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1.
An Acad Bras Cienc ; 92(suppl 1): e20180426, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32159585

RESUMEN

Effective microorganisms (EM) are inoculants formed by fungi and bacteria isolated from soil. EM are commonly used by farmers on agronomic crops to stimulate plant growth, but their composition and their benefits has been controverted. This study aimed to analyze the diversity of microorganisms growing in three EM inoculants, as well as to evaluate their efficiency in the germination of palisade grass seeds. The total DNA of the three EM inoculants was extracted, the 16S rRNA and ITS genes were amplified by PCR and sequenced on the Illumina MiSeq platform. Germination tests were conducted with three type of the EM, in three concentration and two times of the immersion. The bacterial group was the most abundant in EM, followed by fungi. Bacterial operational taxonomic units OTUs were shared by all EMs. Pre-treatments of palisade grass seeds with EMs resulted in a higher germination percentage (% G) and germination speed index (IVG) when EM was used at concentration of 1 or 2% in water. Seed immersion for 5 min was more efficient than immersion for 24 h. We can conclude that EM of different origin can share microbial groups and diversity of microorganisms, besides being an alternative to increase palisade grass seeds germination.


Asunto(s)
Inoculantes Agrícolas/genética , ADN Bacteriano/aislamiento & purificación , ADN de Hongos/aislamiento & purificación , Germinación/fisiología , Poaceae/crecimiento & desarrollo , Semillas/crecimiento & desarrollo , Biodiversidad , Productos Agrícolas/genética , Germinación/efectos de los fármacos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Semillas/efectos de los fármacos , Análisis de Secuencia de ADN , Análisis de Secuencia de ARN , Ácidos Sulfúricos/farmacología
2.
Medicine (Baltimore) ; 99(12): e19577, 2020 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-32195970

RESUMEN

RATIONALE: The diagnosis of anaplastic lymphoma kinase (ALK)-negative inflammatory myofibroblastic tumors (IMT) remains challenging because of their morphological resemblance with spindle cell sarcoma with myofibroblastic characteristics. PATIENT CONCERNS: A 69-year-old female patient presented with loco-regional recurrent IMT several times within 8 years after primary treatment and neck lymph node metastasis 3.5 years after last recurrence. DIAGNOSIS: The primary, recurrence, and lymph node metastasis lesions were diagnosed as ALK-negative IMTs based on the histopathological features. INTERVENTIONS: Biopsy samples were obtained during repeated surgeries and evaluated for genomic alterations during first and recurrent presentations. The evaluation was done using pathway-driven massive parallel sequencing, and genomic alterations between primary and recurrent tumors were compared. OUTCOMES: Copy number gains and overexpression of mouse double minute 2 homolog (MDM2) and cyclin dependent kinase 4 (CDK4) were observed in the primary lesion, and additional gene amplification of Discoidin Domain Receptor Tyrosine Kinase 2 (DDR2), Succinate Dehydrogenase Complex II subunit C (SDHC), and thyroid stimulating hormone receptor (TSHR) Q720H were found in the recurrent tumors. Metastases to the neck lymph node were observed 3.5 years after recurrence. LESSONS: Our results indicated genetic evolution in a microscopically benign condition and highlighted the importance of molecular characterization of fibro-inflammatory lesions of uncertain malignant potential.


Asunto(s)
Granuloma de Células Plasmáticas/metabolismo , Neoplasias de Cabeza y Cuello/secundario , Recurrencia Local de Neoplasia/patología , Neoplasias de Tejido Muscular/metabolismo , Quinasa de Linfoma Anaplásico/metabolismo , Quinasa 4 Dependiente de la Ciclina/metabolismo , Diagnóstico Diferencial , Femenino , Amplificación de Genes , Granuloma de Células Plasmáticas/patología , Neoplasias de Cabeza y Cuello/patología , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Metástasis Linfática , Mediastino/patología , Persona de Mediana Edad , Miofibroblastos/patología , Neoplasias de Tejido Muscular/patología , Neoplasias de Tejido Muscular/radioterapia , Proteínas Proto-Oncogénicas c-mdm2/metabolismo
3.
Med Sci Monit ; 26: e920854, 2020 Mar 22.
Artículo en Inglés | MEDLINE | ID: mdl-32200387

RESUMEN

BACKGROUND Hepatocellular carcinoma (HCC) is one of the most prevalent cancers in the world. Bioinformatics studies have been widely used for screening genes involved in the initiation and progression of HCC. MATERIAL AND METHODS We obtained liver cancer microarray raw data from the GEO database (GSE54238). Next, weighted gene co-expression network analysis (WGCNA) was used to assess the critical modules. Then, we assessed the gene significance by calculating survival, expression level, and receiver operating characteristic (ROC) in the TCGA database. We also validated the expression of selected genes in the Oncomine database and calculated the relationship between 4 hub genes and immune infiltration. Finally, GSEA enrichment analysis was used to explore the potential mechanism. RESULTS We identified the red and blue modules as the critical modules, and found 176 candidate genes by assessing gene significance. GO and KEEG results suggested that the candidate genes are involved in the cell cycle. Four hub genes - SOX4, STK39, TARBP1, and TDRKH - were eventually screened after validating their expression and power in diagnosing HCC in the TCGA database. Immune infiltration analysis and GSEA enrichment analysis showed that these 4 hub genes were correlated with the immune cell populations infiltration and that multiple mechanisms were involved, such as angiogenesis and epithelial-mesenchymal transition. CONCLUSIONS Our findings revealed that these 4 genes can be regarded as potential prognosticators and therapeutic targets for HCC.


