Deep sequencing of New World screw-worm transcripts to discover genes involved in insecticide resistance

Background: The New World screw-worm (NWS), Cochliomyia hominivorax, is one of the most important myiasis-causing flies, causing severe losses to the livestock industry. In its current geographical distribution, this species has been controlled by the application of insecticides, mainly organophosphate (OP) compounds, but a number of lineages have been identified that are resistant to such chemicals. Despite its economic importance, only limited genetic information is available for the NWS. Here, as a part of an effort to characterize the C. hominivorax genome and identify putative genes involved in insecticide resistance, we sampled its transcriptome by deep sequencing of polyadenylated transcripts using the 454 sequencing technology. Results: Deep sequencing on the 454 platform of three normalized libraries (larval, adult male and adult female) generated a total of 548,940 reads. Eighteen candidate genes coding for three metabolic detoxification enzyme families, cytochrome P450 monooxygenases, glutathione S transferases and carboxyl/cholinesterases were selected and gene expression levels were measured using quantitative real-time polymerase chain reaction (qRT-PCR). Of the investigated candidates, only one gene was expressed differently between control and resistant larvae with, at least, a 10-fold down-regulation in the resistant larvae. The presence of mutations in the acetylcholinesterase (target site) and carboxylesterase E3 genes was investigated and all of the resistant flies presented E3 mutations previously associated with insecticide resistance. Conclusions: Here, we provided the largest database of NWS expressed sequence tags that is an important resource, not only for further studies on the molecular basis of the OP resistance in NWS fly, but also for functional and comparative studies among Calliphoridae flies. Among our candidates, only one gene was found differentially expressed in resistant individuals, and its role on insecticide resistance should be further investigated. Furthermore, the absence of mutations in the OP target site and the high frequency of mutant carboxylesterase E3 indicate that metabolic resistance mechanisms have evolved predominantly in this species.

Chrysotile effects on human lung cell carcinoma in culture: 3-D reconstruction and DNA quantification by image analysis

Background: Chrysotile is considered less harmful to human health than other types of asbestos fibers. Its clearance from the lung is faster and, in comparison to amphibole forms of asbestos, chrysotile asbestos fail to accumulate in the lung tissue due to a mechanism involving fibers fragmentation in short pieces. Short exposure to chrysotile has not been associated with any histopathological alteration of lung tissue. Methods: The present work focuses on the association of small chrysotile fibers with interphasic and mitotic human lung cancer cells in culture, using for analyses confocal laser scanning microscopy and 3D reconstructions. The main goal was to perform the analysis of abnormalities in mitosis of fibers-containing cells as well as to quantify nuclear DNA content of treated cells during their recovery in fiber-free culture medium. Results: HK2 cells treated with chrysotile for 48 h and recovered in additional periods of 24, 48 and 72 h in normal medium showed increased frequency of multinucleated and apoptotic cells. DNA ploidy of the cells submitted to the same chrysotile treatment schedules showed enhanced aneuploidy values. The results were consistent with the high frequency of multipolar spindles observed and with the presence of fibers in the intercellular bridge during cytokinesis. Conclusion: The present data show that 48 h chrysotile exposure can cause centrosome amplification, apoptosis and aneuploid cell formation even when long periods of recovery were provided. Internalized fibers seem to interact with the chromatin during mitosis, and they could also interfere in cytokinesis, leading to cytokinesis failure which forms aneuploid or multinucleated cells with centrosome amplification.

The Liver Plays a Major Role in Clearance and Destruction of Blood Trypomastigotes in Trypanosoma cruzi Chronically Infected Mice

