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Studying the molecular mechanisms of the pathogenesis of Parkinson's disease (PD) is critical to improve PD treatment. We used OpenArray technology to assess gene expression in the substantia nigra (SN) cells of mice in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of PD and in controls. Among the 11 housekeeping genes tested, Rps27a was taken as the reference gene due to its most stable expression in normal and experimental conditions. From 101 genes encoding functionally significant proteins of nigrostriatal dopaminergic neurons, 57 highly expressed genes were selected to assess their expressions in the PD model and in the controls. The expressions of Th, Ddc, Maoa, Comt, Slc6a3, Slc18a2, Drd2, and Nr4a2 decreased in the experiment compared to the control, indicating decreases in the synthesis, degradation, and transport of dopamine and the impaired autoregulation of dopaminergic neurons. The expressions of Tubb3, Map2, Syn1, Syt1, Rab7, Sod1, Cib1, Gpx1, Psmd4, Ubb, Usp47, and Ctsb genes were also decreased in the MPTP-treated mice, indicating impairments of axonal and vesicular transport and abnormal functioning of the antioxidant and ubiquitin-proteasome systems in the SN. The detected decreases in the expressions of Snca, Nsf, Dnm1l, and Keap1 may serve to reduce pathological protein aggregation, increase dopamine release in the striatum, prevent mitophagy, and restore the redox status of SN cells.
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Doença de Parkinson , Animais , Camundongos , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Dopamina/metabolismo , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Modelos Animais de Doenças , Fator 2 Relacionado a NF-E2/metabolismo , Substância Negra/metabolismo , Substância Negra/patologia , Proteínas de Ligação a RNA/metabolismoRESUMO
Sepsis is a dysregulated immune response to infections that frequently precipitates multiple organ dysfunction and death despite intensive supportive therapy. The aim of the present study was to identify sepsis-induced alterations in the signaling transcriptome of peripheral blood leukocytes that might shed light on the elusive transition from proinflammatory to anti-inflammatory responses and underlie long-term post-sepsis immunosuppression. Peripheral blood leukocytes were collected from subjects (i) with systemic inflammation, (ii) with sepsis in the acute phase and (iii) 6 months after recovery from sepsis, corresponding to progressive stages of the disease. Transcriptomic analysis was performed with the QuantStudio 12K Flex OpenArray Human Signal Transduction Panel analyzing transcripts of 573 genes playing a significant role in signaling. Of them, 145 genes exhibited differential expression in sepsis as compared to systemic inflammation. Pathway analysis revealed enhanced expression levels of genes involved in primary immune responses (proinflammatory cytokines, neutrophil and macrophage activation markers) and signatures characteristic of immunosuppression (increased expression of anti-inflammatory cytokines and proapoptotic genes; diminished expression of T and B cell receptor dependent activating and survival pathways). Importantly, sepsis-induced expression patterns of 39 genes were not normalized by the end of the 6-month follow-up period, indicating expression aberrations persisting long after clinical recovery. Functional analysis of these transcripts revealed downregulation of the antiapoptotic Wnt and mTOR signaling pathways that might explain the post-sepsis immunosuppression commonly seen in sepsis survivors.
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Sepse , Transcriptoma , Humanos , Projetos Piloto , Leucócitos , Inflamação , Citocinas/metabolismo , Transdução de Sinais , Sepse/genética , Anti-InflamatóriosRESUMO
Single-nucleotide polymorphism (SNP) analysis is a powerful tool for population genetics, pedigree reconstruction and phenotypic trait mapping. However, the untapped potential of SNP markers to discriminate the sex of individuals in species with reduced sexual dimorphism or of individuals during immature stages remains a largely unexplored avenue. Here, we developed a novel protocol for molecular sexing of birds based on the detection of unique Z- and W-linked SNP markers. Our method is based on the identification of two unique loci, one in each sexual chromosome. Individuals are considered males when they show no calls for the W-linked SNP and are heterozygous or homozygous for the Z-linked SNP, while females exhibit both Z- and W-linked SNP calls. We validated the method in the Jackdaw (Corvus monedula). The reduced sexual dimorphism in this species makes it difficult to identify the sex of individuals in the wild. We assessed the reliability of the method using 36 individuals of known sex and found that their sex was correctly assigned in 100% of cases. The sex-linked markers also proved to be widely applicable for discriminating males and females from a sample of 927 genotyped individuals at different maturity stages, with an accuracy of 99.5%. Since SNP markers are increasingly used in quantitative genetic analyses of wild populations, the approach we propose has great potential to be integrated into broader genetic research programmes without the need for additional sexing techniques.
