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A smartphone-based electrochemical aptasensing platform was developed for the point-of-care testing (POCT) of carcinoembryonic antigen (CEA) based on the ferrocene (Fc) and PdPt@PCN-224 dual-signal labeled strategy. The prepared PdPt@PCN-224 nanocomposite showed a strong catalytic property for the reduction of H2O2. Phosphate group-labeled aptamer could capture PdPt@PCN-224 by Zr-O-P bonds to form PdPt@PCN-224-P-Apt. Therefore, a dual signal labeled probe was formed by the hybridization between Fc-DNA and PdPt@PCN-224-P-Apt. The presence of CEA forced PdPt@PCN-224-P-Apt to leave the electrode surface due to the specific affinity, leading to the decrease of the reduction current of H2O2. At the same time, the Fc-DNA strand changed to hairpin structure, which made Fc closer to the electrode and resulted in the increase of the oxidation current of Fc. Thus, CEA can be accurately determined through both signals: the decrease of H2O2 reduction current and the increase of Fc oxidation current, which could avoid the false positive signal. Under the optimal conditions, the prepared aptasensor exhibited a wide linear range from 1 pg·mL-1 to 100 ng·mL-1 and low detection limits of 0.98 pg·mL-1 and 0.27 pg·mL-1 with Fc and PdPt@PCN-224 as signal labels, respectively. The aptasensor developed in this study has successfully demonstrated its capability to detect CEA in real human serum samples. These findings suggest that the proposed sensing platform will hold great potential for clinical tumor diagnosis and monitoring.
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Aptámeros de Nucleótidos , Técnicas Biosensibles , Antígeno Carcinoembrionario , Técnicas Electroquímicas , Compuestos Ferrosos , Peróxido de Hidrógeno , Límite de Detección , Paladio , Pruebas en el Punto de Atención , Teléfono Inteligente , Antígeno Carcinoembrionario/sangre , Antígeno Carcinoembrionario/análisis , Aptámeros de Nucleótidos/química , Técnicas Electroquímicas/métodos , Técnicas Electroquímicas/instrumentación , Humanos , Técnicas Biosensibles/métodos , Peróxido de Hidrógeno/química , Paladio/química , Compuestos Ferrosos/química , Metalocenos/química , Platino (Metal)/químicaRESUMEN
As a prognostic biomarker for breast cancer, human epidermal growth factor receptor 2 (HER-2) is of crucial diagnostic value. Here, a label-free electrochemical aptasensor was established for the ultrasensitive detection of HER-2 using a modified electrode of Bi-Sb alloy materials (Bi-Sb AMs). The performance of the aptasensor was enhanced greatly due to the introduction of Bi-Sb alloy materials (Bi-Sb AMs) with high conductivity. Furthermore, by integrating the aptasensor with the Sensit Smart U-disk electrochemical analyzer, the point-of-care testing (POCT) for HER-2 was realized. Under the optimal experimental parameters, the POCT analyzer showed a wide linear response from 0.01 pg mL-1 to 100 ng mL-1, with a low detection limit (LOD) of 5.96 fg mL-1 for the detection of HER-2. The presented POCT analyzer exhibited good specificity, stability, and reproducibility. Benefiting from the simple operation and rapid testing, the developed analyzer will have potential application in the prognostic diagnosis and treatment of breast cancer.
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Aptámeros de Nucleótidos , Técnicas Biosensibles , Humanos , Técnicas Electroquímicas , Aleaciones , Reproducibilidad de los Resultados , Límite de Detección , OroRESUMEN
With the study of human diseases and biological processes increasing, a large number of non-coding variants have been identified and facilitated. The rapid accumulation of genetic and epigenomic information has resulted in an urgent need to collect and process data to explore the regulation of non-coding variants. Here, we developed a comprehensive variation annotation database for human (VARAdb, http://www.licpathway.net/VARAdb/), which specifically considers non-coding variants. VARAdb provides annotation information for 577,283,813 variations and novel variants, prioritizes variations based on scores using nine annotation categories, and supports pathway downstream analysis. Importantly, VARAdb integrates a large amount of genetic and epigenomic data into five annotation sections, which include 'Variation information', 'Regulatory information', 'Related genes', 'Chromatin accessibility' and 'Chromatin interaction'. The detailed annotation information consists of motif changes, risk SNPs, LD SNPs, eQTLs, clinical variant-drug-gene pairs, sequence conservation, somatic mutations, enhancers, super enhancers, promoters, transcription factors, chromatin states, histone modifications, chromatin accessibility regions and chromatin interactions. This database is a user-friendly interface to query, browse and visualize variations and related annotation information. VARAdb is a useful resource for selecting potential functional variations and interpreting their effects on human diseases and biological processes.
