Delineation of vertebral area on the coronal plane using three-dimensional ultrasonography advanced volume contrast imaging (VCI) Omni view: intrarater reliability and agreement using standard mouse, high definition mouse, and pen-tablet

Objective: To assess the fetal lumbosacral spine by three-dimensional (3D) ultrasonography using volume contrast imaging (VCI) omni view method and compare reproducibility and agreement between three different measurement techniques: standard mouse, high definition mouse and pen-tablet. Methods: A comparative and prospective study with 40 pregnant women between 20 and 34+6 weeks was realized. 3D volume datasets of the fetal spine were acquired using a convex transabdominal transducer. Starting scan plane was the coronal section of fetal lumbosacral spine by VCI-C function. Omni view manual trace was selected and a parallel plane of fetal spine was drawn including interest region. Intraclass correlation coefficient (ICC) was used for reproducibility analysis. The relative difference between three used techniques was compared by chi-square test and Fischer test. Results: Pen-tablet showed better reliability (ICC = 0.987). In the relative proportion of differences, this was significantly higher for the pen-tablet (82.14%; p < 0.01). In paired comparison, the relative difference was significantly greater for the pen-tablet (p < 0.01). Conclusion: The pen-tablet showed to be the most reproductive and concordant method in the measurement of body vertebral area of fetal lumbosacral spine by 3D ultrasonography using the VCI.

Evaluation of the agreement by examiners according to classifications of third molars

Objectives. This study recorded and evaluated the intra-and inter-group agreement degree by different examiners for the classification of lower third molars according to both the Winter's and Pell & Gregory's systems. Study Design. An observational and cross-sectional study was realized with forty lower third molars analyzed from twenty digital panoramic radiographs. Four examiner groups (undergraduates, maxillofacial surgeons, oral radiologists and clinical dentists) from Aracaju, Sergipe, Brazil, classified them in relation to angulation, class and position. The variance test (ANOVA) was applied in the examiner findings with significance level of p<0.05 and confidence intervals of 95%. Results. Intra- and inter-group agreement was observed in Winter's classification system among all examiners. Pell & Gregory's classification system showed an average intra-group agreement and a statistical significant difference to position variable in inter-group analysis with greater disagreement to the clinical dentists group (p<0.05). Conclusions. High reproducibility was associated to Winter's classification, whereas the system proposed by Pell & Gregory did not demonstrate appropriate levels of reliability.

Incomplete fissures in severe emphysematous patients evaluated with MDCT: Incidence and interobserver agreement among radiologists and pneumologists

Objective: Pulmonary fissures completeness predicts efficacy in endobronchial valves (EBV) implantation, a new lobar volume reduction therapy for severe emphysematous patients. We assessed the incidence of incomplete fissures and the interobserver agreement in its evaluation with MDCT, in severe emphysematous patients prior to EBV implantation. Materials and Methods: Volumetric thin-section CT scans of 35 patients (CODP GOLD 3/4, heterogeneous emphysema) were retrospectively reviewed by 2 pneumologists, 1 general and 2 experienced chest radiologists, independently and blinded for treatment outcome, and the pulmonary fissures were classified as either complete or incomplete. Interobserver agreement was assessed with Kappa index (KI). Results: Agreement between all readers for the left oblique, right oblique and horizontal fissure was, respectively, moderate (KI = 0.53), fair (KI = 0.37) and moderate (KI = 0.42). Highest agreement (99/105 fissures) was observed among experienced radiologists, being for left oblique, right oblique and horizontal, respectively, almost perfect (KI = 0.79), perfect (KI = 1.0) and moderate (KI = 0.52). These 2 reviewers found that all of 35 patients had at least one incomplete fissure, with a proportion of incomplete fissures assigned as 74/65%, 85/85% and 91/88%, respectively for the left oblique, right oblique and horizontal fissures. Conclusions: Pneumologists and radiologists agreed fairly to moderately in fissures analysis, while the experienced chest radiologists reached the highest clinically adequate agreement of 94%. We believe that clinical routine visual analysis of the fissures integrity can be done with a good degree of confidence in MDCT images, and experienced readers might be required. Also, a higher than expected incidence of incomplete fissures was described in our studied population. (C) 2012 Elsevier Ireland Ltd. All rights reserved.

