Selection of suitable housekeeping genes for expression analysis in glioblastoma using quantitative RT-PCR

Background: Considering the broad variation in the expression of housekeeping genes among tissues and experimental situations, studies using quantitative RT-PCR require strict definition of adequate endogenous controls. For glioblastoma, the most common type of tumor in the central nervous system, there was no previous report regarding this issue. Results: Here we show that amongst seven frequently used housekeeping genes TBP and HPRT1 are adequate references for glioblastoma gene expression analysis. Evaluation of the expression levels of 12 target genes utilizing different endogenous controls revealed that the normalization method applied might introduce errors in the estimation of relative quantities. Genes presenting expression levels which do not significantly differ between tumor and normal tissues can be considered either increased or decreased if unsuitable reference genes are applied. Most importantly, genes showing significant differences in expression levels between tumor and normal tissues can be missed. We also demonstrated that the Holliday Junction Recognizing Protein, a novel DNA repair protein over expressed in lung cancer, is extremely over-expressed in glioblastoma, with a median change of about 134 fold. Conclusion: Altogether, our data show the relevance of previous validation of candidate control genes for each experimental model and indicate TBP plus HPRT1 as suitable references for studies on glioblastoma gene expression.

Transmission electron microscopy for characterization of acrosomal damage after Percoll gradient centrifugation of cryopreserved bovine spermatozoa

The objective of this study was to characterize acrosomal ultrastructure following discontinuous Percoll gradient centrifugation of cryopreserved bovine sperm. Semen was collected from six bulls of different breeds and three ejaculates per bull were evaluated. Frozen semen samples were thawed and the acrosomal region of sperm cells was evaluated by transmission electron microscopy (TEM) before (n = 18) and after (n = 18) Percoll centrifugation. The evaluation of 20 sperm heads from each of the 36 samples analyzed ensured that a large number of cells were investigated. The data were subjected to analysis of variance at a level of significance of 5%. Percoll centrifugation reduced the percentage of sperm exhibiting normal acrosomes (from 61.77 to 30.24%), reduced the percentage of sperm presenting atypical acrosome reactions (from 28.38 to 4.84%) and increased the percentage of sperm exhibiting damage in the acrosome (from 6.14 to 64.26%). The percentage of sperm with typical acrosome reactions was not significantly different before (3.70%) and after (0.67%) centrifugation. TEM distinguished four different types of acrosomal status and enabled ultrastructural characterization of acrosomal injuries. The percentage of sperm exhibiting normal acrosomes decreased and damage in the acrosome was the most frequent acrosomal injury with the Percoll gradient centrifugation protocol utilized.

Severity of Oral Mucositis in Patients Undergoing Hematopoietic Cell Transplantation and an Oral Laser Phototherapy Protocol: A Survey of 30 Patients

Background Data and Objective: Oral mucositis (OM) is one of the worst cytotoxic effects of chemotherapy and radiotherapy in patients undergoing hematopoietic cell transplantation (HCT), and it causes severe morbidity. Laser phototherapy has been considered as an alternative therapy for prevention and treatment of OM. The aim of this study was to describe the incidence and severity of OM in HCT patients subjected to laser phototherapy, and to discuss its effect on the oral mucosa. Patients and Methods: Information concerning patient age and gender, type of basic disease, conditioning regimen, type of transplant, absence or presence of pain related to the oral cavity, OM grade, and adverse reactions or unusual events were collected from 30 patients undergoing HCT (allogeneic or autologous). These patients were given oral laser phototherapy with a InGaAIP laser (660 nm and 40 mW) daily. The data were tabulated and their frequency expressed as percentages. Results: In the analysis of those with OM, it was observed that 33.4% exhibited grade I, 40% grade II, 23.3% grade III, and 3.3% grade IV disease. On the most critical post-HCT days (D+5 and D+8), it was observed that 63.3% of patients had grade I and 33.3% had grade II disease; no patients had grade III or IV disease in this period. This severity of OM was similar to that seen in other studies of laser phototherapy and OM. Conclusion: The low grades of OM observed in this survey show the beneficial effects of laser phototherapy, but randomized clinical trials are necessary to confirm these findings.