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Aves , Caracteres Sexuais , Humanos , Masculino , Feminino , Animais , Reprodutibilidade dos Testes , Genótipo , Aves/genética , Heterozigoto , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Lymphocystis disease is one of the main viral pathologies affecting cultured gilthead seabream (Sparus aurata) in the Mediterranean region. Recently, we have developed a DNA vaccine based on the major capsid protein (MCP) of the Lymphocystis disease virus 3 (LCDV-Sa). The immune response triggered by either LCDV-Sa infection or vaccination have been previously studied and seem to be highly related to the modulation of the inflammatory and the IFN response. However, a comprehensive evaluation of immune-related gene expression in vaccinated fish after viral infection to identify immunogenes involved in vaccine-induced protection have not been carried out to date. The present study aimed to fulfill this objective by analyzing samples of head-kidney, spleen, intestine, and caudal fin from fish using an OpenArray® platform containing targets related to the immune response of gilthead seabream. The results obtained showed an increase of deregulated genes in the hematopoietic organs between vaccinated and non-vaccinated fish. However, in the intestine and fin, the results showed the opposite trend. The global effect of fish vaccination was a significant decrease (p<0.05) of viral replication in groups of fish previously vaccinated, and the expression of the following immune genes related to viral recognition (tlr9), humoral and cellular response (rag1 and cd48), inflammation (csf1r, elam, il1ß, and il6), antiviral response (isg15, mx1, mx2, mx3), cell-mediated cytotoxicity (nccrp1), and apoptosis (prf1). The exclusive modulation of the immune response provoked by the vaccination seems to control the progression of the infection in the experimentally challenged gilthead seabream.
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Infecções por Vírus de DNA , Iridoviridae , Dourada , Animais , Iridoviridae/fisiologia , DNA , ImunidadeRESUMO
Parkinson's disease (PD) is a neurodegenerative disorder caused by the selective destruction of dopaminergic neurons (DA-nergic). Clinically, PD is diagnosed based on developing signs and symptoms. A neurological and physical examination and sometimes medical and family history also help in the diagnosis of PD. However, most of these features are visible when more than 80% of the dopaminergic neurons have degenerated. An understanding of the selective degeneration process at the cellular and molecular level and the development of new biomarkers are required for effective PD management. Several studies have been carried out using a selected set of miRNAs/ mRNAs and proteins to develop biomarkers of PD; however, an unbiased and combined miRNA-protein profiling study was required to identify the markers of progressive and selected degeneration of dopaminergic neurons in PD patients. In the present study, we have carried out global protein profiling through LC-MS/MS and miRNA profiling by using a "brain-specific" miRNA array panel of 112 miRNAs in PD patients and healthy controls to find the unprejudiced group of proteins and miRNAs that are deregulating in PD. In the whole blood samples of PD patients compared to healthy controls, the expression of 23 miRNAs and 289 proteins was significantly increased, whereas the expression of 4 miRNAs and 132 proteins was considerably downregulated. Network analysis, functional enrichment, annotation, and analysis of miRNA-protein interactions were also performed as part of the bioinformatics investigation of the discovered miRNAs and proteins revealing several pathways that lead to PD development and pathogenesis. Based on the analysis of miRNA and protein profiling, we have identified four miRNAs (hsa-miR-186-5p, miR-29b, miR-139 & has-miR-150-5p) and four proteins (YWHAZ, PSMA4, HYOU1, & SERPINA1), which can be targeted for the development of new biomarkers of PD. In vitro studies have identified the role of miR-186-5p in regulating the levels of the YWHAZ/YWHAB & CALM2 gene, which has shown maximum downregulation in PD patients and is known for its role in neuroprotection from apoptotic cell death & calcium regulation. In conclusion, our research has identified a group of miRNA-proteins that can be developed as PD biomarkers; however, future studies on the release of these miRNAs and proteins in extracellular vesicles circulating in the blood of PD patients can further validate these as specific biomarkers of PD.