Intravenous challenge with Trypanosoma cruzi can be used to investigate the process and consequences of blood parasite clearance in experimental Chagas disease. One hour after intravenous challenge of chronically infected mice with 5610 6 trypomastigotes, the liver constituted a major site of parasite accumulation, as revealed by PCR. Intact parasites and/or parasite remnants were visualized at this time point scattered in the liver parenchyma. Moreover, at this time, many of liver-cleared parasites were viable, as estimated by the frequency of positive cultures, which considerably diminished after 48 h. Following clearance, the number of infiltrating cells in the hepatic tissue notably increased: initially (at 24 h) as diffuse infiltrates affecting the whole parenchyma, and at 48 h, in the form of large focal infiltrates in both the parenchyma and perivascular spaces. Phenotypic characterization of liver-infiltrating cells 24 h after challenge revealed an increase in Mac1(+), CD8(+) and CD4(+) cells, followed by natural killer (NK) cells. As evidence that liver-infiltrating CD4(+) and CD8(+) cells were activated, increased frequencies of CD69(+) CD8(+), CD69(+) CD4(+) and CD25(+) CD122(+) CD4(+) cells were observed at 24 and 48 h after challenge, and of CD25(-)CD122(+)CD4(+) cells at 48 h. The major role of CD4(+) cells in liver protection was suggested by data showing a very high frequency of interferon (IFN)-gamma-producing CD4(+) cells 24 h after challenge. In contrast, liver CD8(+) cells produced little IFN-gamma, even though they showed an enhanced potential for secreting this cytokine, as revealed by in vitro T cell receptor (TCR) stimulation. Confirming the effectiveness of the liver immune response in blood parasite control during the chronic phase of infection, no live parasites were detected in this organ 7 days after challenge.

Absorption of planar waves in a draining bathtub

We present an analysis of the absorption of acoustic waves by a black hole analogue in (2 + 1) dimensions generated by a fluid flow in a draining bathtub. We show that the low-frequency absorption length is equal to the acoustic hole circumference and that the high-frequency absorption length is 4 times the ergoregion radius. For intermediate values of the wave frequency, we compute the absorption length numerically and show that our results are in excellent agreement with the low-and high-frequency limits. We analyze the occurrence of superradiance, manifested as negative partial absorption lengths for corotating modes at low frequencies.

Electromagnetic absorption cross section of Reissner-Nordstrom black holes

The absorption cross section of Reissner-Nordstrom black holes for the electromagnetic field is computed numerically for arbitrary frequencies. The numerical results are in excellent agreement with the low- and high-frequency limits, which are obtained with analytical methods. Special emphasis is given to the extreme Reissner-Nordstrom black hole case.

Exercise training associated with diet improves heart rate recovery and cardiac autonomic nervous system activity in obese children

The purpose of this study was to test the hypotheses that in obese children: 1) hypocaloric diet (D) improves both heart rate recovery at 1 min (Delta HRR1) cfter an exercise test, and cardiac autonomic nervous system activity (CANSA) in obese children; 2) Diet and exercise training (DET) combined leads to greater improvement in both Delta HRR1 after an exercise test and in CANSA, than D alone. Moreover, we examined the relationships among Delta HRR1, CANSA, cardiorespiratory fitness and anthropometric variables (AV) in obese children submitted to D and to DET. 33 obese children (10 +/- 0.2 years; body mass index (BMI) >95(th) percentile) were divided into 2 groups: D (n = 15; BMI = 31 +/- 1 kg/m(2)) and DET (n = 18; 29 +/- 1 kg/m(2)). All children performed a maximal cardiopulmonary exercise test on a treadmill. The Delta HRR1 was defined as the difference between heart rate at peak and at 1-min post-exercise. CANSA was assessed using power spectral analysis of heart rate variability at rest. The sympathovagal balance (low frequency and high frequency ratio, LF/HF) was measured. After interventions, all obese children showed reduced body weight (P < 0.05). The D group did not improve in terms of peak VO(2), Delta HRR1 or LF/HF ratio (P > 0.05). In contrast, the DET group showed increased peak VO(2) (P = 0.01) and improved Delta HRR1 (Delta HRR1 = 37.3 +/- 2.6; P = 0.01) and LF/HF ratio (P = 0.001). The DET group demonstrated significant relationships among Delta HRR1, peak VO(2) and CANSA (P < 0.05). In conclusion, DET, in contrast to D, promoted improved Delta HRR1 and CANSA in obese children, suggesting a positive influence of increased levels of cardiorespiratory fitness by exercise training on cardiac autonomic activity.