Biometry and fetal weight estimation by two-dimensional and three-dimensional ultrasonography: an intraobserver and interobserver reliability and agreement study

Objective To evaluate and compare the intraobserver and interobserver reliability and agreement for the biparietal diameter (BPD), abdominal circumference (AC), femur length (FL) and estimated fetal weight (EFW) obtained by two-dimensional ultrasound (2D-US) and three-dimensional ultrasound (3D-US). Methods Singleton pregnant women between 24 and 40 weeks were invited to participate in this study. They were examined using 2D-US in a blinded manner, twice by one observer, intercalated by a scan by a second observer, to determine BPD, AC and FL. In each of the three examinations, three 3D-US datasets (head, abdomen and thigh) were acquired for measurements of the same parameters. We determined EFW using Hadlock's formula. Systematic errors between 3D-US and 2D-US were examined using the paired t-test. Reliability and agreement were assessed by intraclass correlation coefficients (ICCs), limits of agreement (LoA), SD of differences and proportion of differences below arbitrary points. Results We evaluated 102 singleton pregnancies. No significant systematic error between 2D-US and 3D-US was observed. The ICC values were higher for 3D-US in both intra- and interobserver evaluations; however, only for FL was there no overlap in the 95% CI. The LoA values were wider for 2D-US, suggesting that random errors were smaller when using 3D-US. Additionally, we observed that the SD values determined from 3D-US differences were smaller than those obtained for 2D-US. Higher proportions of differences were below the arbitrarily defined cut-off points when using 3D-US. Conclusion 3D-US improved the reliability and agreement of fetal measurements and EFW compared with 2D-US.

Identification of unannotated exons of low abundance transcripts in Drosophila melanogaster and cloning of a new serine protease gene upregulated upon injury

Abstract Background The sequencing of the D.melanogaster genome revealed an unexpected small number of genes (~ 14,000) indicating that mechanisms acting on generation of transcript diversity must have played a major role in the evolution of complex metazoans. Among the most extensively used mechanisms that accounts for this diversity is alternative splicing. It is estimated that over 40% of Drosophila protein-coding genes contain one or more alternative exons. A recent transcription map of the Drosophila embryogenesis indicates that 30% of the transcribed regions are unannotated, and that 1/3 of this is estimated as missed or alternative exons of previously characterized protein-coding genes. Therefore, the identification of the variety of expressed transcripts depends on experimental data for its final validation and is continuously being performed using different approaches. We applied the Open Reading Frame Expressed Sequence Tags (ORESTES) methodology, which is capable of generating cDNA data from the central portion of rare transcripts, in order to investigate the presence of hitherto unnanotated regions of Drosophila transcriptome. Results Bioinformatic analysis of 1,303 Drosophila ORESTES clusters identified 68 sequences derived from unannotated regions in the current Drosophila genome version (4.3). Of these, a set of 38 was analysed by polyA+ northern blot hybridization, validating 17 (50%) new exons of low abundance transcripts. For one of these ESTs, we obtained the cDNA encompassing the complete coding sequence of a new serine protease, named SP212. The SP212 gene is part of a serine protease gene cluster located in the chromosome region 88A12-B1. This cluster includes the predicted genes CG9631, CG9649 and CG31326, which were previously identified as up-regulated after immune challenges in genomic-scale microarray analysis. In agreement with the proposal that this locus is co-regulated in response to microorganisms infection, we show here that SP212 is also up-regulated upon injury. Conclusion Using the ORESTES methodology we identified 17 novel exons from low abundance Drosophila transcripts, and through a PCR approach the complete CDS of one of these transcripts was defined. Our results show that the computational identification and manual inspection are not sufficient to annotate a genome in the absence of experimentally derived data.

Comparison of disc diffusion, Etest and broth microdilution for testing susceptibility of carbapenem-resistant P. aeruginosa to polymyxins

Abstract Background Considering the increasing use of polymyxins to treat infections due to multidrug resistant Gram-negative in many countries, it is important to evaluate different susceptibility testing methods to this class of antibiotic. Methods Susceptibility of 109 carbapenem-resistant P. aeruginosa to polymyxins was tested comparing broth microdilution (reference method), disc diffusion, and Etest using the new interpretative breakpoints of Clinical and Laboratory Standards Institute. Results Twenty-nine percent of isolates belonged to endemic clone and thus, these strains were excluded of analysis. Among 78 strains evaluated, only one isolate was resistant to polymyxin B by the reference method (MIC: 8.0 μg/mL). Very major and major error rates of 1.2% and 11.5% were detected comparing polymyxin B disc diffusion with the broth microdilution (reference method). Agreement within 1 twofold dilution between Etest and the broth microdilution were 33% for polymyxin B and 79.5% for colistin. One major error and 48.7% minor errors were found comparing polymyxin B Etest with broth microdilution and only 6.4% minor errors with colistin. The concordance between Etest and the broth microdilution (reference method) was respectively 100% for colistin and 90% for polymyxin B. Conclusion Resistance to polymyxins seems to be rare among hospital carbapenem-resistant P. aeruginosa isolates over a six-year period. Our results showed, using the new CLSI criteria, that the disc diffusion susceptibility does not report major errors (false-resistant results) for colistin. On the other hand, showed a high frequency of minor errors and 1 very major error for polymyxin B. Etest presented better results for colistin than polymyxin B. Until these results are reproduced with a large number of polymyxins-resistant P. aeruginosa isolates, susceptibility to polymyxins should be confirmed by a reference method.