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Enfermedad de Alzheimer/genética , Bases de Datos Genéticas , Diabetes Mellitus Tipo 2/genética , Variación Genética , Genoma Humano , Sitios de Carácter Cuantitativo , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Cromatina , Ensamble y Desensamble de Cromatina , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Elementos de Facilitación Genéticos , Humanos , Internet , Anotación de Secuencia Molecular , Polimorfismo de Nucleótido Simple , Regiones Promotoras Genéticas , Programas InformáticosRESUMEN
Multiple sclerosis (MS) is an autoimmune and neurodegenerative disease driven by inflammation and demyelination in the brain, spinal cord, and optic nerve. Optic neuritis, characterized by inflammation and demyelination of the optic nerve, is a symptom in many patients with MS. The optic nerve is the highway for visual information transmitted from the retina to the brain. It contains axons from the retinal ganglion cells (RGCs) that reside in the retina, myelin forming oligodendrocytes and resident microglia and astrocytes. Inflammation, demyelination, and axonal degeneration are also present in the optic nerve of mice subjected to experimental autoimmune encephalomyelitis (EAE), a preclinical mouse model of MS. Monitoring the optic nerve in EAE is a useful strategy to study the presentation and progression of pathology in the visual system; however, current approaches have relied on sectioning, staining and manual quantification. Further, information regarding the spatial load of lesions and inflammation is dependent on the area of sectioning. To better characterize cellular pathology in the EAE model, we employed a tissue clearing and 3D immunolabelling and imaging protocol to observe patterns of immune cell infiltration and activation throughout the optic nerve. Increased density of TOPRO staining for nuclei captured immune cell infiltration and Iba1 immunostaining was employed to monitor microglia and macrophages. Axonal degeneration was monitored by neurofilament immunolabelling to reveal axonal swellings throughout the optic nerve. In parallel, we developed a convolutional neural network with a UNet architecture (CNN-UNet) called BlebNet for automated identification and quantification of axonal swellings in whole mount optic nerves. Together this constitutes a toolkit for 3-dimensional immunostaining to monitor general optic nerve pathology and fast automated quantification of axonal defects that could also be adapted to monitor axonal degeneration and inflammation in other neurodegenerative disease models.
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Aprendizaje Profundo , Encefalomielitis Autoinmune Experimental , Esclerosis Múltiple , Enfermedades Neurodegenerativas , Neuritis Óptica , Ratones , Animales , Ratones Endogámicos C57BL , Neuritis Óptica/patología , Encefalomielitis Autoinmune Experimental/patología , Esclerosis Múltiple/patología , Degeneración Nerviosa , Inflamación , Modelos Animales de EnfermedadRESUMEN
Lung adenocarcinoma is a common histologic type of lung cancer with a high death rate globally. Increasing evidence shows that long non-coding RNA H19 (lncRNA H19) and CDH1 methylation are involved in multiple tumours. Here, we tried to investigate whether lncRNA H19 or CDH1 methylation could affect the development of lung adenocarcinoma. First, lung adenocarcinoma tissues were collected to detect CDH1 methylation. Then, the regulatory mechanisms of lncRNA H19 were detected mainly in concert with the treatment of overexpression of lncRNA H19, siRNA against lncRNA H19, overexpression of CDH1 and demethylating agent A-5az in lung adenocarcinoma A549 cell. The expression of lncRNA H19 and epithelial-mesenchymal transition (EMT)-related factors as well as cell proliferation, sphere-forming ability, apoptosis, migration and invasion were detected. Finally, we observed xenograft tumour in nude mice so as to ascertain tumorigenicity of lung adenocarcinoma cells. LncRNA H19 and methylation of CDH1 were highly expressed in lung adenocarcinoma tissues. A549 cells with silencing of lncRNA H19, overexpression of CDH1 or reduced CDH1 methylation by demethylating agent 5-Az had suppressed cell proliferation, sphere-forming ability, apoptosis, migration and invasion, in addition to inhibited EMT process. Silencing lncRNA H19 could reduce methylation level of CDH1. In vivo, A549 cells with silencing lncRNA H19, overexpression of CDH1 or reduced CDH1 methylation exhibited low tumorigenicity, reflected by the smaller tumour size and lighter tumour weight. Taken together, this study demonstrates that silencing of lncRNA H19 inhibits EMT and proliferation while promoting apoptosis of lung adenocarcinoma cells by inhibiting methylation of CDH1 promoter.
