Validation of reference genes for gene expression studies in the honey bee, Apis mellifera, by quantitative real-time RT-PCR

For obtaining accurate and reliable gene expression results it is essential that quantitative real-time RT-PCR (qRT-PCR) data are normalized with appropriate reference genes. The current exponential increase in postgenomic studies on the honey bee, Apis mellifera, makes the standardization of qRT-PCR results an important task for ongoing community efforts. For this aim we selected four candidate reference genes (actin, ribosomal protein 49, elongation factor 1-alpha, tbp-association factor) and used three software-based approaches (geNorm, BestKeeper and NormFinder) to evaluate the suitability of these genes as endogenous controls. Their expression was examined during honey bee development, in different tissues, and after juvenile hormone exposure. Furthermore, the importance of choosing an appropriate reference gene was investigated for two developmentally regulated target genes. The results led us to consider all four candidate genes as suitable genes for normalization in A. mellifera. However, each condition evaluated in this study revealed a specific set of genes as the most appropriated ones.

Time Changes with the Embodiment of Another's Body Posture

The aim of the present study was to investigate whether the perception of presentation durations of pictures of different body postures was distorted as function of the embodied movement that originally produced these postures. Participants were presented with two pictures, one with a low-arousal body posture judged to require no movement and the other with a high-arousal body posture judged to require considerable movement. In a temporal bisection task with two ranges of standard durations (0.4/1.6 s and 2/8 s), the participants had to judge whether the presentation duration of each of the pictures was more similar to the short or to the long standard duration. The results showed that the duration was judged longer for the posture requiring more movement than for the posture requiring less movement. However the magnitude of this overestimation was relatively greater for the range of short durations than for that of longer durations. Further analyses suggest that this lengthening effect was mediated by an arousal effect of limited duration on the speed of the internal clock system.

Electromagnetic energy within magnetic spheres

Consider that an incident plane wave is scattered by a homogeneous and isotropic magnetic sphere of finite radius. We determine, by means of the rigorous Mie theory, an exact expression for the time-averaged electromagnetic energy within this particle. For magnetic scatterers, we find that the value of the average internal energy in the resonance picks is much larger than the one associated with a scatterer with the same nonmagnetic medium properties. This result is valid even, and especially, for low size parameter values. Expressions for the contributions of the radial and angular field components to the internal energy are determined. For the analytical study of the weak absorption regime, we derive an exact expression for the absorption cross section in terms of the magnetic Mie internal coefficients. We stress that, although the electromagnetic scattering by particles is a well-documented topic, almost no attention has been devoted to magnetic scatterers. Our aim is to provide some new analytical results, which can be used for magnetic particles, and emphasize the unusual properties of the magnetic scatters, which could be important in some applications. (C) 2010 Optical Society of America

TRAVELLING THROUGH PITCH SPACE SPEEDS UP MUSICAL TIME

SEVERAL MODELS OF TIME ESTIMATION HAVE BEEN developed in psychology; a few have been applied to music. In the present study, we assess the influence of the distances travelled through pitch space on retrospective time estimation. Participants listened to an isochronous chord sequence of 20-s duration. They were unexpectedly asked to reproduce the time interval of the sequence. The harmonic structure of the stimulus was manipulated so that the sequence either remained in the same key (CC) or travelled through a closely related key (CFC) or distant key (CGbC). Estimated times were shortened when the sequence modulated to a very distant key. This finding is discussed in light of Lerdahl's Tonal Pitch Space Theory (2001), Firmino and Bueno's Expected Development Fraction Model (in press), and models of time estimation.

Time-resolved fluorescence spectroscopy of white-spot caries in human enamel

The objective is to differentiate noncavitated caries enamel through time-resolved fluorescence and to find excitation and emission parameters that can be applied in future clinical practice for detection of caries lesions that are not clearly visible to the professional. Sixteen human teeth with noncavitiated white-spot caries were selected for this work. Fluorescence intensity decay was measured by using an apparatus based on the time-correlated single-photon counting method. An optical fiber bundle was employed for sample excitation (440 nm), and the fluorescence collected by the same bundle (500 nm) was registered. The average lifetime for sound enamel was 7: 93 +/- 0: 09, 2: 46 +/- 0: 04, and 0: 51 +/- 0: 02 ns, whereas for the carious enamel the lifetimes were 4: 84 +/- 0: 06, 1: 35 +/- 0: 02, and 0: 16 +/- 0: 01 ns. It was concluded that it is possible to differentiate between carious and sound regions by time-resolved fluorescence and that, although the origin of enamel fluorescence is still uncertain, the lifetime values seem to be typical of fluorophores like collagen I. (C) 2010 Optical Society of America

