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1.
Nat Commun ; 10(1): 2879, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253767

RESUMO

Drug development often relies on high-throughput cell-based screening of large compound libraries. However, the lack of miniaturized and parallelized methodologies in chemistry as well as strict separation and incompatibility of the synthesis of bioactive compounds from their biological screenings makes this process expensive and inefficient. Here, we demonstrate an on-chip platform that combines solution-based synthesis of compound libraries with high-throughput biological screenings (chemBIOS). The chemBIOS platform is compatible with both organic solvents required for the synthesis and aqueous solutions necessary for biological screenings. We use the chemBIOS platform to perform 75 parallel, three-component reactions to synthesize a library of lipidoids, followed by characterization via MALDI-MS, on-chip formation of lipoplexes, and on-chip cell screening. The entire process from the library synthesis to cell screening takes only 3 days and about 1 mL of total solutions, demonstrating the potential of the chemBIOS technology to increase efficiency and accelerate screenings and drug development.


Assuntos
Técnicas de Química Combinatória , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Células HEK293 , Humanos , Dispositivos Lab-On-A-Chip , Lipossomos , Bibliotecas de Moléculas Pequenas , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
2.
Nat Commun ; 10(1): 2209, 2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-31101809

RESUMO

Changes in bulk transcriptional profiles of heterogeneous samples often reflect changes in proportions of individual cell types. Several robust techniques have been developed to dissect the composition of such mixed samples given transcriptional signatures of the pure components or their proportions. These approaches are insufficient, however, in situations when no information about individual mixture components is available. This problem is known as the  complete deconvolution problem, where the composition is revealed without any a priori knowledge about cell types and their proportions. Here, we identify a previously unrecognized property of tissue-specific genes - their mutual linearity - and use it to reveal the structure of the topological space of mixed transcriptional profiles and provide a noise-robust approach to the complete deconvolution problem. Furthermore, our analysis reveals systematic bias of all deconvolution techniques due to differences in cell size or RNA-content, and we demonstrate how to address this bias at the experimental design level.


Assuntos
Biologia Computacional/métodos , Modelos Genéticos , Transcriptoma , Algoritmos , Animais , Tamanho Celular , Conjuntos de Dados como Assunto , Perfilação da Expressão Gênica/métodos , Células HEK293 , Humanos , Células Jurkat , Modelos Lineares , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Análise de Sequência de RNA/métodos
3.
Cytogenet Genome Res ; 157(4): 203-212, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31108493

RESUMO

Essential hypertension (EH), a major cause of cardiovascular diseases, is an important public health issue. However, the molecular mechanisms involved in EH remain unknown. Circular RNA (circRNA) is a novel promising biomarker for the disease. The purpose of the present study was to determine the expression of circRNAs in the blood of EH patients and to evaluate the performance of circRNA for early diagnosis of EH. A total of 178 subjects were recruited in the case-control study. Initial screening was done by using the Agilent human circRNA microarray followed by qRT-PCR validation. Finally, miRNAs were combined with circRNAs to create a new early prediction model for EH. The expression level of hsa_circ_0126991 in EH patients was significantly higher in comparison with healthy controls (p < 0.0001). Using the interaction of miR-10a-5p in combination with hsa_circ_0126991 led to a sensitivity of 0.708, a specificity of 0.764, and combined area under the curve of 0.774 (95% CI: 0.705-0.843) for early diagnosis of EH. In summary, the present study uncovered a novel perspective that hyperexpression of hsa_circ_0126991 is correlated with the risk of EH and may serve as a stable biomarker for early diagnosis of EH.


Assuntos
Biomarcadores/sangue , Hipertensão Essencial/diagnóstico , MicroRNAs/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , RNA/genética , Regulação para Cima , Idoso , Estudos de Casos e Controles , Diagnóstico Precoce , Hipertensão Essencial/sangue , Hipertensão Essencial/genética , Feminino , Perfilação da Expressão Gênica/métodos , Predisposição Genética para Doença , Humanos , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , RNA/sangue , Estudos Retrospectivos , Sensibilidade e Especificidade
4.
Nat Commun ; 10(1): 2045, 2019 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-31053733