Bone Marrow Concentrate and Bovine Bone Mineral for Sinus Floor Augmentation: A Controlled, Randomized, Single-Blinded Clinical and Histological Trial-Per-Protocol Analysis

Purpose: The purpose of this work was to evaluate the potential of substituting autogenous bone (AB) by bone marrow aspirate concentrate (BMAC). Both AB and BMAC were tested in combination with a bovine bone mineral (BBM) for their ability of new bone formation (NBF) in a multicentric, randomized, controlled, clinical and histological noninferiority trial. Materials and Methods: Forty-five severely atrophied maxillary sinus from 26 patients were evaluated in a partial cross-over design. As test arm, 34 sinus of 25 patients were augmented with BBM and BMAC containing mesenchymal stem cells. Eleven control sinus from 11 patients were augmented with a mixture of 70% BBM and 30% AB. Biopsies were obtained after a 3-4-month healing period at time of implant placement and histomorphometrically analyzed for NBF. Results: NBF was 14.3%+/- 1.8% for the control and nonsignificantly lower (12.6%+/- 1.7%) for the test (90% confidence interval: -4.6 to 1.2). Values for BBM (31.3%+/- 2.7%) were significantly higher for the test compared with control (19.3%+/- 2.5%) (p < 0.0001). Nonmineralized tissue was lower by 3.3% in the test compared with control (57.6%; p = 0.137). Conclusions: NBF after 3-4 months is equivalent in sinus, augmented with BMAC and BBM or a mixture of AB and BBM. This technique could be an alternative for using autografts to stimulate bone formation.

Sequential analysis of global gene expression profiles in immature and in vitro matured bovine oocytes: potential molecular markers of oocyte maturation

Background: Without intensive selection, the majority of bovine oocytes submitted to in vitro embryo production (IVP) fail to develop to the blastocyst stage. This is attributed partly to their maturation status and competences. Using the Affymetrix GeneChip Bovine Genome Array, global mRNA expression analysis of immature (GV) and in vitro matured (IVM) bovine oocytes was carried out to characterize the transcriptome of bovine oocytes and then use a variety of approaches to determine whether the observed transcriptional changes during IVM was real or an artifact of the techniques used during analysis. Results: 8489 transcripts were detected across the two oocyte groups, of which similar to 25.0% (2117 transcripts) were differentially expressed (p < 0.001); corresponding to 589 over-expressed and 1528 under-expressed transcripts in the IVM oocytes compared to their immature counterparts. Over expression of transcripts by IVM oocytes is particularly interesting, therefore, a variety of approaches were employed to determine whether the observed transcriptional changes during IVM were real or an artifact of the techniques used during analysis, including the analysis of transcript abundance in oocytes in vitro matured in the presence of a-amanitin. Subsets of the differentially expressed genes were also validated by quantitative real-time PCR (qPCR) and the gene expression data was classified according to gene ontology and pathway enrichment. Numerous cell cycle linked (CDC2, CDK5, CDK8, HSPA2, MAPK14, TXNL4B), molecular transport (STX5, STX17, SEC22A, SEC22B), and differentiation (NACA) related genes were found to be among the several over-expressed transcripts in GV oocytes compared to the matured counterparts, while ANXA1, PLAU, STC1and LUM were among the over-expressed genes after oocyte maturation. Conclusion: Using sequential experiments, we have shown and confirmed transcriptional changes during oocyte maturation. This dataset provides a unique reference resource for studies concerned with the molecular mechanisms controlling oocyte meiotic maturation in cattle, addresses the existing conflicting issue of transcription during meiotic maturation and contributes to the global goal of improving assisted reproductive technology.