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MicroRNAs , Doença de Parkinson , Humanos , Doença de Parkinson/diagnóstico , Doença de Parkinson/genética , Transcriptoma , Proteômica , Cromatografia Líquida , Espectrometria de Massas em Tandem , MicroRNAs/metabolismo , Perfilação da Expressão Gênica , Biomarcadores , Proteínas Sanguíneas/genéticaRESUMO
Parkinson's disease (PD) is the age-related neurological disorder characterized by the degeneration of dopamine (DA) neurons in the substantia nigra pars compacta (SNpc). PD is based on motor deficits which start to appear when up to 80% of the DA neurons of SNpc have been lost. Effective management of PD requires the development of novel biomarkers. Therefore, the present study aimed to characterize biomarkers of PD using miRNomics, proteomics, and bioinformatics approaches. Rats exposed to rotenone (2.5 mg/kg b.wt) for 2 months were used as an animal model to identify the unbiased set of miRNAs and proteins deregulated in blood samples. OpenArray, a real-time PCR-based array, is used for high-throughput profiling of miRNAs, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to carry out the global protein profiling. Systematic bioinformatics analysis of miRNAs and proteins was also performed, including annotation, functional classification and functional enrichment, network analysis, and miRNA-protein interaction analysis. Expression of 19 miRNAs and 96 proteins was significantly upregulated in the blood, while 22 proteins were significantly downregulated in blood samples of rotenone-exposed rats. In silico pathway analysis of deregulated proteins and miRNAs in rotenone-exposed rats has identified multiple pathways leading to PD. In summary, we have identified a set of miRNAs (miR-144, miR-96, and miR-29a) and proteins (PLP1, TUBB4A, and TUBA1C), which can be used as a potential biomarker of PD, while further validation required large human population studies.
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MicroRNAs , Doença de Parkinson , Animais , Proteínas Sanguíneas , Cromatografia Líquida , MicroRNAs/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Ratos , Espectrometria de Massas em TandemRESUMO
Nervous necrosis virus (NNV), genus Betanodavirus, the etiological agent of the viral encephalopathy and retinopathy (VER), presents a genome with two positive-sense single-stranded RNA segments. Striped jack nervous necrosis virus (SJNNV) and red-spotted grouper nervous necrosis virus (RGNNV), together with reassortants RGNNV/SJNNV, are the betanodaviruses predominantly isolated in Southern Europe. An RGNNV/SJNNV reassortant isolated from Senegalese sole (wt160) causes high mortalities in this fish species. This virus presents differences in the sequence of the 3' non-coding region (NCR) of both segments compared to RGNNV and SJNNV reference strains. Previously, it has been reported that the reversion of two of these differences (nucleotides 1408 and 1412) in the RNA2 3'NCR to the SJNNV-type (recombinant r1408-1412) resulted in a decrease in sole mortality. In the present study, we have applied an OpenArray® to analyse the involvement of sole immune response in the virulence of several recombinants: the r1408-1412 and two recombinants, developed in the present study, harbouring mutations at positions 3073 and 3093 of RNA1 3'NCR to revert them to RGNNV-type. According to the correlation values and to the number of expressed genes, the infection with the RNA2-mutant provoked the most different immune response compared to the immune response triggered after the infection with the rest of the viruses, and the exclusive and high upregulation of genes related to the complement system. The infection with the RNA1-mutants also provoked a decrease in mortality and their replication was delayed at least 24 h compared to the wt160 replication, which could provoke the lag observed in the immune response. Furthermore, the infection with the RNA1-mutants provoked the exclusive expression of pkr and the downregulation of il17rc.
