Claudia B. Angeli

Multilocus Analyses of Seven Candidate Genes Suggest Interacting Pathways for Obesity‐Related Traits in Brazilian Populations

We investigated whether variants in major candidate genes for food intake and body weight regulation contribute to obesity‐related traits under a multilocus perspective. We studied 375 Brazilian subjects from partially isolated African‐derived populations (quilombos). Seven variants displaying conflicting results in previous reports and supposedly implicated in the susceptibility of obesity‐related phenotypes were investigated: β2‐adrenergic receptor (ADRB2) (Arg16Gly), insulin induced gene 2 (INSIG2) (rs7566605), leptin (LEP) (A19G), LEP receptor (LEPR) (Gln223Arg), perilipin (PLIN) (6209T > C), peroxisome proliferator‐activated receptor‐γ (PPARG) (Pro12Ala), and resistin (RETN) (−420C > G). Regression models as well as generalized multifactor dimensionality reduction (GMDR) were employed to test the contribution of individual effects and higher‐order interactions to BMI and waist‐hip ratio (WHR) variation and risk of overweight/obesity. The best multilocus association signal identified in the quilombos was further examined in an independent sample of 334 Brazilian subjects of European ancestry. In quilombos, only the PPARG polymorphism displayed significant individual effects (WHR variation, P = 0.028). No association was observed either with the risk of overweight/obesity (BMI ≥ 25 kg/m2), risk of obesity alone (BMI ≥ 30 kg/m2) or BMI variation. However, GMDR analyses revealed an interaction between the LEPR and ADRB2 polymorphisms (P = 0.009) as well as a third‐order effect involving the latter two variants plus INSIG2 (P = 0.034) with overweight/obesity. Assessment of the LEPR‐ADRB2 interaction in the second sample indicated a marginally significant association (P = 0.0724), which was further verified to be limited to men (P = 0.0118). Together, our findings suggest evidence for a two‐locus interaction between the LEPR Gln223Arg and ADRB2 Arg16Gly variants in the risk of overweight/obesity, and highlight further the importance of multilocus effects in the genetic component of obesity.

Differences and similarities among parotoid macrogland secretions in South American toads: a preliminary biochemical delineation.

Amphibians are known by cutaneous glands, spread over the skin, containing toxins (proteins, peptides, biogenic amines, steroidal bufadienolides, and alkaloids) used as chemical defense against predators and microbial infection. Toads are characterized by the presence of parotoid macroglands. The common toads have lately been divided into two genera: Bufo (Europe, Asia, and Africa) and Rhinella (South America). Basal Rhaebo genus is exclusively of Central America and Amazon region. Although Rhinella and Rhaebo are related, species may share differences due to the diversity of environments that they live in. In this work, we have performed a biochemical characterization of the components of the poison of eight Rhinella species and one Rhaebo by means of RP-HPLC with either UV or MS detection and by SDS-PAGE, in order to verify whether phylogenetic and biological differences, such as habitat, diet, and defensive strategies, between them may also be reflected in poison composition. Although some components were common among the secretions, we were able to identify exclusive molecules to some species. The fact that closely related animals living in different habitats secrete different molecules into the skin is an indication that biological features, and not only evolution, seem to directly influence the skin secretion composition.

Intraerythrocytic stages of Plasmodium falciparum biosynthesize menaquinone.

Herein, we show that intraerythrocytic stages of Plasmodium falciparum have an active pathway for biosynthesis of menaquinone. Kinetic assays confirmed that plasmodial menaquinone acts at least in the electron transport. Similarly to Escherichia coli, we observed increased levels of menaquinone in parasites kept under anaerobic conditions. Additionally, the mycobacterial inhibitor of menaquinone synthesis Ro 48‐8071 also suppressed menaquinone biosynthesis and growth of parasites, although off‐targets may play a role in this growth‐inhibitory effect. Due to its absence in humans, the menaquinone biosynthesis can be considered an important drug target for malaria.

Multilocus Family-Based Association Analysis of Seven Candidate Polymorphisms with Essential Hypertension in an African-Derived Semi-Isolated Brazilian Population

Background. It has been widely suggested that analyses considering multilocus effects would be crucial to characterize the relationship between gene variability and essential hypertension (EH). Objective. To test for the presence of multilocus effects between/among seven polymorphisms (six genes) on blood pressure-related traits in African-derived semi-isolated Brazilian populations (quilombos). Methods. Analyses were carried out using a family-based design in a sample of 652 participants (97 families). Seven variants were investigated: ACE (rs1799752), AGT (rs669), ADD2 (rs3755351), NOS3 (rs1799983), GNB3 (rs5441 and rs5443), and GRK4 (rs1801058). Sensitivity analyses were further performed under a case-control design with unrelated participants only. Results. None of the investigated variants were associated individually with both systolic and diastolic BP levels (SBP and DBP, respectively) or EH (as a binary outcome). Multifactor dimensionality reduction-based techniques revealed a marginal association of the combined effect of both GNB3 variants on DBP levels in a family-based design (P = 0.040), whereas a putative NOS3-GRK4 interaction also in relation to DBP levels was observed in the case-control design only (P = 0.004). Conclusion. Our results provide limited support for the hypothesis of multilocus effects between/among the studied variants on blood pressure in quilombos. Further larger studies are needed to validate our findings.

