Genetic transformation of three sweet orange cultivars from explants of adult plants

The difficulty in adult tissue genetic transformation in woody species is still an obstacle to be overcome, including in most sweet orange cultivars of the Brazilian citrus industry. This work reports that, after in vitro culture adjustments, transgenic adventitious buds of 'Hamlin', 'Pra', and 'Valencia' sweet oranges (Citrus sinensis L. Osbeck) were recovered using adult material as explant source, in genetic transformation experiments via Agrobacterium tumefaciens. The transgenic buds were identified by the GUS histochemical analysis and confirmed by PCR analysis, which indicated the presence of an amplified fragment of 817 bp corresponding to the uidA gene sequence. The efficiencies of genetic transformation for 'Hamlin', 'Pra', and 'Valencia' sweet orange cultivars were 2.5, 1.4, and 3.7%, respectively. Media supplemented with auxins and cytokinins during co-culture, and media with high concentrations of cytokinins (3 mg L-1) during transgenic selection led to the transformation and, consequently, the regeneration of adequate number of adventitious buds for the three cultivars. The use of sonication during the explant disinfection was not effective to reduce endophytic contamination and reduced transformation efficiency.

Identical sequence patterns in the ends of exons and introns of human protein-coding genes

Intron splicing is one of the most important steps involved in the maturation process of a pre-mRNA. Although the sequence profiles around the splice sites have been studied extensively, the levels of sequence identity between the exonic sequences preceding the donor sites and the intronic sequences preceding the acceptor sites has not been examined as thoroughly. In this study we investigated identity patterns between the last 15 nucleotides of the exonic sequence preceding the 5' splice site and the intronic sequence preceding the 3' splice site in a set of human protein-coding genes that do not exhibit intron retention. We found that almost 60% of consecutive exons and introns in human protein-coding genes share at least two identical nucleotides at their 3' ends and, on average, the sequence identity length is 2.47 nucleotides. Based on our findings we conclude that the 3' ends of exons and introns tend to have longer identical sequences within a gene than when being taken from different genes. Our results hold even if the pairs are non-consecutive in the transcription order. (C) 2012 Elsevier Ltd. All rights reserved.

Interaction among nitric oxide (NO)-related genes in migraine susceptibility

The pathogenic mechanisms involved in migraine are complex and not completely clarified. Because there is evidence for the involvement of nitric oxide (NO) in migraine pathophysiology, candidate gene approaches focusing on genes affecting the endothelial function have been studied including the genes encoding endothelial NO synthase (eNOS), inducible NO synthase (iNOS), and vascular endothelial growth factor (VEGF). However, investigations on gene-gene interactions are warranted to better elucidate the genetic basis of migraine. This study aimed at characterizing interactions among nine clinically relevant polymorphisms in eNOS (T-786C/rs2070744, the 27 bp VNTR in intron 4, the Glu298Asp/rs1799983, and two additional tagSNPs rs3918226 and rs743506), iNOS (C(-1026)A/rs2779249 and G2087A/rs2297518), and VEGF (C(-2578)A/rs699947 and G(-634)C/rs2010963) in migraine patients and control group. Genotypes were determined by real-time polymerase chain reaction using the Taqman(A (R)) allele discrimination assays or PCR and fragment separation by electrophoresis in 99 healthy women without migraine (control group) and in 150 women with migraine divided into two groups: 107 with migraine without aura and 43 with aura. The multifactor dimensionality reduction method was used to detect and characterize gene-gene interactions. We found a significant interaction between eNOS rs743506 and iNOS 2087G/A polymorphisms in migraine patients compared to control group (P < 0.05), suggesting that this combination affect the susceptibility to migraine. Further studies are needed to determine the molecular mechanisms explaining this interaction.

