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1.
BMC Bioinformatics ; 21(1): 271, 2020 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-32605541

RESUMO

BACKGROUND: Systematic technical effects-also called batch effects-are a considerable challenge when analyzing DNA methylation (DNAm) microarray data, because they can lead to false results when confounded with the variable of interest. Methods to correct these batch effects are error-prone, as previous findings have shown. RESULTS: Here, we demonstrate how using the R function ComBat to correct simulated Infinium HumanMethylation450 BeadChip (450 K) and Infinium MethylationEPIC BeadChip Kit (EPIC) DNAm data can lead to a large number of false positive results under certain conditions. We further provide a detailed assessment of the consequences for the highly relevant problem of p-value inflation with subsequent false positive findings after application of the frequently used ComBat method. Using ComBat to correct for batch effects in randomly generated samples produced alarming numbers of false discovery rate (FDR) and Bonferroni-corrected (BF) false positive results in unbalanced as well as in balanced sample distributions in terms of the relation between the outcome of interest variable and the technical position of the sample during the probe measurement. Both sample size and number of batch factors (e.g. number of chips) were systematically simulated to assess the probability of false positive findings. The effect of sample size was simulated using n = 48 up to n = 768 randomly generated samples. Increasing the number of corrected factors led to an exponential increase in the number of false positive signals. Increasing the number of samples reduced, but did not completely prevent, this effect. CONCLUSIONS: Using the approach described, we demonstrate, that using ComBat for batch correction in DNAm data can lead to false positive results under certain conditions and sample distributions. Our results are thus contrary to previous publications, considering a balanced sample distribution as unproblematic when using ComBat. We do not claim completeness in terms of reporting all technical conditions and possible solutions of the occurring problems as we approach the problem from a clinician's perspective and not from that of a computer scientist. With our approach of simulating data, we provide readers with a simple method to assess the probability of false positive findings in DNAm microarray data analysis pipelines.


Assuntos
Metilação de DNA , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Ilhas de CpG , Reações Falso-Positivas , Humanos , Dispositivos Lab-On-A-Chip , Probabilidade , Tamanho da Amostra
2.
NPJ Syst Biol Appl ; 6(1): 17, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32518234

RESUMO

Numerous time-course gene expression datasets have been generated for studying the biological dynamics that drive disease progression; and nearly as many methods have been proposed to analyse them. However, barely any method exists that can appropriately model time-course data while accounting for heterogeneity that entails many complex diseases. Most methods manage to fulfil either one of those qualities, but not both. The lack of appropriate methods hinders our capability of understanding the disease process and pursuing preventive treatments. We present a method that models time-course data in a personalised manner using Gaussian processes in order to identify differentially expressed genes (DEGs); and combines the DEG lists on a pathway-level using a permutation-based empirical hypothesis testing in order to overcome gene-level variability and inconsistencies prevalent to datasets from heterogenous diseases. Our method can be applied to study the time-course dynamics, as well as specific time-windows of heterogeneous diseases. We apply our personalised approach on three longitudinal type 1 diabetes (T1D) datasets, where the first two are used to determine perturbations taking place during early prognosis of the disease, as well as in time-windows before autoantibody positivity and T1D diagnosis; and the third is used to assess the generalisability of our method. By comparing to non-personalised methods, we demonstrate that our approach is biologically motivated and can reveal more insights into progression of heterogeneous diseases. With its robust capabilities of identifying disease-relevant pathways, our approach could be useful for predicting events in the progression of heterogeneous diseases and even for biomarker identification.


Assuntos
Perfilação da Expressão Gênica/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Medicina de Precisão/métodos , Algoritmos , Biomarcadores , Simulação por Computador , Progressão da Doença , Humanos , Dinâmica não Linear , Distribuição Normal , Prognóstico , Software , Transcriptoma/genética
3.
NPJ Syst Biol Appl ; 6(1): 20, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561750

RESUMO

Differential coexpression has recently emerged as a new way to establish a fundamental difference in expression pattern among a group of genes between two populations. Earlier methods used some scoring techniques to detect changes in correlation patterns of a gene pair in two conditions. However, modeling differential coexpression by means of finding differences in the dependence structure of the gene pair has hitherto not been carried out. We exploit a copula-based framework to model differential coexpression between gene pairs in two different conditions. The Copula is used to model the dependency between expression profiles of a gene pair. For a gene pair, the distance between two joint distributions produced by copula is served as differential coexpression. We used five pan-cancer TCGA RNA-Seq data to evaluate the model that outperforms the existing state of the art. Moreover, the proposed model can detect a mild change in the coexpression pattern across two conditions. For noisy expression data, the proposed method performs well because of the popular scale-invariant property of copula. In addition, we have identified differentially coexpressed modules by applying hierarchical clustering on the distance matrix. The identified modules are analyzed through Gene Ontology terms and KEGG pathway enrichment analysis.