Asunto(s)
Carcinoma Hepatocelular/genética , Regulación Neoplásica de la Expresión Génica/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Neoplasias Hepáticas/genética , Biomarcadores de Tumor/genética , Carcinoma Hepatocelular/mortalidad , Biología Computacional/métodos , Perfilación de la Expresión Génica/métodos , Redes Reguladoras de Genes , Humanos , Neoplasias Hepáticas/mortalidad , Curva ROC
4.
Gene ; 736: 144412, 2020 Apr 30.
Artículo en Inglés | MEDLINE | ID: mdl-32007586

RESUMEN

The emergence of somaclonal variability in in vitro cultures is undesirable during micropropagation, but this phenomenon may be a source of genetic variability sought by breeders. The main factors that affect the appearance of variability are known, but the exact mechanism has not yet been determined. In this paper, we used next-generation sequencing and comparative genomics to study changes in the genomes of cucumber lines resulting from in vitro regeneration and somaclonal mutation in comparison to a reference, the highly inbred B10 line. The total number of obtained polymorphisms differed between the three somaclonal lines S1, S2 and S3, with 8369, 7591 and 44510, respectively. Polymorphisms occurred most frequently in non-coding regions and were mainly SNPs. High-impact changes accounted for 1%-3% of all polymorphisms and most often caused an open reading frame shift. Functional analysis of genes affected by high impact variants showed that they were related to transport, biosynthetic processes, nucleotide-containing compounds and cellular protein modification processes. The obtained results indicated significant factors affecting somaclonal variability and the appearance of changes in the genome, and demonstrated a lack of dependence between phenotype and the number of genomic polymorphisms.


Asunto(s)
Cucumis sativus/genética , ADN/genética , Genoma de Planta/genética , Mutación/genética , Polimorfismo de Nucleótido Simple/genética , Estudio de Asociación del Genoma Completo , Genómica/métodos , Genotipo , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Sistemas de Lectura Abierta/genética , Fenotipo , Análisis de Secuencia de ADN/métodos
5.
Lancet ; 395(10224): 565-574, 2020 02 22.
Artículo en Inglés | MEDLINE | ID: mdl-32007145

RESUMEN

BACKGROUND: In late December, 2019, patients presenting with viral pneumonia due to an unidentified microbial agent were reported in Wuhan, China. A novel coronavirus was subsequently identified as the causative pathogen, provisionally named 2019 novel coronavirus (2019-nCoV). As of Jan 26, 2020, more than 2000 cases of 2019-nCoV infection have been confirmed, most of which involved people living in or visiting Wuhan, and human-to-human transmission has been confirmed. METHODS: We did next-generation sequencing of samples from bronchoalveolar lavage fluid and cultured isolates from nine inpatients, eight of whom had visited the Huanan seafood market in Wuhan. Complete and partial 2019-nCoV genome sequences were obtained from these individuals. Viral contigs were connected using Sanger sequencing to obtain the full-length genomes, with the terminal regions determined by rapid amplification of cDNA ends. Phylogenetic analysis of these 2019-nCoV genomes and those of other coronaviruses was used to determine the evolutionary history of the virus and help infer its likely origin. Homology modelling was done to explore the likely receptor-binding properties of the virus. FINDINGS: The ten genome sequences of 2019-nCoV obtained from the nine patients were extremely similar, exhibiting more than 99·98% sequence identity. Notably, 2019-nCoV was closely related (with 88% identity) to two bat-derived severe acute respiratory syndrome (SARS)-like coronaviruses, bat-SL-CoVZC45 and bat-SL-CoVZXC21, collected in 2018 in Zhoushan, eastern China, but were more distant from SARS-CoV (about 79%) and MERS-CoV (about 50%). Phylogenetic analysis revealed that 2019-nCoV fell within the subgenus Sarbecovirus of the genus Betacoronavirus, with a relatively long branch length to its closest relatives bat-SL-CoVZC45 and bat-SL-CoVZXC21, and was genetically distinct from SARS-CoV. Notably, homology modelling revealed that 2019-nCoV had a similar receptor-binding domain structure to that of SARS-CoV, despite amino acid variation at some key residues. INTERPRETATION: 2019-nCoV is sufficiently divergent from SARS-CoV to be considered a new human-infecting betacoronavirus. Although our phylogenetic analysis suggests that bats might be the original host of this virus, an animal sold at the seafood market in Wuhan might represent an intermediate host facilitating the emergence of the virus in humans. Importantly, structural analysis suggests that 2019-nCoV might be able to bind to the angiotensin-converting enzyme 2 receptor in humans. The future evolution, adaptation, and spread of this virus warrant urgent investigation. FUNDING: National Key Research and Development Program of China, National Major Project for Control and Prevention of Infectious Disease in China, Chinese Academy of Sciences, Shandong First Medical University.


