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Within Internet of Things (IoT) sensors, the challenge is how to dig out the potentially valuable information from the collected data to support decision making. This paper proposes a method based on machine learning to predict long cycle maintenance time of wind turbines for efficient management in the power company. Long cycle maintenance time prediction makes the power company operate wind turbines as cost-effectively as possible to maximize the profit. Sensor data including operation data, maintenance time data, and event codes are collected from 31 wind turbines in two wind farms. Data aggregation is performed to filter out some errors and get significant information from the data. Then, the hybrid network is built to train the predictive model based on the convolutional neural network (CNN) and support vector machine (SVM). The experimental results show that the prediction of the proposed method reaches high accuracy, which helps drive up the efficiency of wind turbine maintenance.
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OBJECTIVE: The present study aimed to establish an induced pluripotent stem cell (iPSC) line from acute myelogenous leukemia (AML) cells in vitro and identify their biological characteristics. METHODS: Cells from the AML-infiltrated skin from an M6 patient were infected with a lentivirus carrying OCT4, SOX2, KLF4 and C-MYC to induce iPSCs. The characteristics of the iPSCs were confirmed by alkaline phosphatase (ALP) staining. The proliferation ability of iPSCs was detected with a CCK-8 assay. The expression of pluripotency markers was measured by immunostaining, and the expression of stem cell-related genes was detected by qRT-PCR; distortion during the induction process was detected by karyotype analysis; the differentiation potential of iPSCs was determined by embryoid body-formation and teratoma-formation assays. ALP staining confirmed that these cells exhibited positive staining and had the characteristics of iPSCs. RESULTS: The CCK-8 assay showed that the iPSCs had the ability to proliferate. Immunostaining demonstrated that iPSC clones showed positive expression of NANOG, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. qRT-PCR results revealed that the mRNA expression of Nanog, Lin28, Cripto, FOX3, DNMT3b, DPPA2, and DPPA4 significantly increased in iPSCs. Karyotype analysis found no chromosome aberration in the iPSCs. The results of the embryoid body-formation and teratoma-formation assays indicated that the iPSCs had the potential to differentiate into all three germ layers. CONCLUSION: Our study provided evidence that an iPSC line derived from AML cells was successfully established.
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Regulación Leucémica de la Expresión Génica , Células Madre Pluripotentes Inducidas/metabolismo , Leucemia Eritroblástica Aguda/metabolismo , Proteínas de Neoplasias/biosíntesis , Neoplasias Cutáneas/metabolismo , Factores de Transcripción/biosíntesis , Adulto , Humanos , Células Madre Pluripotentes Inducidas/patología , Factor 4 Similar a Kruppel , Leucemia Eritroblástica Aguda/genética , Leucemia Eritroblástica Aguda/patología , Masculino , Proteínas de Neoplasias/genética , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Factores de Transcripción/genéticaRESUMEN
Underwater image processing has been shown to exhibit significant potential for exploring underwater environments. It has been applied to a wide variety of fields, such as underwater terrain scanning and autonomous underwater vehicles (AUVs)-driven applications, such as image-based underwater object detection. However, underwater images often suffer from degeneration due to attenuation, color distortion, and noise from artificial lighting sources as well as the effects of possibly low-end optical imaging devices. Thus, object detection performance would be degraded accordingly. To tackle this problem, in this article, a lightweight deep underwater object detection network is proposed. The key is to present a deep model for jointly learning color conversion and object detection for underwater images. The image color conversion module aims at transforming color images to the corresponding grayscale images to solve the problem of underwater color absorption to enhance the object detection performance with lower computational complexity. The presented experimental results with our implementation on the Raspberry pi platform have justified the effectiveness of the proposed lightweight jointly learning model for underwater object detection compared with the state-of-the-art approaches.
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This study developed a novel surface-enhanced Raman spectroscopy (SERS) method coupled with cellulose nanofiber (CNF)-based SERS wipers that were fabricated on quartz papers coated with a mixture of silver nanoparticle (AgNP) and gold nanostar (AuNS). A "drop-wipe-test" protocol was developed for rapid detection of pesticide residues in vegetables by SERS. Tremendously enhanced Raman scattering signals were obtained from the quartz/CNF/mixture (AgNP + AuNS) substrate, which were much higher than the paper/mixture (AgNP + AuNS) substrate. This method was used to detect ferbam on kale leaves within a few minutes and the detection limit was 50 µg/kg based on the PLS models (R2 = 0.89). The enhancement factor of the SERS substrate was calculated to be ~ 104 with satisfactory reproducibility. Satisfactory SERS performance could be achieved within 1-month storage period. These results demonstrate that this CNF-based SERS/wiper method is a practical approach for rapid detection of chemical contaminants in fresh produce.