Hand-carried ultrasound performed at bedside in cardiology inpatient setting – a comparative study with comprehensive echocardiography

Abstract Background Hand-carried ultrasound (HCU) devices have been demonstrated to improve the diagnosis of cardiac diseases over physical examination, and have the potential to broaden the versatility in ultrasound application. The role of these devices in the assessment of hospitalized patients is not completely established. In this study we sought to perform a direct comparison between bedside evaluation using HCU and comprehensive echocardiography (CE), in cardiology inpatient setting. Methods We studied 44 consecutive patients (mean age 54 ± 18 years, 25 men) who underwent bedside echocardiography using HCU and CE. HCU was performed by a cardiologist with level-2 training in the performance and interpretation of echocardiography, using two-dimensional imaging, color Doppler, and simple calliper measurements. CE was performed by an experienced echocardiographer (level-3 training) and considered as the gold standard. Results There were no significant differences in cardiac chamber dimensions and left ventricular ejection fraction determined by the two techniques. The agreement between HCU and CE for the detection of segmental wall motion abnormalities was 83% (Kappa = 0.58). There was good agreement for detecting significant mitral valve regurgitation (Kappa = 0.85), aortic regurgitation (kappa = 0.89), and tricuspid regurgitation (Kappa = 0.74). A complete evaluation of patients with stenotic and prosthetic dysfunctional valves, as well as pulmonary hypertension, was not possible using HCU due to its technical limitations in determining hemodynamic parameters. Conclusion Bedside evaluation using HCU is helpful for assessing cardiac chamber dimensions, left ventricular global and segmental function, and significant valvular regurgitation. However, it has limitations regarding hemodynamic assessment, an important issue in the cardiology inpatient setting.

HPV type infection in different anogenital sites among HIV-positive Brazilian women

Abstract Objectives To evaluate the prevalence of human papillomavirus (HPV) types, and risk factors for HPV positivity across cervix, vagina and anus, we conducted a study among 138 women with human immunodeficiency virus (HIV). Goal Compare the prevalence of different HPV types and the risk factors for HPV positivity in three sites. Results The most frequently detected HPV types in all sites were, in decreasing order, HPV16, 53, 18, 61 and 81. Agreement between the cervix and vagina was good (kappa 0.60 – 0.80) for HPV16 and 53 and excellent (Kappa > 0.80) for HPV18 and 61. HPV positivity was inversely associated with age for all combinations including the anal site. Conclusion In HIV positive women, HPV18 is the most spread HPV type found in combinations of anal and genital sites. The relationship of anal to genital infection has implications for the development of anal malignancies. Thus, the efficacy of the current HPV vaccine may be considered not only for the cervix, but also for prevention of HPV18 anal infection among immunossuppressed individuals.

SAG2A protein from Toxoplasma gondii interacts with both innate and adaptive immune compartments of infected hosts

Abstract Background Toxoplasma gondii is an intracellular parasite that causes relevant clinical disease in humans and animals. Several studies have been performed in order to understand the interactions between proteins of the parasite and host cells. SAG2A is a 22 kDa protein that is mainly found in the surface of tachyzoites. In the present work, our aim was to correlate the predicted three-dimensional structure of this protein with the immune system of infected hosts. Methods To accomplish our goals, we performed in silico analysis of the amino acid sequence of SAG2A, correlating the predictions with in vitro stimulation of antigen presenting cells and serological assays. Results Structure modeling predicts that SAG2A protein possesses an unfolded C-terminal end, which varies its conformation within distinct strain types of T. gondii. This structure within the protein shelters a known B-cell immunodominant epitope, which presents low identity with its closest phyllogenetically related protein, an orthologue predicted in Neospora caninum. In agreement with the in silico observations, sera of known T. gondii infected mice and goats recognized recombinant SAG2A, whereas no serological cross-reactivity was observed with samples from N. caninum animals. Additionally, the C-terminal end of the protein was able to down-modulate pro-inflammatory responses of activated macrophages and dendritic cells. Conclusions Altogether, we demonstrate herein that recombinant SAG2A protein from T. gondii is immunologically relevant in the host-parasite interface and may be targeted in therapeutic and diagnostic procedures designed against the infection.