Asunto(s)
Betacoronavirus/genética , Infecciones por Coronavirus/epidemiología , Infecciones por Coronavirus/virología , Genoma Viral , Neumonía Viral/epidemiología , Neumonía Viral/virología , Receptores Virales/metabolismo , Betacoronavirus/metabolismo , Líquido del Lavado Bronquioalveolar/virología , China/epidemiología , Infecciones por Coronavirus/diagnóstico , Infecciones por Coronavirus/transmisión , ADN Viral/genética , Reservorios de Enfermedades/virología , Genómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Filogenia , Neumonía Viral/diagnóstico , Neumonía Viral/transmisión , Alineación de Secuencia
6.
mSphere ; 5(1)2020 01 29.
Artículo en Inglés | MEDLINE | ID: mdl-31996413

RESUMEN

Coronaviruses (CoVs) of bat origin have caused two pandemics in this century. Severe acute respiratory syndrome (SARS)-CoV and Middle East respiratory syndrome (MERS)-CoV both originated from bats, and it is highly likely that bat coronaviruses will cause future outbreaks. Active surveillance is both urgent and essential to predict and mitigate the emergence of these viruses in humans. Next-generation sequencing (NGS) is currently the preferred methodology for virus discovery to ensure unbiased sequencing of bat CoVs, considering their high genetic diversity. However, unbiased NGS is an expensive methodology and is prone to missing low-abundance CoV sequences due to the high background level of nonviral sequences present in surveillance field samples. Here, we employ a capture-based NGS approach using baits targeting most of the CoV species. Using this technology, we effectively reduced sequencing costs by increasing the sensitivity of detection. We discovered nine full genomes of bat CoVs in this study and revealed great genetic diversity for eight of them.IMPORTANCE Active surveillance is both urgent and essential to predict and mitigate the emergence of bat-origin CoV in humans and livestock. However, great genetic diversity increases the chance of homologous recombination among CoVs. Performing targeted PCR, a common practice for many surveillance studies, would not reflect this diversity. NGS, on the other hand, is an expensive methodology and is prone to missing low-abundance CoV sequences. Here, we employ a capture-based NGS approach using baits targeting all CoVs. Our work demonstrates that targeted, cost-effective, large-scale, genome-level surveillance of bat CoVs is now highly feasible.


Asunto(s)
Quirópteros/virología , Coronavirus/clasificación , Coronavirus/aislamiento & purificación , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Animales , Variación Genética , Genoma Viral
7.
Cancer Invest ; 38(2): 85-93, 2020 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-31939681

RESUMEN

The identification and quantification of actionable mutations are critical for guiding targeted therapy and monitoring drug response in colorectal cancer. Liquid biopsy (LB) based on plasma cell-free DNA analysis has emerged as a noninvasive approach with many clinical advantages over conventional tissue sampling. Here, we developed a LB protocol using ultra-deep massive parallel sequencing and validated its clinical performance for detection and quantification of actionable mutations in three major driver genes (KRAS, NRAS and BRAF). The assay showed a 92% concordance for mutation detection between plasma and paired tissues and great reliability in quantification of variant allele frequency.


Asunto(s)
ADN Tumoral Circulante/genética , Neoplasias Colorrectales/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Biopsia Líquida/métodos , Neoplasias Colorrectales/sangre , GTP Fosfohidrolasas/genética , Humanos , Proteínas de la Membrana/genética , Mutación , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Reproducibilidad de los Resultados
8.
Gene ; 726: 144176, 2020 Feb 05.
Artículo en Inglés | MEDLINE | ID: mdl-31669641

RESUMEN

Gastric cancer is a serious problem for human health. As part of noncoding RNA, circular RNA (circRNA) plays a key role in the occurrence and development of malignant tumor. We used next generation sequencing technology to detect circRNA expression profiles in 5 paired human gastric cancer tissues. Then, bioinformatics analysis was carried out to analyze the function of dysregulated circRNAs. Hsa_circ_0058092 was selected as the object of follow-up analysis. After using the Cistrome DB dataset the data was used to predict specific transcription factors of hsa_circ_0058092. The relationship between hsa_circ_0058092 and PODXL was further validated using RT-PCR and immunohistochemical techniques. Survival data were collected using a Kaplan-Meier analysis of hsa_circ_0058092. We identified 319 aberrantly expressed circRNAs, Hsa_circ_0058092 was selected for our studies. Functional analysis of hsa_circ_0058092 revealed that it was related to metabolic processes. The prediction results suggested that hsa_circ_0058092 has a relationship with hsa-miR-4269 which could specifically bind to the PODXL sequence. Transcription factor CEBPB may regulate the transcription process of hsa_circ_0058092. The expression of hsa_circ_0058092 was positively correlated with PODXL expression. Immunohistochemical analysis of PODXL showed that the expression of PODXL protein in cancer tissues is higher than that in adjacent tissues. Kaplan-Meier analysis suggested that hsa_circ_0058092 was associated with survival of gastric cancer patients. All of these results showed that hsa_circ_0058092 was a potential oncogene.