A reliable measure of similarity based on dependency for short time series: an application to gene expression networks

Background: Microarray techniques have become an important tool to the investigation of genetic relationships and the assignment of different phenotypes. Since microarrays are still very expensive, most of the experiments are performed with small samples. This paper introduces a method to quantify dependency between data series composed of few sample points. The method is used to construct gene co-expression subnetworks of highly significant edges. Results: The results shown here are for an adapted subset of a Saccharomyces cerevisiae gene expression data set with low temporal resolution and poor statistics. The method reveals common transcription factors with a high confidence level and allows the construction of subnetworks with high biological relevance that reveals characteristic features of the processes driving the organism adaptations to specific environmental conditions. Conclusion: Our method allows a reliable and sophisticated analysis of microarray data even under severe constraints. The utilization of systems biology improves the biologists ability to elucidate the mechanisms underlying celular processes and to formulate new hypotheses.

Development of Hepatitis C Virus Genotyping by Real-Time PCR Based on the NS5B Region

Background: Hepatitis C virus (HCV) genotyping is the most significant predictor of the response to antiviral therapy. The aim of this study was to develop and evaluate a novel real-time PCR method for HCV genotyping based on the NS5B region. Methodology/Principal Findings: Two triplex reaction sets were designed, one to detect genotypes 1a, 1b and 3a; and another to detect genotypes 2a, 2b, and 2c. This approach had an overall sensitivity of 97.0%, detecting 295 of the 304 tested samples. All samples genotyped by real-time PCR had the same type that was assigned using LiPA version 1 (Line in Probe Assay). Although LiPA v. 1 was not able to subtype 68 of the 295 samples (23.0%) and rendered different subtype results from those assigned by real-time PCR for 12/295 samples (4.0%), NS5B sequencing and real-time PCR results agreed in all 146 tested cases. Analytical sensitivity of the real-time PCR assay was determined by end-point dilution of the 5000 IU/ml member of the OptiQuant HCV RNA panel. The lower limit of detection was estimated to be 125 IU/ml for genotype 3a, 250 IU/ml for genotypes 1b and 2b, and 500 IU/ml for genotype 1a. Conclusions/Significance: The total time required for performing this assay was two hours, compared to four hours required for LiPA v. 1 after PCR-amplification. Furthermore, the estimated reaction cost was nine times lower than that of available commercial methods in Brazil. Thus, we have developed an efficient, feasible, and affordable method for HCV genotype identification.

Half-Pringle Maneuver: A Useful Tool in Laparoscopic Liver Resection

Introduction: Laparoscopic liver resections are becoming a common procedure, and bleeding remains the major concern during parenchymal transection. Total vascular inflow occlusion can be performed, but ischemic reperfusion injuries can lead to postoperative morbidity. On the other hand, hemihepatic inflow occlusion, leading to hemiliver ischemia, decreases the amount of liver parenchyma submitted to reperfusion damage and offers the advantage of reduced blood loss. Objective: The aim of this work was to describe our experience with laparoscopic the half-Pringle maneuver for segmentar or nonanatomic liver resctions. Patients and Methods: Eight patients submitted to laparoscopic liver resection in a single tertiary center. Results: There were 5 women and 3 men with a mean age of 40.2 years (range, 26-54). Mean tumor size was 4.1 cm (range, 2.6-6.0), and mean hospital stay was 3.1 days (1-5). There were 3 liver adenomas, 2 hepatocellular carcinomas, 1 metastatic melanoma, 1 metastatic colorectal carcinoma, and 1 peripheral colangiocarcinoma. No postoperative complications or mortalities were observed. Conclusions: Results demonstrate that laparoscopic liver resection with the half-Pringle maneuver is feasible and safe and may be included in the technical armamentarium of laparoscopic liver resections for a selected group of patients.

