Analysis of peptides in prohormone convertase 1/3 null mouse brain using quantitative peptidomics

P>Neuropeptides are produced from larger precursors by limited proteolysis, first by endopeptidases and then by carboxypeptidases. Major endopeptidases required for these cleavages include prohormone convertase (PC) 1/3 and PC2. In this study, quantitative peptidomics analysis was used to characterize the specific role PC1/3 plays in this process. Peptides isolated from hypothalamus, amygdala, and striatum of PC1/3 null mice were compared with those from heterozygous and wild-type mice. Extracts were labeled with stable isotopic tags and fractionated by HPLC, after which relative peptide levels were determined using tandem mass spectrometry. In total, 92 peptides were found, of which 35 were known neuropeptides or related peptides derived from 15 distinct secretory pathway proteins: 7B2, chromogranin A and B, cocaine- and amphetamine-regulated transcript, procholecystokinin, proenkephalin, promelanin concentrating hormone, proneurotensin, propituitary adenylate cyclase-activating peptide, proSAAS, prosomatosatin, provasoactive intestinal peptide, provasopressin, secretogranin III, and VGF. Among the peptides derived from these proteins, similar to 1/3 were decreased in the PC1/3 null mice relative to wild-type mice, similar to 1/3 showed no change, and similar to 1/3 increased in PC1/3 null. Cleavage sites were analyzed in peptides that showed no change or that decreased in PC1/3 mice, and these results were compared with peptides that showed no change or decreased in previous peptidomic studies with PC2 null mice. Analysis of these sites showed that while PC1/3 and PC2 have overlapping substrate preferences, there are particular cleavage site residues that distinguish peptides preferred by each PC.

Spatial and temporal distribution of tadpole assemblages (Amphibia, Anura) in a seasonal dry tropical forest of southeastern Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Analysis of Climatic Factors Impact on RSV Infection Distribution in Children Attending Childcare at Northwest Region of São Paulo, Brazil

Respiratory syncytial virus (RSV) was detected in samples collected from children from 0 to 6 years of age with acute respiratory infection, attending public childcare on Northwest region of São Paulo, Brazil. RSV distribution was associated to seasonal climatic variables as temperature, rainfall and relative air humidity. We utilized samples of nasopharyngeal aspirate collected during the period of July 2003 to September 2005. RT-PCR was the chosen method for viral identification. Results showed that from the 817 samples (collected from 179 children), 7.7% (63/817) were RSV positive. In 2003, RSV was detected from July until October. In 2004, RSV infections occurred in March, May, June, July, October, November, and December. In 2005, RSV was detected in March, April, May, August, and September. RSV circulation patterns in childcare children showed seasonal distribution associated to decreases in temperature and relative air humidity. RSV was detected in childcare children as an important viral agent causing respiratory infections, with varying patterns of circulation into the cohort during the study period. Moreover, RSV distribution showed to be associated with the dry season on Northwest region of São Paulo, Brazil.

Methylenedioxymethamphetamine (Ecstasy) Decreases Neutrophil Activity and Alters Leukocyte Distribution in Bone Marrow, Spleen and Blood

Objective: Looking for possible neuroimmune relationships, we analyzed the effects of methylenedioxymethamphetamine (MDMA) administration on neuroendocrine, neutrophil activity and leukocyte distribution in mice. Methods: Five experiments were performed. In the first, mice were treated with MDMA (10 mg/kg) 30, 60 min and 24 h prior to blood sample collection for neutrophil activity analysis. In the second experiment, the blood of nave mice was collected and incubated with MDMA for neutrophil activity in vitro analysis. In the third and fourth experiments, mice were injected with MDMA (10 mg/kg) and 60 min later, blood and brain were collected to analyze corticosterone serum levels and hypothalamic noradrenaline (NA) levels and turnover. In the last experiment, mice were injected with MDMA 10 mg/kg and 60 min later, blood, bone marrow and spleen were collected for leukocyte distribution analysis. Results: Results showed an increase in hypothalamic NA turnover and corticosterone serum levels 60 min after MDMA (10 mg/kg) administration, a decrease in peripheral blood neutrophil oxidative burst and a decrease in the percentage and intensity of neutrophil phagocytosis. It was further found that MDMA (10 mg/kg) treatment also altered leukocyte distribution in blood, bone marrow and spleen. In addition, no effects were observed for MDMA after in vitro exposure both in neutrophil oxidative burst and phagocytosis. Conclusion: The effects of MDMA administration (10 mg/kg) on neutrophil activity and leukocyte distribution might have been induced indirectly through noradrenergic neurons and/or hypothalamic-pituitary-adrenal axis activations. Copyright (C) 2009 S. Karger AG, Basel