Assuntos
Biologia Computacional/métodos , Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes/genética , Algoritmos , Análise por Conglomerados , Ontologia Genética , Humanos , Neoplasias/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Análise de Sequência de RNA/métodos , Análise de Sistemas
4.
PLoS One ; 15(5): e0232955, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32413060

RESUMO

Whole-genome expression data generated by microarray studies have shown promise for quantitative human health risk assessment. While numerous approaches have been developed to determine benchmark doses (BMDs) from probeset-level dose responses, sensitivity of the results to methods used for normalization of the data has not yet been systematically investigated. Normalization of microarray data converts raw hybridization signals to expression estimates that are expected to be proportional to the amounts of transcripts in the profiled specimens. Different approaches to normalization have been shown to greatly influence the results of some downstream analyses, including biological interpretation. In this study we evaluate the influence of microarray normalization methods on the transcriptomic BMDs. We demonstrate using in vivo data that the use of alternative pipelines for normalization of Affymetrix microarray data can have a considerable impact on the number of detected differentially expressed genes and pathways (processes) determined to be treatment responsive, which may lead to alternative interpretations of the data. In addition, we found that normalization can have a considerable effect (as much as ~30-fold in this study) on estimation of the minimum biological potency (transcriptomic point of departure). We argue for consideration of alternative normalization methods and their data-informed selection to most effectively interpret microarray data for use in human health risk assessment.


Assuntos
Benchmarking/métodos , Biologia Computacional/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Perfilação da Expressão Gênica/métodos , Genoma/genética , Humanos , Hibridização de Ácido Nucleico/métodos , Medição de Risco/métodos , Transcriptoma/genética
5.
BMC Bioinformatics ; 21(1): 135, 2020 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-32264950

RESUMO

BACKGROUND: Microarray technology provides the expression level of many genes. Nowadays, an important issue is to select a small number of informative differentially expressed genes that provide biological knowledge and may be key elements for a disease. With the increasing volume of data generated by modern biomedical studies, software is required for effective identification of differentially expressed genes. Here, we describe an R package, called ORdensity, that implements a recent methodology (Irigoien and Arenas, 2018) developed in order to identify differentially expressed genes. The benefits of parallel implementation are discussed. RESULTS: ORdensity gives the user the list of genes identified as differentially expressed genes in an easy and comprehensible way. The experimentation carried out in an off-the-self computer with the parallel execution enabled shows an improvement in run-time. This implementation may also lead to an important use of memory load. Results previously obtained with simulated and real data indicated that the procedure implemented in the package is robust and suitable for differentially expressed genes identification. CONCLUSIONS: The new package, ORdensity, offers a friendly and easy way to identify differentially expressed genes, which is very useful for users not familiar with programming. AVAILABILITY: https://github.com/rsait/ORdensity.


Assuntos
Interface Usuário-Computador , Doença/genética , Regulação da Expressão Gênica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , RNA-Seq/métodos
6.
Proc Natl Acad Sci U S A ; 117(16): 8719-8726, 2020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32241887

RESUMO

Rapid methods for diagnosis of bacterial infections are urgently needed to reduce inappropriate use of antibiotics, which contributes to antimicrobial resistance. In many rapid diagnostic methods, DNA oligonucleotide probes, attached to a surface, bind to specific nucleotide sequences in the DNA of a target pathogen. Typically, each probe binds to a single target sequence; i.e., target-probe binding is monovalent. Here we show using computer simulations that the detection sensitivity and specificity can be improved by designing probes that bind multivalently to the entire length of the pathogen genomic DNA, such that a given probe binds to multiple sites along the target DNA. Our results suggest that multivalent targeting of long pieces of genomic DNA can allow highly sensitive and selective binding of the target DNA, even if competing DNA in the sample also contains binding sites for the same probe sequences. Our results are robust to mild fragmentation of the bacterial genome. Our conclusions may also be relevant for DNA detection in other fields, such as disease diagnostics more broadly, environmental management, and food safety.