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The management of neuropsychiatric disorders involves different pharmacological treatments. In order to perform efficacious drug treatments, the metabolism of CYP genes can help to foresee potential drug-drug interactions. The NeuroPGx software is an open-source web-based tool for genotype/diplotype/phenotype interpretation for neuropharmacogenomic purposes. The software provides information about: (i) the genotypes of evaluated SNPs (single nucleotide polymorphisms); (ii) the main diplotypes in CYP genes and corresponding metabolization phenotypes; (iii) the list of neuropsychiatric drugs with recommended dosage adjustment (according to CPIC and DPWG guidelines); (iv) the list of possible (rare) diplotypes and corresponding metabolization phenotypes. The combined application of NeuroPGx software to the OpenArray technology results in an easy, quick, and highly automated device ready to be used in routine clinical practice.
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BACKGROUND: The study of circulating miRNAs in CSF has gained tremendous attention during the last years, as these molecules might be promising candidates to be used as biomarkers and provide new insights into the disease pathology of neurological disorders. OBJECTIVE: The main aim of this study was to describe an OpenArray panel of CSF-enriched miRNAs to offer a suitable tool to identify and characterize new molecular signatures in different neurological diseases. METHODS: Two hundred and fifteen human miRNAs were selected to be included in the panel, and their expression and abundance in CSF samples were analyzed. In addition, their stability was studied in order to propose suitable endogenous controls for CSF miRNA studies. RESULTS: miR-143-3p and miR-23a-3p were detected in all CSF samples, while another 80 miRNAs were detected in at least 70% of samples. miR-770-5p was the most abundant miRNA in CSF, presenting the lowest mean Cq value. In addition, miR-26b-5p, miR-335-5p and miR-92b-3p were the most stable miRNAs and could be suitable endogenous normalizers for CSF miRNA studies. CONCLUSIONS: These OpenArray plates might be a suitable and efficient tool to identify and characterize new molecular signatures in different neurological diseases and would improve the yield of miRNA detection in CSF.
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BACKGROUND: Pharmacogenomics has the potential to improve patient outcomes through predicting drug response. We designed and evaluated the analytical performance of a custom OpenArray® pharmacogenomics panel targeting 478 single-nucleotide variants (SNVs). METHODS: Forty Coriell Institute cell line (CCL) DNA samples and DNA isolated from 28 whole-blood samples were used for accuracy evaluation. Genotyping calls were compared to at least 1 reference method: next-generation sequencing, Sequenom MassARRAY®, or Sanger sequencing. For precision evaluation, 23 CCL samples were analyzed 3 times and reproducibility of the assays was assessed. For sensitivity evaluation, 6 CCL samples and 5 whole-blood DNA samples were analyzed at DNA concentrations of 10 ng/µL and 50 ng/µL, and their reproducibility and genotyping call rates were compared. RESULTS: For 443 variants, all samples assayed had concordant calls with at least 1 reference genotype and also demonstrated reproducibility. However, 6 of these 443 variants showed an unsatisfactory performance, such as low PCR amplification or insufficient separation of genotypes in scatter plots. Call rates were comparable between 50 ng/µL DNA (99.6%) and 10 ng/µL (99.2%). Use of 10 ng/µL DNA resulted in an incorrect call for a single sample for a single variant. Thus, as recommended by the manufacturer, 50 ng/µL is the preferred concentration for patient genotyping. CONCLUSIONS: We evaluated a custom-designed pharmacogenomics panel and found that it reliably interrogated 437 variants. Clinically actionable results from selected variants on this panel are currently used in clinical studies employing pharmacogenomics for clinical decision-making.