Inference of Evolutionary Forces Acting on Human Biological Pathways.

Because natural selection is likely to act on multiple genes underlying a given phenotypic trait, we study here the potential effect of ongoing and past selection on the genetic diversity of human biological pathways. We first show that genes included in gene sets are generally under stronger selective constraints than other genes and that their evolutionary response is correlated. We then introduce a new procedure to detect selection at the pathway level based on a decomposition of the classical McDonald-Kreitman test extended to multiple genes. This new test, called 2DNS, detects outlier gene sets and takes into account past demographic effects and evolutionary constraints specific to gene sets. Selective forces acting on gene sets can be easily identified by a mere visual inspection of the position of the gene sets relative to their two-dimensional null distribution. We thus find several outlier gene sets that show signals of positive, balancing, or purifying selection but also others showing an ancient relaxation of selective constraints. The principle of the 2DNS test can also be applied to other genomic contrasts. For instance, the comparison of patterns of polymorphisms private to African and non-African populations reveals that most pathways show a higher proportion of nonsynonymous mutations in non-Africans than in Africans, potentially due to different demographic histories and selective pressures.

Size and power properties of some tests in the Birnbaum-Saunders regression model

The Birnbaum-Saunders distribution has been used quite effectively to model times to failure for materials subject to fatigue and for modeling lifetime data. In this paper we obtain asymptotic expansions, up to order n(-1/2) and under a sequence of Pitman alternatives, for the non-null distribution functions of the likelihood ratio, Wald, score and gradient test statistics in the Birnbaum-Saunders regression model. The asymptotic distributions of all four statistics are obtained for testing a subset of regression parameters and for testing the shape parameter. Monte Carlo simulation is presented in order to compare the finite-sample performance of these tests. We also present two empirical applications. (C) 2010 Elsevier B.V. All rights reserved.

Structural studies of prephenate dehydratase from Mycobacterium tuberculosis H37Rv by SAXS, ultracentrifugation, and computational analysis

Tuberculosis (TB) is one of the most common infectious diseases known to man and responsible for millions of human deaths in the world. The increasing incidence of TB in developing countries, the proliferation of multidrug resistant strains, and the absence of resources for treatment have highlighted the need of developing new drugs against TB. The shikimate pathway leads to the biosynthesis of chorismate, a precursor of aromatic amino acids. This pathway is absent from mammals and shown to be essential for the survival of Mycobacterium tuberculosis, the causative agent of TB. Accordingly, enzymes of aromatic amino acid biosynthesis pathway represent promising targets for structure-based drug design. The first reaction in phenylalanine biosynthesis involves the conversion of chorismate to prephenate, catalyzed by chorismate mutase. The second reaction is catalyzed by prephenate dehydratase (PDT) and involves decarboxylation and dehydratation of prephenate to form phenylpyruvate, the precursor of phenylalanine. Here, we describe utilization of different techniques to infer the structure of M. tuberculosis PDT (MtbPDT) in solution. Small angle X-ray scattering and ultracentrifugation analysis showed that the protein oligomeric state is a tetramer and MtbPDT is a flat disk protein. Bioinformatics tools were used to infer the structure of MtbPDT A molecular model for MtbPDT is presented and molecular dynamics simulations indicate that MtbPDT i.s stable. Experimental and molecular modeling results were in agreement and provide evidence for a tetrameric state of MtbPDT in solution.