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Brassica/química , Dimetilditiocarbamato/análisis , Nanopartículas del Metal/química , Nanofibras/química , Espectrometría Raman/métodos , Celulosa/química , Oro/química , Residuos de Plaguicidas/análisis , Reproducibilidad de los Resultados , Plata/químicaRESUMEN
OBJECTIVE: To investigate the role of nucleophosmin (NPM) in the proliferation of chronic myeloid leukemia cells (K562 cells) and its mechanism by RNAi technology. METHODS: shRNA was used to inhibit the expression of NPM. The expression of NPM gene was detected by real-time quantitative PCR. The effect of inhibiting NPM gene on cell proliferation was detected by MTS assay. Change of cell cycle was detected by flow cytometry. Western blot was used to detect the expression of cell cycle-related proteins. RESULTS: The shRNA lentiviral vector targeting at NPM gene was successfully constructed and used to transfect the K562 cells. The results showed that compared with the control groups, suppression of NPM gene expression in K562 cells could inhibit the cell proliferation and decrease the cell colony formation. Moreover, interference of NPM gene could prolong G0/G1 phase and arrest cell cycle, which may be related to the down-regulation of NPM gene expression and activation of p21 protein expression, thereby inhibited the formation of CDK2/ Cyclin E complex. CONCLUSION: Down-regulation of NPM gene expression in K562 cells can induce cell cycle arrest and inhibit cell proliferation.
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Leucemia Mielógena Crónica BCR-ABL Positiva , Apoptosis , Proliferación Celular , Técnicas de Silenciamiento del Gen , Humanos , Células K562 , Proteínas Nucleares , NucleofosminaRESUMEN
OBJECTIVE: To construct a K562 and adriamycin-resistant K562 (KAR) cell line with stably down-regulation of NCL (nucleolin) expression, and to investigate the effect of NCL down-regulation on the drug resistance in K562 and KAR cells. METHODS: K562 and KAR cells were infected with lentivirus, and stably transfected cell clones were obtained by puromycin screening. The cell proliferation was detected by MTS assay, the cell apoptosis was detected by flow cytometry, and the expression level of drug resistance related genes was detected by real-time PCR. RESULTS: The K562 and KAR cells with stable down-regulation of NCL were successfully constructed. Compared with the control group, the proliferation of K562 and KAR cells with down-regulating NCL expression decreased significantly (P ï¼0.05), the apoptosis of cells increased significantly (P ï¼0.05), and cell resistance to adriamycin was down-regulated. CONCLUSION: Inhibition of NCL expression may increase the sensitivity of cells to adriamycin, which may be related with the promotion of apoptosis of K562 and KAR cells.
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Resistencia a Antineoplásicos , Leucemia Mielógena Crónica BCR-ABL Positiva , Apoptosis , Doxorrubicina , Humanos , Células K562 , Fosfoproteínas , Proteínas de Unión al ARN , NucleolinaRESUMEN
OBJECTIVE: To investigate the effect of baicalin derivative 02-036 on proliferation and apoptosis human Burkitt lymphoma cell line CA46 and its related mechanisms. METHODS: The MTT assay and cell colony formation assay were used to measure the growth inhibition of CA46 cells after 02-036 treatment. The flow cytometry with AnnexinV-FITC/PI double staining was employed to detect the apoptosis induction effect of 02-036 on CA46 cells. Cell cycle distribution of CA46 cells was estimeted by using DNA ploid analysis. Western blot was used to determine the changes of apoptosis-related proteins, including C-MYC, BCL-2, Procaspase-9, Procaspase-3, PARP and Cleaved-PARP. RESULTS: Baicalin derivative 02-036 obviously inhibited the proliferation of CA46 cells, with dose- and time-dependent manner (r=0.963, r=0.992). The averaged IC50 value of CA46 cells was (6.04±0.11) µmol/L after 48-hour treatment. Low concentration of 02-036 could significantly inhibit the colony formation of CA46 cells. Flow cytometry analysis confirmed that 02-036 could effectively induce CA46 cell apoptosis. The apoptosis rate correlated with drug concentrations (r=0.959). Also, DNA ploid analysis showed that the cell cycle of CA46 was arrested in the S phase. The expression levels of BCL-2, Pro-caspase-9, Pro-caspase-3, PARP and C-MYC proteins decreased with a 02-036-dose dependent manner (r values were -0.990, -0.939, -0.971 and -0.967, respectively). In contrast, the expression level of cleaved-PARP increased with the same manner (r=0.920). CONCLUSION: Baicalin derivative 02-036 can effectively inhibit the proliferation and induce apoptosis of CA46 cells, and its related mechanisms may be correlated with the down-regulation of apoptosis-related molecule expression levels, such as BCL-2, Pro-caspase-9, Pro-caspase-3, PARP and C-MYC.