Endosymbiosis in trypanosomatids: the genomic cooperation between bacterium and host in the synthesis of essential amino acids is heavily influenced by multiple horizontal gene transfers

Background Trypanosomatids of the genera Angomonas and Strigomonas live in a mutualistic association characterized by extensive metabolic cooperation with obligate endosymbiotic Betaproteobacteria. However, the role played by the symbiont has been more guessed by indirect means than evidenced. Symbiont-harboring trypanosomatids, in contrast to their counterparts lacking symbionts, exhibit lower nutritional requirements and are autotrophic for essential amino acids. To evidence the symbiont’s contributions to this autotrophy, entire genomes of symbionts and trypanosomatids with and without symbionts were sequenced here. Results Analyses of the essential amino acid pathways revealed that most biosynthetic routes are in the symbiont genome. By contrast, the host trypanosomatid genome contains fewer genes, about half of which originated from different bacterial groups, perhaps only one of which (ornithine cyclodeaminase, EC:4.3.1.12) derived from the symbiont. Nutritional, enzymatic, and genomic data were jointly analyzed to construct an integrated view of essential amino acid metabolism in symbiont-harboring trypanosomatids. This comprehensive analysis showed perfect concordance among all these data, and revealed that the symbiont contains genes for enzymes that complete essential biosynthetic routes for the host amino acid production, thus explaining the low requirement for these elements in symbiont-harboring trypanosomatids. Phylogenetic analyses show that the cooperation between symbionts and their hosts is complemented by multiple horizontal gene transfers, from bacterial lineages to trypanosomatids, that occurred several times in the course of their evolution. Transfers occur preferentially in parts of the pathways that are missing from other eukaryotes. Conclusion We have herein uncovered the genetic and evolutionary bases of essential amino acid biosynthesis in several trypanosomatids with and without endosymbionts, explaining and complementing decades of experimental results. We uncovered the remarkable plasticity in essential amino acid biosynthesis pathway evolution in these protozoans, demonstrating heavy influence of horizontal gene transfer events, from Bacteria to trypanosomatid nuclei, in the evolution of these pathways.

Genetic diversity and population structure of Musa accessions in ex situ conservation

Abstract Background Banana cultivars are mostly derived from hybridization between wild diploid subspecies of Musa acuminata (A genome) and M. balbisiana (B genome), and they exhibit various levels of ploidy and genomic constitution. The Embrapa ex situ Musa collection contains over 220 accessions, of which only a few have been genetically characterized. Knowledge regarding the genetic relationships and diversity between modern cultivars and wild relatives would assist in conservation and breeding strategies. Our objectives were to determine the genomic constitution based on Internal Transcribed Spacer (ITS) regions polymorphism and the ploidy of all accessions by flow cytometry and to investigate the population structure of the collection using Simple Sequence Repeat (SSR) loci as co-dominant markers based on Structure software, not previously performed in Musa. Results From the 221 accessions analyzed by flow cytometry, the correct ploidy was confirmed or established for 212 (95.9%), whereas digestion of the ITS region confirmed the genomic constitution of 209 (94.6%). Neighbor-joining clustering analysis derived from SSR binary data allowed the detection of two major groups, essentially distinguished by the presence or absence of the B genome, while subgroups were formed according to the genomic composition and commercial classification. The co-dominant nature of SSR was explored to analyze the structure of the population based on a Bayesian approach, detecting 21 subpopulations. Most of the subpopulations were in agreement with the clustering analysis. Conclusions The data generated by flow cytometry, ITS and SSR supported the hypothesis about the occurrence of homeologue recombination between A and B genomes, leading to discrepancies in the number of sets or portions from each parental genome. These phenomenons have been largely disregarded in the evolution of banana, as the “single-step domestication” hypothesis had long predominated. These findings will have an impact in future breeding approaches. Structure analysis enabled the efficient detection of ancestry of recently developed tetraploid hybrids by breeding programs, and for some triploids. However, for the main commercial subgroups, Structure appeared to be less efficient to detect the ancestry in diploid groups, possibly due to sampling restrictions. The possibility of inferring the membership among accessions to correct the effects of genetic structure opens possibilities for its use in marker-assisted selection by association mapping.