RESUMO

Long noncoding RNAs (lncRNAs) have emerged as new regulatory molecules implicated in diverse biological processes, including therapeutic resistance. However, the mechanisms underlying lncRNA-mediated temozolomide (TMZ) resistance in glioblastoma (GBM) remain largely unknown. To illustrate the role of lncRNA in TMZ resistance, we induce TMZ-resistant GBM cells, perform a lncRNA microarray of the parental and TMZ-resistant cells, and find an unreported lncRNA in GBM, lnc-TALC (temozolomide-associated lncRNA in glioblastoma recurrence), correlated with TMZ resistance via competitively binding miR-20b-3p to facilitate c-Met expression. A phosphorylated AKT/FOXO3 axis regulated lnc-TALC expression in TMZ-resistant GBM cells. Furthermore, lnc-TALC increased MGMT expression by mediating the acetylation of H3K9, H3K27 and H3K36 in MGMT promoter regions through the c-Met/Stat3/p300 axis. In clinical patients, lnc-TALC is required for TMZ resistance and GBM recurrence. Our results reveal that lnc-TALC in GBM could serve as a therapeutic target to overcome TMZ resistance, enhancing the clinical benefits of TMZ chemotherapy.


Assuntos
Antineoplásicos Alquilantes/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Glioblastoma/tratamento farmacológico , MicroRNAs/metabolismo , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , RNA Longo não Codificante/metabolismo , Temozolomida/farmacologia , Adulto , Animais , Antineoplásicos Alquilantes/uso terapêutico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Feminino , Perfilação da Expressão Gênica/métodos , Glioblastoma/genética , Glioblastoma/mortalidade , Glioblastoma/patologia , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Análise de Sobrevida , Temozolomida/uso terapêutico , Proteínas Supressoras de Tumor/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
5.
BMC Genomics ; 20(1): 366, 2019 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-31088362

RESUMO

BACKGROUND: There has been a steady increase in the number of studies aiming to identify DNA methylation differences associated with complex phenotypes. Many of the challenges of epigenetic epidemiology regarding study design and interpretation have been discussed in detail, however there are analytical concerns that are outstanding and require further exploration. In this study we seek to address three analytical issues. First, we quantify the multiple testing burden and propose a standard statistical significance threshold for identifying DNA methylation sites that are associated with an outcome. Second, we establish whether linear regression, the chosen statistical tool for the majority of studies, is appropriate and whether it is biased by the underlying distribution of DNA methylation data. Finally, we assess the sample size required for adequately powered DNA methylation association studies. RESULTS: We quantified DNA methylation in the Understanding Society cohort (n = 1175), a large population based study, using the Illumina EPIC array to assess the statistical properties of DNA methylation association analyses. By simulating null DNA methylation studies, we generated the distribution of p-values expected by chance and calculated the 5% family-wise error for EPIC array studies to be 9 × 10- 8. Next, we tested whether the assumptions of linear regression are violated by DNA methylation data and found that the majority of sites do not satisfy the assumption of normal residuals. Nevertheless, we found no evidence that this bias influences analyses by increasing the likelihood of affected sites to be false positives. Finally, we performed power calculations for EPIC based DNA methylation studies, demonstrating that existing studies with data on ~ 1000 samples are adequately powered to detect small differences at the majority of sites. CONCLUSION: We propose that a significance threshold of P < 9 × 10- 8 adequately controls the false positive rate for EPIC array DNA methylation studies. Moreover, our results indicate that linear regression is a valid statistical methodology for DNA methylation studies, despite the fact that the data do not always satisfy the assumptions of this test. These findings have implications for epidemiological-based studies of DNA methylation and provide a framework for the interpretation of findings from current and future studies.


Assuntos
Metilação de DNA , Epigenômica/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Ilhas de CpG , Epigênese Genética , Estudo de Associação Genômica Ampla , Humanos , Modelos Lineares
6.
BMC Genomics ; 20(1): 376, 2019 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-31088363

RESUMO

BACKGROUND: Copy number variations (CNVs), which are genetic variations present throughout mammalian genomes, are a vital source of phenotypic variation that can lead to the development of unique traits. In this study we used the Illunima BovineHD BeadChip to conduct genome-wide detection of CNVs in 215 polled yaks. RESULTS: A total of 1066 CNV regions (CNVRs) were detected with a total length of 181.6 Mb, comprising ~ 7.2% of the bovine autosomal genome. The size of these CNVRs ranged from 5.53 kb to 1148.45 kb, with an average size of 170.31 kb. Eight out of nine randomly chosen CNVRs were successfully validated by qPCR. A functional enrichment analysis of the CNVR-associated genes indicated their relationship to a number of molecular adaptations that enable yaks to thrive at high altitudes. One third of the detected CNVRs were mapped to QTLs associated with six classes of economically important traits, indicating that these CNVRs may play an important role in variations of these traits. CONCLUSIONS: Our genome-wide yak CNV map may thus provide valuable insights into both the molecular mechanisms of high altitude adaptation and the potential genomic basis of economically important traits in yak.