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Linfoma de Burkitt , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Flavonoides , HumanosRESUMEN
Entropy images, representing the complexity of original fundus photographs, may strengthen the contrast between diabetic retinopathy (DR) lesions and unaffected areas. The aim of this study is to compare the detection performance for severe DR between original fundus photographs and entropy images by deep learning. A sample of 21,123 interpretable fundus photographs obtained from a publicly available data set was expanded to 33,000 images by rotating and flipping. All photographs were transformed into entropy images using block size 9 and downsized to a standard resolution of 100 × 100 pixels. The stages of DR are classified into 5 grades based on the International Clinical Diabetic Retinopathy Disease Severity Scale: Grade 0 (no DR), Grade 1 (mild nonproliferative DR), Grade 2 (moderate nonproliferative DR), Grade 3 (severe nonproliferative DR), and Grade 4 (proliferative DR). Of these 33,000 photographs, 30,000 images were randomly selected as the training set, and the remaining 3,000 images were used as the testing set. Both the original fundus photographs and the entropy images were used as the inputs of convolutional neural network (CNN), and the results of detecting referable DR (Grades 2-4) as the outputs from the two data sets were compared. The detection accuracy, sensitivity, and specificity of using the original fundus photographs data set were 81.80%, 68.36%, 89.87%, respectively, for the entropy images data set, and the figures significantly increased to 86.10%, 73.24%, and 93.81%, respectively (all p values <0.001). The entropy image quantifies the amount of information in the fundus photograph and efficiently accelerates the generating of feature maps in the CNN. The research results draw the conclusion that transformed entropy imaging of fundus photographs can increase the machinery detection accuracy, sensitivity, and specificity of referable DR for the deep learning-based system.
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Activation-induced cytidine deaminase (AID) is essential for somatic hypermutation and class switch recombination in mature B-cells, while AID was also shown to play a role in developing pre-BCR/BCR-positive B-cells of the bone marrow. To further elucidate a potential function of Aid in the bone marrow prior to V(D)J-recombination, we utilized an in vivo model which exerts a B-cell developmental arrest at the pro-B cell stage with low frequencies of pro-B cell acute lymphoblastic leukemia (pro-B ALL) development. Therefore, p19Arf-/-Rag1-/- (AR) mice were crossed with Aid-deficient mice (ARA). Surprisingly, loss of Aid expression in pro-B cells accelerated pro-B ALL incidence from 30% (AR) to 98% (ARA). This effect was Aid dose dependent, since Aid+/- animals of the same background displayed a significantly lower incidence (83%). Furthermore, B-cell-specific Aid up-regulation was observed in Aid-competent pro-B ALLs. Additional whole exome/sanger sequencing of murine pro-B ALLs revealed an accumulation of recurrent somatic Jak3 (p.R653H, p.V670A) and Dnm2 (p.G397R) mutations, which highlights the importance of active IL7R signaling in the pro-B ALL blast cells. These findings were further supported by an enhanced proliferative potential of ARA pro-B cells compared to Aid-competent cells from the same genetic background. In summary, we show that both Aid and Rag1 act as a negative regulators in pro-B cells, preventing pro-B ALL.