Assuntos
Desenho Assistido por Computador , Sondas de DNA , DNA Bacteriano/isolamento & purificação , Genoma Bacteriano , Sondas de Oligonucleotídeos , Biologia Computacional/métodos , Simulação por Computador , DNA Bacteriano/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Sensibilidade e Especificidade , Análise de Sequência de DNA/métodos
7.
Int J Oncol ; 56(3): 807-820, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32124947

RESUMO

Previous studies have demonstrated that long non­coding RNAs (lncRNAs) are involved in breast cancer development, progression and metastasis. However, the association between lncRNAs and breast cancer stem cells (BCSCs) has been poorly explored. To address this issue, microarray analyses were performed to detect the lncRNA profile of BCSCs. In addition, bioinformatics analyses, including Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes pathway analyses, were performed to explore the functional roles of lncRNAs in BCSCs. Lastly, loss of function assays were used to explore the potential function of lncRNA CUE domain containing 1 (lncCUEDC1). A total of 142 differentially expressed lncRNAs were identified. Among these, 25 were downregulated and 117 were upregulated in BCSCs compared with in non­BCSCs. In addition, the present study revealed that the lncRNAs were largely associated with stemness­related signaling pathways. Furthermore, it was demonstrated that lncCUEDC1 negatively regulated the phenotype and biological functions of BCSCs in vitro. Mechanistically, lncCUEDC1 could bind NANOG to inhibit the stemness. To the best of our knowledge, the present study was the first to established the lncRNA profile of BCSCs. These findings provided evidence for exploring the functions of lncRNAs in BCSCs and indicated that lncCUEDC1 is a prospective target in BCSCs.


Assuntos
Neoplasias da Mama/genética , Perfilação da Expressão Gênica/métodos , Células-Tronco Neoplásicas/química , Análise de Sequência com Séries de Oligonucleotídeos/métodos , RNA Longo não Codificante/genética , Linhagem Celular Tumoral , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Células MCF-7 , Estudos Prospectivos , Análise de Sequência de DNA
8.
PLoS One ; 15(2): e0229659, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32101588

RESUMO

The cultivation of genetically modified organisms (GMO) continues to expand worldwide. Still, many consumers express concerns about the use of GMO in food or feed, and many countries have legislated on labelling systems to indicate the presence of GMO in commercial products. To deal with the increased number of GMO events and to address related regulations, alternative detection methods for GMO inspection are required. In this work, a genosensor based on Surface Plasmon Resonance under continuous flow was developed for the detection and quantification of a genetically modified soybean (event GTS 40-3-2). In a single chip, the simultaneous detection of the event-specific and the taxon-specific samples were achieved, whose detection limits were 20 pM and 16 pM, respectively. The reproducibility was 1.4%, which supports the use of the chip as a reliable and cost-effective alternative to other DNA-based techniques. The results indicate that the proposed method is a versatile tool for GMO quantification in food and feed samples.


Assuntos
Soja/genética , Ressonância de Plasmônio de Superfície/métodos , DNA de Plantas/genética , Alimentos Geneticamente Modificados/classificação , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Organismos Geneticamente Modificados/química , Organismos Geneticamente Modificados/genética , Plantas Geneticamente Modificadas/genética , Reprodutibilidade dos Testes
9.
PLoS One ; 15(2): e0229485, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32109938

RESUMO

Periodontal disease, the most prevalent infectious disease in the world, is caused by biofilms formed in periodontal pockets. No specific bacterial species that can cause periodontitis alone has been found in any study to date. Several periodontopathic bacteria are associated with the progress of periodontal disease. Consequently, it is hypothesized that dysbiosis of subgingival microbiota may be a cause of periodontal disease. This study aimed to investigate the relationship between the subgingival microbiota and the clinical status of periodontal pockets in a quantitative and clinically applicable way with the newly developed Oral Care Chip. The Oral Care Chip is a DNA microarray tool with improved quantitative performance, that can be used in combination with competitive PCR to quantitatively detect 17 species of subgingival bacteria. Cluster analysis based on the similarity of each bacterial quantity was performed on 204 subgingival plaque samples collected from periodontitis patients and healthy volunteers. A significant difference in the number of total bacteria, Treponema denticola, Campylobacter rectus, Fusobacterium nucleatum, and Streptococcus intermedia bacteria in any combination of the three clusters indicated that these bacteria gradually increased in number from the stage before the pocket depth deepened. Conversely, Porphyromonas gingivalis, Tannerella forsythia, Prevotella intermedia, and Streptococcus constellatus, which had significant differences only in limited clusters, were thought to increase in number as the pocket depth deepened, after periodontal pocket formation. Furthermore, in clusters where healthy or mild periodontal disease sites were classified, there was no statistically significant difference in pocket depth, but the number of bacteria gradually increased from the stage before the pocket depth increased. This means that quantitative changes in these bacteria can be a predictor of the progress of periodontal tissue destruction, and this novel microbiological test using the Oral Care Chip could be effective at detecting dysbiosis.