Assuntos
Variações do Número de Cópias de DNA , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Locos de Características Quantitativas , Animais , Bovinos , Mapeamento Cromossômico , Feminino , Fenótipo , Polimorfismo de Nucleotídeo Único
7.
World J Gastroenterol ; 25(13): 1580-1591, 2019 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-30983818

RESUMO

BACKGROUND: Early gastric cancer (EGC), compared with advanced gastric cancer (AGC), has a higher 5-year survival rate. However, due to the lack of typical symptoms and the difficulty in diagnosing EGC, no effective biomarkers exist for the detection of EGC, and gastroscopy is the only detection method. AIM: To provide new biomarkers with high specificity and sensitivity through analyzed the differentially expressed microRNAs (miRNAs) in EGC and AGC and compared them with those in benign gastritis (BG). METHODS: We examined the differentially expressed miRNAs in the plasma of 30 patients with EGC, AGC, and BG by miRNA chip analysis. Then, we analyzed and selected the significantly different miRNAs using bioinformatics. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) confirmed the relative transcription level of these miRNAs in another 122 patients, including patients with EGC, AGC, Helicobacter pylori (H. pylori)-negative gastritis (Control-1), and H. pylori-positive atrophic gastritis (Control-2). To establish a diagnostic model for the detection of plasma miRNA in EGC, we chose miRNAs that can be used to determine EGC and AGC from Control-1 and Control-2 and miRNAs in EGC from all other groups. RESULTS: Among the expression profiles of the miRNA chips in the three groups in the discovery set, of 117 aberrantly expressed miRNAs, 30 confirmed target prediction, whereas 14 were included as potential miRNAs. The RT-qPCR results showed that 14 potential miRNAs expression profiles in the two groups exhibited no differences in terms of H. pylori-negative gastritis (Control-1) and H. pylori-positive atrophic gastritis (Control-2). Hence, these two groups were incorporated into the Control group. A combination of four types of miRNAs, miR-7641, miR-425-5p, miR-1180-3p and miR-122-5p, were used to effectively distinguish the Cancer group (EGC + AGC) from the Control group [area under the curve (AUC) = 0.799, 95% confidence interval (CI): 0.691-0.908, P < 0.001]. Additionally, miR-425-5p, miR-24-3p, miR-1180-3p and miR-122-5p were utilized to distinguish EGC from the Control group (AUC = 0.829, 95%CI: 0.657-1.000, P = 0.001). Moreover, the miR-24-3p expression level in EGC was lower than that in the AGC (AUC = 0.782, 95%CI: 0.571-0.993, P = 0.029), and the miR-4632-5p expression level in EGC was significantly higher than that in AGC (AUC = 0.791, 95%CI: 0.574-1.000, P = 0.024). CONCLUSION: The differentially expressed circulatory plasma miR-425-5p, miR-1180-3p, miR-122-5p, miR-24-3p and miR-4632-5p can be regarded as a new potential biomarker panel for the diagnosis of EGC. The prediction and early diagnosis of EGC can be considerably facilitated by combining gastroscopy with the use of these miRNA biomarkers, thereby optimizing the strategy for effective detection of EGC. Nevertheless, larger-scale human experiments are still required to confirm our findings.


Assuntos
Biomarcadores Tumorais/sangue , Detecção Precoce de Câncer/métodos , MicroRNAs/sangue , Neoplasias Gástricas/diagnóstico , Adulto , Idoso , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/isolamento & purificação , Biópsia , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Masculino , MicroRNAs/genética , MicroRNAs/isolamento & purificação , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Curva ROC , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Estômago/patologia , Neoplasias Gástricas/sangue , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia
8.
Transplant Proc ; 51(3): 722-728, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30979456

RESUMO

TruGraf v1 is a laboratory-developed DNA microarray-based gene expression blood test to enable proactive noninvasive serial assessment of kidney transplant recipients with stable renal function. It has been previously validated in patients identified as Transplant eXcellence (TX: stable serum creatinine, normal biopsy results, indicative of immune quiescence), and not-TX (renal dysfunction and/or rejection on biopsy results). TruGraf v1 is intended for use in subjects with stable renal function to measure the immune status as an alternative to invasive, expensive, and risky surveillance biopsies. MATERIALS AND METHODS: In this study, simultaneous blood tests and clinical assessments were performed in 192 patients from 7 transplant centers to evaluate TruGraf v1. The molecular testing laboratory was blinded to renal function and biopsy results. RESULTS: Overall, TruGraf v1 accuracy (concordance between TruGraf v1 result and clinical and/or histologic assessment) was 74% (142/192), and a result of TX was accurate in 116 of 125 (93%). The negative predictive value for TruGraf v1 was 90%, with a sensitivity 74% and specificity of 73%. Results did not significantly differ in patients with a biopsy-confirmed diagnosis vs those without a biopsy. CONCLUSIONS: TruGraf v1 can potentially support a clinical decision enabling unnecessary surveillance biopsies with high confidence, making it an invaluable addition to the transplant physician's tool kit for managing patients. TruGraf v1 testing can potentially avoid painful and risky invasive biopsies, reduce health care costs, and enable frequent assessment of patients with stable renal function to confirm the presence of immune quiescence in the peripheral blood.