Evolutionary history of exon shuffling

Exon shuffling has been characterized as one of the major evolutionary forces shaping both the genome and the proteome of eukaryotes. This mechanism was particularly important in the creation of multidomain proteins during animal evolution, bringing a number of functional genetic novelties. Here, genome information from a variety of eukaryotic species was used to address several issues related to the evolutionary history of exon shuffling. By comparing all protein sequences within each species, we were able to characterize exon shuffling signatures throughout metazoans. Intron phase (the position of the intron regarding the codon) and exon symmetry (the pattern of flanking introns for a given exon or block of adjacent exons) were features used to evaluate exon shuffling. We confirmed previous observations that exon shuffling mediated by phase 1 introns (1-1 exon shuffling) is the predominant kind in multicellular animals. Evidence is provided that such pattern was achieved since the early steps of animal evolution, supported by a detectable presence of 1-1 shuffling units in Trichoplax adhaerens and a considerable prevalence of them in Nematostella vectensis. In contrast, Monosiga brevicollis, one of the closest relatives of metazoans, and Arabidopsis thaliana, showed no evidence of 1-1 exon or domain shuffling above what it would be expected by chance. Instead, exon shuffling events are less abundant and predominantly mediated by phase 0 introns (0-0 exon shuffling) in those non-metazoan species. Moreover, an intermediate pattern of 1-1 and 0-0 exon shuffling was observed for the placozoan T. adhaerens, a primitive animal. Finally, characterization of flanking intron phases around domain borders allowed us to identify a common set of symmetric 1-1 domains that have been shuffled throughout the metazoan lineage.

A new portal for the analysis of human splicing variants

Understanding alternative splicing is crucial to elucidate the mechanisms behind several biological phenomena, including diseases. The huge amount of expressed sequences available nowadays represents an opportunity and a challenge to catalog and display alternative splicing events (ASEs). Although several groups have faced this challenge with relative success, we still lack a computational tool that uses a simple and straightforward method to retrieve, name and present ASEs. Here we present SPLOOCE, a portal for the analysis of human splicing variants. SPLOOCE uses a method based on regular expressions for retrieval of ASEs. We propose a simple syntax that is able to capture the complexity of ASEs.

Epistasis among eNOS, MMP-9 and VEGF maternal genotypes in hypertensive disorders of pregnancy

Polymorphisms of the endothelial nitric oxide synthase (eNOS), matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF) genes were shown to be associated with hypertensive disorders of pregnancy. However, epistasis is suggested to be an important component of the genetic susceptibility to preeclampsia (PE). The aim of this study was to characterize the interactions among these genes in PE and gestational hypertension (GH). Seven clinically relevant polymorphisms of eNOS (T-786C, rs2070744, a variable number of tandem repeats in intron 4 and Glu298Asp, rs1799983), MMP-9 (C-1562T, rs3918242 and -90(CA)(13-25), rs2234681) and VEGF (C-2578A, rs699947 and G-634C, rs2010963) were genotyped by TaqMan allelic discrimination assays or PCR and fragment separation by electrophoresis in 122 patients with PE, 107 patients with GH and a control group of 102 normotensive pregnant (NP) women. A robust multifactor dimensionality reduction analysis was used to characterize gene-gene interactions. Although no significant genotype combinations were observed for the comparison between the GH and NP groups (P>0.05), the combination of MMP-9-1562CC with VEGF-634GG was more frequent in NP women than in women with PE (P<0.05). Moreover, the combination of MMP-9-1562CC with VEGF-634CC or MMP-9-1562CT with VEGF-634CC or-634GG was more frequent in women with PE than in NP women (P<0.05). These results are obscured when single polymorphisms in these genes are considered and suggest that specific genotype combinations of MMP-9 and VEGF contribute to PE susceptibility. Hypertension Research (2012) 35, 917-921; doi:10.1038/hr.2012.60; published online 10 May 2012

Phylogeography of an Atlantic forest passerine reveals demographic stability through the last glacial maximum