Assuntos
Bactérias/isolamento & purificação , DNA Bacteriano/análise , Microbiota , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Doenças Periodontais/microbiologia , Bolsa Periodontal/microbiologia , Adulto , Campylobacter rectus/isolamento & purificação , Feminino , Fusobacterium nucleatum/isolamento & purificação , Humanos , Masculino , Pessoa de Meia-Idade , Doenças Periodontais/diagnóstico , Índice Periodontal , Porphyromonas gingivalis/isolamento & purificação , Prevotella intermedia/isolamento & purificação , Streptococcus constellatus/isolamento & purificação , Tannerella forsythia/isolamento & purificação , Treponema denticola/isolamento & purificação , Adulto Jovem
10.
Eur J Paediatr Neurol ; 25: 106-112, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32014392

RESUMO

OBJECTIVES: To systematically investigate chromosomal abnormalities and copy number variants (CNVs) in fetuses with different types of ventriculomegaly (VM) by karyotyping and/or chromosomal microarray analysis (CMA). METHODS: This retrospective study included 312 fetuses diagnosed with VM. Amniotic fluid and umbilical blood samples were collected by amniocentesis and cordocentesis, respectively, and subjected to karyotyping and/or CMA. Subgroup analysis by VM type, including mild VM (MVM) and severe VM (SVM), unilateral and bilateral VM, isolated VM (IVM), and non-isolated VM (NIVM), was performed. RESULTS: The detection rate of chromosomal abnormalities was 12.1% (34/281) by karyotyping and 20.6% when CMA was additionally performed (P < 0.05). Abnormalities were identified by CMA in 17.4% (38/218) of fetuses and pathogenic CNVs in 5.0% (11/218). Notably, CMA detected CNVs in 10.6% (23/218) of fetuses with normal karyotypes. The incidence of chromosomal abnormalities by karyotyping was higher in bilateral than in unilateral VM (20.5% versus 6.5%), whereas the incidence detected by CMA was higher in NIVM than in IVM (21.4% versus 10.3%; both P < 0.05). In NIVM, CMA provided an additional detection rate of 11.4% (16/140) and a detection rate of 10.0% for pathogenic CNVs and aneuploidies. Central nervous system (CNS) abnormalities were the most common other ultrasonic abnormalities. CONCLUSIONS: CMA is highly recommended for prenatal diagnosis of fetal VM together with karyotyping, especially in fetuses with bilateral VM and NIVM with abnormal CNS findings. Further study is necessary to explore the relationships between genotypes and phenotypes to facilitate prenatal diagnosis of fetal VM.


Assuntos
Ventrículos Cerebrais/anormalidades , Malformações do Sistema Nervoso/diagnóstico , Malformações do Sistema Nervoso/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Diagnóstico Pré-Natal/métodos , Aberrações Cromossômicas , Variações do Número de Cópias de DNA , Feminino , Feto/anormalidades , Humanos , Hidrocefalia/diagnóstico , Hidrocefalia/etiologia , Cariotipagem/métodos , Malformações do Sistema Nervoso/complicações , Gravidez , Estudos Retrospectivos
11.
Prostate ; 80(6): 491-499, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32068909

RESUMO

BACKGROUND: Prostate cancer (PC) research has relied heavily on patient-derived cell lines, which may be used for in vitro (two-dimensional [2D]) studies or cultivated as three-dimensional (3D) xenografts in mice. These approaches are likely to have differential impacts on cell phenotypes, with implications for experimental outcomes. Therefore, defining and comparing the transcriptional signatures associated with 2D and 3D approaches may be useful for designing experiments and interpreting research results. METHODS: In this study, LNCaP, VCaP, and 22Rv1 human PC cells were either cultivated in monolayers or as xenografts in NOD SCID mice, and their gene transcription profiles were quantitated and compared using microarray and real-time polymerase chain reaction techniques. Immunohistochemistry was used to evaluate protein expression in cancer cell xenografts. RESULTS: Comparisons of gene expression profiles of tumor cells grown in 2D vs 3D environments identified gene sets featuring similar expression patterns in all three cancer cell lines and unique transcriptional signatures associated with 3D vs 2D growth. Pathways related to cell-cell interactions, differentiation, and the extracellular matrix were enriched in 3D conditions. Immunohistochemical analyses confirmed that gene upregulation in xenografts occurred in implanted cancer cells and not in mouse stromal cells. Cultivating cells in vitro in the presence of mouse, rather than bovine serum failed to elicit the gene transcription profile observed in xenografts, further supporting the hypothesis that this profile reflects 3D growth and enhanced microenvironmental interactions, rather than exposure to species-specific serum factors. CONCLUSIONS: Overall, these findings define the expression profiles observed in PC cells cultivated in 2D monolayers and in 3D xenografts, highlighting differentially regulated pathways in each setting and providing information for interpreting research results in model systems.