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Adenocarcinoma del Pulmón/genética , Antígenos CD/genética , Cadherinas/genética , Neoplasias Pulmonares/genética , Regiones Promotoras Genéticas/genética , ARN Largo no Codificante/genética , Células A549 , Adulto , Anciano , Animales , Apoptosis/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Progresión de la Enfermedad , Transición Epitelial-Mesenquimal/genética , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Masculino , Metilación , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , ARN Interferente Pequeño/genética , Adulto JovenRESUMEN
The involvement of several microRNAs (miRs) in the initiation and development of tumors through the suppression of the target gene expression has been highlighted. The aberrant expression of miR-181d-5p and cyclin-dependent kinase inhibitor 3 (CDKN3) in non-small-cell lung cancer (NSCLC) was then screened by microarray analysis. In the present study, we performed a series of in vivo and in vitro experiments for the purpose of investigating their roles in NSCLC and the underlying mechanism. There was a high expression of CDKN3, whereas miR-181d-5p was downregulated in NSCLC. Quantitative RT-PCR, Western blot analysis, and dual-luciferase reporter gene assay further identified that CDKN3 could be negatively regulated by miR-181d-5p. Moreover, the upregulation of miR-181d-5p or silencing of CDKN3 could inactivate the Akt signaling pathway. A549 with the lowest miR-181d-5p and H1975 with the highest CDKN3 among the five NSCLC cell lines (H1299, A549, H1975, NCI-H157, and GLC-82) were adopted for in vitro experiments, in which expression of miR-181d-5p and CDKN3 was altered by transfection of miR-181d-5p mimic/inhibitor or siRNA-targeting CDKN3. Afterwards, cell proliferation, apoptosis, invasion, migration, and angiogenesis, as well as epithelial-mesenchymal transition (EMT), were evaluated, and tumorigenicity was assessed. In addition, an elevation in miR-181d-5p or depletion in CDKN3 led to significant reductions in proliferation, invasion, migration, angiogenesis, EMT, and tumorigenicity of NSCLC cells, coupling with increased cell apoptosis. In conclusion, this study highlights the tumor-suppressive effects of miR-181d-5p on NSCLC via Akt signaling pathway inactivation by suppressing CDKN3, thus providing a promising therapeutic strategy for the treatment of NSCLC.
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Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina/metabolismo , Fosfatasas de Especificidad Dual/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , MicroARNs/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Apoptosis/genética , Apoptosis/fisiología , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Proliferación Celular/genética , Proliferación Celular/fisiología , Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina/antagonistas & inhibidores , Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina/genética , Fosfatasas de Especificidad Dual/antagonistas & inhibidores , Fosfatasas de Especificidad Dual/genética , Transición Epitelial-Mesenquimal/genética , Transición Epitelial-Mesenquimal/fisiología , Regulación Neoplásica de la Expresión Génica , Genes Supresores de Tumor , Xenoinjertos , Humanos , Neoplasias Pulmonares/patología , Masculino , Ratones , Ratones Desnudos , MicroARNs/antagonistas & inhibidores , MicroARNs/metabolismo , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , Transducción de SeñalRESUMEN
The selection of a suitable reference gene is an important prerequisite for the precise analysis of target gene expression by real-time quantitative PCR (qPCR). The present study aims to explore the expression pattern of the Macrobrachium nipponense (M. nipponense) ß-actin gene under Aeromonas hydrophila bacterial infection conditions. The complete sequence of the ß-actin gene from M. nipponense was cloned by PCR. Identified and named ß-actin genes were searched in the NCBI database, and the characteristics of the ß-actin gene were analyzed using bioinformatics methods. The expression profiles of ß-actin under stresses challenged by bacteria after 3, 6, 12, 24 and 48 h were investigated by measuring Ct values by qPCR. The prokaryotic expression vector pET-30a-actin was constructed by PCR and recombinant DNA techniques. Fused protein was induced by IPTG in the transformed Escherichia coli BL21 (DE3). Recombinant rActin was purified by nickel column. The bioinformatics analysis result revealed that the deduced protein encoded by the ß-actin gene from M. nipponense had the highest homology with other prawns in the homologous assay (99%). The phylogenetic tree indicates that the ß-actin from M. nipponense and other crustaceans have a single cluster. The qPCR results revealed that a stable expression of ß-actin was observed in response to the A. hydrophila challenge for 3-48 h, and the Ct value was 22 ± 1.5. ß-actin was ranked as a stable gene after the bacterial challenge, which was selected as the appropriate reference gene in M. nipponense.