Caracterização biológica e molecular de quatro cepas de Trypanosoma cruzi Chagas,1909 (Kinetoplastida, Trypanosomatidae) isoladas de pacientes chagásicos crônicos

Pós-graduação em Biociências e Biotecnologia Aplicadas à Farmácia - FCFAR

The effect of pulsed jet lavage in vertebroplasty on injection forces of polymethylmethacrylate bone cement, material distribution, and potential fat embolism: a cadaver study

STUDY DESIGN: In vitro testing of vertebroplasty techniques including pulsed jet-lavage for fat and marrow removal in human cadaveric lumbar and thoracic vertebrae. OBJECTIVE: To develop jet-lavage techniques for vertebroplasty and investigate their effect on cement distribution, injection forces, and fat embolism. SUMMARY OF BACKGROUND DATA: The main complications of cement vertebroplasty are cement leakage and pulmonary fat embolism, which can have fatal consequences and are difficult to prevent reliably by current vertebroplasty techniques. METHODS: Twenty-four vertebrae (Th8-L04) from 5 osteoporotic cadaver spines were grouped in triplets depending on bone mineral density (BMD). Before polymethylmethacrylate (PMMA) vertebroplasty, a pulsatile jet-lavage for removal of intertrabecular fat and bone marrow was performed in 2 groups with 8 specimens each, performing radial and axial irrigation from the biopsy needles. One hundred mL of Ringer solution were injected through 1 pedicle and regained by low vacuum via the contralateral pedicle. Eight control vertebrae were not irrigated. All specimens underwent standardized PMMA cement augmentation injecting 20% of the vertebral volume. Injection forces, cement distribution, and extravasations were quantified. RESULTS: All irrigation solution could be retrieved with the vacuum applied. A Kruskal-Wallis test revealed significantly higher injection forces of the control group as compared with the irrigated groups (P = 0.021). Dilatation of the syringe at forces above 300 N occurred in 75% of the untreated compared with 12.5% of the lavaged specimens. CT distribution analysis showed more homogenous cement distribution of the cement and significantly less extravasation in the irrigated specimens. CONCLUSION: The developed lavage technique for vertebroplasty showed to be feasible and reproducible. The reduction of injection forces would allow the use of more viscous PMMA cement lowering the risk for cement embolization and results in a safer procedure. The wash-out of bone marrow and the possible reduction of pulmonary fat embolism have to be verified with in vivo models.

Mutual Information for Testing Gene-Environment Interaction

Despite current enthusiasm for investigation of gene-gene interactions and gene-environment interactions, the essential issue of how to define and detect gene-environment interactions remains unresolved. In this report, we define gene-environment interactions as a stochastic dependence in the context of the effects of the genetic and environmental risk factors on the cause of phenotypic variation among individuals. We use mutual information that is widely used in communication and complex system analysis to measure gene-environment interactions. We investigate how gene-environment interactions generate the large difference in the information measure of gene-environment interactions between the general population and a diseased population, which motives us to develop mutual information-based statistics for testing gene-environment interactions. We validated the null distribution and calculated the type 1 error rates for the mutual information-based statistics to test gene-environment interactions using extensive simulation studies. We found that the new test statistics were more powerful than the traditional logistic regression under several disease models. Finally, in order to further evaluate the performance of our new method, we applied the mutual information-based statistics to three real examples. Our results showed that P-values for the mutual information-based statistics were much smaller than that obtained by other approaches including logistic regression models.