Assuntos
Perfilação da Expressão Gênica/métodos , Rejeição de Enxerto/diagnóstico , Transplante de Rim , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Adulto , Biópsia , Feminino , Rejeição de Enxerto/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Sensibilidade e Especificidade , Transplantados
9.
Transplant Proc ; 51(3): 729-733, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30979457

RESUMO

BACKGROUND: TruGraf v1 is a well-validated DNA microarray-based test that analyzes blood gene expression profiles as an indicator of immune status in kidney transplant recipients with stable renal function. METHODS: In this study, investigators assessed clinical utility of the TruGraf test in patient management. In a retrospective study, simultaneous blood tests and clinical assessments were performed in 192 patients at 7 transplant centers, and in a prospective observational study they were performed in 45 subjects at 5 transplant centers. RESULTS: When queried regarding whether or not the TruGraf test result impacted their decision regarding patient management, in 168 of 192 (87.5%) cases the investigator responded affirmatively. The prospective study indicated that TruGraf results supported physicians' decisions on patient management 87% (39/45) of the time, and in 93% of cases physicians indicated that they would use serial TruGraf testing in future patient management. A total of 21 of 39 (54%) reported results confirmed their decision that no intervention was needed, and 17 of 39 (44%) reported that results specifically informed them that a decision not to perform a surveillance biopsy was correct. CONCLUSIONS: TruGraf is the first and only noninvasive test to be evaluated for clinical utility in determining rejection status of patients with stable renal function and shows promise of providing support for clinical decisions to avoid unnecessary surveillance biopsies with a high degree of confidence. TruGraf is an invaluable addition to the transplant physician's tool kit for managing patient health by avoiding painful and invasive biopsies, reducing health care costs, and enabling frequent assessment of patients with stable renal function to confirm immune quiescence.


Assuntos
Perfilação da Expressão Gênica/métodos , Rejeição de Enxerto/diagnóstico , Transplante de Rim , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Biópsia , Tomada de Decisões , Feminino , Rejeição de Enxerto/sangue , Rejeição de Enxerto/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Patologia Molecular/métodos , Médicos , Estudos Prospectivos , Estudos Retrospectivos
10.
Med Sci Monit ; 25: 2488-2504, 2019 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-30948703

RESUMO

BACKGROUND Globally, gastric cancer (GC) is the third most common source of cancer-associated mortality. The aim of this study was to identify key genes and circular RNAs (circRNAs) in GC diagnosis, prognosis, and therapy and to further explore the potential molecular mechanisms of GC. MATERIAL AND METHODS Differentially expressed genes (DEGs) and circRNAs (DE circRNAs) between GC tissues and adjacent non-tumor tissues were identified from 3 mRNA and 3 circRNA expression profiles. Functional analyses were performed, and protein-protein interaction (PPI) networks were constructed. The significant modules and key genes in the PPI networks were identified. Kaplan-Meier analysis was performed to evaluate the prognostic value of these key genes. Potential miRNA-binding sites of the DE circRNAs and target genes of these miRNAs were predicted and used to construct DE circRNA-miRNA-mRNA networks. RESULTS A total of 196 upregulated and 311 downregulated genes were identified in GC. The results of functional analysis showed that these DEGs were significantly enriched in a variety of functions and pathways, including extracellular matrix-related pathways. Ten hub genes (COL1A1, COL3A1, COL1A2, COL5A2, FN1, THBS1, COL5A1, SPARC, COL18A1, and COL11A1) were identified via PPI network analysis. Kaplan-Meier analysis revealed that 7 of these were associated with a poor overall survival in GC patients. Furthermore, we identified 2 DE circRNAs, hsa_circ_0000332 and hsa_circ_0021087. To reveal the potential molecular mechanisms of circRNAs in GC, DE circRNA-microRNA-mRNA networks were constructed. CONCLUSIONS Key candidate genes and circRNAs were identified, and novel PPI and circRNA-microRNA-mRNA networks in GC were constructed. These may provide useful information for the exploration of potential biomarkers and targets for the diagnosis, prognosis, and therapy of GC.