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Farmacogenética , Polimorfismo de Nucleotídeo Único , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Polimorfismo de Nucleotídeo Único/genética , Reprodutibilidade dos TestesRESUMO
A custom plate of OpenArray™ technology was evaluated to test 60 single-nucleotide polymorphisms (SNPs) validated for the prediction of eye color, hair color, and skin pigmentation, and for personal identification. The SNPs were selected from already validated subsets (Hirisplex-s, Precision ID Identity SNP Panel, and ForenSeq DNA Signature Prep Kit). The concordance rate and call rate for every SNP were calculated by analyzing 314 sequenced DNA samples. The sensitivity of the assay was assessed by preparing a dilution series of 10.0, 5.0, 1.0, and 0.5 ng. The OpenArray™ platform obtained an average call rate of 96.9% and a concordance rate near 99.8%. Sensitivity testing performed on serial dilutions demonstrated that a sample with 0.5 ng of total input DNA can be correctly typed. The profiles of the 19 SNPs selected for human identification reached a random match probability (RMP) of, on average, 10-8. An analysis of 21 examples of biological evidence from 8 individuals, that generated single short tandem repeat profiles during the routine workflow, demonstrated the applicability of this technology in real cases. Seventeen samples were correctly typed, revealing a call rate higher than 90%. Accordingly, the phenotype prediction revealed the same accuracy described in the corresponding validation data. Despite the reduced discrimination power of this system compared to STR based kits, the OpenArray™ System can be used to exclude suspects and prioritize samples for downstream analyses, providing well-established information about the prediction of eye color, hair color, and skin pigmentation. More studies will be needed for further validation of this technology and to consider the opportunity to implement this custom array with more SNPs to obtain a lower RMP and to include markers for studies of ancestry and lineage.
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Cor de Olho/genética , Antropologia Forense/métodos , Técnicas de Genotipagem , Cor de Cabelo/genética , Pigmentação da Pele/genética , Impressões Digitais de DNA , Genética Forense/tendências , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Polimorfismo de Nucleotídeo Único/genéticaRESUMO
OpenArray is one of the most high-throughput qPCR platforms available but its efficiency can be limited by sample preparation methods that are slow and costly. To optimize the sample workflow for high-throughput qPCR processing by OpenArray, small-scale sample preparation methods were compared for compatibility with this system to build confidence in a method that maintains quality and accuracy while using less starting material and saving time and money. This study is the first to show that the Cells-to-CT kit can be used to prepare samples within the dynamic range of OpenArray directly from cultured cells in a single well of a 96-well plate when used together with a cDNA preamplification PCR step. Use of Cells-to-CT produced results of similar quality and accuracy to that of a preparation method using purified RNA in less than half the sample preparation time. While Cells-to-CT samples also exhibited slightly increased variance, which affects the ability of OpenArray to distinguish small differences in gene expression, overall gene expression mean results correlated well between small-scale methods. This work demonstrates that Cells-to-CT with preamplification can be used to reliably prepare samples for OpenArray analysis while saving time, money, and starting material.
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The transcriptomic response of Senegalese sole (Solea senegalensis) triggered by two betanodaviruses with different virulence to that fish species has been assessed using an OpenArray® platform based on TaqMan™ quantitative PCR. The transcription of 112 genes per sample has been evaluated at two sampling times in two organs (head kidney and eye/brain-pooled samples). Those genes were involved in several roles or pathways, such as viral recognition, regulation of type I (IFN-1)-dependent immune responses, JAK-STAT cascade, interferon stimulated genes, protein ubiquitination, virus responsive genes, complement system, inflammatory response, other immune system effectors, regulation of T-cell proliferation, and proteolysis and apoptosis. The highly virulent isolate, wSs160.3, a wild type reassortant containing a RGNNV-type RNA1 and a SJNNV-type RNA2 segments, induced the expression of a higher number of genes in both tested organs than the moderately virulent strain, a recombinant harbouring mutations in the protruding domain of the capsid protein. The number of differentially expressed genes was higher 2 days after the infection with the wild type isolate than at 3 days post-inoculation. The wild type isolate also elicited an exacerbated interferon 1 response, which, instead of protecting sole against the infection, increases the disease severity by the induction of apoptosis and inflammation-derived immunopathology, although inflammation seems to be modulated by the complement system. Furthermore, results derived from this study suggest a potential important role for some genes with high expression after infection with the highly virulent virus, such as rtp3, sacs and isg15. On the other hand, the infection with the mutant does not induce immune response, probably due to an altered recognition by the host, which is supported by a different viral recognition pathway, involving myd88 and tbkbp1.