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Actinas/genética , Perfilación de la Expresión Génica/métodos , Palaemonidae/genética , Actinas/fisiología , Aeromonas hydrophila/patogenicidad , Animales , Infecciones Bacterianas/genética , Infecciones Bacterianas/metabolismo , Clonación Molecular/métodos , Biología Computacional/métodos , Perfilación de la Expresión Génica/normas , Palaemonidae/microbiología , Palaemonidae/fisiología , Filogenia , Reacción en Cadena en Tiempo Real de la Polimerasa/métodosRESUMEN
This study aimed to investigate the association of SDH gene mutations and promoter methylation with succinate dehydrogenase-deficient gastrointestinal stromal tumors (SDH-deficient GISTs) and to further discuss the potential molecular mechanisms underlying SDHB expression loss in these tumors. First, a total of 26 patients with SDH-deficient GISTs were selected by identifying the loss of SDHB protein expression and wild-type for KIT and PDGFRa mutations. Then SDH gene mutations and promoter methylation were detected by DNA sequencing and methylation-specific polymerase chain reaction, respectively, and the clinical and pathological data of SDH-deficient GISTs patients were collected and analyzed accordingly. The results of genetic testing demonstrated that 38.46% (10/26) of these patients harbored mutations in SDHB, SDHC, and SDHD genes (3 cases with double mutations). Besides, aberrant promoter methylation of SDH genes was detected in 10 out of 26 cases (38.46%), including 8 cases in SDHA gene, 3 cases in SDHB gene, 1 case in both SDHA and SDHB genes. It is suggested that SDH gene mutations and promoter methylation may contribute to the loss of SDH protein expression in sporadic SDH-deficient GISTs. This study indicated that the genetic and epigenetic alterations of SDH genes may occur during tumor formation.
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Epigénesis Genética/genética , Tumores del Estroma Gastrointestinal/patología , Succinato Deshidrogenasa/genética , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Tumores del Estroma Gastrointestinal/genética , Humanos , Masculino , Proteínas de la Membrana/metabolismo , Persona de Mediana Edad , Mutación/genética , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-kit/genética , Succinato Deshidrogenasa/deficienciaRESUMEN
Forsythiaside A, a phenylethanoid glycoside monomer extracted from Forsythia suspensa, shows anti-inflammatory, anti-infective, anti-oxidative, and antiviral pharmacological effects. The precise mechanism underlying the antiviral action of forsythiaside A is not completely clear. Therefore, in this study, we aimed to determine whether the anti-influenza action of forsythiaside A occurs via the retinoic acid-inducible gene-I-like receptors (RLRs) signaling pathway in the lung immune cells. Forsythiaside A was used to treat C57BL/6J mice and MAVS-/- mice infected with mouse-adapted influenza A virus FM1 (H1N1, A/FM1/1/47 strain), and the physical parameters (body weight and lung index) and the expression of key factors in the RLRs/NF-κB signaling pathway were evaluated. At the same time, the level of virus replication and the ratio of Th1/Th2 and Th17/Treg of T cell subsets were measured. Compared with the untreated group, the weight loss in the forsythiaside A group in the C57BL/6J mice decreased, and the histopathological sections showed less inflammatory damage after the infection with the influenza A virus FM1 strain. The gene and protein expression of retinoic acid-inducible gene-I (RIG-I), MAVS, and NF-κB were significantly decreased in the forsythiaside A group. Flow cytometry showed that Th1/Th2 and Th17/Treg differentiated into Th2 cells and Treg cells, respectively, after treatment with forsythiaside A. In conclusion, forsythiaside A reduces the inflammatory response caused by influenza A virus FM1 strain in mouse lungs by affecting the RLRs signaling pathway in the mouse lung immune cells.
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Glicósidos/farmacología , Subtipo H1N1 del Virus de la Influenza A/inmunología , Pulmón/inmunología , FN-kappa B/inmunología , Infecciones por Orthomyxoviridae/inmunología , Transducción de Señal/efectos de los fármacos , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/inmunología , Animales , Proteína 58 DEAD Box/genética , Proteína 58 DEAD Box/inmunología , Femenino , Subtipo H1N1 del Virus de la Influenza A/genética , Pulmón/patología , Pulmón/virología , Ratones , Ratones Noqueados , Ratones Transgénicos , FN-kappa B/genética , Infecciones por Orthomyxoviridae/genética , Infecciones por Orthomyxoviridae/patología , Transducción de Señal/genética , Transducción de Señal/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Colaboradores-Inductores/patologíaRESUMEN
Both Xp11 translocation renal cell carcinomas and the corresponding mesenchymal neoplasms are characterized by a variety of gene fusions involving TFE3. It has been known that tumors with different gene fusions may have different clinicopathologic features; however, further in-depth investigations of subtyping Xp11 translocation-associated cancers are needed in order to explore more meaningful clinicopathologic correlations. A total of 22 unusual cases of Xp11 translocation-associated cancers were selected for the current study; 20 cases were further analyzed by RNA sequencing to explore their TFE3 gene fusion partners. RNA sequencing identified 17 of 20 cases (85%) with TFE3-associated gene fusions, including 4 ASPSCR1/ASPL-TFE3, 3 PRCC-TFE3, 3 SFPQ/PSF-TFE3, 1 NONO-TFE3, 4 MED15-TFE3, 1 MATR3-TFE3, and 1 FUBP1-TFE3. The results have been verified by fusion fluorescence in situ hybridization (FISH) assays or reverse transcriptase polymerase chain reaction (RT-PCR). The remaining 2 cases with specific pathologic features highly suggestive of MED15-TFE3 renal cell carcinoma were identified by fusion FISH assay. We provide the detailed morphologic and immunophenotypic description