Asunto(s)
Oncogenes/genética , Neoplasias Gástricas/genética , Biomarcadores de Tumor/genética , Biología Computacional/métodos , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Masculino , MicroARNs/genética , Persona de Mediana Edad , ARN no Traducido/genética , Sialoglicoproteínas/genética , Factores de Transcripción/genética , Transcripción Genética/genética
9.
Chemosphere ; 242: 125078, 2020 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-31704520

RESUMEN

The widespread use of tetrabromobisphenol A (TBBPA) in industries has resulted in its frequent detection in environmental matrices, and the mechanisms of its associated hazards need further investigation. In this study, the nematode Caenorhabditis elegans (C. elegans) was exposed to environmentally relevant concentrations of TBBPA (0, 0.1, 1, 10, 100, 200 µg/L) to determine its effects. At TBBPA concentrations above 1 µg/L, the number of head thrashes, as the most sensitive physiological indicator, decreased significantly. Using the Illumina HiSeq™ 2000 sequencer, differentially expressed genes (DEGs) were determined, and 52 were down regulated and 105 were up regulated in the 200 µg/L TBBPA treatment group versus the control group. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database analysis demonstrated that dorso-ventral axis formation is related to neurotoxicity; metabolism of xenobiotics by Cytochrome P450 (CYP450) and glutathione-S-transferase (GST) was found to be the vital metabolic mechanisms and were confirmed by quantitative real-time polymerase chain reaction (qRT-PCR). GST was ascribed to the augmentation because mutations in cyp-13A7 were constrained under TBBPA exposure. Additionally, oxidative stress indicators accumulated in a dose-dependent relationship. These results will help understand the molecular basis for TBBPA-induced toxicity in C. elegans and open novel avenues for facilitating the exploration of more efficient strategies against TBBPA toxicity.


Asunto(s)
Caenorhabditis elegans/efectos de los fármacos , Contaminantes Ambientales/toxicidad , Bifenilos Polibrominados/toxicidad , Animales , Sistema Enzimático del Citocromo P-450/metabolismo , Perfilación de la Expresión Génica , Glutatión Transferasa/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Estrés Oxidativo , Reacción en Cadena en Tiempo Real de la Polimerasa
10.
Int J Radiat Oncol Biol Phys ; 106(2): 349-357, 2020 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-31678224

RESUMEN

PURPOSE: NCT01725165 was a phase II prospective trial in which patients with non-small cell lung cancer were randomized to local consolidative therapy (LCT) versus maintenance therapy or observation (MT/O). METHODS AND MATERIALS: Peripheral blood from patients enrolled on NCT01725165 were labeled as (1) baseline, (2) early follow-up (FU) if obtained in the first or second FU evaluation (6-18 weeks), and (3) late FU if obtained in the third to sixth FU evaluations (22-50 weeks). All patients who underwent LCT and were included in this analysis received radiation. Among 49 randomized patients, 21 patients underwent T cell CDR3 variable region sequencing using immunoSEQ, 31 patients underwent circulating tumor DNA (ctDNA) analysis using next-generation sequencing with a 1021 cancer gene panel, and cytokine concentration was assayed in 19 patients using enzyme-linked immunosorbent assay. All analyses were exploratory and not corrected for multiple testing. RESULTS: No associations were identified between baseline T cell repertoire and ctDNA metrics with patient outcomes. Among baseline cytokines, interleukin 1α was the only cytokine associated with both overall survival (hazard ratio, 0.02; 95% confidence interval, 0.1-0.5; P = .0006) and progression-free survival (hazard ratio, 0.5; 95% confidence interval, 0.2-0.9; P = .03). At early FU, LCT was associated with decreased ctDNA burden, including lower number of detected mutations (median, 2 [interquartile range {IQR}, 1-6] vs 6 [IQR, 4-18]) and decreased average variable allele frequency (VAF; median, 0.006 [IQR, 0.003-0.010] vs 0.011 [IQR, 0.007-0.014]) compared with MT/O. Among 6 patients with serial ctDNA analysis, a rise in ctDNA detected mutation burden preceded clinical progression by 6.7 months. At early FU, LCT was associated with changes in T cell clonality that suggested oligoclonal expansion specifically increased T cell clonality (median, 0.15 [IQR, 0.12-0.24] vs 0.10 [IQR, 0.05-0.13]) and frequency of top 10 clones (median, 0.14 [IQR, 0.06-0.18] vs 0.21[IQR, 0.19-0.28]). CONCLUSION: LCT was associated with decreased ctDNA burden and oligoclonal expansion at early FU timepoints. Baseline interleukin 1α was associated with improved patient outcomes.