DXS548/FRAXAC1 haplotypes in fragile X chromosomes in the Brazilian population.

In order to investigate the origin of the fragile X mutation in the Brazilian population, we assessed the size of the microsatellite markers DXS548, FRAXAC1 and FRAXAC2 in 72 X chromosomes from unrelated affected males and 64 control chromosomes. We found a significantly different distribution of alleles between fragile X and controls for loci DXS548 and FRAXAC1, but no apparent linkage disequilibrium was detected for the sequence FRAXAC2. The most frequent DXS548/FRAXAC1 haplotypes in affected males were haplotypes 204/158 bp (2-1) and 196/152 bp (6-4). These findings are in accordance with the proposed two main mutational pathways for the generation of FMR-1 alleles that predispose to instability and hyperexpansion.

AGG interspersion patterns in the CGG repeat of the FMR1 gene and linked DXS548/FRAXAC1 haplotypes in Brazilian populations

We have recently described in this journal the distribution of CGG repeats of the FMR1 gene, the allelic frequencies at DXS548 and FRAXAC1 microsatellite loci, and DXS548/FRAXAC1 haplotype frequencies in African-derived, European-derived, and Amerindian populations from South America [Mingroni-Netto et al., 2002]. The CGG repeat sequences and AGG interspersion patterns were not available at that time. A number of haplotype studies in affected males with the fragile X syndrome pointed to two main pathways for the origin of the fragile X mutation in European-derived populations. The two most frequent DXS548/FRAXAC1 haplotypes linked to the fragile X mutation have been found to be associated with CGG repeat structures in the general population, which would favor expansions: the haplotype 2-1 (204/158 bp) is associated to long repeats with uninterrupted CGG arrays, leading to predisposed chromosomes, which would increase in size gradually; the haplotype 6-4 (196/152 bp) appears associated to smaller repeats particularly prone to expansions because of the loss of AGG interruptions at the 30 end of the repeat, also giving rise to long uninterspersed CGG arrays [Eichler et al., 1996]. In the city of São Paulo, Brazil, we observed that these two haplotypes were the most frequent among fragile X patients [MingroniNetto et al., 1999]. More recently, Crawford et al. [2000a] found that the two most prevalent haplotypes in African-American fragile X patients were 4-4 (200/152 bp) and 3-4 (202/152 bp). In normal African-American populations, the haplotype 4-4 was linked to CGG repeats devoid of AGG interruptions near the 50 end of the repeat [Crawford et al., 2000b]. These findings suggested that the pathways leading to fragile X mutations in Africans might be distinct from those leading to mutations in Europeans. To investigate the pathways leading to fragile X mutations in Africans, we expanded our previous sample of partially isolated communities of African ancestry (quilombos) investigating the populations of Abobral, Pedro Cubas, Galvão, and São Pedro in the Ribeira River Valley, state of São Paulo, South-Easthern Brazil (Fig. 1). Runaway slaves who reached the region between 1750 and 1850 founded these communities. A predominantly European-derived sample of 55 unrelated non-carrier brothers of fragile X males, collected in the city of São Paulo, was included in the study for comparison. All individuals were genotyped for DXS548 and FRAXAC1 alleles, as described by Mingroni-Netto et al. [2002]. Allelic frequencies at the two loci are shown in Table I. The frequencies of DXS548/FRAXAC1 haplotypes are in Table II. For AGG interruption analysis, a subset of 32 AfricanBrazilian males was selected, including individuals from Pontal, Cajual, and Rio das Rãs, previously studied by Mingroni-Netto et al. [2002]. Repeats on haplotypes known to be in linkage disequilibrium with the fragile X mutation either in Europeans (2-1 and 6-4) or in African-Americans (4-4 and 34) were preferentially sequenced, as well as those associated to uncommon haplotypes in European-derived populations. The 55 alleles from São Paulo city population were all sequenced, regardless of the linked haplotypes. In brief, the CGG repeat and surrounding DNA sequences were amplified from genomic DNA by Pfx polymerase (Invitrogen, Carlsbad, CA) with primers c and f [Fu et al., 1991]. In most samples, the forward and reverse strands were sequenced. Whenever sequencing of only one strand was obtained, the pattern was validated by re-sequencing. In the instances of rare or not previously reported patterns, sequencing was performed at least twice with each primer. After purification, approximately 60 ng of PCR products were sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Forster City, CA), in an ABI PRISM 310 Genetic Analyzer (Perkin-Elmer, Forster City, CA). CGG repeat interspersion patterns are presented in Figure 2. Haplotype 2-1 was not found among the four AfricanBrazilian populations from the state of São Paulo. In one sample, from Rio das Rãs, [Mingroni-Netto et al., 2002], this haplotype was linked to a 23 CGG repeat with one AGG interruption at the 50 end (Fig. 2a). In the predominantly Europeanderived sample from São Paulo city, the 2-1 haplotype was associated to four alleles with high repeat numbers ranging from 39–48 repeats. The 39 repeat had three interruptions: 9þ 9þ 9þ 9 (9 representing the number of CGG’s, and the signalþ one AGG interruption). The other alleles had two AGG interruptions: 9þ 9þ 22, 9þ 9þ 23, and 9þ 9þ 28 (Fig. 2b). Haplotype 6-4 was relatively frequent in the African-derived communities (8.3%–19.6%). It was less frequent in São Paulo city (1.8%, present study and 8.3%, Mingroni-Netto et al., 2002]. This is not a rare haplotype in populations hereto studied [Chiurazzi et al., 1996; Bonaventure et al., 1998; Jara Claudia B. Angeli and Leonardo P. Capelli equally contributed to this study.