Expression, purification and molecular analysis of the human ZNF706 protein

Background: The ZNF706 gene encodes a protein that belongs to the zinc finger family of proteins and was found to be highly expressed in laryngeal cancer, making the structure and function of ZNF706 worthy of investigation. In this study, we expressed and purified recombinant human ZNF706 that was suitable for structural analysis in Escherichia coli BL21(DH3). Findings: ZNF706 mRNA was extracted from a larynx tissue sample, and cDNA was ligated into a cloning vector using the TOPO method. ZNF706 protein was expressed according to the E. coli expression system procedures and was purified using a nickel-affinity column. The structural qualities of recombinant ZNF706 and quantification alpha, beta sheet, and other structures were obtained by spectroscopy of circular dichroism. ZNF706's structural modeling showed that it is composed of α-helices (28.3%), β-strands (19.4%), and turns (20.9%), in agreement with the spectral data from the dichroism analysis. Conclusions: We used circular dichroism and molecular modeling to examine the structure of ZNF706. The results suggest that human recombinant ZNF706 keeps its secondary structures and is appropriate for functional and structural studies. The method of expressing ZNF706 protein used in this study can be used to direct various functional and structural studies that will contribute to the understanding of its function as well as its relationship with other biological molecules and its putative role in carcinogenesis.

Effects of pretreatment on morphology, chemical composition and enzymatic digestibility of eucalyptus bark: a potentially valuable source of fermentable sugars for biofuel production – part 1

Abstract Background In recent years, the growing demand for biofuels has encouraged the search for different sources of underutilized lignocellulosic feedstocks that are available in sufficient abundance to be used for sustainable biofuel production. Much attention has been focused on biomass from grass. However, large amounts of timber residues such as eucalyptus bark are available and represent a potential source for conversion to bioethanol. In the present paper, we investigate the effects of a delignification process with increasing sodium hydroxide concentrations, preceded or not by diluted acid, on the bark of two eucalyptus clones: Eucalyptus grandis (EG) and the hybrid, E. grandis x urophylla (HGU). The enzymatic digestibility and total cellulose conversion were measured, along with the effect on the composition of the solid and the liquor fractions. Barks were also assessed using Fourier-transform infrared spectroscopy (FTIR), solid-state nuclear magnetic resonance (NMR), X-Ray diffraction, and scanning electron microscopy (SEM). Results Compositional analysis revealed an increase in the cellulose content, reaching around 81% and 76% of glucose for HGU and EG, respectively, using a two-step treatment with HCl 1%, followed by 4% NaOH. Lignin removal was 84% (HGU) and 79% (EG), while the hemicellulose removal was 95% and 97% for HGU and EG, respectively. However, when we applied a one-step treatment, with 4% NaOH, higher hydrolysis efficiencies were found after 48 h for both clones, reaching almost 100% for HGU and 80% for EG, in spite of the lower lignin and hemicellulose removal. Total cellulose conversion increased from 5% and 7% to around 65% for HGU and 59% for EG. NMR and FTIR provided important insight into the lignin and hemicellulose removal and SEM studies shed light on the cell-wall unstructuring after pretreatment and lignin migration and precipitation on the fibers surface, which explain the different hydrolysis rates found for the clones. Conclusion Our results show that the single step alkaline pretreatment improves the enzymatic digestibility of Eucalyptus bark. Furthermore, the chemical and physical methods combined in this study provide a better comprehension of the pretreatment effects on cell-wall and the factors that influence enzymatic digestibility of this forest residue.

Comparison of child self-reports and parent proxy-reports on quality of life of children with attention deficit hyperactivity disorder

Abstract Background Attention deficit hyperactivity disorder (ADHD) is a neurobiological condition that affects 3%–7% of the pediatric population and significantly compromises the quality of life (QoL) of these individuals. The aim of the current study was to compare child self-reports and parent proxy reports on the QoL of children with ADHD. Methods Forty-five children with ADHD, combined type, aged 8–12 years without comorbidities, were compared with 43 typically developing children. PedsQL™ 4.0 (Pediatric QoL Inventory™) Generic Core Scales (physical, emotional, social, and school functioning) were completed by families and children self-reporting their health-related QoL. Results Children with ADHD reported themselves significantly lowered their PedsQL™ scores on all dimensions in comparison to typically developing children. Statistically significant differences were observed in social functioning (p = 0.010), school functioning (p <0.001), psychosocial health (p <0.001), and total score (p = 0.002). The physical functioning and emotional functioning dimensions did not differ significantly between groups, with p = 0.841 and p = 0.070, respectively. Parents of children with ADHD also reported lower PedsQL™ scores, with statistically significant differences in all dimensions. The relationship between child self-reports and parent proxy reports indicated that there is greater agreement among children with ADHD, except for the school functioning. Conclusions This suggests that children with the disorder and their parents have a perception of the functional limitations the disorder brings. It is therefore important to undertake studies to verify the QoL in children with ADHD that aim to provide and measure the scope of the well-being of these children.