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OBJECTIVE: To explore the effect of a novel emodin derivative E19 on proliferation inhibition and apoptosis induction of human chronic myelogenous leukemia (CML) cell line K562 and imatinib-resistant CML cell line (K562/G01), and to clarify the involved mechanisms. METHODS: MTT and colony formation test were used to detect the cell proliferation. Apoptotic induction effects were examined by DAPI staining method and DNA ladder assay. Western blot was performed to detect the changes of P210(Bcr-Abl) protein. RESULTS: The emodin derivative E19 could efficiently inhibit proliferation and induce apoptosis in K562 and K562/G01 cells. IC50 of K562 cells and IC50 of K562/G01 cells were (1.20 ± 0.19) µmol/L and (1.22 ± 0.16) µmol/L, respectively. DNA fragmentation in K562 cells and K562/G01 cells confirmed that the E19 induced apoptosis in dose-dependent manner. Western blot showed that emodin derivative inhibited phosphorylation of P210 protein in K562 cells and K562/G01 cells and down-regulated the expression level of P210 in dose- and time-dependent manners. CONCLUSION: The emodin derivative E19 can efficiently inhibit growth and induce apoptosis of K562 cells and K562/G01 cells, while the inhibition of phosphorylation of P210 protein and down-regulation of P210 protein expression may be involved in these processes.
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Apoptosis/efectos de los fármacos , Emodina/farmacología , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Proliferación Celular , Regulación hacia Abajo , Resistencia a Antineoplásicos , Emodina/análogos & derivados , Proteínas de Fusión bcr-abl/metabolismo , Humanos , Mesilato de Imatinib/farmacología , Células K562/efectos de los fármacos , FosforilaciónRESUMEN
OBJECTIVE: To investigate the apoptosis-inducing effects of emodin on multidrug resistant leukemia cell line K562/Adr, and to explore the role of Akt-Caspase 3 signal pathway in apoptosis of K562/Adr cells treated with emodin. METHODS: K562/Adr cells were exposed to emodin of different doses. The ability of emodin to induce apoptosis of K562/Adr cells was detected by Annexin V/PI double labeled flow cytometry and DNA ploidy analysis, the expressions of procaspase-3, PARP, Akt, p-Akt protein were determined by Western blot. RESULTS: Apoptosis in K562/Adr cells could be induced by emodin in a dose dependent manner, Western blot results showed that emodin down-regulated the expression levels of procaspase-3, Akt, p-Akt, PARA 116 KD in treated K562/Adr cells, up-regulated expressions leves of PARP 85 KD in a time-dependent manner. CONCLUSION: The Akt-Caspase 3 signal pathway may be involved in these processes.
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Apoptosis , Transducción de Señal , Caspasa 3 , Regulación hacia Abajo , Emodina , Humanos , Células K562RESUMEN
This study was aimed to investigate the expression of stathmin1 mRNA and stathmin1 protein in de novo patients with acute leukemia (AL), relapsed patients with AL and complete remission patients with AL, and its clinical significance. The expression of stathmin1 mRNA and stathmin1 protein in peripheral blood samples from 76 cases of AL and 25 healthy persons were examined by fluorescent quantitative PCR (FQ-PCR) and Western blot, respectively. The results showed that the stathmin1 protein expression could not be detected in healthy persons, only the low level of its mRNA could be observed in them. The stathmin1 mRNA expression level in de novo AL patients was higher than that in healthy persons (P < 0.05), the stathmin1 mRNA expression level in relapsed patients with AL was higher than that in de novo patients (P < 0.05), and there was no significant difference of stathmin1 mRNA expression between patients with AML and patients with ALL. The positive rate of stathmin1 protein expression in de novo patients with AL was 89%, while it obviously decreased or did not express in complete remission patients with AL. The stathmin1 protein expression in relapsed patients with AL did not display significant difference as compared with that in de novo patients (P > 0.05). There was no significant difference in stathmin1 protein expression between patients with AML and patients with ALL (P > 0.05). It is concluded that stathmin1 protein and mRNA are overexpressed in de novo patients and relapsed patients, and lowly expressed in complete remission patients. Therefore, the stathmin1 may be a new biological marker for evaluation of minimal residual disease.
