Abstract Next-generation sequencing (NGS) platforms allow the analysis of hundreds of millions of molecules in a single sequencing run, revolutionizing many research areas. NGS-based microRNA studies enable expression quantification in unprecedented scale without the limitations of closed-platforms. Yet, whereas a massive amount of data produced by these platforms is available, comparisons of quantification/discovery capabilities between platforms are still lacking. Here we compare two NGS-platforms: SOLiD and PGM, by evaluating their microRNA identification/quantification capabilities using two breast-derived cell-lines. A high expression correlation (R2 > 0.9) was achieved, encompassing 97% of the miRNAs, and the few discrepancies in miRNA counts were attributable to molecules that have very low expression. Quantification divergences indicative of artefactual representation were seen for 14 miRNAs (higher in SOLiD-reads) and another 10 miRNAs more abundant in PGM-data. An inspection of these revealed an increased and statistically significant count of uracyls and uracyl-stretches for PGM-enriched miRNAs, compared to SOLiD and to the miRBase. In parallel, adenines and adenine-stretches were enriched for SOLiDderived miRNA reads. We conclude that, whereas both platforms are overall consistent and can be used interchangeably for microRNA expression studies, particular sequence features appear to be indicative of specific platform bias, and their presence in microRNAs should be considered for database-analyses.

ABSTRACT Approximately 240 million people worldwide are infected by Schistosoma. In Brazil, one of the main intermediate hosts of this parasite is Biomphalaria glabrata snails. The early detection of larval stages in intermediate hosts is an important challenge to public health, but it also represents an opportunity as a new alternative to indicate earlier natural infections before cercariae differentiation and emergence. In this context, we demonstrated that PCR amplification of a 28S gene fragment from the parasite does demonstrate S. mansoni infection in snails 14 days post infection. This conventional polymerase chain reaction amplified clear bands and was able to detect parasitic infection in the intermediate host B. glabrata under experimental conditions. However, we reinforce that this approach requires deeper investigations and further comparisons to confirm its specificity and sensitivity in earlier time points after miracidia infection. This approach has relevant potential as an effective molecular-based strategy for the monitoring of schistosomiasis transmission.

CONTEXTO: A performance musical requer alto nível de habilidade em diversos parâmetros, como coordenação motora, atenção e memória, o que a torna particularmente suscetível aos estados de ansiedade. Pesquisas nessa área têm avançado com a introdução de instrumentos específicos para abordar a ansiedade na performance musical, como é o caso da Kenny Music Performance Anxiety Inventory (K-MPAI). OBJETIVOS: O presente estudo teve como objetivo traduzir, adaptar e validar a K-MPAI para a língua portuguesa. MÉTODOS: Após autorização da autora, a escala K-MPAI foi traduzida e validada. A escala em língua portuguesa foi aplicada a 218 músicos de ambos os sexos, amadores e profissionais. Para a validação concorrente, foi utilizado o Inventário de Ansiedade Traço-Estado (IDATE), versão validada na língua portuguesa da State Trait Anxiety Inventory (STAI). RESULTADOS: A análise da consistência interna apresentou alfa de Cronbach = 0,957 com p < 0,001, reprodutibilidade com p = 0,378 e validação concorrente com a IDATE com alfa de Cronbach = 0,642 e p < 0,001. CONCLUSÃO: O estudo permite considerar a amostra com graus de confiabilidade e reprodutibilidade elevados, o que traduz este estudo como provindo de uma amostra não tendenciada e replicável a outras populações. A validação concorrente entre a K-MPAI e a IDATE permite inferir que ambas as escalas são comparáveis na capacidade de medir os níveis de ansiedade em musicistas.

BACKGROUND: Musical performance demands high-leveled coordination, concentration, motor- and memory-skills, making it particularly susceptible to anxiety states. Researches in this field have advanced significantly with the development of specific instruments to evaluate music performance anxiety, such as the Kenny Music Performance Anxiety Inventory (K-MPAI). OBJECTIVES: The present study has the objective of translating, adapting and validating the K-MPAI to the Portuguese language. METHODS: After the written consent given by the author of the original K-MPAI scale, the K-MPAI scale was translated and validated for Portuguese idiom. The Portuguese-version of K-MPAI was then applied to 218 amateur and professional musicians of both genders. For the concurrent validation, the validated Portuguese-version of the State Trait Anxiety Inventory (STAI) was used. RESULTS: Analysis of the internal consistency demonstrated a Cronbach's alpha = 0.957, with p < 0.001, replicated with p = 0.378 and the concurrent validation with the State Trait Anxiety Inventory, demonstrated a Cronbach's alpha = 0.642 and p < 0.001. DISCUSSION: The study allows evaluating data samples with high levels of reliability and replicability, which translates this study based on an unbiased sample and replicable to other populations. The concurrent validation between K-MPAI and IDATE, allows inferring that the scales are comparable in their capability of measuring anxiety levels in musicians.