Assuntos
RNA/genética , Neoplasias Gástricas/genética , Biomarcadores , Biologia Computacional/métodos , Regulação Neoplásica da Expressão Gênica/genética , Redes Reguladoras de Genes/genética , Humanos , Estimativa de Kaplan-Meier , MicroRNAs/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Prognóstico , Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas/genética , RNA/metabolismo , RNA Mensageiro/genética
11.
Med Sci Monit ; 25: 2896-2907, 2019 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-31004080

RESUMO

Worldwide, metastatic melanoma of the skin has an aggressive course with high morbidity and mortality. Therefore, an increased understanding of the pathogenesis of metastatic melanoma has gained increasing attention, including the role of epigenetic modification and competing endogenous RNA (ceRNA). This study aimed to used bioinformatics data to undertake an integrative analysis of long noncoding RNA (lncRNA), microRNA (miRNA) and mRNA expression to construct a ceRNA network in metastatic melanoma. Data from the Cancer Genome Atlas (TCGA), the Gene Ontology (GO) database, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway were analyzed. There were 471 cases that included 103 primary solid tumors and 368 cases of metastatic melanoma that included transcriptome sequencing data (including lncRNA and mRNA); 452 cases had miRNA sequencing data. Analysis of chip data identified 85 6 mRNAs, 67 miRNAs, and 250 lncRNAs that were differentially expressed in cases of metastatic melanoma, of which 25 miRNAs, 18 lncRNAs, and 18 mRNAs participated in the formation of ceRNAs. Survival analysis identified seven differentially expressed mRNAs, five differentially expressed miRNAs (miRNA-29c, miRNA-100, miR-142-3p, miR-150, miR-516a-2), and six differentially expressed lncRNAs (AC068594.1, C7orf71, FAM41C, GPC5-AS1, MUC19, LINC00402) that were correlated with survival time in patients with metastatic melanoma. Bioinformatics data and integrative analysis identified lncRNA, miRNA, and mRNA expression to construct a ceRNA and patient survival network in metastatic melanoma. These findings support the need for further studies on the mechanisms involved in the regulation of metastatic melanoma by ceRNAs.


Assuntos
Melanoma/genética , MicroRNAs/biossíntese , RNA Mensageiro/biossíntese , Neoplasias Cutâneas/genética , Biologia Computacional/métodos , Bases de Dados Genéticas , Ontologia Genética , Redes Reguladoras de Genes , Humanos , Estimativa de Kaplan-Meier , Melanoma/metabolismo , MicroRNAs/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , RNA Longo não Codificante/genética , RNA Mensageiro/genética , Neoplasias Cutâneas/metabolismo , Análise de Sobrevida , Transcriptoma
12.
Methods Mol Biol ; 1979: 227-234, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31028641

RESUMO

Single-cell genome sequencing can detect low-frequency genetic alterations present in complex tissues. However, the experimental procedures are technically challenging. This includes dissociation of the tissue, isolation of single cells, whole-genome amplification, sequencing library preparation, and an optional target enrichment. Here we describe how to perform each of these processes to obtain high-quality single-cell genome sequencing data.


Assuntos
Separação Celular/métodos , Genômica/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Análise de Célula Única/métodos , Animais , DNA/genética , Variações do Número de Cópias de DNA , Biblioteca Gênica , Genoma , Humanos , Técnicas de Amplificação de Ácido Nucleico/métodos
13.
Comput Biol Chem ; 80: 121-127, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30947070

RESUMO

DNA microarray data has been widely used in cancer research due to the significant advantage helped to successfully distinguish between tumor classes. However, typical gene expression data usually presents a high-dimensional imbalanced characteristic, which poses severe challenge for traditional machine learning methods to construct a robust classifier performing well on both the minority and majority classes. As one of the most successful feature weighting techniques, Relief is considered to particularly suit to handle high-dimensional problems. Unfortunately, almost all relief-based methods have not taken the class imbalance distribution into account. This study identifies that existing Relief-based algorithms may underestimate the features with the discernibility ability of minority classes, and ignore the distribution characteristic of minority class samples. As a result, an additional bias towards being classified into the majority classes can be introduced. To this end, a new method, named imRelief, is proposed for efficiently handling high-dimensional imbalanced gene expression data. imRelief can correct the bias towards to the majority classes, and consider the scattered distributional characteristic of minority class samples in the process of estimating feature weights. This way, imRelief has the ability to reward the features which perform well at separating the minority classes from other classes. Experiments on four microarray gene expression data sets demonstrate the effectiveness of imRelief in both feature weighting and feature subset selection applications.