Assuntos
Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Animais , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Genoma Humano , Xenoenxertos , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Transcriptoma , Células Tumorais Cultivadas , Microambiente Tumoral/genética
12.
Nat Protoc ; 15(2): 479-512, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31932775

RESUMO

DNA methylation data-based precision cancer diagnostics is emerging as the state of the art for molecular tumor classification. Standards for choosing statistical methods with regard to well-calibrated probability estimates for these typically highly multiclass classification tasks are still lacking. To support this choice, we evaluated well-established machine learning (ML) classifiers including random forests (RFs), elastic net (ELNET), support vector machines (SVMs) and boosted trees in combination with post-processing algorithms and developed ML workflows that allow for unbiased class probability (CP) estimation. Calibrators included ridge-penalized multinomial logistic regression (MR) and Platt scaling by fitting logistic regression (LR) and Firth's penalized LR. We compared these workflows on a recently published brain tumor 450k DNA methylation cohort of 2,801 samples with 91 diagnostic categories using a 5 × 5-fold nested cross-validation scheme and demonstrated their generalizability on external data from The Cancer Genome Atlas. ELNET was the top stand-alone classifier with the best calibration profiles. The best overall two-stage workflow was MR-calibrated SVM with linear kernels closely followed by ridge-calibrated tuned RF. For calibration, MR was the most effective regardless of the primary classifier. The protocols developed as a result of these comparisons provide valuable guidance on choosing ML workflows and their tuning to generate well-calibrated CP estimates for precision diagnostics using DNA methylation data. Computation times vary depending on the ML algorithm from <15 min to 5 d using multi-core desktop PCs. Detailed scripts in the open-source R language are freely available on GitHub, targeting users with intermediate experience in bioinformatics and statistics and using R with Bioconductor extensions.


Assuntos
Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/genética , Metilação de DNA , Aprendizado de Máquina , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Medicina de Precisão , Fluxo de Trabalho , Humanos , Probabilidade
13.
J Endocrinol ; 245(1): 65-78, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31990671

RESUMO

Despite extensive investigation, the mechanisms underlying adipogenesis are not fully understood. We previously identified proliferative cells in adipose tissue expressing adipocyte-specific genes, which were named small proliferative adipocytes (SPA). In this study, we investigated the characteristics and roles of SPA in adipose tissue. Epididymal and inguinal fat was digested by collagenase, and then SPA were separated by centrifugation from stromal vascular cells (SVC) and mature white adipocytes. To clarify the feature of gene expression in SPA, microarray and real-time PCR were performed. The expression of adipocyte-specific genes and several neuronal genes was increased in the order of SVC < SPA < mature white adipocytes. In addition, proliferin was detected only in SPA. SPA differentiated more effectively into lipid-laden cells than SVC. Moreover, differentiated SPA expressed uncoupling protein 1 and mitochondria-related genes more than differentiated SVC. Treatment of SPA with pioglitazone and CL316243, a specific ß3-adrenergic receptor agonist, differentiated SPA into beige-like cells. Therefore, SPA are able to differentiate into beige cells. SPA isolated from epididymal fat (epididymal SPA), but not SPA from inguinal fat (inguinal SPA), expressed a marker of visceral adipocyte precursor, WT1. However, no significant differences were detected in the expression levels of adipocyte-specific genes or neuronal genes between epididymal and inguinal SPA. The ability to differentiate into lipid-laden cells in epididymal SPA was a little superior to that in inguinal SPA, whereas the ability to differentiate into beige-like cells was greater in inguinal SPA than epididymal SPA. In conclusion, SPA may be progenitors of beige cells.