Single nucleotide polymorphism (SNP) selection for genotype-phenotype association studies

With hundreds of single nucleotide polymorphisms (SNPs) in a candidate gene and millions of SNPs across the genome, selecting an informative subset of SNPs to maximize the ability to detect genotype-phenotype association is of great interest and importance. In addition, with a large number of SNPs, analytic methods are needed that allow investigators to control the false positive rate resulting from large numbers of SNP genotype-phenotype analyses. This dissertation uses simulated data to explore methods for selecting SNPs for genotype-phenotype association studies. I examined the pattern of linkage disequilibrium (LD) across a candidate gene region and used this pattern to aid in localizing a disease-influencing mutation. The results indicate that the r2 measure of linkage disequilibrium is preferred over the common D′ measure for use in genotype-phenotype association studies. Using step-wise linear regression, the best predictor of the quantitative trait was not usually the single functional mutation. Rather it was a SNP that was in high linkage disequilibrium with the functional mutation. Next, I compared three strategies for selecting SNPs for application to phenotype association studies: based on measures of linkage disequilibrium, based on a measure of haplotype diversity, and random selection. The results demonstrate that SNPs selected based on maximum haplotype diversity are more informative and yield higher power than randomly selected SNPs or SNPs selected based on low pair-wise LD. The data also indicate that for genes with small contribution to the phenotype, it is more prudent for investigators to increase their sample size than to continuously increase the number of SNPs in order to improve statistical power. When typing large numbers of SNPs, researchers are faced with the challenge of utilizing an appropriate statistical method that controls the type I error rate while maintaining adequate power. We show that an empirical genotype based multi-locus global test that uses permutation testing to investigate the null distribution of the maximum test statistic maintains a desired overall type I error rate while not overly sacrificing statistical power. The results also show that when the penetrance model is simple the multi-locus global test does as well or better than the haplotype analysis. However, for more complex models, haplotype analyses offer advantages. The results of this dissertation will be of utility to human geneticists designing large-scale multi-locus genotype-phenotype association studies. ^

Rank test of location optimal for hyperbolic secant distribution

There are at least two reasons for a symmetric, unimodal, diffuse tailed hyperbolic secant distribution to be interesting in real-life applications. It displays one of the common types of non normality in natural data and is closely related to the logistic and Cauchy distributions that often arise in practice. To test the difference in location between two hyperbolic secant distributions, we develop a simple linear rank test with trigonometric scores. We investigate the small-sample and asymptotic properties of the test statistic and provide tables of the exact null distribution for small sample sizes. We compare the test to the Wilcoxon two-sample test and show that, although the asymptotic powers of the tests are comparable, the present test has certain practical advantages over the Wilcoxon test.

Ecologia de comunidades e comportamento reprodutivo de anfíbios anuros em savana amazônica

The Amazon savannas occur as isolated patches throughout extensive areas of forest in the states of Amapá, Amazonas, Pará, and Roraima. There is a considerable variation in the composition of anuran assemblages in the localities and phytophysiognomies of Amazon savannas and given the absence of studies on reproductive behavior, a systematic and geographically wide sampling has been carried out in the Amapá savanna, located in the Eastern Amazon. The study was conducted in a savanna area in the state of Amapá to examine the composition, ecology, and reproductive behavior of anuran amphibians. We carried out 24 field trips in each phytophysiognomy (gramineous-woody savana, gramineous-herbaceous-woody savana, park savana, and arboreal savanna); for analysis of reproductive behavior observations were made during the period January to December 2013, lasting four consecutive days. Samples were collected by active and acoustic search along 20 plots of 100x50 meters. Twenty-one anuran species were recorded, of which four are new records for the state of Amapá: Dendropsophus walfordi, Scinax fuscomarginatus, Pseudopaludicola boliviana e Elachistocleis helianneae. The KruskalWallis ANOVA revealed significant differences between richness and species diversity in the phytophysiognomies (p < 0.05). The Bray-Curtis similarity coefficient divided the phytophysiognomies into three groups: arboreal savana, gramineous-woody savanna and gramineous-herbaceous-woody savanna, and park savanna. According to the non-metric multidimensional scaling, the structure of the anuran community resulted in a separation into three phytophysiognomies, with significant differences in the structure of communities (ANOSIM, R = 0.823; p < 0.001). In the study of community ecology, the results obtained for spatial, temporal, and trophic niche breadth suggest that the assemblage of anurans of the Amapá savanna is not composed of predominantly generalist species. Also, the presence of other specialist anurans may explain the processes of speciation associated with the isolation of habitats, resulting in heterogeneity and spatial discontinuity in the phytophysiognomies with open formations. The null model analysis revealed that the community is structured based on temporal and trophic niche, indicating a significant influence of contemporary ecological factors on the assemblage. The absence of structure based on spatial niche might be explained by the spatial segregation in the distribution and occupation of anurans in the different phytophysiognomies of the Amapá savanna. Regarding the reproductive behavior of anurans, 11 species were classified as having a long breeding season, intrinsically associated with the rainy season and the reproductive mode of most species that lay egg clutches in lentic water bodies. Six reproductive modes were recorded and parental care was observed in Leptodactylus macrosternum and L. podicipinus, whose reproductive mode is characterized by foam nests. Regarding behavioral reproductive strategies, calling males were observed in all species of anurans, satellite males were recorded only for D. walfordi, Hypsiboas multifasciatus, S. nebulosus and S. fuscomarginatus; active search for females was observed for Phyllomedusa hypochondrialis and L. fuscus, and male displacement was recorded only for Rhinella major and R. margaritifera. Of the reproductive behaviors observed, throat and vocal sac display is associated with courtship and territorial behavior exhibited by males. In addition to courtship behavior, visual signals associated with courtship strategies were recorded for the anurans of the Amapá savanna.