Real-Time PCR in HIV/Trypanosoma cruzi Coinfection with and without Chagas Disease Reactivation: Association with HIV Viral Load and CD4(+) Level

Background: Reactivation of chronic Chagas disease, which occurs in approximately 20% of patients coinfected with HIV/Trypanosoma cruzi (T. cruzi), is commonly characterized by severe meningoencephalitis and myocarditis. The use of quantitative molecular tests to monitor Chagas disease reactivation was analyzed. Methodology: Polymerase chain reaction (PCR) of kDNA sequences, competitive (C-) PCR and real-time quantitative (q) PCR were compared with blood cultures and xenodiagnosis in samples from 91 patients (57 patients with chronic Chagas disease and 34 with HIV/T. cruzi coinfection), of whom 5 had reactivation of Chagas disease and 29 did not. Principal Findings: qRT-PCR showed significant differences between groups; the highest parasitemia was observed in patients infected with HIV/T. cruzi with Chagas disease reactivation (median 1428.90 T. cruzi/mL), followed by patients with HIV/T. cruzi infection without reactivation (median 1.57 T. cruzi/mL) and patients with Chagas disease without HIV (median 0.00 T. cruzi/mL). Spearman's correlation coefficient showed that xenodiagnosis was correlated with blood culture, C-PCR and qRT-PCR. A stronger Spearman correlation index was found between C-PCR and qRT-PCR, the number of parasites and the HIV viral load, expressed as the number of CD4(+) cells or the CD4(+)/CD8(+) ratio. Conclusions: qRT-PCR distinguished the groups of HIV/T. cruzi coinfected patients with and without reactivation. Therefore, this new method of qRT-PCR is proposed as a tool for prospective studies to analyze the importance of parasitemia (persistent and/or increased) as a criterion for recommending pre-emptive therapy in patients with chronic Chagas disease with HIV infection or immunosuppression. As seen in this study, an increase in HIV viral load and decreases in the number of CD4(+) cells/mm(3) and the CD4(+)/CD8(+) ratio were identified as cofactors for increased parasitemia that can be used to target the introduction of early, pre-emptive therapy.

Time variation of some selected topics in bioethical publications

Objective: To analyse the time variation of topics in bioethical publications as a proxy of the relative importance. Methods: We searched the Medline database for bioethics publications using the words ""ethics or bioethics'', and for 360 specific topics publications, associating Medical Subject Heading topic descriptors to those words. We calculated the ratio of bioethics publications to the total publications of Medline, and the ratio of each topic publications to the total bioethics publications, for five-year intervals, from 1970 to 2004. We calculated the time variation of ratios, dividing the difference between the highest and lowest ratio of each topic by its highest ratio. Four topics were described, selected to illustrate different patterns of variation: ""Induced Abortion'', ""Conflict of Interest'', ""Acquired Immunodeficiency Syndrome'', ""Medical Education.'' Results: The ratio of bioethics publications to total Medline publications increased from 0.003 to 0.012. The variation of the topic's ratios was higher than 0.7 for 68% of the topics. The Induced Abortion ratios decreased from 0.12 to 0.02. Conflict of Interest ratios increased from zero to 0.07. The Acquired Immunodeficiency Syndrome ratios were nearly zero in the first three intervals, had a peak of 0.06 during 1985-9, followed by a decrease to 0.01. Medical Education ratios varied few, from 0.04 to 0.03. Conclusions: There was an increase of bioethical publications in the Medline database. The topics in bioethics literature have an important time variation. Some factors were suggested to explain this variation: current legal cases, resolution of the issue, saturation of a discussion and epidemiologic importance.

Detection of dengue virus in saliva and urine by real time RT-PCR

Early diagnosis of dengue virus (DENV) infection is important for patient management and control of dengue outbreaks. The objective of this study was to analyze the usefulness of urine and saliva samples for early diagnosis of DENV infection by real time RT-PCR. Two febrile patients, who have been attended at the General Hospital of the School of Medicine of Ribeirao Preto, Sao Paulo University were included in the study. Serum, urine and saliva samples collected from both patients were subjected to real time RT-PCR for DENV detection and quantification. Dengue RNA was detected in serum, urine and saliva samples of both patients. Patient 1 was infected with DENV-2 and patient 2 with DENV-3. Data presented in this study suggest that urine and saliva could be used as alternative samples for early diagnosis of dengue virus infection when blood samples are difficult to obtain, e.g.,in newborns and patients with hemorrhagic syndromes.

Viral mutation and its influence in the time evolution of the immunizations

In this paper, we study the behavior of immune memory against antigenic mutation. Using a dynamic model proposed by one of the authors in a previous study (A. de Castro [Phys. J. Appl. Phys. 33, 147 (2006) and Simul. Mod. Pract. Theory. 15, 831 (2007)]), we have performed simulations of several inoculations, where in each virtual sample the viral population undergoes mutations. Our results suggest that the sustainability of the immunizations is dependent on viral variability and that the memory lifetimes are not random, what contradicts what was suggested by Tarlinton et al. [Curr. Opin. Immunol. 20, 162 (2008)]. We show that what may cause an apparent random behavior of the immune memory is the antigenic variability.