Assuntos
Adipócitos Bege/metabolismo , Adipócitos Brancos/metabolismo , Adipócitos/metabolismo , Perfilação da Expressão Gênica/métodos , Células-Tronco/metabolismo , Adipócitos/citologia , Adipócitos Bege/citologia , Adipócitos Brancos/citologia , Adipogenia/genética , Animais , Diferenciação Celular/genética , Células Cultivadas , Camundongos Endogâmicos C57BL , Mitocôndrias/genética , Mitocôndrias/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Células-Tronco/citologia , Proteína Desacopladora 1/genética , Proteína Desacopladora 1/metabolismo
14.
Vet Microbiol ; 240: 108539, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31902492

RESUMO

The objective of our study was to provide a molecular analysis using DNA-microarray based assays of commensal E. coli populations from apparently healthy livestock and their attendants to assess the virulence potential as well as multidrug resistance (MDR) genotypes. We randomly collected 132 fecal samples from seemingly healthy smallholder´s food producing animals [buffalo (n = 32) and cattle (n = 50)] as well as from contacting farmers (n = 50). Bacterial isolation and identification were performed using standard protocols, while E. coli isolates were characterized using a DNA microarray system targeting 60 different virulence and 47 antibiotic resistance genes of clinical importance and allowing assignment to most common H and O types. From the fecal samples examined, 47 E. coli isolates were obtained. The array predicted serotypes for 14 out of the 47 E. coli isolates. Six E. coli isolates were identified as STEC since Shiga toxin genes were detected. In summary, 36 different virulence genes were identified; of which, hemL, lpfA and iss were most prevalent. Thirty-four E. coli isolates were found to carry at least one antimicrobial resistance gene. Of these, 20 did exhibit genes allowing strain classification as MDR. More than half of the isolates contained antimicrobial resistance genes associated with beta lactam resistance 27/47 (57.5 %). The 13 remaining isolates did not contain any resistance gene tested with the array. Our study demonstrated the presence of antimicrobial resistance genes and virulence genotypes among commensal E. coli of human and animal sources.


Assuntos
Farmacorresistência Bacteriana Múltipla/genética , Escherichia coli/genética , Fazendeiros , Gado/microbiologia , Simbiose , Fatores de Virulência/genética , Animais , Antibacterianos/farmacologia , Búfalos/microbiologia , Bovinos/microbiologia , Egito , Escherichia coli/efeitos dos fármacos , Escherichia coli/patogenicidade , Infecções por Escherichia coli/microbiologia , Fezes/microbiologia , Genótipo , Humanos , Testes de Sensibilidade Microbiana , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Toxina Shiga/genética
15.
Genes (Basel) ; 11(1)2020 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-31947936

RESUMO

Knowledge of population structure is essential to improve the management and conservation of farm animal genetic resources. Microsatellites, which have long been popular for this type of analysis, are more and more neglected in favor of whole-genome single nucleotide polymorphism (SNP) chips that are now available for the main farmed animal species. In this study, we compared genetic patterns derived from microsatellites to that inferred by SNPs, considering three pairs of datasets of sheep and cattle. Population genetic differentiation analyses (Fixation index, FST), as well as STRUCTURE analyses showed a very strong consistency between the two types of markers. Microsatellites gave pictures that were largely concordant with SNPs, although less accurate. The best concordance was found in the most complex dataset, which included 17 French sheep breeds (with a Pearson correlation coefficient of 0.95 considering the 136 values of pairwise FST, obtained with both types of markers). The use of microsatellites reduces the cost and the related analyses do not require specific computer equipment (i.e., information technology (IT) infrastructure able to provide adequate computing and storage capacity). Therefore, this tool may still be a very appropriate solution to evaluate, in a first stage, the general state of livestock at national scales. At a time when local breeds are disappearing at an alarming rate, it is urgent to improve our knowledge of them, in particular by promoting tools accessible to the greatest number.


Assuntos
Animais Domésticos/genética , Repetições de Microssatélites/genética , Polimorfismo de Nucleotídeo Único/genética , Criação de Animais Domésticos/métodos , Animais , Bovinos/genética , Variação Genética/genética , Genoma/genética , Genótipo , Desequilíbrio de Ligação/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Filogenia , Ovinos/genética
16.
Food Chem ; 311: 125884, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31810726

RESUMO

Seafood is particularly susceptible to the substitution of species. In order to guarantee authentic seafood products, seafood processors and traders must perform self-checks on the authenticity of imported and purchased goods. However, the conventional Sanger sequencing of PCR products for the authentication of seafood species is time-consuming and requires advanced infrastructure. DNA microarrays (DNA chips) with species-specific oligonucleotide probes represent a rapid alternative to sequencing-based species authentication. So far, though, only DNA microarrays for the authentication of land vertebrate species have achieved market success. In this work, a user-friendly DNA microarray assay was developed for the authentication of ten important food fish species that can be performed in four to five hours from start to end. The assay was tested with authenticated specimens from 67 different fish species, and by comparing the probe signal patterns all target species and even closely related non-target species could be distinguished.