In this study we analyzed the phylogeographic pattern and historical demography of an endemic Atlantic forest (AF) bird, Basileuterus leucoblepharus, and test the influence of the last glacial maximum (LGM) on its population effective size using coalescent simulations. We address two main questions: (i) Does B. leucoblepharus present population genetic structure congruent with the patterns observed for other AF organisms? (ii) How did the LGM affect the effective population size of B. leucoblepharus? We sequenced 914 bp of the mitochondrial gene cytochrome b and 512 bp of the nuclear intron 5 of beta-fibrinogen of 62 individuals from 15 localities along the AF. Both molecular markers revealed no genetic structure in B. leucoblepharus. Neutrality tests based on both loci showed significant demographic expansion. The extended Bayesian skyline plot showed that the species seems to have experienced demographic expansion starting around 300,000 years ago, during the late Pleistocene. This date does not coincide with the LGM and the dynamics of population size showed stability during the LGM. To further test the effect of the LGM on this species, we simulated seven demographic scenarios to explore whether populations suffered specific bottlenecks. The scenarios most congruent with our data were population stability during the LGM with bottlenecks older than this period. This is the first example of an AF organism that does not show phylogeographic breaks caused by vicariant events associated to climate change and geotectonic activities in the Quaternary. Differential ecological, environmental tolerances and habitat requirements are possibly influencing the different evolutionary histories of these organisms. Our results show that the history of organism diversification in this megadiverse Neotropical forest is complex. Crown Copyright (c) 2012 Published by Elsevier Inc. All rights reserved.

Novel organization of the mitochondrial genome in the deep-sea coral, Madrepora oculata (Hexacorallia, Scleractinia, Oculinidae) and its taxonomic implications

Madrepora is one of the most ecologically important genera of reef-building scleractinians in the deep sea, occurring from tropical to high-latitude regions. Despite this, the taxonomic affinities and relationships within the genus Madrepora remain unclear. To clarify these issues, we sequenced the mitochondrial (mt) genome of the most widespread Madrepora species, M. oculata, and compared this with data for other scleractinians. The architecture of the M. oculara mt genome was very similar to that of other scleractinians, except for a novel gene rearrangement affecting only cox2 and cox3. This pattern of gene organization was common to four geographically distinct M. oculata individuals as well as the congeneric species M. minutiseptum, but was not shared by other genera that are closely related on the basis of cox1 sequence analysis nor other oculinids, suggesting that it might be unique to Madrepora. (C) 2012 Elsevier Inc. All rights reserved.

A comparison between diuretics and angiotensin-receptor blocker agents in patients with stage I hypertension (PREVER-treatment trial): study protocol for a randomized double-blind controlled trial

Abstract Background Cardiovascular disease is the leading cause of death in Brazil, and hypertension is its major risk factor. The benefit of its drug treatment to prevent major cardiovascular events was consistently demonstrated. Angiotensin-receptor blockers (ARB) have been the preferential drugs in the management of hypertension worldwide, despite the absence of any consistent evidence of advantage over older agents, and the concern that they may be associated with lower renal protection and risk for cancer. Diuretics are as efficacious as other agents, are well tolerated, have longer duration of action and low cost, but have been scarcely compared with ARBs. A study comparing diuretic and ARB is therefore warranted. Methods/design This is a randomized, double-blind, clinical trial, comparing the association of chlorthalidone and amiloride with losartan as first drug option in patients aged 30 to 70 years, with stage I hypertension. The primary outcomes will be variation of blood pressure by time, adverse events and development or worsening of microalbuminuria and of left ventricular hypertrophy in the EKG. The secondary outcomes will be fatal or non-fatal cardiovascular events: myocardial infarction, stroke, heart failure, evidence of new subclinical atherosclerosis and sudden death. The study will last 18 months. The sample size will be of 1200 participants for group in order to confer enough power to test for all primary outcomes. The project was approved by the Ethics committee of each participating institution. Discussion The putative pleiotropic effects of ARB agents, particularly renal protection, have been disputed, and they have been scarcely compared with diuretics in large clinical trials, despite that they have been at least as efficacious as newer agents in managing hypertension. Even if the null hypothesis is not rejected, the information will be useful for health care policy to treat hypertension in Brazil. Clinical trials registration number ClinicalTrials.gov: NCT00971165

Identification of protein-coding and non-coding RNA expression profiles in CD34+ and in stromal cells in refractory anemia with ringed sideroblasts