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Leucemia/sangre , Neoplasia Residual/sangre , Estatmina/sangre , Enfermedad Aguda , Adolescente , Adulto , Estudios de Casos y Controles , Humanos , Leucemia/patología , Persona de Mediana Edad , Neoplasia Residual/diagnóstico , Adulto JovenRESUMEN
This study was aimed to construct model cell line of NPM1-RNAi in HL-60 cells and its resistant line (HL-60/ADR) so as to provide a experimental basis for investigating the potential role of NPM1 gene in leukemia drug resistance. The shRNA targeting to NPM1 was ligated into linear pGCSIL-GFP vector, and transformed into E.coli DH5α. Positive clone was identified by PCR and DNA sequencing. pHelper 1.0, pHelper 2.0 and pGCSIL-GFP-NPM1-shRNA were cotransformed into 293T cells by lentivirus vector system. NPM1-RNAi-LV was transfected into HL-60 and HL-60/ADR cell lines. The efficiency of NPM-RNAi-LV was detected by using real-time quantitative RT-PCR and Western blot. The results showed that the recombinant eukaryotic expression vector pGCSIL-GFP-NPM1-shRNA was constructed. pGCSIL-GFP-NPM1-shRNA was packed into NPM1-RNAi-LV by lentivirus vector system, and transfected into HL-60 and HL-60/ADR cell lines. At mRNA level, the efficiency of NPM1 mRNA knockdown was more than 90% (p < 0.05). At protein level, obvious down-regulation of NPM protein was noted, indicating that NPM1 gene in HL-60 and HL-60/ADR cell lines was knocked down after transfected with NPM1-RNAi-LV. The resistance of HL-60/ADR cell line to adriamycin decreased to a certain degree after NPM1 gene silencing. It is concluded that the model cell lines of NPM1-RNAi in HL-60 and HL-60/ADR are successfully constructed, which can be used for investigating the potential role of NPM1 gene in drug resistance of leukemia.
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Doxorrubicina/farmacología , Proteínas Nucleares/genética , Interferencia de ARN , Línea Celular Tumoral , Resistencia a Antineoplásicos/genética , Vectores Genéticos , Células HL-60 , Humanos , Lentivirus/genética , NucleofosminaRESUMEN
The purpose of this study was to compare the differences of the protein expression profiles between human myeloid leukemia K562 cells and adriamycin-resistant K562/A02 cells, as well as to select novel resistance-related proteins in myeloid leukemia by means of proteomics. The total cellular proteins were separated from K562 and adriamycin-resistant K562/A02 cells by using technique of two dimensional difference in gel electrophoresis (2D-DIGE). Differentially expressed proteins were analyzed by matrix-assisted laser desorption ionization/time of flight-mass spectrometry (MALDI-TOF/MS), and by protein database searching. Moreover, the differentially expressed proteins were verified at protein and mRNA levels by Western blot assay and quantitative real time PCR. The results showed that 8 proteins differentially expressed in adriamycin-resistant K562/A02 cells, among them 2 proteins were identified to be down-regulated and 6 to be up-regulated. These identified proteins involved in the cell energy metabolism, cell proliferation, cell apoptosis, signal transduction, gene transcription and translation respectively. The results assayed by Western blot were similar to those detected by 2D-PAGE. Two up-regulated proteins Stathmin and CrkL were selected for verification in K562 and K562/A02 cells. As a result, the results detected by Western blot were identical with results from 2D-DIGE; real time quantitative PCR assay showed that the changes of CrkL at mRNA level were identical with changes at protein level, but no complete identity of Stathmin changes at mRNA level and protein level was observed. It is concluded that the difference of protein expression profile exists in K562 and K562/A02 cells. Stathmin and CrkL proteins may be involved in the drug resistance and suggest a novel clue for the resistant mechanisms in myeloid leukemia, which is worth further to explore.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Leucemia/metabolismo , Proteínas Nucleares/metabolismo , Estatmina/metabolismo , Doxorrubicina/farmacología , Resistencia a Antineoplásicos , Humanos , Células K562RESUMEN
In order to clone the full-length cDNA of a novel EST which is probably related to acute leukemia relapse and to analyse the sequences, the electronic cloning technique combined with RT-PCR was used to clone the full-length cDNA, and the sequences were analyzed by bioinformatics. The results showed that the two novel splicing variants of eIF4E named as splicing variant 1 and 2 of eIF4E were obtained. Bioinformatics analysis showed that variant 1 and 2 exhibited 84% and 47% similarity to eIF4E mRNA, and were localized on eIF4E locus on chromosome 4. The lengths of 2 variants are 1 904 bp and 3 393 bp, encoding 245 amino acids and 132 amino acids respectively. BLAST results showed that the both variants mentioned above contains seven exons. Among them, the sequences of the three exons at 5' end of variant 1, variant 2 and eIF4E mRNA were different from each other. Protein BLAST showed that they are partially different from eIF4E protein. It is concluded that the two novel splicing variants of eIF4E were cloned, and their relation to acute leukemia relapse needs to be further investigated.