A low carbohydrate diet affects autonomic modulation during heavy but not moderate exercise

The aim of this study was to examine the effects of low carbohydrate (CHO) availability on heart rate variability (HRV) responses during moderate and severe exercise intensities until exhaustion. Six healthy males (age, 26.5 +/- 6.7 years; body mass, 78.4 +/- 7.7 kg; body fat %, 11.3 +/- 4.5%; (V) over dotO(2max), 39.5 +/- 6.6 mL kg(-1) min(-1)) volunteered for this study. All tests were performed in the morning, after 8-12 h overnight fasting, at a moderate intensity corresponding to 50% of the difference between the first (LT(1)) and second (LT(2)) lactate breakpoints and at a severe intensity corresponding to 25% of the difference between the maximal power output and LT(2). Forty-eight hours before each experimental session, the subjects performed a 90-min cycling exercise followed by 5-min rest periods and subsequent 1-min cycling bouts at 125% (V) over dotO(2max) (with 1-min rest periods) until exhaustion, in order to deplete muscle glycogen. A diet providing 10% (CHO(low)) or 65% (CHO(control)) of energy as carbohydrates was consumed for the following 2 days until the experimental test. The Poicare plots (standard deviations 1 and 2: SD1 and SD2, respectively) and spectral autoregressive model (low frequency LF, and high frequency HF) were applied to obtain HRV parameters. The CHO availability had no effect on the HRV parameters or ventilation during moderate-intensity exercise. However, the SD1 and SD2 parameters were significantly higher in CHO(low) than in CHO(control), as taken at exhaustion during the severe-intensity exercise (P < 0.05). The HF and LF frequencies (ms(2)) were also significantly higher in CHO(low) than in CHO(control) (P < 0.05). In addition, ventilation measured at the 5 and 10-min was higher in CHO(low) (62.5 +/- 4.4 and 74.8 +/- 6.5 L min(-1), respectively, P < 0.05) than in CHO(control) (70.0 +/- 3.6 and 79.6 +/- 5.1 L min(-1), respectively; P < 0.05) during the severe-intensity exercise. These results suggest that the CHO availability alters the HRV parameters during severe-, but not moderate-, intensity exercise, and this was associated with an increase in ventilation volume.

Combined solution for fault location in three-terminal lines based on wavelet transforms

This work presents the study and development of a combined fault location scheme for three-terminal transmission lines using wavelet transforms (WTs). The methodology is based on the low- and high-frequency components of the transient signals originated from fault situations registered in the terminals of a system. By processing these signals and using the WT, it is possible to determine the time of travelling waves of voltages and/or currents from the fault point to the terminals, as well as estimate the fundamental frequency components. A new approach presents a reliable and accurate fault location scheme combining some different solutions. The main idea is to have a decision routine in order to select which method should be used in each situation presented to the algorithm. The combined algorithm was tested for different fault conditions by simulations using the ATP (Alternative Transients Program) software. The results obtained are promising and demonstrate a highly satisfactory degree of accuracy and reliability of the proposed method.

GOOAL: A routine based on Genetic Algorithms to track harmonics in electric power systems

Voltage and current waveforms of a distribution or transmission power system are not pure sinusoids. There are distortions in these waveforms that can be represented as a combination of the fundamental frequency, harmonics and high frequency transients. This paper presents a novel approach to identifying harmonics in power system distorted waveforms. The proposed method is based on Genetic Algorithms, which is an optimization technique inspired by genetics and natural evolution. GOOAL, a specially designed intelligent algorithm for optimization problems, was successfully implemented and tested. Two kinds of representations concerning chromosomes are utilized: binary and real. The results show that the proposed method is more precise than the traditional Fourier Transform, especially considering the real representation of the chromosomes.

Improved effective charge density in MOS capacitors with PECVD SiOxNy dielectric layer obtained at low RF power

In this work SiOxNy films are produced and characterized. Series of samples were deposited by the plasma enhanced chemical vapor deposition (PECVD) technique at low temperatures from silane (SiH4), nitrous oxide (N2O) and helium (He) precursor gaseous mixtures, at different deposition power in order to analyze the effect of this parameter on the films structural properties, on the SiOxNy/Si interface quality and on the SiOxNy effective charge density. In order to compare the film structural properties with the interface (SiOxNy/Si) quality and effective charge density, MOS capacitors were fabricated using these films as dielectric layer. X-ray absorption near-edge spectroscopy (XANES), at the Si-K edge, was utilized to investigate the structure of the films and the material bonding characteristics were analyzed through Fourier transform infrared spectroscopy (FTIR). The MOS capacitors were characterized by low and high frequency capacitance (C-V) measurements, in order to obtain the interface state density (D-it) and the effective charge density (N-ss). An effective charge density linear reduction for decreasing deposition power was observed, result that is attributed to the smaller amount of ions present in the plasma for low RF power. (C) 2008 Elsevier B.V. All rights reserved.