Asunto(s)
Biomarcadores de Tumor/sangre , Carcinoma de Pulmón de Células no Pequeñas/radioterapia , ADN Tumoral Circulante/sangre , Citocinas/sangre , Neoplasias Pulmonares/radioterapia , Alelos , Carcinoma de Pulmón de Células no Pequeñas/sangre , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/inmunología , ADN Tumoral Circulante/genética , Dermatoglifia del ADN , Ensayo de Inmunoadsorción Enzimática , Estudios de Seguimiento , Perfilación de la Expresión Génica/métodos , Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Neoplasias Pulmonares/sangre , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/inmunología , Mutación , Supervivencia sin Progresión , Estudios Prospectivos , Linfocitos T/citología , Linfocitos T/inmunología , Linfocitos T/efectos de la radiación , Factores de Tiempo , Investigación en Medicina Traslacional , Espera Vigilante
11.
Arch Virol ; 165(1): 105-114, 2020 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-31741095

RESUMEN

Piwi-interacting RNAs (piRNAs) play pivotal roles in spermatogenesis and are widely distributed among somatic tissues. However, little is known about piRNAs in HeLa cells infected with coxsackievirus B3 (CVB3). In this study, we systematically investigated changes in piRNA expression in HeLa cells infected with CVB3 using high-throughput sequencing technology. piRNA expression profiles in CVB3-infected HeLa cells were examined at 3, 6 and 9 h postinfection (pi). Of the 32,826 piRNAs that were annotated in the NCBI database, 151,571, 89,698 and 76,626 piRNAs were detected in CVB3-infected HeLa cells at 3, 6 and 9 h pi, respectively. Compared with normal cells, 211, 72 and 94 piRNAs were differentially expressed in CVB3-infected HeLa cells at 3, 6 and 9 h pi, respectively. Thirteen piRNAs, including four novel piRNAs, exhibited concurrent changes in CVB3-infected HeLa cells. The changes in the expression of these 13 piRNAs was confirmed in CVB3-infected HeLa cells and 293T cells by stem-loop RT-qPCR at 3, 6 and 9 h pi. The target genes of 13 piRNAs were predicted. The four novel piRNAs were associated with LTR/ERV, LINE/L1 and LTR/ERVK repetitive elements located on different chromosomes. These findings may promote a better understanding of the regulatory mechanism of pathophysiological changes induced by CVB3 infection.


Asunto(s)
Infecciones por Coxsackievirus/genética , Enterovirus Humano B/patogenicidad , ARN Interferente Pequeño/genética , Regulación de la Expresión Génica , Células HEK293 , Células HeLa , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Análisis de Secuencia de ARN/métodos
12.
Arch Virol ; 165(1): 127-135, 2020 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-31741097

RESUMEN

In clinical virome research, whole-genome/transcriptome amplification is required when starting material is limited. An improved method, named "template-dependent multiple displacement amplification" (tdMDA), has recently been developed in our lab (Wang et al. in BioTechniques 63:21-25. https://doi.org/10.2144/000114566, 2017). In combination with Illumina sequencing and bioinformatics pipelines, its application in virome sequencing was explored using a serum sample from a patient with chronic hepatitis C virus (HCV) infection. In comparison to an amplification-free procedure, virome sequencing via tdMDA showed a 9.47-fold enrichment for HCV-mapped reads and, accordingly, an increase in HCV genome coverage from 28.5% to 70.1%. Eight serum samples from acute patients liver failure (ALF) with or without known etiology were then used for virome sequencing with an average depth at 94,913x. Both similarity-based (mapping, NCBI BLASTn, BLASTp, and profile hidden Markov model analysis) and similarity-independent methods (machine-learning algorithms) identified viruses from multiple families, including Herpesviridae, Picornaviridae, Myoviridae, and Anelloviridae. However, their commensal nature and cross-detection ruled out an etiological interpretation. Together with a lack of detection of novel viruses in a comprehensive analysis at a resolution of single reads, these data indicate that viral agents might be rare in ALF cases with indeterminate etiology.