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Clonación Molecular , Factor 4E Eucariótico de Iniciación/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Secuencia de Bases , Biología Computacional , ADN Complementario/genética , ADN Recombinante , Humanos , Datos de Secuencia Molecular , Isoformas de Proteínas/genéticaRESUMEN
The study was aimed to investigate the effects of emodin on proliferation inhibition and apoptosis induction in human chronic myeloid leukemia cell line K562 cells, and to explore the role of P210 protein and activation of caspase 3 in these processes. K562 cells were exposed to emodin at different doses. The proliferation inhibition was detected by MTT assay and colony formation test. The ability of emodin to induce apoptosis and DNA fragmentation were examined by flow cytometry. The expressions of P210, procaspase-3 and PARP protein were determined by Western blot. The results indicated that the emodin remarkably inhibited the K562 cell proliferation, with IC(50) value of 38.25 micromol/L after treatment for 48 hours. Meanwhile induced apoptosis, Annexin V-FITC positive cells, sub-G(1) apoptotic peak and DNA fragmentation in K562 cells confirmed that emodin induced apoptosis in K562 cells in dose-dependent manner. Western blot results showed that emodin inhibited phosphorylation of P210 protein in K562 cells and down-regulated the expression levels of P210. The procaspase-3 level in treated K562 cells decreased with increased expressions of PARP in time-dependent manner. It is concluded that the emodin efficiently inhibits growth and induces apoptosis of K562 cells, while the inhibition of phosphorylation of P210 protein, down-regulation of P210 protein expression and activation of caspase-3 may be involved in these processes.
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Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Proliferación Celular/efectos de los fármacos , Emodina/farmacología , Proteínas de Fusión bcr-abl/metabolismo , Regulación Leucémica de la Expresión Génica , Humanos , Células K562 , Fosforilación , Poli(ADP-Ribosa) Polimerasas/metabolismoRESUMEN
Vps4 is a host factor known to be involved in cellular vacuolar protein sorting. We report here that HBV replication and secretion can be significantly inhibited by Vps4 dominant negative, ATPase-defective, mutants K173Q and E228Q. In contrast, wild-type Vps4 at low dose can inhibit HBV replication more effectively in human hepatoblastoma cell line HepG2, than in human hepatoma cell line Huh7. To our knowledge, this is the first demonstration of an anti-HBV potential of dominant negative mutants of a protein sorting host factor Vps4.
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Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/fisiología , Virus de la Hepatitis B/fisiología , Proteínas Represoras/genética , Proteínas Represoras/fisiología , Replicación Viral , ATPasas Asociadas con Actividades Celulares Diversas , Sustitución de Aminoácidos , Línea Celular Tumoral , Complejos de Clasificación Endosomal Requeridos para el Transporte , Humanos , Mutación , ATPasas de Translocación de Protón Vacuolares , Proteínas de Transporte VesicularRESUMEN
Recently, a rat pancreatic cell line (AR42J-B13) was shown to transdifferentiate to hepatocyte-like cells upon induction with dexamethasone (Dex). The aim of this study is to determine whether transdifferentiated hepatocytes can indeed function like bona fide liver cells and support replication of hepatotropic hepatitis B virus (HBV). We stably transfected AR42J-B13 cells with HBV DNA and examined the expression of hepatocyte markers and viral activities in control and transdifferentiated cells. A full spectrum of HBV replicative intermediates, including covalently closed circular DNA (cccDNA) and Dane particles, were detected only after induction with Dex and oncostatin M. Strikingly, the small envelope protein and RNA of HBV were increased by 40- to 100-fold upon induction. When HBV RNAs were examined by primer extension analysis, novel core- and precore-specific transcripts were induced by Dex which initiated at nucleotide (nt) 1820 and nt 1789, respectively. Most surprisingly, another species of core-specific RNA, which initiates at nt 1825, is always present at almost equal intensity before and after Dex treatment, a result consistent with Northern blot analysis. The fact that HBV core protein is dramatically produced only after transdifferentiation suggests the possibility of both transcriptional and translational regulation of HBV core antigen in HBV-transfected AR42J-B13 cells. Upon withdrawal of Dex, HBV replication and gene expression decreased rapidly-less than 50% of the cccDNA remained detectable in 1.5 days. Our studies demonstrate that the transdifferentiated AR42J-B13 cells can function like bona fide hepatocytes. This system offers a new opportunity for basic research of virus-host interactions and pancreatic transdifferentiation.