of the MED15-TFE3 renal cell carcinomas, which frequently demonstrate extensively cystic architecture, similar to multilocular cystic renal neoplasm of low malignant potential, and expressed cathepsin K and melanotic biomarker Melan A. This is the first time to correlate the MED15-TFE3 renal cell carcinoma with specific clinicopathologic features. We also report the first case of the corresponding mesenchymal neoplasm with MED15-TFE3 gene fusion. Additional novel TFE3 gene fusion partners, MATR3 and FUBP1, were identified. Cases with ASPSCR1-TFE3, SFPQ-TFE3, PRCC-TFE3, and NONO-TFE3 gene fusion showed a wide variability in morphologic features, including invasive tubulopapillary pattern simulating collecting duct carcinoma, extensive calcification and ossification, and overlapping and high columnar cells with nuclear grooves mimicking tall cell variant of papillary thyroid carcinoma. Furthermore, we respectively evaluated the ability of TFE3 immunohistochemistry, TFE3 FISH, RT-PCR, and RNA sequencing to subclassify Xp11 translocation-associated cancers. In summary, our study expands the list of TFE3 gene fusion partners and the clinicopathologic features of Xp11 translocation-associated cancers, and highlights the importance of subtyping Xp11 translocation-associated cancers combining morphology, immunohistochemistry, and multiple molecular techniques.
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Carcinoma de Células Renales/genética , Cromosomas Humanos Par 11 , Cromosomas Humanos X , Neoplasias Renales/genética , Fusión de Oncogenes/genética , Proteínas de Fusión Oncogénica/genética , Translocación Genética , Adulto , Carcinoma de Células Renales/patología , Femenino , Humanos , Hibridación Fluorescente in Situ , Neoplasias Renales/patología , Masculino , Persona de Mediana Edad , Análisis de Secuencia de ARN , Adulto JovenRESUMEN
AIMS: MITF, TFE3, TFEB and TFEC belong to the same microphthalmia-associated transcription factor family (MiT). Two transcription factors in this family have been identified in two unusual types of renal cell carcinoma (RCC): Xp11 translocation RCC harbouring TFE3 gene fusions and t(6;11) RCC harbouring a MALAT1-TFEB gene fusion. The 2016 World Health Organisation classification of renal neoplasia grouped these two neoplasms together under the category of MiT family translocation RCC. RCCs associated with the other two MiT family members, MITF and TFEC, have rarely been reported. Herein, we identify a case of MITF translocation RCC with the novel PRCC-MITF gene fusion by RNA sequencing. METHODS AND RESULTS: Histological examination of the present tumour showed typical features of MiT family translocation RCCs, overlapping with Xp11 translocation RCC and t(6;11) RCC. However, this tumour showed negative results in TFE3 and TFEB immunochemistry and split fluorescence in-situ hybridisation (FISH) assays. The other MiT family members, MITF and TFEC, were tested further immunochemically and also showed negative results. RNA sequencing and reverse transcription-polymerase chain reaction confirmed the presence of a PRCC-MITF gene fusion: a fusion of PRCC exon 5 to MITF exon 4. We then developed FISH assays covering MITF break-apart probes and PRCC-MITF fusion probes to detect the MITF gene rearrangement. CONCLUSIONS: This study both proves the recurring existence of MITF translocation RCC and expands the genotype spectrum of MiT family translocation RCCs.
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Carcinoma de Células Renales/genética , Proteínas de Ciclo Celular/genética , Neoplasias Renales/genética , Factor de Transcripción Asociado a Microftalmía/genética , Proteínas de Neoplasias/genética , Proteínas de Fusión Oncogénica/genética , Carcinoma de Células Renales/patología , Humanos , Hibridación Fluorescente in Situ , Neoplasias Renales/patología , Masculino , Persona de Mediana Edad , Análisis de Secuencia de ARNRESUMEN
Xp11 translocation renal cell carcinomas are characterized by several different translocations involving the TFE3 gene. Tumors with different specific gene fusions may have different clinicopathological manifestations. Fewer than 10 renal cell carcinoma cases with NONO-TFE3 have been described. Here we examined eight additional cases of this rare tumor using clinicopathological, immunohistochemical, and molecular analyses. The male-to-female ratio of our study cohort was 1:1, and the median age was 30 years. The most distinctive feature of the tumors was that they exhibited glandular/tubular or papillary architecture that was lined with small-to-medium cuboidal to high columnar cells with indistinct cell borders and an abundantly clear or flocculent eosinophilic cytoplasm. The nuclei were oriented toward the luminal surface and were round and uniform in shape, which resulted in the appearance of secretory endometrioid subnuclear vacuolization. The distinct glandular/tubular or papillary architecture was often accompanied by sheets of epithelial cells that presented a biphasic pattern. Immunohistochemically, all eight cases demonstrated moderate (2+) or strong (3+) positive staining for TFE3, CD10, RCC marker, and PAX-8. None of the tumors were immunoreactive for CK7, Cathepsin K, Melan-A, HMB45, Ksp-cadherin, Vimentin, CA9, 34ßE12 or CD117. NONO-TFE3 fusion transcripts were identified in six cases by RT-PCR. All eight cases showed equivocal split signals with a distance of nearly 2 signal diameters and sometimes had false-negative results. Furthermore, we developed a fluorescence in situ hybridization (FISH) assay to serve as an adjunct diagnostic tool for the detection of the NONO-TFE3 fusion gene and used this method to detect the fusion gene in all eight cases. Long-term follow-up (range, 10-102 months) was available for 7 patients. All 7 patients were alive with no evidence of recurrent disease or disease progression after their initial resection. This report adds to the known data regarding NONO-TFE3 renal cell carcinoma.