Assuntos
Algoritmos , Biologia Computacional/métodos , Expressão Gênica , Neoplasias/classificação , Neoplasias/genética , Bases de Dados Genéticas/estatística & dados numéricos , Humanos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Máquina de Vetores de Suporte
14.
Sensors (Basel) ; 19(7)2019 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-30987195

RESUMO

Temperature control is the most important and fundamental part of a polymerase chain reaction (PCR). To date, there have been several methods to realize the periodic heating and cooling of the thermal-cycler system for continuous-flow PCR reactions, and three of them were widely used: the thermo-cycled thermoelectric cooler (TEC), the heating block, and the thermostatic heater. In the present study, a new approach called open-loop controlled single thermostatic TEC was introduced to control the thermal cycle during the amplification process. Differing from the former three methods, the size of this microdevice is much smaller, especially when compared to the microdevice used in the heating block method. Furthermore, the rising and cooling speed of this method is much rapider than that in a traditional TEC cycler, and is nearly 20-30% faster than a single thermostatic heater. Thus, a portable PCR system was made without any external heat source, and only a Teflon tube-wrapped TEC chip was used to achieve the continuous-flow PCR reactions. This provides an efficient way to reduce the size of the system and simplify it. In addition, through further experiments, the microdevice is not only found to be capable of amplification of a PCR product from Human papillomavirus type 49 (Genbank ref: X74480.1) and Rubella virus (RUBV), but also enables clinical diagnostics, such as a test for hepatitis B virus.


Assuntos
Vírus da Hepatite B/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Vírus da Rubéola/isolamento & purificação , Viroses/diagnóstico , Fontes de Energia Elétrica , Calefação , Vírus da Hepatite B/genética , Vírus da Hepatite B/patogenicidade , Humanos , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reação em Cadeia da Polimerase/instrumentação , Vírus da Rubéola/genética , Vírus da Rubéola/patogenicidade , Temperatura Ambiente , Viroses/virologia
15.
BMC Infect Dis ; 19(1): 289, 2019 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-30922257

RESUMO

BACKGROUND: Purulent meningitis (PM) is a serious life-threatening infection of the central nervous system (CNS) by bacteria or fungi and associated with high mortality and high incidence of CNS sequelae in children. However, the conventional cerebrospinal fluid (CSF) culture method is time-consuming and has a low sensitivity. METHODS: Our study developed a real-time PCR-based purulent meningitis-TaqMan array card (PM-TAC) that targeted 21 PM-related pathogens and could produce results within 3 h. Primers and probes were adapted from published sources possibly. The performance of them were evaluated and optimized and then they were spotted on TAC. RESULTS: The PM-TAC showed a sensitivity and specificity of 95 and 96%, respectively. For all of the 21 targeted pathogens, the PM-TAC assay had a LOD ranging from 5 copies/reaction to 100 copies/reaction, an intra-assay variation of 0.07-4.45%, and an inter-assay variation of 0.11-6.81%. Of the 15 CSF samples collected from patients with PM after empiric antibiotic therapies, the positive rate was 53.3% (8/15) for our PM-TAC assay but was only 13.3% (2/15) for the CSF culture method. Of the 17 CSF samples showing negative CSF culture, the PM-TAC assay identified a case of Neisseria meningitidis infection. Furthermore, all of the 10 CSF samples from patients without CNS infection showed negative for the PM-TAC assay. CONCLUSIONS: Our PM-TAC assay also demonstrated that the pathogen loads in the CSF samples correlated with the severity of PM. Thus, the PM-TAC may be helpful to improve the prognosis of PM and clinical outcomes from antibiotic therapies.


Assuntos
Bactérias/genética , Fungos/genética , Meningites Bacterianas/microbiologia , Meningite Fúngica/microbiologia , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Bactérias/classificação , Bactérias/isolamento & purificação , Bactérias/patogenicidade , Líquido Cefalorraquidiano/microbiologia , Criança , Primers do DNA/genética , Feminino , Fungos/isolamento & purificação , Fungos/patogenicidade , Humanos , Masculino , Meningites Bacterianas/líquido cefalorraquidiano , Meningite Fúngica/líquido cefalorraquidiano , Neisseria meningitidis/genética , Neisseria meningitidis/isolamento & purificação , Sensibilidade e Especificidade
16.
Oncol Rep ; 41(5): 3089-3099, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30896887