The Coupled Aerosol and Tracer Transport model to the Brazilian developments on the Regional Atmospheric Modeling System (CATT-BRAMS) - Part 1: Model description and evaluation

We introduce the Coupled Aerosol and Tracer Transport model to the Brazilian developments on the Regional Atmospheric Modeling System (CATT-BRAMS). CATT-BRAMS is an on-line transport model fully consistent with the simulated atmospheric dynamics. Emission sources from biomass burning and urban-industrial-vehicular activities for trace gases and from biomass burning aerosol particles are obtained from several published datasets and remote sensing information. The tracer and aerosol mass concentration prognostics include the effects of sub-grid scale turbulence in the planetary boundary layer, convective transport by shallow and deep moist convection, wet and dry deposition, and plume rise associated with vegetation fires in addition to the grid scale transport. The radiation parameterization takes into account the interaction between the simulated biomass burning aerosol particles and short and long wave radiation. The atmospheric model BRAMS is based on the Regional Atmospheric Modeling System (RAMS), with several improvements associated with cumulus convection representation, soil moisture initialization and surface scheme tuned for the tropics, among others. In this paper the CATT-BRAMS model is used to simulate carbon monoxide and particulate material (PM(2.5)) surface fluxes and atmospheric transport during the 2002 LBA field campaigns, conducted during the transition from the dry to wet season in the southwest Amazon Basin. Model evaluation is addressed with comparisons between model results and near surface, radiosondes and airborne measurements performed during the field campaign, as well as remote sensing derived products. We show the matching of emissions strengths to observed carbon monoxide in the LBA campaign. A relatively good comparison to the MOPITT data, in spite of the fact that MOPITT a priori assumptions imply several difficulties, is also obtained.

SMALL MAMMAL PREY SELECTION BY TWO OWL SPECIES IN SOUTHEASTERN BRAZIL

A variety of factors influence prey selection by predators. Because Barn Owls (Tyto alba) and Burrowing Owls (Athene cunicularia) differ in size and foraging tactics, we expected differential predation on small mammal prey. We hypothesized that the Barn Owl, all active predator, would prey on smaller and younger individuals than the Burrowing Owl, a sit-and-wait predator. We used pellet analyses to evaluate selection of small mammals by the two owls in relation to prey), species, age, and size at the Ecological Station of Itirapina, state of Sao Paulo, in southeastern Brazil. Small mammals constituted most of the prey individuals and biomass in the diet of Barn Owls. Although Burrowing Owls consumed a wider range of taxa, small mammals represented one-third of all biomass consumed. With respect. to small mammals, Barn Owls foraged selectively relative to prey species, size, and age. Burrowing Owls foraged opportunistically relative to prey species, but selectively relative to prey size and age. Barn Owls selected smaller and younger (juvenile and subadult) individuals of the delicate vesper mouse (Calomys tener) and Burrowing Owls preyed more oil larger and older (subadult only) individuals. morphology and behavior of both prey and predators may explain this differential predation. Our data suggest that the active predator feeds oil smaller and younger prey, and the sit-and-wait predator took relatively larger and older prey.

Positive Selection Results in Frequent Reversible Amino Acid Replacements in the G Protein Gene of Human Respiratory Syncytial Virus

Human respiratory syncytial virus (HRSV) is the major cause of lower respiratory tract infections in children under 5 years of age and the elderly, causing annual disease outbreaks during the fall and winter. Multiple lineages of the HRSVA and HRSVB serotypes co-circulate within a single outbreak and display a strongly temporal pattern of genetic variation, with a replacement of dominant genotypes occurring during consecutive years. In the present study we utilized phylogenetic methods to detect and map sites subject to adaptive evolution in the G protein of HRSVA and HRSVB. A total of 29 and 23 amino acid sites were found to be putatively positively selected in HRSVA and HRSVB, respectively. Several of these sites defined genotypes and lineages within genotypes in both groups, and correlated well with epitopes previously described in group A. Remarkably, 18 of these positively selected tended to revert in time to a previous codon state, producing a ""flipflop'' phylogenetic pattern. Such frequent evolutionary reversals in HRSV are indicative of a combination of frequent positive selection, reflecting the changing immune status of the human population, and a limited repertoire of functionally viable amino acids at specific amino acid sites.