Site-specific characterization of N-linked glycosylation in human urinary glycoproteins and endogenous glycopeptides

Glycosylation is a very important post-translational modification involved in various cellular processes, such as cell adhesion, signal transduction and immune response. Urine is a rich source of glycoproteins and attractive biological fluid for biomarker discovery, owing to its availability, ease of collection, and correlation with pathophysiology of diseases. Although the urinary proteomics have been explored previously, the urinary glycoproteome characterization remains challenging requiring the development and optimization of analytical and bioinformatics methods for protein glycoprofiling. This study describes the high confident identification of 472 unique N-glycosylation sites covering 256 urinary glycoproteins. Besides, 202 unique N-glycosylation sites were identified in low molecular weight endogenous glycopeptides, which belong to 90 glycoproteins. Global site-specific characterization of the N-linked glycan heterogeneity was achieved by intact glycopeptide analysis, revealing 303 unique glycopeptides most of them displaying complex/hybrid glycans composed by sialic acid and fucose. These datasets consist in a valuable resource of glycoproteins and N-glycosylation sites found in healthy human urine that can be further explored in different disorders, in which the N-linked glycosylation may be aberrant.

Inhibition of histone methyltransferase EZH2 in Schistosoma mansoni in vitro by GSK343 reduces egg laying and decreases the expression of genes implicated in DNA replication and noncoding RNA metabolism.

Background The possibility of emergence of praziquantel-resistant Schistosoma parasites and the lack of other effective drugs demand the discovery of new schistosomicidal agents. In this context the study of compounds that target histone-modifying enzymes is extremely promising. Our aim was to investigate the effect of inhibition of EZH2, a histone methyltransferase that is involved in chromatin remodeling processes and gene expression control; we tested different developmental forms of Schistosoma mansoni using GKS343, a selective inhibitor of EZH2 in human cells. Methodology/Principal findings Adult male and female worms and schistosomula were treated with different concentrations of GSK343 for up to two days in vitro. Western blotting showed a decrease in the H3K27me3 histone mark in all three developmental forms. Motility, mortality, pairing and egg laying were employed as schistosomicidal parameters for adult worms. Schistosomula viability was evaluated with propidium iodide staining and ATP quantification. Adult worms showed decreased motility when exposed to GSK343. Also, an approximate 40% reduction of egg laying by GSK343-treated females was observed when compared with controls (0.1% DMSO). Scanning electron microscopy showed the formation of bulges and bubbles throughout the dorsal region of GSK343-treated adult worms. In schistosomula the body was extremely contracted with the presence of numerous folds, and growth was markedly slowed. RNA-seq was applied to identify the metabolic pathways affected by GSK343 sublethal doses. GSK343-treated adult worms showed significantly altered expression of genes related to transmembrane transport, cellular homeostasis and egg development. In females, genes related to DNA replication and noncoding RNA metabolism processes were downregulated. Schistosomula showed altered expression of genes related to cell adhesion and membrane synthesis pathways. Conclusions/Significance The results indicated that GSK343 presents in vitro activities against S. mansoni, and the characterization of EZH2 as a new potential molecular target establishes EZH2 inhibitors as part of a promising new group of compounds that could be used for the development of schistosomicidal agents.

Distribution and biological role of the oligopeptide-binding protein (OppA) in Xanthomonas species

In this study we investigated the prevalence of the oppA gene, encoding the oligopeptide binding protein (OppA) of the major bacterial oligopeptide uptake system (Opp), in different species of the genus Xanthomonas. The oppA gene was detected in two Xanthomonas axonopodis strains among eight tested Xanthomonas species. The generation of an isogenic oppA-knockout derivative of the Xac 306 strain, showed that the OppA protein neither plays a relevant role in oligopeptide uptake nor contributes to the infectivity and multiplication of the bacterial strain in leaves of sweet orange (Citrus sinensis) and Rangpur lime (Citrus limonia). Taken together these results suggest that the oppA gene has a recent evolutionary history in the genus and does not contribute in the physiology or pathogenesis of X. axonopodis.