Asunto(s)
Biología Computacional/métodos , Hepatitis C Crónica/diagnóstico , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Fallo Hepático Agudo/virología , Suero/virología , Anelloviridae/aislamiento & purificación , Anelloviridae/fisiología , Perfilación de la Expresión Génica/métodos , Hepacivirus/genética , Hepacivirus/aislamiento & purificación , Hepatitis C Crónica/sangre , Herpesviridae/aislamiento & purificación , Herpesviridae/fisiología , Humanos , Fallo Hepático Agudo/sangre , Myoviridae/aislamiento & purificación , Myoviridae/fisiología , Picornaviridae/aislamiento & purificación , Picornaviridae/fisiología , Especificidad de la Especie , Simbiosis , Secuenciación Completa del Genoma/métodos
13.
Cancer Sci ; 111(2): 580-591, 2020 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-31804030

RESUMEN

Patients with lower-risk myelodysplastic syndromes (LR-MDS) as defined by the International Prognostic Scoring System (IPSS) have more favorable prognosis in general, but significant inter-individual heterogeneity exists. In this study, we examined the molecular profile of 15 MDS-relevant genes in 159 patients with LR-MDS using next-generation sequencing. In univariate COX regression, shorter overall survival (OS) was associated with mutation status of ASXL1 (P = .001), RUNX1 (P = .031), EZH2 (P = .049), TP53 (P = .016), SRSF2 (P = .046), JAK2 (P = .040), and IDH2 (P = .035). We also found significantly shorter OS in patients with an adjusted TET2 variant allele frequency (VAF) ≥18% versus those with either an adjusted TET2 VAF <18% or without TET2 mutations (median: 20.4 vs 47.8 months; P = .020; HR = 2.183, 95%CI: 1.129-4.224). After adjustment for IPSS, shorter OS was associated with mutation status of ASXL1 (P < .001; HR = 4.306, 95% CI: 2.144-8.650), TP53 (P = .004; HR = 4.863, 95% CI: 1.662-14.230) and JAK2 (P = .002; HR = 5.466, 95%CI: 1.848-16.169), as well as adjusted TET2 VAF ≥18% (P = .008; HR = 2.492, 95% CI: 1.273-4.876). Also, OS was increasingly shorter as the number of mutational factors increased (P < .001). A novel prognostic scoring system incorporating the presence/absence of the four independent mutational factors into the IPSS further stratified LR-MDS patients into three prognostically different groups (P < .001). The newly developed scoring system redefined 10.1% (16/159) of patients as a higher-risk group, who could not be predicted by the currently prognostic models. In conclusion, integration of the IPSS with mutation status/burden of certain MDS-relevant genes may improve the prognostication of patients with LR-MDS and could help identify those with worse-than-expected prognosis for more aggressive treatment.


Asunto(s)
Redes Reguladoras de Genes , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Mutación , Síndromes Mielodisplásicos/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Frecuencia de los Genes , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Análisis de Secuencia de ADN/métodos , Análisis de Supervivencia , Adulto Joven
14.
Immunogenetics ; 72(1-2): 89-100, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-31713647

RESUMEN

The IPD-MHC Database represents the official repository for non-human major histocompatibility complex (MHC) sequences, overseen and supported by the Comparative MHC Nomenclature Committee, providing access to curated MHC data and associated analysis tools. IPD-MHC gathers allelic MHC class I and class II sequences from classical and non-classical MHC loci from various non-human animals including pets, farmed and experimental model animals. So far, Atlantic salmon and rainbow trout are the only teleost fish species with MHC class I and class II sequences present. For the remaining teleost or ray-finned species, data on alleles originating from given classical locus is scarce hampering their inclusion in the database. However, a fast expansion of sequenced genomes opens for identification of classical loci where high-throughput sequencing (HTS) will enable typing of allelic variants in a variety of new teleost or ray-finned species. HTS also opens for large-scale studies of salmonid MHC diversity challenging the current database nomenclature and analysis tools. Here we establish an Illumina approach to identify allelic MHC diversity in Atlantic salmon, using animals from an endangered wild population, and alter the salmonid MHC nomenclature to accommodate the expected sequence expansions.


Asunto(s)
Complejo Mayor de Histocompatibilidad/genética , Salmo salar/genética , Salmo salar/inmunología , Alelos , Animales , Bases de Datos Factuales , Evolución Molecular , Variación Genética , Genoma , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Datos de Secuencia Molecular , Filogenia , Alineación de Secuencia , Análisis de Secuencia de Proteína
15.
Forensic Sci Int ; 306: 110052, 2020 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-31778923

RESUMEN

Metabarcoding through Next Generation Sequencing (NGS) has revolutionized environmental biological studies. The availability of this technical approach has opened the opportunity for a systematic implementation of fungal metabarcoding analysis in forensics, where standardized, sensitive and reproducible protocols are highly desirable. In the present paper, a pipeline including a semi-automated molecular protocol and user-friendly bioinformatics tools are applied to several kinds of environmental samples and forensic caseworks. The identification of fungi that characterize specific environments (like Aspergillus for indoor walls, or Penicillium, Debaryomices and Wickerhamomyces for food storage) can be informative for the provenance of samples. In some situations, fungal analysis cannot allow the identification of a defined environment but seems useful to cluster samples with similar provenance. Based on these considerations, fungal analysis can be included in a wider process of non-human DNA identification in order to provide clues on sample provenance.