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Doenças dos Peixes/genética , Doenças dos Peixes/virologia , Linguados/genética , Linguados/virologia , Fenômenos Imunogenéticos/genética , Nodaviridae , Animais , Encéfalo/metabolismo , Olho/metabolismo , Doenças dos Peixes/imunologia , Linguados/imunologia , Perfilação da Expressão Gênica , Rim Cefálico/metabolismo , Interferon Tipo I/metabolismo , Nodaviridae/imunologia , Nodaviridae/patogenicidade , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/veterinária , RNA-Seq , Virulência , Replicação ViralRESUMO
Several lines of epidemiological and biochemical evidence support the association of type 2 diabetes mellitus (T2DM) and colorectal cancer (CRC). T2DM has been shown to impinge on the transcriptome of colon tumor cells, promoting their proliferation and invasion. In order to gain insight into diabetes-specific modulation of colon cancer signaling, we analyzed gene expression patterns of more than five hundred genes encoding signaling proteins on TaqMan OpenArray panels from colonoscopic colorectal tumor samples of type 2 diabetic and non-diabetic patients. In total, 48 transcripts were found to be differentially expressed in tumors of T2DM patients as compared to healthy colon samples. Enrichment analysis with the g:GOSt (Gene Ontology Statistics) functional profiling tool revealed that the underlying genes can be classified into five signaling pathways (in decreasing order of significance: Wnt (wingless-type)/ß-catenin; Hippo; TNF (tumor necrosis factor); PI3K/Akt (phosphoinositide-3 kinase/protein kinase B), and platelet activation), implying that targeted downregulation of these signaling cascades might help combat CRC in diabetic patients. Transcript levels of some of the differentially expressed genes were also measured from surgically removed diabetic and non-diabetic CRC specimens by individual qPCR (quantitative real-time PCR) assays using the adjacent normal tissue mRNA levels as an internal control. The most significantly altered genes in diabetic tumor samples were largely different from those in non-diabetic ones, implying that T2DM profoundly alters the expression of signaling genes and presumably the biological characteristics of CRC.
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European sea bass is highly susceptible to the nervous necrosis virus, RGNNV genotype, whereas natural outbreaks caused by the SJNNV genotype have not been recorded. The onset and severity of an infectious disease depend on pathogen virulence factors and the host immune response. The importance of RGNNV capsid protein amino acids 247 and 270 as virulence factors has been previously demonstrated in European sea bass; however, sea bass immune response against nodaviruses with different levels of virulence has been poorly characterized. Knowing the differences between the immune response against both kinds of isolates may be key to get more insight into the host mechanisms responsible for NNV virulence. For this reason, this study analyses the transcription of immunogenes differentially expressed in European sea bass inoculated with nodaviruses with different virulence: a RGNNV virus obtained by reverse genetics (rDl956), highly virulent to sea bass, and a mutated virus (Mut247+270Dl956, RGNNV virus displaying SJNNV-type amino acids at positions 247 and 270 of the capsid protein), presenting lower virulence. This study has been performed in brain and head kidney, and the main differences between the immunogene responses triggered by both viruses have been observed in brain. The immunogene response in this organ is stronger after inoculation with the most virulent virus, and the main differences involved genes related with IFN I system, inflammatory response, cell-mediated response, and apoptosis. The lower virulence of Mut247+270Dl956 to European sea bass can be associated with a delayed IFN I response, as well as an early and transitory inflammation and cell-mediated responses, suggesting that those can be pivotal elements in controlling the viral infection, and therefore, their functional activity could be analysed in future studies. In addition, this study supports the role of capsid amino acids at positions 247 and 270 as important determinants of RGNNV virulence to European sea bass.