Spectral characteristics of atrial electrograms in sinus rhythm correlates with sites of ganglionated plexuses in patients with paroxysmal atrial fibrillation

Aims To verify whether spectral components of atrial electrograms (AE) during sinus rhythm (SR) correlate with cardiac ganglionated plexus (GP) sites. Methods and results Thirteen patients undergoing atrial fibrillation (AF) ablation were prospectively enrolled. Prior to radio frequency application, endocardial AE were recorded with a sequential point-by-point approach. Electrical stimuli were delivered at 20 Hz, amplitude 100 V, and pulse width of 4 ms. A vagal response was defined as a high-frequency stimulation (HFS) evoked atrioventricular block or a prolongation of RR interval. Spectral analysis was performed on single AE during SR, sampling rate of 1000 Hz, Hanning window. Overall, 1488 SR electrograms were analysed from 186 different left atrium sites, 129 of them corresponding to negative vagal response sites, and 57 to positive response sites. The electrogram duration and the number of deflections were similar in positive and negative response sites. Spectral power density of sites with vagal response was lower between 26 and 83 Hz and higher between 107 and 200 Hz compared with negative response sites. The area between 120 and 170 Hz normalized to the total spectrum area was tested as a diagnostic parameter. Receiver operating characteristic curve analysis demonstrated that an area120-170/area(total) value >0.14 identified vagal sites with 70.9% sensitivity and 72.1% specificity. Conclusion Spectral analysis of AE during SR in sites that correspond to the anatomical location of the GP is feasible and may be a simpler method of mapping the cardiac autonomic nervous system, compared with the HFS technique.

Genome structure of a Saccharomyces cerevisiae strain widely used in bioethanol production

Bioethanol is a biofuel produced mainly from the fermentation of carbohydrates derived from agricultural feedstocks by the yeast Saccharomyces cerevisiae. One of the most widely adopted strains is PE-2, a heterothallic diploid naturally adapted to the sugar cane fermentation process used in Brazil. Here we report the molecular genetic analysis of a PE-2 derived diploid (JAY270), and the complete genome sequence of a haploid derivative (JAY291). The JAY270 genome is highly heterozygous (similar to 2 SNPs/kb) and has several structural polymorphisms between homologous chromosomes. These chromosomal rearrangements are confined to the peripheral regions of the chromosomes, with breakpoints within repetitive DNA sequences. Despite its complex karyotype, this diploid, when sporulated, had a high frequency of viable spores. Hybrid diploids formed by outcrossing with the laboratory strain S288c also displayed good spore viability. Thus, the rearrangements that exist near the ends of chromosomes do not impair meiosis, as they do not span regions that contain essential genes. This observation is consistent with a model in which the peripheral regions of chromosomes represent plastic domains of the genome that are free to recombine ectopically and experiment with alternative structures. We also explored features of the JAY270 and JAY291 genomes that help explain their high adaptation to industrial environments, exhibiting desirable phenotypes such as high ethanol and cell mass production and high temperature and oxidative stress tolerance. The genomic manipulation of such strains could enable the creation of a new generation of industrial organisms, ideally suited for use as delivery vehicles for future bioenergy technologies.

Occurrence of AFM(1) in urine samples of a Brazilian population and association with food consumption

This survey evaluated the presence of AFM(1) in human urine samples from a specific Brazilian population, as well as corn, peanut, and milk consumption measured by two types of food inquiry. Urine samples from donors who live in the city of Piracicaba, State of Sao Paulo, Brazil were analyzed to detect the presence of aflatoxin M(1) (AFM(1)). an aflatoxin B(1) metabolite, which may be used as aflatoxin B(1) exposure biomarker. The AFM(1) analysis was performed using immunoaffinity clean-up and detection by high-performance-liquid chromatography with fluorescence detector. A total of 69 samples were analyzed and 45 of them (65%) presented contaminations >= 1.8 pg ml(-1), which was the limit of quantification (LOQ). Seventy eight percent (n = 54) of the samples presented detectable concentrations of AFM(1) (>0.6 pg ml(-1)). The AFM(1) concentration among samples above LOQ ranged from 1.8 to 39.9 pg ml(-1). There were differences in food consumption profile among donors, although no association was found between food consumption and AFM(1) concentration in urine. The high frequency of positive samples suggests exposure of the populations studied to aflatoxins. (C) 2009 Elsevier Ltd. All rights reserved.