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Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Carcinoma de Células Renales/genética , Reordenamiento Génico , Neoplasias Renales/genética , Proteínas Asociadas a Matriz Nuclear/genética , Factores de Transcripción de Octámeros/genética , Fusión de Oncogenes , Proteínas de Unión al ARN/genética , Adulto , Carcinoma de Células Renales/patología , Proteínas de Unión al ADN , Femenino , Humanos , Neoplasias Renales/patología , Masculino , Persona de Mediana Edad , Pronóstico , Adulto JovenRESUMEN
AIMS: The aim of this study was to evaluate the mutation status of epidermal growth factor receptor (EGFR) in gastrointestinal stromal tumours (GISTs) and its association with various clinicopathological variables, as well as to discuss further the effects of EGFR mutations on tumour formation and progression. METHODS AND RESULTS: A well-characterized cohort of 323 GISTs, obtained between 2010 and 2015 from the surgical pathology files of at the Department of Pathology of the Nanjing Jinling Hospital, was screened for mutations in exons 19 and 21 of the EGFR gene. Patient clinical data and clinicopathological features were collected if available in the medical records. Among the 323 primary GISTs, we identified three cases (0.93%) of EGFR mutations; these mutations never occurred together with KIT, PDGFRα, KRAS or BRAF mutations. In two cases, tumour cells exhibited spindle cell morphology and, in one case, epithelioid cell morphology. Additionally, the morphology and immunophenotype of these three cases did not show significant differences compared to common GISTs. The clinical results in summary were that two cases of EGFR-mutated GISTs occurred in females and in the stomach. The mean age of EGFR-mutated cases was 54.33 years, and the follow-up data indicated that these tumours were low risk and exhibited low recurrence. CONCLUSIONS: We first established that GISTs carrying EGFR mutation are relatively benign tumours. Although EGFR mutations were rarely present in GIST, EGFR seems to play a significant role in the development and progression of GIST.
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Receptores ErbB/genética , Tumores del Estroma Gastrointestinal/genética , Adulto , Estudios de Cohortes , Femenino , Tumores del Estroma Gastrointestinal/patología , Humanos , Inmunoquímica , Masculino , Persona de Mediana Edad , MutaciónRESUMEN
AIMS: The aim of this study was to investigate potential molecular mechanisms associated with loss of BRM expression in poorly differentiated clear cell renal cell carcinoma (ccRCC). METHODS AND RESULTS: Nineteen previously selected BRM-negative RCC tissues were examined by DNA sequencing, fluorescence in-situ hybridization (FISH) and methylation-specific polymerase chain reaction (PCR) of the BRM gene. BRM mutation was identified in 78.9% (15 of 19) cases, chromosome 9 monosomy or BRM deletion in 43.8% (seven of 16) and BRM promoter region cytosine-phosphate-guanine (CpG) methylation in 42.8% (six of 14). These results indicated that 89.5% (17 of 19) of the cases harboured at least one type of BRM genetic alteration, with two or more types of alteration in 47.4% (nine of 19). Such alterations were found rarely in adjacent non-neoplastic tissues and low-grade areas of composite tumours. CONCLUSIONS: BRM gene mutation, chromosome 9 monosomy or BRM deletion and CpG methylation contribute collectively to the loss of BRM expression in ccRCC. This work focusing on composite tumours indicated that BRM abnormality occurred during tumour progression.