RESUMO

Piwi­interacting RNAs (piRNAs) comprise the largest class of non­coding RNAs. They represent a molecular feature shared by all non­aging biological systems, including germline and somatic cancer stem cells, which display an indefinite capacity of renewal and proliferation and are potentially immortal. They have been identified in animal stomachs, but their relationship with human gastric cancers remains largely unclear. The present study aimed to identify the piRNAs associated with human gastric cancers across the whole transcriptome. Fresh tumor tissues and adjacent non­tumorous tissues from stomachs were examined using a piRNA microarray (23,677 piRNAs) that was then validated by qPCR. The differential expression of piRNAs between cases and controls was analyzed. The transposable elements (TEs) that are potentially targeted by the risk piRNAs were searched. The expression of the nearest genes that are complementary to the sequences of the piRNAs was examined in the stomach tissue. The regulatory effects of genome­wide significant and replicated cancer­risk DNA variants on the piRNA expression in stomach were tested. Based on the findings, we identified a total of 8,759 piRNAs in human stomachs. Of all, 50 were significantly (P<0.05) and differentially (>2­fold change) expressed between the cases and controls, and 64.7% of the protein­coding genes potentially regulated by the gastric cancer­associated piRNAs were expressed in the human stomach. The expression of many cancer­associated piRNAs was correlated with the genome­wide and replicated cancer­risk SNPs. In conclusion, we conclude that piRNAs are abundant in human stomachs and may play important roles in the etiological processes of gastric cancers.


Assuntos
Regulação Neoplásica da Expressão Gênica , RNA Interferente Pequeno/genética , Neoplasias Gástricas/genética , Transcriptoma/genética , Adulto , Idoso , Animais , Estudos de Coortes , Elementos de DNA Transponíveis/genética , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Polimorfismo de Nucleotídeo Único/genética , RNA Interferente Pequeno/metabolismo , Estômago/patologia , Neoplasias Gástricas/patologia
17.
Genes Genomics ; 41(5): 573-581, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30830681

RESUMO

BACKGROUND: Short hairpin RNAs (shRNAs) expressed from vectors have been used as an effective means of exploiting the RNA interference (RNAi) pathway in mammalian cells. Genome-scale screening with shRNA libraries has been used to investigate the relationship between genotypes and phenotypes on a large scale. Although several methods have been developed to construct shRNA libraries, their broad application has been limited by the high cost of constructing these libraries. OBJECTIVE: We develop a new method that efficiently constructs a shRNA library at low cost, using treatments with several enzymes and an oligonucleotide library. METHODS: The library of shRNA expression cassettes, which were cloned into a lentiviral plasmid, was produced through several enzymatic reactions, starting from a library of 20,000 different short oligonucleotides produced by microarray-based oligonucleotide synthesis. RESULTS: The NGS sequence analysis of the library shows that 99.8% of them (19,956 from 20,000 sequences) were contained in the library: 63.2% of them represent the correct sequences and the rest showed one or two base pair differences from the expected sequences. CONCLUSION: Considering the ease of our method, shRNA libraries of new genomes and of specific populations of genes can be prepared in a short period of time for genome-scale RNAi library screening.


Assuntos
Técnicas de Química Combinatória/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , RNA Interferente Pequeno/genética , Animais , Biblioteca Gênica , Humanos , Oligonucleotídeos , Interferência de RNA/fisiologia , RNA Interferente Pequeno/síntese química
18.
Int J Oncol ; 54(5): 1579-1590, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30896785

RESUMO

The incidence of recurrent t(6;9) translocation of the MYB proto­oncogene to NFIB (the gene that encodes nuclear factor 1 B­type) in adenoid cystic carcinoma (ACC) tumour tissues is high. However, MYB [the gene that encodes transcriptional activator Myb (MYB)] overexpression is more common, indicating that MYB serves a key role in ACC. The current study aimed to investigate the role of MYB in salivary (S)ACC growth and metastasis. A total of 50 fresh­frozen SACC tissues and 41 fresh­frozen normal submandibular gland (SMG) tissues were collected to measure MYB mRNA expression, and to analyse the associations between MYB and epithelial­mesenchymal transition (EMT) markers. Compared with normal SMG tissue, SACC tissues demonstrated significantly increased MYB expression, with a high expression rate of 90%. Interestingly, MYB tended to be negatively correlated with CDH1 [the gene that encodes cadherin­1 (E­cadherin)] and positively correlated with VIM (the gene that encodes vimentin), suggesting that MYB is associated with SACC metastasis. To explore the role of MYB in SACC, the authors stably overexpressed and knocked down MYB in SACC cells. The authors of the current study demonstrated that MYB overexpression promoted SACC cell proliferation, migration and invasion, whereas its knockdown inhibited these activities. Additionally, when MYB was overexpressed, CDH1 expression was downregulated, and CDH2 (the gene that encodes cadherin­2), VIM and ACTA2 (the gene that encodes actin, aortic smooth muscle) expression was upregulated. Then, the effect of MYB on lung tumour metastasis was investigated in vivo in non­obese diabetic/severe combined immunodeficiency mice. MYB overexpressing and control cells were injected into the mice through the tail vein. The results revealed that MYB promoted SACC lung metastasis. Collectively, these results demonstrated that MYB is aberrantly overexpressed in SACC tissues, and promotes SACC cell proliferation and metastasis, indicating that MYB may be a novel therapeutic target for SACC.