O seqüenciamento de nosso genoma representa um passo essencial no entendimento da biologia humana e no planejamento racional de pesquisas biomédicas. Contudo, é importante notar que o seqüenciamento de um dado genoma é apenas uma parte de um complexo quebra-cabeças. A informação genética deve ser usada como um "mapa", a partir do qual começamos a compreender a base das doenças e a importância da variação genética através da análise da complexidade e do comportamento das regiões reguladoras, genes e proteínas, funções gênicas e sistemas celulares. Apesar dos enormes esforços para identificar genes de susceptibilidade, os resultados de estudos de genética molecular de esquizofrenia até o momento têm sido modestos. O uso apropriado da genômica poderá ajudar imensamente na elucidação das causas da esquizofrenia, permitindo avaliar o papel de novos genes, das variações genéticas, das formas de splicing alternativo, das variações de expressão gênica e de vias metabólicas de interesse. A convergência de dados bioquímicos, de imagem, de neuroanatomia, farmacológicos, clínicos e genéticos permite prever que estamos muito próximos de uma melhor compreensão das bases biológicas da esquizofrenia. A disponibilidade desses avanços terá um enorme impacto na pesquisa desta doença.

The sequencing of our genome represents an essential step in the comprehension of the human biology and in the rational planning of biomedical research. However, it is important to realize that the sequencing of a genome is only a piece of a complex puzzle. The genetic information must be used as a "map", the starting-point to understand the basis of the diseases and the importance of the genetic variation through the analysis of the complexity and behavior of the regulatory regions, genes and proteins, gene functions and cellular systems. Despite the enormous efforts made towards the identification of susceptibility genes for schizophrenia, the results of molecular genetics studies have been modest. The appropriate use of genomics should add more information to help the elucidation of the causes of the schizophrenia, supporting areas such as gene discovery, determination of genetic variations, alternative splicing, analysis of diferential gene expression and in the study of metabolic pathways of interest. The convergence of data from studies of biochemistry, imaging, neuroanatomy, pharmacology, clinical and genetics allows to foresee that we should be near to a better understanding of the biological basis of schizophrenia. The availability of these advances will have a dramatic impact on the research of this disease.

Com o aumento da expectativa de vida, visto hoje em todo o planeta, um maior número de indivíduos alcança uma idade avançada em que a manifestação de doenças neurodegenerativas é mais freqüente. Entre essas, a doença de Alzheimer (DA) é a causa mais freqüente de demência. Os achados mais marcantes na DA, em cérebros de pacientes acometidos pela doença, são as placas senis, os emaranhados neurofibrilares e a extensa perda neuronal. No entanto, existe uma carência generalizada de marcadores biológicos preditivos ou com valor diagnóstico para a DA. Estudos de genética molecular permitiram identificar quatro genes consistentemente associados com o maior risco de desenvolvimento da doença: APP, apoE, PSEN1 e PSEN2. No entanto, inúmeros estudos apontam para papel importante de outros genes, fortalecendo a hipótese de uma doença poligênica e multifatorial. Neste sentido, novas abordagens de estudo têm um futuro promissor, podendo indicar uma vasta população de genes ou alterações moleculares que possam explicar o surgimento da doença, vindo a fornecer as bases para a compreensão da DA e também para o delineamento de novas e mais eficazes abordagens de tratamento ou prevenção da doença.

With the increase of the life expectancy that is seen today in the entire planet, a larger number of individuals reaches an advanced age when the manifestation of neurodegenerative illnesses is more frequent. Among these, the Alzheimer's disease (AD) is the most common cause of dementia. The most substantial findings in the brains of patients suffering from AD are the senile plaques, the neurofibrillary tangles and an extensive neuronal loss. However, a general deficiency of predictive biological markers for AD impairs the correct diagnosis and the better understanding of the disease. Studies of molecular genetics allowed the identification of four genes that are consistently linked to AD: APP, apoE, PSEN1 and PSEN2. However, diverse studies demonstrate that other genes also play important roles in the development of AD, strengthening the notion that it is a polygenic illness. Modern large-scale techniques can now be applied in the study of AD, permitting the study of a vast population of genes or molecular alterations that can explain the origin of the illness, offering the basis for the understanding of the pathogenesis of AD and enabling the delineation of new and more efficient approaches to treat and to prevent the sprouting of the disease.

Sabemos hoje que os polimorfismos no gene da apolipoproteína E (apoE) são importantes fatores de risco para o desenvolvimento da doença de Alzheimer (DA). O gene apoE humano, mapeado no braço longo do cromossomo 19 (19q13.2), codifica uma glicoproteína com 317 aminoácidos, a qual desempenha um papel fundamental para o catabolismo de componentes ricos em triglicérides no corpo humano. Em humanos, existem três alelos principais do gene apoE, decorrentes de apenas duas alterações no DNA, chamados de épsilon2, épsilon3 e épsilon4. A identificação da variante épsilon4 do gene apoE como o fator genético de risco mais comum para a DA de início tardio sugere que o colesterol deva ter um papel direto na patogênese da doença. Contudo, a simples presença do alelo apoE épsilon4 não é necessária nem suficiente para causar DA; este alelo apenas aumenta o risco de o indivíduo vir a desenvolver a doença, indicando que existem outros fatores ambientais e genéticos importantes no desenvolvimento da mesma.