Assuntos
DNA/química , Peixes/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Alimentos Marinhos/análise , Animais , Citocromos b/química , Citocromos b/genética , Citocromos b/metabolismo , DNA/genética , DNA/metabolismo , Hibridização de Ácido Nucleico , Sondas de Oligonucleotídeos/metabolismo , Reação em Cadeia da Polimerase , RNA Ribossômico 16S/química , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , Análise de Sequência de DNA , Especificidade da Espécie
17.
Biochem Genet ; 58(1): 74-101, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31273557

RESUMO

Chromosomal microarray (CMA) has emerged as a robust tool for identifying microdeletions and microduplications, termed copy number variants (CNVs). Nevertheless, data regarding its utility in different patient populations with developmental delay (DD), dysmorphic features (DF) and congenital anomalies (CA), is a matter of dense debate. Although regions of homozygosity (ROH) are not diagnostic of a specific condition, they may have pathogenic implications. Certain CNVs and ROH have ethnically specific occurrences and frequencies. We aimed to determine whether CMA testing offers additional diagnostic information over classical cytogenetics for identifying genomic imbalances in a pediatric cohort with idiopathic DD, DF, or CA. One hundred sixty-nine patients were offered cytogenetics and CMA simultaneously for etiological diagnosis of DD (n = 67), DF (n = 52) and CA (n = 50). CMA could identify additional, clinically significant anomalies as compared with cytogenetics. CMA detected 61 CNVs [21 (34.4%) pathogenic CNVs, 37 (60.7%) variants of uncertain clinical significance and 3 (4.9%) benign CNVs] in 44 patients. CMA identified one or more ROH in 116/169 (68.6%) patients. When considering pathogenic CNVs and aneuploidies as positive findings, 9/169 (5.3%) received a genetic diagnosis from cytogenetics, while 25/169 (14.8%) could have a genetic diagnosis from CMA. The identification of ROH was clinically significant in two cases (2/169), thereby, adding 1.2% to the diagnostic yield of CMA (16% vs. 5.3%, p < 0.001). CMA uncovers additional genetic diagnoses over cytogenetics, thereby, offering a much higher diagnostic yield. Our findings convincingly demonstrate the additive diagnostic value of clinically significant ROH identified during CMA testing, highlighting the need for careful clinical interpretation of these ROH.


Assuntos
Síndrome de Down/diagnóstico , Homozigoto , Síndrome de Klinefelter/diagnóstico , Síndrome da Trissomia do Cromossomo 13/diagnóstico , Síndrome da Trissomía do Cromossomo 18/diagnóstico , Síndrome de Turner/diagnóstico , Adolescente , Criança , Pré-Escolar , Quebra Cromossômica , Estudos de Coortes , Variações do Número de Cópias de DNA , Deficiências do Desenvolvimento/genética , Síndrome de Down/genética , Feminino , Humanos , Lactente , Recém-Nascido , Síndrome de Klinefelter/genética , Masculino , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Síndrome da Trissomia do Cromossomo 13/genética , Síndrome da Trissomía do Cromossomo 18/genética , Síndrome de Turner/genética
18.
Mol Carcinog ; 59(2): 202-214, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31793078

RESUMO

Circular RNAs (circRNAs) are involved in the regulation of gene expression in different physiological and pathological processes. These macromolecules can act as microRNA (miRNA) sponges and play an important role as gene regulators throughout the circRNA-miRNA pathway. In this study, we established a radioresistance model with the nasopharyngeal carcinoma cell line CNE-2, and then analyzed the differences in the circRNAs between radioresistant and normal nasopharyngeal carcinoma cell lines using a high-throughput microarray. Tested circRNAs included 1042 upregulated and 1558 downregulated circRNAs. Relevant signaling pathways associated with the circRNAs and their target miRNAs were analyzed using bioinformatics analysis to determine the radioresistance of the differentially expressed circRNAs. Curcumin was used to treat irradiated cell lines, and changes in the circRNA before and after curcumin treatment were analyzed to investigate the radiosensitization effects of curcumin. The results showed that curcumin could regulate the circRNA-miRNA-messenger RNA network and inhibit the epidermal growth factor receptor (EGFR), signal transducers and activators of transcription 3 (STAT3), and growth factor receptor-bound protein 2 (GRB2) to achieve radiosensitization. Thus, circRNA acted as a miRNA sponge and regulated the expression of miRNA, thereby affecting EGFR, STAT3, and GRB2 expression and radiosensitization.