Abstract Background Myelodysplastic syndromes (MDS) are a group of clonal hematological disorders characterized by ineffective hematopoiesis with morphological evidence of marrow cell dysplasia resulting in peripheral blood cytopenia. Microarray technology has permitted a refined high-throughput mapping of the transcriptional activity in the human genome. Non-coding RNAs (ncRNAs) transcribed from intronic regions of genes are involved in a number of processes related to post-transcriptional control of gene expression, and in the regulation of exon-skipping and intron retention. Characterization of ncRNAs in progenitor cells and stromal cells of MDS patients could be strategic for understanding gene expression regulation in this disease. Methods In this study, gene expression profiles of CD34+ cells of 4 patients with MDS of refractory anemia with ringed sideroblasts (RARS) subgroup and stromal cells of 3 patients with MDS-RARS were compared with healthy individuals using 44 k combined intron-exon oligoarrays, which included probes for exons of protein-coding genes, and for non-coding RNAs transcribed from intronic regions in either the sense or antisense strands. Real-time RT-PCR was performed to confirm the expression levels of selected transcripts. Results In CD34+ cells of MDS-RARS patients, 216 genes were significantly differentially expressed (q-value ≤ 0.01) in comparison to healthy individuals, of which 65 (30%) were non-coding transcripts. In stromal cells of MDS-RARS, 12 genes were significantly differentially expressed (q-value ≤ 0.05) in comparison to healthy individuals, of which 3 (25%) were non-coding transcripts. Conclusions These results demonstrated, for the first time, the differential ncRNA expression profile between MDS-RARS and healthy individuals, in CD34+ cells and stromal cells, suggesting that ncRNAs may play an important role during the development of myelodysplastic syndromes.

Genome mapping and expression analyses of human intronic noncoding RNAs reveal tissue-specific patterns and enrichment in genes related to regulation of transcription

Abstract Background RNAs transcribed from intronic regions of genes are involved in a number of processes related to post-transcriptional control of gene expression. However, the complement of human genes in which introns are transcribed, and the number of intronic transcriptional units and their tissue expression patterns are not known. Results A survey of mRNA and EST public databases revealed more than 55,000 totally intronic noncoding (TIN) RNAs transcribed from the introns of 74% of all unique RefSeq genes. Guided by this information, we designed an oligoarray platform containing sense and antisense probes for each of 7,135 randomly selected TIN transcripts plus the corresponding protein-coding genes. We identified exonic and intronic tissue-specific expression signatures for human liver, prostate and kidney. The most highly expressed antisense TIN RNAs were transcribed from introns of protein-coding genes significantly enriched (p = 0.002 to 0.022) in the 'Regulation of transcription' Gene Ontology category. RNA polymerase II inhibition resulted in increased expression of a fraction of intronic RNAs in cell cultures, suggesting that other RNA polymerases may be involved in their biosynthesis. Members of a subset of intronic and protein-coding signatures transcribed from the same genomic loci have correlated expression patterns, suggesting that intronic RNAs regulate the abundance or the pattern of exon usage in protein-coding messages. Conclusion We have identified diverse intronic RNA expression patterns, pointing to distinct regulatory roles. This gene-oriented approach, using a combined intron-exon oligoarray, should permit further comparative analysis of intronic transcription under various physiological and pathological conditions, thus advancing current knowledge about the biological functions of these noncoding RNAs.

SLCO1B1 rs4149056 polymorphism associated with statin-induced myopathy is differently distributed according to ethnicity in the Brazilian general population: Amerindians as a high risk ethnic group

Background Recent studies reported the association between SLCO1B1 polymorphisms and the development of statin-induced myopathy. In the scenario of the Brazilian population, being one of the most heterogeneous in the world, the main aim here was to evaluate SLCO1B1 polymorphisms according to ethnic groups as an initial step for future pharmacogenetic studies. Methods One hundred and eighty-two Amerindians plus 1,032 subjects from the general urban population were included. Genotypes for the SLCO1B1 rs4149056 (c.T521C, p.V174A, exon 5) and SLCO1B1 rs4363657 (g.T89595C, intron 11) polymorphisms were detected by polymerase chain reaction followed by high resolution melting analysis with the Rotor Gene 6000® instrument. Results The frequencies of the SLCO1B1 rs4149056 and rs4363657 C variant allele were higher in Amerindians (28.3% and 26.1%) and were lower in African descent subjects (5.7% and 10.8%) compared with Mulatto (14.9% and 18.2%) and Caucasian descent (14.8% and 15.4%) ethnic groups (p < 0.001 and p < 0.001, respectively). Linkage disequilibrium analysis show that these variant alleles are in different linkage disequilibrium patterns depending on the ethnic origin. Conclusion Our findings indicate interethnic differences for the SLCO1B1 rs4149056 C risk allele frequency among Brazilians. These data will be useful in the development of effective programs for stratifying individuals regarding adherence, efficacy and choice of statin-type.