Genome-wide SNP genotyping highlights the role of natural selection in Plasmodium falciparum population divergence

Background: The malaria parasite Plasmodium falciparum exhibits abundant genetic diversity, and this diversity is key to its success as a pathogen. Previous efforts to study genetic diversity in P. falciparum have begun to elucidate the demographic history of the species, as well as patterns of population structure and patterns of linkage disequilibrium within its genome. Such studies will be greatly enhanced by new genomic tools and recent large-scale efforts to map genomic variation. To that end, we have developed a high throughput single nucleotide polymorphism (SNP) genotyping platform for P. falciparum. Results: Using an Affymetrix 3,000 SNP assay array, we found roughly half the assays (1,638) yielded high quality, 100% accurate genotyping calls for both major and minor SNP alleles. Genotype data from 76 global isolates confirm significant genetic differentiation among continental populations and varying levels of SNP diversity and linkage disequilibrium according to geographic location and local epidemiological factors. We further discovered that nonsynonymous and silent (synonymous or noncoding) SNPs differ with respect to within-population diversity, interpopulation differentiation, and the degree to which allele frequencies are correlated between populations. Conclusions: The distinct population profile of nonsynonymous variants indicates that natural selection has a significant influence on genomic diversity in P. falciparum, and that many of these changes may reflect functional variants deserving of follow-up study. Our analysis demonstrates the potential for new high-throughput genotyping technologies to enhance studies of population structure, natural selection, and ultimately enable genome-wide association studies in P. falciparum to find genes underlying key phenotypic traits.

Genetic variability and natural selection at the ligand domain of the Duffy binding protein in brazilian Plasmodium vivax populations

Background: Plasmodium vivax malaria is a major public health challenge in Latin America, Asia and Oceania, with 130-435 million clinical cases per year worldwide. Invasion of host blood cells by P. vivax mainly depends on a type I membrane protein called Duffy binding protein (PvDBP). The erythrocyte-binding motif of PvDBP is a 170 amino-acid stretch located in its cysteine-rich region II (PvDBP(II)), which is the most variable segment of the protein. Methods: To test whether diversifying natural selection has shaped the nucleotide diversity of PvDBP(II) in Brazilian populations, this region was sequenced in 122 isolates from six different geographic areas. A Bayesian method was applied to test for the action of natural selection under a population genetic model that incorporates recombination. The analysis was integrated with a structural model of PvDBP(II), and T-and B-cell epitopes were localized on the 3-D structure. Results: The results suggest that: (i) recombination plays an important role in determining the haplotype structure of PvDBP(II), and (ii) PvDBP(II) appears to contain neutrally evolving codons as well as codons evolving under natural selection. Diversifying selection preferentially acts on sites identified as epitopes, particularly on amino acid residues 417, 419, and 424, which show strong linkage disequilibrium. Conclusions: This study shows that some polymorphisms of PvDBP(II) are present near the erythrocyte-binding domain and might serve to elude antibodies that inhibit cell invasion. Therefore, these polymorphisms should be taken into account when designing vaccines aimed at eliciting antibodies to inhibit erythrocyte invasion.

The Coupled Aerosol and Tracer Transport model to the Brazilian developments on the Regional Atmospheric Modeling System (CATT-BRAMS) - Part 2: Model sensitivity to the biomass burning

We describe an estimation technique for biomass burning emissions in South America based on a combination of remote-sensing fire products and field observations, the Brazilian Biomass Burning Emission Model (3BEM). For each fire pixel detected by remote sensing, the mass of the emitted tracer is calculated based on field observations of fire properties related to the type of vegetation burning. The burnt area is estimated from the instantaneous fire size retrieved by remote sensing, when available, or from statistical properties of the burn scars. The sources are then spatially and temporally distributed and assimilated daily by the Coupled Aerosol and Tracer Transport model to the Brazilian developments on the Regional Atmospheric Modeling System (CATT-BRAMS) in order to perform the prognosis of related tracer concentrations. Three other biomass burning inventories, including GFEDv2 and EDGAR, are simultaneously used to compare the emission strength in terms of the resultant tracer distribution. We also assess the effect of using the daily time resolution of fire emissions by including runs with monthly-averaged emissions. We evaluate the performance of the model using the different emission estimation techniques by comparing the model results with direct measurements of carbon monoxide both near-surface and airborne, as well as remote sensing derived products. The model results obtained using the 3BEM methodology of estimation introduced in this paper show relatively good agreement with the direct measurements and MOPITT data product, suggesting the reliability of the model at local to regional scales.