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Bass/genética , Doenças dos Peixes/imunologia , Nodaviridae/fisiologia , Nodaviridae/patogenicidade , Infecções por Vírus de RNA/veterinária , Transcriptoma/imunologia , Animais , Bass/imunologia , Encéfalo/virologia , Doenças dos Peixes/microbiologia , Perfilação da Expressão Gênica/veterinária , Rim Cefálico/virologia , Infecções por Vírus de RNA/imunologia , Infecções por Vírus de RNA/microbiologia , VirulênciaRESUMO
Real time technology provides great advancements over PCR-based methods for a broad range of applications. With the increased availability of sequencing information, there is a need for the development and application of high-throughput real time PCR genotyping and gene expression methods that significantly broaden the current screening capabilities. Thermo Fisher Scientific (USA) has released a platform (QuantStudio™ 12K Flex system coupled with OpenArray® technology) with key elements required for high-throughput SNP genotyping and gene expression analysis. This allows for a rapid screening of large numbers of TaqMan® assays (up to 256) in many samples (up to 480) per run. This advanced real-time method involves the use of an array composed of 3,000 through-holes running on the QuantStudio™ 12K with OpenArray® block. The aim of this chapter is to outline the OpenArray® approach while providing a comprehensive in-depth review of the scientific literature on this topic. In agreement with a large number of independent studies, we conclude that the use of OpenArray® technology is a rapid and accurate method for high-throughput and large-scale systems biology studies with high specificity and sensitivity.
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Perfilação da Expressão Gênica/instrumentação , Técnicas de Genotipagem/instrumentação , Ensaios de Triagem em Larga Escala/instrumentação , Reação em Cadeia da Polimerase em Tempo Real/instrumentação , Perfilação da Expressão Gênica/métodos , Técnicas de Genotipagem/métodos , Ensaios de Triagem em Larga Escala/métodos , Humanos , Biologia de Sistemas/instrumentação , Biologia de Sistemas/métodosRESUMO
BACKGROUND: A number of human inflammatory diseases and tumors have been shown to cause alterations in the glycosylation pattern of plasma proteins in a specific manner. These highly variable and versatile post-translational modifications finetune protein functions by influencing sorting, folding, enzyme activity and subcellular localization. However, relatively little is known about regulatory factors of this procedure and about the accurate causative connection between glycosylation and disease. OBJECTIVE: The aim of the present study was to investigate whether certain single nucleotide polymorphisms (SNPs) in genes encoding glycosyltransferases and glycosidases could be associated with elevated risk for chronic obstructive pulmonary disease (COPD) and lung adenocarcinoma. METHODS: A total of 32 SNPs localized in genes related to N-glycosylation were selected for the association analysis. Polymorphisms with putative biological functions (missense or regulatory variants) were recruited. SNPs were genotyped by a TaqMan OpenArray platform. A single base extension-based method in combination with capillary gel electrophoresis was used for verification. RESULTS: The TaqMan OpenArray approach provided accurate and reliable genotype data (global call rate: 94.9%, accuracy: 99.6%). No significant discrepancy was detected between the obtained and expected genotype frequency values (Hardy-Weinberg equilibrium) in the healthy control sample group in case of any SNP confirming reliable sampling and genotyping. Allele frequencies of the rs3944508 polymorphism localized in the 3' UTR of the MGAT5 gene significantly differed between the sample groups compared. CONCLUSION: Our results suggest that the rs34944508 SNP might modulate the risk for lung cancer by influencing the expression of MGAT5. This enzyme catalyzes the addition of N-acetylglucosamine (GlcNAc) in beta 1-6 linkage to the alpha-linked mannose of biantennary N-linked oligosaccharides, thus, increasing branching that is the characteristic of invasive malignancies.