Asunto(s)
Código de Barras del ADN Taxonómico , ADN de Hongos/genética , Hongos/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Microbiología Ambiental , Ciencias Forenses , Análisis de Componente Principal , Análisis de Secuencia de ADN , Programas Informáticos
16.
Arch Virol ; 165(1): 21-31, 2020 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-31624917

RESUMEN

To obtain insight into the sequence diversity of strawberry latent ringspot virus (SLRSV), isolates from collections and diagnostic samples were sequenced by high-throughput sequencing. For five SLRSV isolates, the complete genome sequences were determined, and for 18 other isolates nearly complete genome sequences were determined. The sequence data were analysed in relation to sequences of SLRSV and related virus isolates available in the NCBI GenBank database. The genome sequences were annotated, and sequences of the protease-polymerase (Pro-Pol) region and coat proteins (CPs) (large and small CP together) were used for phylogenetic analysis. The amino acid sequences of the Pro-Pol region were very similar, whereas the nucleotide sequences of this region were more variable. The amino acid sequences of the CPs were less similar, which was corroborated by the results of a serological comparison performed using antisera raised against different isolates of SLRSV. Based on these results, we propose that SLRSV and related unassigned viruses be assigned to a new genus within the family Secoviridae, named "Stralarivirus". Based on the phylogenetic analysis, this genus should include at least three viruses, i.e., SLRSV-A, SLRSV-B and lychnis mottle virus. The newly generated sequence data provide a basis for designing molecular tests to screen for SLRSV.


Asunto(s)
Fragaria/virología , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Secoviridae/clasificación , Análisis de Secuencia de ARN/métodos , Proteínas de la Cápside/genética , ARN Polimerasas Dirigidas por ADN/genética , Variación Genética , Anotación de Secuencia Molecular , Péptido Hidrolasas/genética , Filogenia , Virus de Plantas/clasificación , Virus de Plantas/genética , Virus de Plantas/aislamiento & purificación , ARN Viral/genética , Secoviridae/genética , Secoviridae/aislamiento & purificación
17.
Acta Cytol ; 64(1-2): 175-181, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31121596

RESUMEN

Biliary brushing cytology has become the standard of practice for the investigation of strictures of the biliary and pancreatic duct systems. The methodology however has a limitation in that it has low diagnostic sensitivity when only cytologic evaluation is used. A number of testing methodologies have been applied to brushing specimens in an attempt to improve overall sensitivity without loss of specificity. These have included DNA ploidy analysis, immunocytochemistry, individual gene mutational analysis, fluorescence in-situ hybridization (FISH), and next generation sequencing (NGS). Currently, FISH coupled with routine cytology appears to be the method of choice for improving diagnostic sensitivity. NGS shows significant promise for improvement of diagnostic sensitivity.


Asunto(s)
Neoplasias de los Conductos Biliares/diagnóstico , Conductos Biliares Intrahepáticos/patología , Sistema Biliar/patología , Colangiocarcinoma/diagnóstico , Citodiagnóstico/métodos , Neoplasias de los Conductos Biliares/genética , Neoplasias de los Conductos Biliares/metabolismo , Conductos Biliares Intrahepáticos/metabolismo , Sistema Biliar/metabolismo , Colangiocarcinoma/genética , Colangiocarcinoma/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Inmunohistoquímica/métodos , Hibridación Fluorescente in Situ/métodos , Sensibilidad y Especificidad
18.
Arch Virol ; 165(1): 227-231, 2020 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-31659444

RESUMEN

Three viral contig sequences, which represented complete genome of a novel virus with three dsRNAs of 1,712 nucleotides (nt) (dsRNA1), 1,504 nt (dsRNA2) and 1,353 nt (dsRNA3), were found in tea-oil camellia plants by high-throughput sequencing analysis. The three dsRNAs were re-sequenced by RT-PCR cloning. The largest dsRNA, dsRNA1, had a single open reading frame (ORF) that encoded a putative 52.7-kDa protein of a putative viral RNA-dependent RNA polymerase (RdRp). DsRNA2 and dsRNA3 were predicted to encode putative capsid proteins (CPs) of 40.47 kDa and 40.59 kDa, respectively. The virus, which is provisionally named "tea-oil camellia deltapartitivirus 1",  shared amino acid sequence itentities of 36.09-69.18% with members of the genus Deltapartitivirus on RdRp. Phylogenetic analysis based on RdRp also placed the new virus and other deltapartitiviruses together in a group, suggesting that this virus should be considered a new member of the genus Deltapartitivirus.