It is known that polymorphisms in the gene of the apolipoprotein E (apoE) are important risk factors in the development of the Alzheimer's disease (AD). The human gene apoE, which is mapped in the long arm of chromosome 19 (19q13.2), codes for a glycoprotein with 317 amino acids, which plays a basic role for the catabolism of triglyceride-rich components in the human body. In our species the apoE gene appears in the form of three main alleles, produced from two alterations in the DNA sequence, called epsilon2, epsilon3 and epsilon4. The identification of the variant epsilon4 of gene apoE as the most relevant genetic marker for the risk for late-onset AD suggests that cholesterol may have a direct involvement in the pathogenesis of this disease. However, apoE epsilon4 is not necessary nor enough to cause Alzheimer's disease. It only increases the risk of an individual to develop the illness, indicating that other environmental/genetic factors shoud play important roles in the development of the disease.

As Schistosoma sp. control programs are chiefly based on treatment of infected population, adequate case finding has a crucial role. The available diagnostic methods are far from ideal, since the search for eggs in stools and the detection of circulating antigens lack sensitivity in low prevalence and post-treatment situations and antibody detection lacks specificity. In most endemic foci, repeated treatment of infected people leaves a number of non-diagnosed and consequently non-treated persons, enough to maintain a persistent residue of 5 to 10% prevalence. In an attempt to surpass these diagnostic limitations we have developed a polymerase chain reaction (PCR) for the detection of Schistosoma sp. in feces that, in a first population study, has shown to be more sensitive than three-repeated stool Kato-Katz examination. The PCR may constitute a valuable tool for the diagnosis of the Schistosoma sp. infection in special situations, when high sensitivity and specificity are required and infrastructure is available.

Studies based on shell or reproductive organ morphology and genetic considerations suggest extensive intraspecific variation in Biomphalaria snails. The high variability at the morphological and genetic levels, as well as the small size of some specimens and similarities between species complicate the correct identification of these snails. Here we review our work using methods based on polymerase chain reaction (PCR) amplification for analysis of genetic variation and identification of Biomphalaria snails from Brazil, Argentina, Uruguay and Paraguay. Arbitrarily primed-PCR revealed that the genome of B. glabrata exihibits a remarkable degree of intraespecific polymorphism. Low stringency-PCR using primers for 18S rRNA permited the identification of B. glabrata, B. tenagophila and B. occidentalis. The study of individuals obtained from geographically distinct populations exhibits significant intraspecific DNA polymorphism, however specimens from the same species, exhibit some species specific LSPs. We also showed that PCR-restriction fragment of length polymorphism of the internal transcribed spacer region of Biomphalaria rDNA, using DdeI permits the differentiation of the three intermediate hosts of Schistosoma mansoni. The molecular biological techniques used in our studies are very useful for the generation of new knowledge concerning the systematics and population genetics of Biomphalaria snails.

The low stringency-polymerase chain reaction (LS-PCR) with a pair of specific primers for the amplification of the 18S rRNA gene was evaluated as a means of differentiating between the two Schistosoma mansoni intermediate host species in Brazil: Biomphalaria glabrata and B. tenagophila. Individual snails obtained from different states of Brazil were used and the amplification patterns obtained showed a high degree of genetic variability in these species. Nevertheless, 4 and 3 clearly defined specific diagnostic bands was observed in individuals from B. glabrata and B. tenagophila respectively. The detection of snail specific diagnostic bands suggests the possibility of reliable species differentiation at the DNA level using LS-PCR.

Analysis of the genomes of schistosomes and one of their intermediate hosts, Biomphalaria glabrata, using Random Amplified Polymorphic DNA (RAPD) demonstrated that intraspecific genetic polymorphism in the parasite is limited but in the snail is highly pronounced. This suggests an important role for the snail in the determination of the epidemiology of the disease. In addition to their intraspecific stability, schistosome derived RAPDs exhibit a high level of interspecific polymorphism and are thus ideal for the construction of phylogenetic trees. For the detection of intraspecific polymorphisms extensive variation in the mitochondrial DNA is being exploited for the development of a PCR based test for Schistosoma mansoni. Gene level polymorphisms are being analyzed by Low Stringency Single Specific Primer PCR.

A new serological assay dot-dye-immunoassay (dot-DIA) was evaluated for the diagnosis of schistosomiasis mansoni. This method consist of four steps: (a) biding of antigens to a nitrocellulose membrane (NC); (b) blocking of free sites of the NC; (c) incubation in specific primary antibody; (d) detection of primary antibody reactivity by color development using second antibody coupled to textile dyes. Sera from 82 individuals, 61 with Schistosoma mansoni eggs in the stool and 21 stool negative were tested by ELISA, dot-ELISA, and dotDIA. A high level of agreement between the methods tested was observed for all sera tested: ELISA x dot-ELISA: 95.1%, ELISA x dot-DIA: 92.7% and dot-ELISA x dot-DIA: 97.6%. In this study, dot-DIA proved to be a feasible, sensitive, rapid and practical test for the diagnosis of shcistosomiasis.