Methylation patterns revealed by MSAP profiling in genetically stable somatic embryogenic cultures of Ocotea catharinensis (Lauraceae)

Ocotea catharinensis is a rare tree species indigenous to the Atlantic rainforest of South America. In spite of its value as a hardwood species, it is in danger of extinction. The species erratically produces seeds showing irregular flowering and slow growth. Therefore, plants are not easily replaced. Tissue culture-based techniques are commonly used for obtaining living material for tree propagation and in vitro preservation. Therefore, a high-frequency somatic embryogenic system was developed for the species. In the present work, the genetic fidelity of cell aggregates and somatic embryos at various stages of in vitro development of O. catharinensis was investigated using RAPD and AFLP markers. Both analyses confirmed the absence of genetic variation in all developmental stages of O. catharinensis embryogenic cultures, verifying that the in vitro system is genetically stable. The cultures were also analyzed for their methylation profiles at 5`-CCGG-3` sites by identifying methylation-sensitive amplification polymorphisms. Some of these markers differentiated cell aggregates from embryo bodies. The sequencing of ten MSAP markers revealed that four sequences showed significant similarity to genes encoding plant proteins. Particularly, the predicted amino acid sequence of the fragment designated as OcEaggHMttc155 was similar to the enzyme 1-aminocyclopropane-1-carboxylate oxidase (ACO), which is involved in the biosynthesis of ethylene, and its expression was reported to occur from the beginning to the intermediate stages of plant embryo development. Here, we suggest that this enzyme is possibly involved in the control of the earliest stages of somatic embryogenesis of O. catharinensis, and an approach to study ACO expression during somatic embryogenesis is proposed.

Effect of water renewal on dominance hierarchy of juvenile Nile tilapia

Nile tilapia social position (Oreochromis niloticus) can be mediated by multiple channels, including chemical communication. Absence of chemical cues in the environment prevents hierarchical settlement among pairs, and enhances time spent in confrontations. The aim of this study was to test the effect of continuously renewed water flow on the establishment of hierarchical dominance in Nile tilapia juveniles. In this condition, a high frequency of attacks and disruption on hierarchical stability were expected because chemical cues for hierarchy maintenance could be washed out. After 3 days in isolation, the fish were paired by standard size but not by sex, and submitted to two conditions: continuously renewed water flow (RENEWED, n = 7) and non-renewed water flow (NONRENEWED n = 8). The paired fish were placed in an aquarium (40 cm x 30 cm x 40 cm) for 3 h; four 10-min sessions were video-recorded: the first, immediately after the fish were paired and the others 1, 2, and 3 h after pairing. Hierarchy was identified by a dominance index (DI = given attacks/received + given attacks) For each fish. The hierarchical stability was achieved by analyzing the difference between dominant DI and subordinate DI (DI-D). Hierarchy was established in both groups after second session because the DI was significantly higher for one fish of the pair. The frequency of attacks of the dominant fish in RENEWED and NONRENEWED conditions was similar in all observation sessions. The attack frequency by subordinate fish was also similar during the first three sessions (2-h pairing). However, the frequency of attacks by subordinate fish in the RENEWED condition was higher than in the NONRENEWED situation at the fourth observation session (means +/- S.E.: RENEWED = 2.83 +/- 0.94 x 10 min(-1) and NONRENEWED = 0.25 +/- 0.16 x 10 min(-1); Mann-Whitney, p = 0.04). At this point, a significant reduction of the DI-D was observed (means +/- S.E.: RENEWED = 0.70 +/- 0.11 and NONRENEWED = 1,00 +/- 0.002; Mann-Whitney, p = 0.04). The changes in DI-D were related to more frequent attacks by the subordinated fish in renewed water flow. According to our results, the unsteady agonistic interaction under renewed water flow leads to social instability. Thus, continuous water renewing can wash out relevant chemical substances and therefore disturb the dominance recognition by subordinate fish. (C) 2007 Elsevier B.V. All rights reserved.