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Predisposição Genética para Doença , Sequenciamento de Nucleotídeos em Larga Escala , Neoplasias Pulmonares/genética , Polimorfismo de Nucleotídeo Único/genética , Doença Pulmonar Obstrutiva Crônica/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Estudos de Casos e Controles , Feminino , Frequência do Gene/genética , Estudos de Associação Genética , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
OBJECTIVE: A simple, non-invasive sample collection method is key for the integration of pharmacogenetics into clinical practice. The aim of this study was to gain samples for pharmacogenetic testing and evaluate the variation between dry-flocked and sponge-tipped buccal swabs in yield and quality of DNA isolated. RESULTS: Thirty-one participants collected samples using dry-flocked swabs and sponge-tipped swabs. Samples were assessed for DNA yield, quality and genotyping performance on a qPCR OpenArray platform of 28 pharmacogenetic SNPs and a CYP2D6 TaqMan copy number variant. DNA from sponge-tipped swabs had a significantly greater yield compared to DNA collected with dry-flocked swabs (p = 4.4 × 10-7). Moreover, highest genotyping call rates across all assays and highest CNV confidence scores were observed in DNA samples collected from sponge-tipped swabs (97% vs. 54% dry-flocked swabs; 0.99 vs. 0.88 dry-flocked swabs, respectively). Sample collection using sponge-tipped swabs provides a DNA source of sufficient quantity and quality for pharmacogenetic variant detection using qPCR.
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Técnicas de Genotipagem/métodos , Mucosa Bucal , Farmacogenética/métodos , Manejo de Espécimes/métodos , Adulto , Variações do Número de Cópias de DNA , Técnicas de Genotipagem/normas , Humanos , Farmacogenética/normas , Polimorfismo de Nucleotídeo Único , Reação em Cadeia da Polimerase em Tempo Real , Manejo de Espécimes/normasRESUMO
Alzheimer's disease is the most common form of dementia and is characterized by a progressive loss of cognitive functions. As the result of predicted demographic changes over the next decades, Alzheimer's disease is expected to be one of the most pressing medical and social challenges facing our generation. Current treatment strategies remain symptomatic. However, new approaches have shown promise in clinical trials, particularly in patients with only mild or moderate symptoms. Early detection of Alzheimer's disease is therefore of critical importance. Currently available diagnostic approaches (such as protein analysis in cerebrospinal fluid or neuroimaging), however, are expensive and invasive and therefore unsuitable for the screening of a large population. Consequently, Alzheimer's disease is generally diagnosed too late for effective intervention. MicroRNAs-readily measurable in biofluids and resistant to freeze-thaw and pH changes, have shown encouraging diagnostic potential in Alzheimer's disease. Several studies have attempted to correlate changes of specific microRNAs to disease progression using different approaches and profiling platforms including micro-arrays, RNA sequencing, and qPCR-based systems. In the present book chapter, we will describe the different steps involved in how to determine the microRNA profile in plasma samples from patients using the OpenArray platform.
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Doença de Alzheimer/genética , Biomarcadores/análise , MicroRNA Circulante/análise , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Doença de Alzheimer/sangue , Biomarcadores/sangue , Diagnóstico Precoce , Humanos , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de RNARESUMO
BACKGROUND: Ethnic differences exist in the frequencies of genetic variations that contribute to the risk of common disease. This study aimed to analyse the distribution of several genes, previously associated with susceptibility to type 2 diabetes and obesity-related phenotypes, in a Kazakh population. METHODS: A total of 966 individuals belonging to the Kazakh ethnicity were recruited from an outpatient clinic. We genotyped 41 common single nucleotide polymorphisms (SNPs) previously associated with type 2 diabetes in other ethnic groups and 31 of these were in Hardy-Weinberg equilibrium. The obtained allele frequencies were further compared to publicly available data from other ethnic populations. Allele frequencies for other (compared) populations were pooled from the haplotype map (HapMap) database. Principal component analysis (PCA), cluster analysis, and multidimensional scaling (MDS) were used for the analysis of genetic relationship between the populations. RESULTS: Comparative analysis of allele frequencies of the studied SNPs showed significant differentiation among the studied populations. The Kazakh population was grouped with Asian populations according to the cluster analysis and with the Caucasian populations according to PCA. According to MDS, results of the current study show that the Kazakh population holds an intermediate position between Caucasian and Asian populations. CONCLUSION: A high percentage of population differentiation was observed between Kazakh and world populations. The Kazakh population was clustered with Caucasian populations, and this result may indicate a significant Caucasian component in the Kazakh gene pool.