Influence of pulse repetition rate on temperature rise and working time during composite filling removal with the Er : YAG laser

Objective: The purpose of this study was to assess the efficacy of Er:YAG laser energy for composite resin removal and the influence of pulse repetition rate on the thermal alterations occurring during laser ablation. Materials and Methods: Composite resin filling was placed in cavities (1.0 mm deep) prepared in bovine teeth and the specimens were randomly assigned to five groups according to the technique used for composite filling removal. In group I (controls), the restorations were removed using a high-speed diamond bur. In the other groups, the composite fillings were removed using an Er: YAG laser with different pulse repetition rates: group 2-2 Hz; group 3-4 Hz; group 4-6 Hz; and group 5-10 Hz. The time required for complete removal of the restorative material and the temperature changes were recorded. Results: Temperature rise during composite resin removal with the Er: YAG laser occurred in the substrate underneath the restoration and was directly proportional to the increase in pulse repetition rate. None of the groups had a temperature increase during composite filling removal of more than 5.6 degrees C, which is considered the critical point above which irreversible thermal damage to the pulp may result. Regarding the time for composite filling removal, all the laser-ablated groups (except for group 5 [10 Hz]) required more time than the control group for complete elimination of the material from the cavity walls. Conclusion: Under the tested conditions, Er: YAG laser irradiation was efficient for composite resin ablation and did not cause a temperature increase above the limit considered safe for the pulp. Among the tested pulse repetition rates, 6 Hz produced minimal temperature change compared to the control group (high-speed bur), and allowed composite filling removal within a time period that is acceptable for clinical conditions.

Incorporation of Real-Time PCR into Routine Public Health Surveillance of Culture Negative Bacterial Meningitis in Sao Paulo, Brazil

Real-time (RT)-PCR increases diagnostic yield for bacterial meningitis and is ideal for incorporation into routine surveillance in a developing country. We validated a multiplex RT-PCR assay for Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae in Brazil. Risk factors for being culture-negative, RT-PCR positive were determined. The sensitivity of RT-PCR in cerebrospinal fluid (CSF) was 100% (95% confidence limits, 96.0%-100%) for N. meningitidis, 97.8% (85.5%-99.9%) for S. pneumoniae, and 66.7% (9.4%-99.2%) for H. influenzae. Specificity ranged from 98.9% to 100%. Addition of RT-PCR to routine microbiologic methods increased the yield for detection of S. pneumoniae, N. meningitidis, and H. influenzae cases by 52%, 85%, and 20%, respectively. The main risk factor for being culture negative and RT-PCR positive was presence of antibiotic in CSF (odds ratio 12.2, 95% CI 5.9-25.0). RT-PCR using CSF was highly sensitive and specific and substantially added to measures of meningitis disease burden when incorporated into routine public health surveillance in Brazil.

Three-dimensional finite element thermal analysis of dental tissues irradiated with Er,Cr:YSGG laser

In the present study, a finite element model of a half-sectioned molar tooth was developed in order to understand the thermal behavior of dental hard tissues (both enamel and dentin) under laser irradiation. The model was validated by comparing it with an in vitro experiment where a sound molar tooth was irradiated by an Er,Cr:YSGG pulsed laser. The numerical tooth model was conceived to simulate the in vitro experiment, reproducing the dimensions and physical conditions of the typical molar sound tooth, considering laser energy absorption and calculating the heat transfer through the dental tissues in three dimensions. The numerical assay considered the same three laser energy densities at the same wavelength (2.79 mu m) used in the experiment. A thermographic camera was used to perform the in vitro experiment, in which an Er, Cr: YSGG laser (2.79 mu m) was used to irradiate tooth samples and the infrared images obtained were stored and analyzed. The temperature increments in both the finite element model and the in vitro experiment were compared. The distribution of temperature inside the tooth versus time plotted for two critical points showed a relatively good agreement between the results of the experiment and model. The three dimensional model allows one to understand how the heat propagates through the dentin and enamel and to relate the amount of energy applied, width of the laser pulses, and temperature inside the tooth. (C) 2008 American Institute of Physics. [DOI: 10.1063/1.2953526]