Asunto(s)
Camellia/virología , Virus ARN/genética , Secuenciación Completa del Genoma/métodos , Mapeo Contig , Genoma Viral , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Sistemas de Lectura Abierta , Filogenia , Virus ARN/clasificación , ARN Bicatenario/genética
19.
DNA Cell Biol ; 39(1): 126-143, 2020 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-31730374

RESUMEN

Extensive searches for genomic regions harboring various types of candidate human-specific regulatory sequences (HSRS) identified thousands' HSRS using high-resolution next-generation sequencing technologies and methodologically diverse comparative analyses of human and nonhuman primates' (NHPs) reference genomes. In this study, a comprehensive catalogue of 59,732 genomic loci harboring candidate HSRS has been assembled to facilitate the systematic analyses of genomic sequences that were either inherited from extinct common ancestors (ECAs) or created de novo in human genomes. These analyses identified thousands of candidate HSRS and HSRS-harboring loci that appear inherited from ECAs, yet absent in genomes of our closest evolutionary relatives, chimpanzee and bonobo, presumably due to the incomplete lineage sorting and/or species-specific loss or regulatory DNA. This pattern is particularly prominent for HSRS-harboring loci that have been putatively associated with human-specific gene expression changes in cerebral organoid models. A prominent majority of regions harboring human-specific mutations associated with human-specific expression changes during brain development is highly conserved in chimpanzee, bonobo, and gorilla genomes. Among NHPs, dominant fractions of HSRS-harboring loci associated with human-specific gene expression in both excitatory neurons (347 loci; 67%) and radial glia (683 loci; 72%) are highly conserved in the gorilla genome. Analysis of 4433 genes encoding virus-interacting proteins (VIPs) revealed that 95.9% of human VIPs are components of human-specific regulatory networks that appear to operate in distinct types of human cells from preimplantation embryos to adult dorsolateral prefrontal cortex. These analyses demonstrate that modern humans captured unique genome-wide combinations of regulatory sequences, divergent subsets of which are highly conserved in distinct species of six NHP separated by 30 million years of evolution. Concurrently, this unique-to-human mosaic of genomic regulatory patterns inherited from ECAs was supplemented with 12,486 created de novo HSRS. Genes encoding VIPs appear to represent a principal genomic target during evolution of human-specific regulatory networks, which contribute to fitness of Homo sapiens and affect a functionally diverse spectrum of biological and cellular processes controlled by VIP-containing liquid-liquid phase-separated condensates.


Asunto(s)
Encéfalo/metabolismo , Regulación de la Expresión Génica , Genoma Humano/genética , Genoma/genética , Células Madre Pluripotentes/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Animales , Encéfalo/embriología , Encéfalo/crecimiento & desarrollo , Células Madre Embrionarias/metabolismo , Evolución Molecular , Perfilación de la Expresión Génica/métodos , Redes Reguladoras de Genes , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Mutación , Primates , Especificidad de la Especie
20.
Cancer Sci ; 111(1): 266-278, 2020 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-31746520

RESUMEN

According to cancer genome sequences, more than 90% of cases of pancreatic ductal adenocarcinoma (PDAC) harbor active KRAS mutations. Digital PCR (dPCR) enables accurate detection and quantification of rare mutations. We assessed the dynamics of circulating tumor DNA (ct-DNA) in patients with advanced PDAC undergoing chemotherapy using dPCR. KRAS G12/13 mutation was assayed by dPCR in 47 paired tissue- and ct-DNA samples. The 21 patients were subjected to quantitative ct-DNA monitoring at 4 to 8-week intervals during chemotherapy. KRAS mutation was detected in 45 of those 47 patients using tissue DNA. In the KRAS mutation-negative cases, next-generation sequencing revealed KRAS Q61K and NRAS Q61R mutations. KRAS mutation was detected in 23/45 cases using ct-DNA (liver or lung metastasis, 18/19; mutation allele frequency [MAF], 0.1%-31.7%; peritoneal metastasis, 3/9 [0.1%], locally advanced, 2/17 [0.1%-0.2%]). In the ct-DNA monitoring, the MAF value changed in concordance with the disease state. In the 6 locally advanced cases, KRAS mutation appeared concurrently with liver metastasis. Among the 6 cases with liver metastasis, KRAS mutation disappeared during the duration of stable disease or a partial response, and reappeared at the time of progressive disease. The median progression-free survival was longer in cases in which KRAS mutation disappeared after an initial course of chemotherapy than in those in which it was continuously detected (248.5 vs 50 days, P < .001). Therefore, ct-DNA monitoring enables continuous assessment of disease state and could have prognostic utility during chemotherapy.


Asunto(s)
ADN Tumoral Circulante/genética , ADN/sangre , Neoplasias Pancreáticas/sangre , Neoplasias Pancreáticas/genética , Adulto , Anciano , Biomarcadores de Tumor/sangre , Carcinoma Ductal Pancreático/sangre , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Estudios de Evaluación como Asunto , Femenino , Frecuencia de los Genes/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Neoplasias Hepáticas/sangre , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Neoplasias Pulmonares/sangre , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Mutación/genética , Páncreas/patología , Neoplasias Pancreáticas/patología , Pronóstico , Supervivencia sin Progresión
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