Transport of North African dust from the Bod,l, depression to the Amazon Basin: a case study

Through long-range transport of dust, the North-African desert supplies essential minerals to the Amazon rain forest. Since North African dust reaches South America mostly during the Northern Hemisphere winter, the dust sources active during winter are the main contributors to the forest. Given that the Bod,l, depression area in southwestern Chad is the main winter dust source, a close link is expected between the Bod,l, emission patterns and volumes and the mineral supply flux to the Amazon. Until now, the particular link between the Bod,l, and the Amazon forest was based on sparse satellite measurements and modeling studies. In this study, we combine a detailed analysis of space-borne and ground data with reanalysis model data and surface measurements taken in the central Amazon during the Amazonian Aerosol Characterization Experiment (AMAZE-08) in order to explore the validity and the nature of the proposed link between the Bod,l, depression and the Amazon forest. This case study follows the dust events of 11-16 and 18-27 February 2008, from the emission in the Bod,l, over West Africa (most likely with contribution from other dust sources in the region) the crossing of the Atlantic Ocean, to the observed effects above the Amazon canopy about 10 days after the emission. The dust was lifted by surface winds stronger than 14 m s(-1), usually starting early in the morning. The lofted dust, mixed with biomass burning aerosols over Nigeria, was transported over the Atlantic Ocean, and arrived over the South American continent. The top of the aerosol layer reached above 3 km, and the bottom merged with the boundary layer. The arrival of the dusty air parcel over the Amazon forest increased the average concentration of aerosol crustal elements by an order of magnitude.

Transport properties in nontwist area-preserving maps

Nontwist systems, common in the dynamical descriptions of fluids and plasmas, possess a shearless curve with a concomitant transport barrier that eliminates or reduces chaotic transport, even after its breakdown. In order to investigate the transport properties of nontwist systems, we analyze the barrier escape time and barrier transmissivity for the standard nontwist map, a paradigm of such systems. We interpret the sensitive dependence of these quantities upon map parameters by investigating chaotic orbit stickiness and the associated role played by the dominant crossing of stable and unstable manifolds. (C) 2009 American Institute of Physics. [doi: 10.1063/1.3247349]

Reduction of chaotic particle transport driven by drift waves in sheared flows

Investigations of chaotic particle transport by drift waves propagating in the edge plasma of tokamaks with poloidal zonal flow are described. For large aspect ratio tokamaks, the influence of radial electric field profiles on convective cells and transport barriers, created by the nonlinear interaction between the poloidal flow and resonant waves, is investigated. For equilibria with edge shear flow, particle transport is seen to be reduced when the electric field shear is reversed. The transport reduction is attributed to the robust invariant tori that occur in nontwist Hamiltonian systems. This mechanism is proposed as an explanation for the transport reduction in Tokamak Chauffage Alfven Bresilien [R. M. O. Galvao , Plasma Phys. Controlled Fusion 43, 1181 (2001)] for discharges with a biased electrode at the plasma edge.

Suppression Pattern of Neutral Pions at High Transverse Momentum in Au plus Au Collisions at root S(NN)=200 GeV and Constraints on Medium Transport Coefficients

For Au + Au collisions at 200 GeV, we measure neutral pion production with good statistics for transverse momentum, p(T), up to 20 GeV/c. A fivefold suppression is found, which is essentially constant for 5 < p(T) < 20 GeV/c. Experimental uncertainties are small enough to constrain any model-dependent parametrization for the transport coefficient of the medium, e. g., <(q) over cap > in the parton quenching model. The spectral shape is similar for all collision classes, and the suppression does not saturate in Au + Au collisions.