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Carcinoma de Células Renales/genética , Neoplasias Renales/genética , Factores de Transcripción/genética , Metilación de ADN/genética , Análisis Mutacional de ADN , Eliminación de Gen , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Mutación , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas/genéticaRESUMEN
To investigate that whether myoepithelial tumors of salivary glands (MTs) with EWSR1 rearrangement display distinctive morphological characteristics and whether EWSR1 detection aids to distinguish malignant myoepithelial tumors (MMTs) from benign myoepithelial tumors (BMTs) of salivary glands. We examined 37 cases of MTs, including 24 BMTs, 13 MMTs, by histological, immunohistochemical, and molecular analysis. All of 37 cases were immunoreactive for CKpan, and at least one myoepithelial marker. 26 of 37 cases of MTs were available to be analyzed for EWSR1 rearrangement, with the result that EWSR1 gene break was detected in 4 cases of 15 BMTs, and 4 cases of 11 MMTs. In addition, the 8 EWSR1-rearranged cases displayed not exactly similar morphological features, covering 4 clear-cell cases, 1 plasmacytoid-cell case, 1 spindle-cell case, 1 epithelioid-cell case, and 1 chordoid-cell case. Our study proposed that EWSR1 rearrangement was present in a subset of MTs, with variable morphological features. Moreover, the presence of EWSR1 rearrangement could not be a forceful evidence to distinguish MMTs from BBTs.
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Mioepitelioma/genética , Proteína EWS de Unión a ARN/metabolismo , Neoplasias de las Glándulas Salivales/genética , Glándulas Salivales/patología , Biomarcadores de Tumor/análisis , Células Epitelioides/patología , Femenino , Reordenamiento Génico/genética , Humanos , Hibridación Fluorescente in Situ/métodos , Masculino , Proteína EWS de Unión a ARN/genética , Neoplasias de las Glándulas Salivales/metabolismo , Neoplasias de las Glándulas Salivales/patología , Neoplasias de los Tejidos Blandos/genéticaRESUMEN
Many nanomaterials are reported to disrupt lysosomal function and homeostasis, but how cells sense and then respond to nanomaterial-elicited lysosome stress is poorly understood. Nucleus translocation of transcription factor EB (TFEB) plays critical roles in lysosome biogenesis following lysosome stress induced by starvation. The authors previously reported massive cellular vacuolization, along with autophagy induction, in cells treated with rare earth oxide (REO) nanoparticles. Here, the authors identify these giant cellular vacuoles as abnormally enlarged and alkalinized endo/lysosomes whose formation is dependent on macropinocytosis. This vacuolization causes deactivation of mammalian target of rapamycin (mTOR), a TFEB-interacting kinase that resides on the lysosome membrane. Subsequently, TFEB is dephosphorylated at serine 142 and translocated into cell nucleus. This nucleus translocation of TFEB is observed only in vacuolated cells and it is critical for maintaining lysosome homeostasis after REO nanoparticle treatment, as knock-down of TFEB gene significantly compromises lysosome function and enhances cell death in nanoparticle-treated cells. Our results reveal that cellular vacuolization, which is commonly observed in cells treated with REOs and other nanomaterials, represents a condition of profound lysosome stress, and cells sense and respond to this stress by facilitating mTOR-dependent TFEB nucleus translocation in an effort to restore lysosome homeostasis.
Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Núcleo Celular/metabolismo , Lisosomas/metabolismo , Metales de Tierras Raras/química , Nanopartículas/química , Óxidos/química , Serina-Treonina Quinasas TOR/metabolismo , Vacuolas/metabolismo , Álcalis/química , Supervivencia Celular , Endosomas/metabolismo , Células HeLa , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Modelos Biológicos , Pinocitosis , Transporte de ProteínasRESUMEN
Recently, an increasing number of TFE3 rearrangement-associated tumours have been reported, such as TFE3 rearrangement-associated perivascular epithelioid cell tumours (PEComas), melanotic Xp11 translocation renal cancers and melanotic Xp11 neoplasms. We have suggested that these tumours belong to a single clinicopathological spectrum. 'Xp11 neoplasm with melanocytic differentiation' or 'melanotic Xp11 neoplasm' have been proposed to designate this unique neoplasm. Herein, we describe the first case of an Xp11 neoplasm with melanocytic differentiation to be described in the prostate, bearing the novel NONO-TFE3 gene fusion. This study both adds to the spectrum regarding melanotic Xp11 neoplasms and expands its gene fusion spectrum. Moreover, we discuss the relationship of these rare tumours to neoplasms such as conventional PEComas, alveolar soft part sarcomas, malignant melanomas, clear cell sarcomas and Xp11 translocation renal cancers.
Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Proteínas Asociadas a Matriz Nuclear/genética , Factores de Transcripción de Octámeros/genética , Neoplasias de la Próstata/genética , Proteínas de Unión al ARN/genética , Adulto , Biomarcadores de Tumor/análisis , Cromosomas Humanos X/genética , Proteínas de Unión al ADN , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Masculino , Melanocitos/patología , Fusión de Oncogenes/genética , Neoplasias de la Próstata/patología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
AIMS: Malignant rhabdoid tumours (MRTs) are highly aggressive malignancies of early infancy characterized by inactivation of SMARCB1, a core member of the SWI/SNF chromatin-remodelling complex. The aim of this study was to explore the status of multiple key subunits of the SWI/SNF complex in MRTs. METHODS AND RESULTS: We screened the key subunits of the SWI/SNF complex, including SMARCB1, SMARCA2, PBRM1, SMARCA4, and ARID1A, in four MRTs by immunohistochemistry, sequencing, and fluorescence in-situ hybridization (FISH). Complete loss of SMARCB1, SMARCA2 and PBRM1 expression and corresponding mutations in the same genes were observed in all cases. The mutations included seven missense, three same-sense, four frameshift and two truncating mutations. FISH revealed heterozygous deletion of SMARCB1 in one case, and monoploidy of chromosome 22, which harbours SMARCB1, in another case. Furthermore, trisomy of chromosome 9, which harbours SMARCA2, was observed in two cases. Abnormality of PBRM1 was not found in any case. CONCLUSIONS: We report, for the first time, co-inactivation and frequent mutations of SMARCB1, SMARCA2 and PBRM1 in MRTs. Multiple subunit abnormalities of the SWI/SNF complex potentially act together to contribute to the tumorigenesis of MRTs, which provides unique insights into this disease.
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Proteínas Cromosómicas no Histona/genética , Proteínas de Unión al ADN/genética , Silenciador del Gen , Mutación/genética , Proteínas Nucleares/genética , Tumor Rabdoide/genética , Factores de Transcripción/genética , Preescolar , Proteínas Cromosómicas no Histona/metabolismo , Análisis Mutacional de ADN , Proteínas de Unión al ADN/metabolismo , Femenino , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Masculino , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Proteína SMARCB1 , Análisis de Secuencia de ADN , Factores de Transcripción/metabolismoRESUMEN
As we all known, the instantaneous reaction between protein and ligands are very important to adjust the normal playing of biological function. And nitric oxide interactions with iron are the most important biological reactions in which NO participates. Unlike carbon monoxide or oxygen, NO can also bind reversibly to ferric iron. In this paper, UV-Vis absorption and CD spectra were used to study coordination reaction process between horse heart metMb and NO, to demonstrate the coordination reaction mechanism and to explore the influencing factors of metMb with NO. The experimental results showed that metMb could react with NO, and obtained three new peaks at 420 nm, 534 and 568 nm, respectively, which implied metMb and NO have reacted and generated a new complex-nitrosylmetmyoglobin (metMbNO). Then as time went on, NO concentration decreased in the solution, and the Fe-N bond fractured under the attack of H2O, then NO leaves slowly from metMbNO, and met-Mb was regenerated. In this experiment, we also found that external conditions such as buffer medium, ionic strength, pH, temperature, etc, had an important influence on the coordination reaction between metMb and NO. It was favorable for the coordination reaction, when the 0.01 mol x L(-1) phosphate buffer. solution is near neutral condition, the temperature is 280 K, the coordination reaction could reach equilibrium at a fastest speed. In addition, the CD date show that NO only reacts with Fe atom in the center of heme and has less effect on the secondary structuers of protein. The research of metMb and NO played an important role to further study the function of NO. Especially the establish of equilibrium reaction mechanism between NO and heme protein has an important research value on maintaining the balance of NO in vivo and keeping the normal function in the body's cells.
Asunto(s)
Metamioglobina/química , Óxido Nítrico/química , Animales , Hemo/química , Caballos , Concentración de Iones de Hidrógeno , Hierro/química , Soluciones , TemperaturaRESUMEN
AIMS: The aim of this study was to examine the status of Brahma (BRM), a key SWI/SNF complex subunit, in clear cell renal cell carcinomas (RCCs), and to analyse the histopathology, immunophenotype, molecular features and prognosis of the BRM-negative cases. METHODS AND RESULTS: We identified 19 cases of grade 4 tumours lacking BRM expression among 625 clear cell RCCs. All 19 cases exhibited features of poor differentiation: 13 showed pure poorly differentiated morphology, while six were composite tumours with an admixed typical low-grade component. Besides negative BRM expression, the immunophenotype of these cases was similar to clear cell RCC. VHL gene mutations were identified in nine of the 19 patients (47%). Chromosome 3p deletion was detected in 11 of 13 poorly differentiated RCCs and both areas of five of five composite tumours. All poorly differentiated tumour areas showed polysomy of chromosome 3. No losses or gains of chromosome 3 were observed in low-grade tumour areas of five of five composite RCCs. CONCLUSIONS: We have shown that loss of BRM expression is a common feature among poorly differentiated tumours in clear cell RCCs. We hypothesize that loss of BRM expression is involved in tumor de-differentiation in clear cell RCCs and may play an important role during tumour progression.