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Virus de la Hepatitis B/fisiología , Cultivo de Virus/métodos , Animales , Diferenciación Celular , Línea Celular , ADN Viral/genética , Dexametasona/farmacología , Regulación Viral de la Expresión Génica/efectos de los fármacos , Virus de la Hepatitis B/genética , Hepatocitos/virología , Oncostatina M , Páncreas/citología , Péptidos/farmacología , Ratas , Especificidad de la Especie , Transfección , Replicación Viral/efectos de los fármacosRESUMEN
We identified two novel naturally occurring mutations (W74L and L77R) in the small S envelope protein of hepatitis B virus (HBV). Mutation L77R alone resulted in >10-fold-reduced secretion of virions. In addition, the 2.8-fold reduction of the extracellular HBV surface antigen (HBsAg) of mutant L77R from transfected Huh7 cells appeared to be correlated with a 1.7-fold reduction of intracellular HBsAg, as measured by enzyme-linked immunosorbent assay (ELISA). Surprisingly, opposite to the ELISA results, Western blot analysis revealed a near-10-fold-increased level of the intracellular mutant small S envelope protein. The discrepancy between ELISA and Western blot data was due to significant accumulation of the mutant L77R HBsAg in the intracellular pellet fraction. In contrast to HBsAg, the secretion of HBeAg was normal in L77R-transfected cells. The wild-type HBsAg was usually more diffuse and evenly distributed in the cytoplasm, often outside the perinuclear endoplasmic reticulum (ER) and Golgi apparatus, as observed by immunofluorescence assay. In contrast, the L77R mutant HBsAg tends to be highly restricted within the ER and Golgi, often accumulated in the Golgi compartments distal from the nucleus. The almost exclusive retention in the ER-Golgi of L77R HBsAg was similar to what was observed when the large envelope protein was overexpressed. These multiple aberrant phenotypes of mutant L77R can be corrected by a second naturally occurring S envelope mutation, W74L. Despite the accumulation of L77R HBsAg in ER-Golgi of transfected Huh7 cells, we detected no increase in Grp78 mRNA and proteins, which are common markers for ER stress response.
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Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Antígenos de Superficie de la Hepatitis B/metabolismo , Virus de la Hepatitis B/fisiología , Hepatitis B Crónica/virología , Proteínas del Envoltorio Viral/metabolismo , Virión/fisiología , Adolescente , Adulto , Línea Celular , Chaperón BiP del Retículo Endoplásmico , Femenino , Antígenos e de la Hepatitis B/metabolismo , Virus de la Hepatitis B/genética , Hepatitis B Crónica/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Mutación , Fenotipo , Proteínas del Envoltorio Viral/genética , Replicación ViralRESUMEN
Human hepatitis B virus (HBV) variants containing in-frame core internal deletion (CID) have been demonstrated to contain all the functional features of defective interfering (DI) particles (Yuan, T. T.-T., M.-H. Lin, D. S. Chen, and C. Shih, 1998, J. Virol. 72, 578-584). Here, we report that out-of-frame HBV CID variants exhibit defective interfering property similar to in-frame CID variants characterized previously. This result raises the possibility that it may be the deleted pregenomic RNA product, rather than the deleted core protein product, that is responsible for interference. Furthermore, a genomic deletion elsewhere does not cause interference since preS2 deletion variants exhibit no influence on wild-type HBV replication. Consistent with the natural occurrence of HBV CID variants, we recently identified CID variants of woodchuck hepatitis virus (WHV) in natural infection. However, unlike HBV CID variants, functional characterization of WHV CID variants using a human hepatoma cell line has not revealed any interference in tissue culture. In summary, defective interference is a general phenomenon for both in-frame and out-of-frame HBV CID variants.