Herbicidas no alto rio Poxim, Sergipe e os riscos de contaminação dos recursos hídricos

A crescente atividade agrícola em áreas de mananciais tem causado preocupações quanto à contaminação por herbicidas em áreas agrícolas. O problema se torna mais importante quando a contaminação pode afetar a água para uso humano, como ocorre com a água do Rio Poxim, que abastece a cidade de Aracaju, capital do Estado de Sergipe. O objetivo do estudo foi avaliar o risco de contaminação de águas superficiais e subterrâneas por herbicidas no alto da Sub-bacia do Rio Poxim e detectar a presença dos princípios ativos Diuron e Ametrina, à montante das plantações de cana-de-açúcar. A análise de risco foi realizada mediante critérios da Environmental Protection Agency (EPA), índice de GUS e método de GOSS. Observou-se que vários princípios ativos sofrem risco de lixiviação, demonstrando a importância do monitoramento do rio para controle tanto da qualidade da água como da frequência e volume de herbicidas aplicado na região. A partir do resultado, foi realizado um monitoramento bimensal de julho de 2009 a julho de 2010, em dois pontos de amostragem. As amostras de água foram analisadas em laboratório, onde se, constatou a presença de Diuron e Ametrina. A qualidade da água na Sub-bacia do Rio Poxim está sendo influenciada pelo uso de herbicidas na região. Ocorreu um aumento nas concentrações dos herbicidas na água superficial, durante o período chuvoso, provocado possivelmente pelo escoamento superficial.

Exome analysis of HIV patients submitted to dendritic cells therapeutic vaccine reveals an association of CNOT1 gene with response to the treatment

INTRODUCTION: With the aim of searching genetic factors associated with the response to an immune treatment based on autologous monocyte-derived dendritic cells pulsed with autologous inactivated HIV, we performed exome analysis by screening more than 240,000 putative functional exonic variants in 18 HIV-positive Brazilian patients that underwent the immune treatment. METHODS: Exome analysis has been performed using the ILLUMINA Infinium HumanExome BeadChip. zCall algorithm allowed us to recall rare variants. Quality control and SNP-centred analysis were done with GenABEL R package. An in-house implementation of the Wang method permitted gene-centred analysis. RESULTS: CCR4-NOT transcription complex, subunit 1 (CNOT1) gene (16q21), showed the strongest association with the modification of the response to the therapeutic vaccine (p=0.00075). CNOT1 SNP rs7188697 A/G was significantly associated with DC treatment response. The presence of a G allele indicated poor response to the therapeutic vaccine (p=0.0031; OR=33.00; CI=1.74-624.66), and the SNP behaved in a dominant model (A/A vs. A/G+G/G p=0.0009; OR=107.66; 95% CI=3.85-3013.31), being the A/G genotype present only in weak/transient responders, conferring susceptibility to poor response to the immune treatment. DISCUSSION: CNOT1 is known to be involved in the control of mRNA deadenylation and mRNA decay. Moreover, CNOT1 has been recently described as being involved in the regulation of inflammatory processes mediated by tristetraprolin (TTP). The TTP-CCR4-NOT complex (CNOT1 in the CCR4-NOT complex is the binding site for TTP) has been reported as interfering with HIV replication, through post-transcriptional control. Therefore, we can hypothesize that genetic variation occurring in the CNOT1 gene could impair the TTP-CCR4-NOT complex, thus interfering with HIV replication and/or host immune response. CONCLUSIONS: Being aware that our findings are exclusive to the 18 patients studied with a need for replication, and that the genetic variant of CNOT1 gene, localized at intron 3, has no known functional effect, we propose a novel potential candidate locus for the modulation of the response to the immune treatment, and open a discussion on the necessity to consider the host genome as another potential variant to be evaluated when designing an immune therapy study