Assuntos
Carcinoma Adenoide Cístico/patologia , Perfilação da Expressão Gênica/métodos , Neoplasias Pulmonares/secundário , Proteínas Proto-Oncogênicas c-myb/genética , Neoplasias das Glândulas Salivares/patologia , Regulação para Cima , Animais , Carcinoma Adenoide Cístico/genética , Carcinoma Adenoide Cístico/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Metástase Neoplásica , Transplante de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Proteínas Proto-Oncogênicas c-myb/metabolismo , Neoplasias das Glândulas Salivares/genética , Neoplasias das Glândulas Salivares/metabolismo
19.
Methods Mol Biol ; 1955: 203-214, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30868529

RESUMO

MicroRNAs (miRNAs) are a class of small noncoding RNAs (typically 19-23 nucleotides) which act by annealing to partially complementary binding sites present on the 3' untranslated regions (UTR) of messenger RNAs (mRNAs) leading to inhibition of protein translation or by inducing mRNA decay. Since their discovery, miRNAs have come to be recognized as master regulators of gene expression in plant and mammals, controlling tissue-specific protein expression. Up to one-third of mammalian mRNAs are susceptible to miRNA-mediated regulation. It has been shown that miRNAs are determinants of the physiology and pathophysiology of the cardiovascular system, and altered expression of muscle- and/or cardiac-specific miRNAs in myocardial tissue is involved in heart development and cardiovascular diseases, including myocardial hypertrophy, heart failure, and fibrosis. The analysis of miRNA expression pattern provides important information, as well as is a starting point to understand miRNA function in different tissues, during development, and in disease. Several techniques can be used for miRNA profiling analysis like high-throughput sequencing, microarrays, and real-time PCR using microfluidic low-density arrays. This chapter describes the complete methodology to perform miRNA profiling using the stem-loop reverse-transcription (RT)-based TaqMan® MicroRNA low-density arrays (TLDA) method. This methodology was used to perform miRNA profiling in the heart of T. cruzi acutely infected mice.


Assuntos
Doença de Chagas/genética , Perfilação da Expressão Gênica/métodos , Coração/parasitologia , MicroRNAs/genética , Transcriptoma , Trypanosoma cruzi/fisiologia , Animais , Doença de Chagas/parasitologia , Feminino , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Interações Hospedeiro-Parasita , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Trypanosoma cruzi/isolamento & purificação
20.
Gene ; 698: 82-91, 2019 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-30825599

RESUMO

Differential co-expression is a cutting-edge approach to analyze gene expression data and identify both shared and divergent expression patterns. The availability of high-throughput gene expression datasets and efficient computational approaches have unfolded the opportunity to a systems level understanding of functional genomics of different stresses with respect to plants. We performed the meta-analysis of the available microarray data for reoviridae and sequiviridae infection in rice with the aim to identify the shared gene co-expression profile. The microarray data were downloaded from ArrayExpress and analyzed through a modified Weighted Gene Co-expression Network Analysis (WGCNA) protocol. WGCNA clustered the genes based on the expression intensities across the samples followed by identification of modules, eigengenes, principal components, topology overlap, module membership and module preservation. The module preservation analysis identified 4 modules; salmon (638 genes), midnightblue (584 genes), lightcyan (686 genes) and red (562 genes), which are highly preserved in both the cases. The networks in case of reoviridae infection showed neatly packed clusters whereas, in sequiviridae, the clusters were loosely connected which is due to the differences in the correlation values. We also identified 83 common transcription factors targeting the hub genes from all the identified modules. This study provides a coherent view of the comparative aspect of the expression of common genes involved in different virus infections which may aid in the identification of novel targets and development of new intervention strategy against the virus.


Assuntos
Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica de Plantas/genética , Oryza/genética , Biologia Computacional/métodos , Redes Reguladoras de Genes/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reoviridae/patogenicidade , Infecções por Reoviridae/genética , Sequiviridae/patogenicidade , Transcriptoma/genética , Viroses/genética
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