Assuntos
Curcumina/farmacologia , Regulação Neoplásica da Expressão Gênica/genética , Carcinoma Nasofaríngeo/genética , Neoplasias Nasofaríngeas/genética , RNA Circular/genética , Tolerância a Radiação/efeitos dos fármacos , Linhagem Celular Tumoral , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Redes Reguladoras de Genes/efeitos dos fármacos , Redes Reguladoras de Genes/genética , Redes Reguladoras de Genes/efeitos da radiação , Humanos , Carcinoma Nasofaríngeo/patologia , Neoplasias Nasofaríngeas/patologia , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Tolerância a Radiação/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/efeitos da radiação
19.
Mol Genet Genomics ; 295(1): 209-219, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31642957

RESUMO

The objective of this study was to map the quantitative trait loci (QTLs) for chip color after harvest (AH), cold storage (CS) and after reconditioning (RC) in diploid potato and compare them with QTLs for starch-corrected chip color. Chip color traits AH, CS, and RC significantly correlated with tuber starch content (TSC). To limit the effect of starch content, the chip color was corrected for TSC. The QTLs for chip color (AH, CS, and RC) and the starch-corrected chip color determined with the starch content after harvest (SCAH), after cold storage (SCCS) and after reconditioning (SCRC) were compared to assess the extent of the effect of starch and the location of genetic factors underlying this effect on chip color. We detected QTLs for the AH, CS, RC and starch-corrected traits on ten potato chromosomes, confirming the polygenic nature of the traits. The QTLs with the strongest effects were detected on chromosomes I (AH, 0 cM, 11.5% of variance explained), IV (CS, 43.9 cM, 12.7%) and I (RC, 49.7 cM, 14.1%). When starch correction was applied, the QTLs with the strongest effects were revealed on chromosomes VIII (SCAH, 39.3 cM, 10.8% of variance explained), XI (SCCS, 79.5 cM, 10.9%) and IV (SCRC, 43.9 cM, 10.8%). Applying the starch correction changed the landscape of QTLs for chip color, as some QTLs became statistically insignificant, shifted or were refined, and new QTLs were detected for SCAH. The QTLs on chromosomes I and IV were significant for all traits with and without starch correction.


Assuntos
Locos de Características Quantitativas/genética , Solanum tuberosum/genética , Amido/genética , Mapeamento Cromossômico/métodos , Cor , Diploide , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Tubérculos/genética
20.
Oncogene ; 39(2): 385-398, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31477838

RESUMO

Tumor invasion and metastasis are the major causes of treatment failure and mortality in lung cancer patients. In this study, we identified a group of genes with differential expression in in situ and invasive lung adenocarcinoma tissues by expression profiling; among these genes we further characterized the association of the upregulation of PRNP, the gene encoding cellular Prion protein (PrPc), with lung adenocarcinoma invasiveness. Immunohistochemistry on clinical specimens showed an association of PrPc expression with invasive but not in situ lung adenocarcinoma. Consistently, the expression of PrPc was higher in the highly invasive than in the lowly invasive lung adenocarcinoma cell lines. Knockdown of PrPc expression in cultured lung adenocarcinoma cells decreased their lamellipodium formation, in vitro migration and invasion, and in vivo experimental lung metastasis. Phosphorylation of JNKs was found to correlate with PrPc expression and the inhibition of JNKs suppressed the PrPc-induced up-regulation of lamellipodium formation, cell migration, and invasion. Moreover, we identified the nuclear factor, interleukin 3 regulated (NFIL3) protein as a transcriptional activator of the PRNP promoter. Accordingly, NFIL3 promoted lung cancer cell migration and invasion in a PrPc-dependent manner. High NFIL3 expression in clinical specimens of lung adenocarcinoma was also associated with tumor invasiveness. Overall, our observations suggest that the NFIL3/PrPc axis, through regulating lamellipodium formation and cell mobility via JNK signaling, plays a critical role in lung cancer invasiveness and metastasis.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/genética , Neoplasias Pulmonares/genética , Proteínas Priônicas/genética , Pseudópodes/genética , Animais , Movimento Celular/genética , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Hibridização In Situ , Neoplasias Pulmonares/patologia , Sistema de Sinalização das MAP Quinases/genética , Masculino , Camundongos , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Metástase Neoplásica , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Regiões Promotoras Genéticas/genética , Pseudópodes/patologia
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