Co-Infestation and Spatial Distribution of Bactrocera carambolae and Anastrepha spp. (Diptera: Tephritidae) in Common Guava in the Eastern Amazon.

Field infestation and spatial distribution of introduced Bactrocera carambolae Drew and Hancock and native species of Anastrepha in common guavas [Psidium guajava (L.)] were investigated in the eastern Amazon. Fruit sampling was carried out in the municipalities of Calc¸oene and Oiapoque in the state of Amapa, Brazil. The frequency distribution of larvae in fruit was fitted to the negative binomial distribution. Anastrepha striata was more abundant in both sampled areas in comparison to Anastrepha fraterculus (Wiedemann) and B. carambolae. The frequency distribution analysis of adults revealed an aggregated pattern for B. carambolae as well as for A. fraterculus and Anastrepha striata Schiner, described by the negative binomial distribution. Although the populations of Anastrepha spp. may have suffered some impact due to the presence of B. carambolae, the results are still not robust enough to indicate effective reduction in the abundance of Anastrepha spp. caused by B. carambolae in a general sense. The high degree of aggregation observed for both species suggests interspecific co-occurrence with the simultaneous presence of both species in the analysed fruit. Moreover, a significant fraction of uninfested guavas also indicated absence of competitive displacement.

An Integrated Framework To Improve The Concept Of Resource Specialisation.

Resource specialisation, although a fundamental component of ecological theory, is employed in disparate ways. Most definitions derive from simple counts of resource species. We build on recent advances in ecophylogenetics and null model analysis to propose a concept of specialisation that comprises affinities among resources as well as their co-occurrence with consumers. In the distance-based specialisation index (DSI), specialisation is measured as relatedness (phylogenetic or otherwise) of resources, scaled by the null expectation of random use of locally available resources. Thus, specialists use significantly clustered sets of resources, whereas generalists use over-dispersed resources. Intermediate species are classed as indiscriminate consumers. The effectiveness of this approach was assessed with differentially restricted null models, applied to a data set of 168 herbivorous insect species and their hosts. Incorporation of plant relatedness and relative abundance greatly improved specialisation measures compared to taxon counts or simpler null models, which overestimate the fraction of specialists, a problem compounded by insufficient sampling effort. This framework disambiguates the concept of specialisation with an explicit measure applicable to any mode of affinity among resource classes, and is also linked to ecological and evolutionary processes. This will enable a more rigorous deployment of ecological specialisation in empirical and theoretical studies.