controlled thyristor bridge that supplies DC current to the rotor winding. The rectifier bridge is fed by the stator terminals through a step-down transformer. The first control structure, named ""Direct Control"", has a single proportional-integral (PI) regulator that compares stator voltage setpoint with measured voltage and acts directly on the thyristor bridge`s firing angle. This control structure is usually employed in commercial excitation systems for hydrogenerators. The second structure, named ""Cascade Control"", was inspired on control loops of commercial DC motor drives. Such drives employ two PIs in a cascade arrangement, the external PI deals with the motor speed while the internal one regulates the armature current. In the adaptation proposed, the external PI compares setpoint with the actual stator voltage and produces the setpoint to the internal PI-loop which controls the field current.

Perioperative Temporary Occlusion of the Internal Iliac Arteries as Prophylaxis in Cesarean Section at Risk of Hemorrhage in Placenta Accreta

Purpose The purpose of this study was to describe the preliminary results of prophylactic temporary balloon occlusion of the internal iliac arteries for bleeding control in patients with placenta accreta during cesarean hysterectomy. Methods From May 2006 to March 2010, 21 patients diagnosed with placenta accreta using ultrasound and/or magnetic resonance imaging were submitted to prophylactic balloon occlusion before hysterectomy. Fluoroscopy, balloon occlusion time, surgical duration, intraoperative blood loss, transfusion volume, and procedure complications were analyzed. Results The mean age was 30.5 years with a mean of 3.6 previous gestations. Imaging studies revealed that all patients had placenta accreta and all were submitted to cesarean hysterectomy. One hysterectomy was due to previous diagnosis of fetal death and another due to cesarean with uterine curettage. Mean fluoroscopy time was 7.5 min, balloon occlusion time was 164 min, and surgery duration was 260 min. Estimated blood loss was 1,671.5 ml with mean reposition fluids of 3,538 ml of crystalloids, 309.5 ml of colloids, and 1.24 ml of packed red blood cells. Two patients were submitted to thromboembolectomy due to prolonged surgical time. There was no maternal or fetal mortality related to the procedure. Conclusions The results demonstrated that prophylactic balloon occlusion of internal iliac artery is a safe method and appears to reduce blood loss and transfusion requirements in patients diagnosed with placenta accreta who undergo cesarean hysterectomy. Antenatal imaging diagnosis of placenta accreta enables preoperative planning.

Body Posture Evaluations In Subjects with Internal Temporomandibular Joint Derangement

The aim of this study was to verify possible relationships between global body posture and temporomandibular joint internal derangement (TMJ-id), by comparing 30 subjects presenting typical TMJ-id signs to 20 healthy subjects. Body posture was assessed using the analysis of muscle chains on several photographs. Results show a higher frequency of lifted shoulders (p=0.04) and of changes in the antero-internal hip chain (p=0.02) in the test group, but no further differences were found significant between the control and test groups. The test group was then divided into three subgroups according to the Helkimo index of temporomandibular disorder severity. Again, no significant differences were found between the subgroups. However, there was a trend noticed in the group with the most severe dysfunction, to present a forward head and shoulders posture. Results are discussed in light of previous studies using the same sample.

Association between symptom severity and internal capsule volume in obsessive-compulsive disorder

Neurobiological models support an involvement of white matter tracts in the pathophysiology of obsessive-compulsive disorder (OCD), but there has been little systematic evaluation of white matter volumes in OCD using magnetic resonance imaging (MRI). We investigated potential differences in the volume of the cingulum bundle (CB) and anterior limb of internal capsule (ALIC) in OCD patients (n = 19) relative to asymptomatic control subjects (n = 15). White matter volumes were assessed using a 1.5T MRI scanner. Between-group comparisons were carried out after spatial normalization and image segmentation using optimized voxel-based morphometry. Correlations between regional white matter volumes in OCD subjects and symptom severity ratings were also investigated. We found significant global white matter reductions in OCD patients compared to control subjects. The voxel-based search for regional abnormalities (with covariance for total white matter volumes) showed no specific white matter volume deficits in brain portions predicted a priori to be affected in OCD (CB and ALIC). However, large clusters of significant positive correlation with OCD severity scores were found bilaterally on the ALIC. These findings provide evidence of OCD-related ALIC abnormalities and suggest a connectivity dysfunction within frontal-striatal-thalamic-cortical circuits. Further studies are warranted to better define the role of such white matter alterations in the pathophysiology of OCD, and may provide clues for a more effectively targeting of neurosurgical treatments for OCD. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

GAPD and tubulin are suitable internal controls for qPCR analysis of oral squamous cell carcinoma cell lines

The selection of housekeeping genes is critical for gene expression studies. To address this issue, four candidate housekeeping genes, including several commonly used ones, were investigated in oral squamous cell carcinoma cell lines. A simple quantitative RT-PCR approach was employed by comparing relative expression of the four candidate genes within two cancerous cell tines (HN6 and HN31) and one noncancerous cell tine (HaCaT) treated or not with EGF and TGF-beta 1. Data were analyzed using ANOVA followed by the NormFinder software program. On this basis, stability of the candidate housekeeping genes was ranked and non statistical differences were found using ANOVA test. On the other hand, the NormFinder was able to show that GAPD and TUBB presented the less variable results, representing appropriated housekeeping genes for the samples and conditions anatyzed. In conclusion, this study suggests that the GAPD and the TUBB represent adequate normalizers for gene profiting studies in OSCC cell. tines, covering, respectively, high and low expression levels genes. (C) 2008 Elsevier Ltd. All rights reserved.

Use of checkerboard DNA-DNA hybridization to evaluate the internal contamination of dental implants and comparison of bacterial leakage with cast or pre-machined abutments

To evaluate the checkerboard DNA-DNA hybridization method for detection and quantitation of bacteria from the internal parts of dental implants and to compare bacterial leakage from implants connected either to cast or to pre-machined abutments. Nine plastic abutments cast in a Ni-Cr alloy and nine pre-machined Co-Cr alloy abutments with plastic sleeves cast in Ni-Cr were connected to Branemark-compatible implants. A group of nine implants was used as control. The implants were inoculated with 3 mu l of a solution containing 10(8) cells/ml of Streptococcus sobrinus. Bacterial samples were immediately collected from the control implants while assemblies were completely immersed in 5 ml of sterile Tripty Soy Broth (TSB) medium. After 14 days of anaerobic incubation, occurrence of leakage at the implant-abutment interface was evaluated by assessing contamination of the TSB medium. Internal contamination of the implants was evaluated with the checkerboard DNA-DNA hybridization method. DNA-DNA hybridization was sensitive enough to detect and quantify the microorganism from the internal parts of the implants. No differences in leakage and in internal contamination were found between cast and pre-machined abutments. Bacterial scores in the control group were significantly higher than in the other groups (P < 0.05). Bacterial leakage through the implant-abutment interface does not significantly differ when cast or pre-machined abutments are used. The checkerboard DNA-DNA hybridization technique is suitable for the evaluation of the internal contamination of dental implants although further studies are necessary to validate the use of computational methods for the improvement of the test accuracy. To cite this article:do Nascimento C, Barbosa RES, Issa JPM, Watanabe E, Ito IY, Albuquerque Junior RF. Use of checkerboard DNA-DNA hybridization to evaluate the internal contamination of dental implants and comparison of bacterial leakage with cast or pre-machined abutments.Clin. Oral Impl. Res. 20, 2009; 571-577.doi: 10.1111/j.1600-0501.2008.01663.x.