ABSTRACT
Glioblastoma (GBM) is the most common brain tumor and remains incurable. Primary GBM cultures are widely used tools for drug screening, but there is a lack of genomic and pharmacological characterization for these primary GBM cultures. Here, we collect 50 patient-derived glioma cell (PDGC) lines and characterize them by whole genome sequencing, RNA sequencing, and drug response screening. We identify three molecular subtypes among PDGCs: mesenchymal (MES), proneural (PN), and oxidative phosphorylation (OXPHOS). Drug response profiling reveals that PN subtype PDGCs are sensitive to tyrosine kinase inhibitors, whereas OXPHOS subtype PDGCs are sensitive to histone deacetylase inhibitors, oxidative phosphorylation inhibitors, and HMG-CoA reductase inhibitors. PN and OXPHOS subtype PDGCs stably form tumors in vivo upon intracranial transplantation into immunodeficient mice, whereas most MES subtype PDGCs fail to form tumors in vivo. In addition, PDGCs cultured by serum-free medium, especially long-passage PDGCs, carry MYC/MYCN amplification, which is rare in GBM patients. Our study provides a valuable resource for understanding primary glioma cell cultures and clinical translation and highlights the problems of serum-free PDGC culture systems that cannot be ignored.
Subject(s)
Brain Neoplasms , Glioma , Humans , Animals , Brain Neoplasms/pathology , Brain Neoplasms/genetics , Brain Neoplasms/drug therapy , Brain Neoplasms/metabolism , Cell Line, Tumor , Mice , Glioma/genetics , Glioma/pathology , Glioma/drug therapy , Glioma/metabolism , Oxidative Phosphorylation/drug effects , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Glioblastoma/genetics , Glioblastoma/pathology , Glioblastoma/drug therapy , Glioblastoma/metabolism , Female , Male , Whole Genome Sequencing , Xenograft Model Antitumor Assays , Genomics/methods , Gene Expression Regulation, Neoplastic/drug effects , MultiomicsABSTRACT
Glioblastoma is one of the most common malignant brain tumors. Vitamin D, primarily its hormonally active form calcitriol, has been reported to have anti-cancer activity. In the present study, we used patient-derived glioma cell lines to examine the effect of vitamin D3 and calcitriol on glioblastoma. Surprisingly, vitamin D3 showed a more significant inhibitory effect than calcitriol on cell viability and proliferation. Vitamin D receptor (VDR) mediates most of the cellular effects of vitamin D, and thus we examined the expression level and function of VDR via gene silencing and gene knockout experiments. We observed that VDR does not affect the sensitivity of patient-derived glioma cell lines to vitamin D3, and the gene encoding VDR is not essential for growth of patient-derived glioma cell lines. RNA sequencing data analysis and sterolomics analysis revealed that vitamin D3 inhibits cholesterol synthesis and cholesterol homeostasis by inhibiting the expression level of 7-dehydrocholesterol reductase, which leads to the accumulation of 7-dehydrocholesterol and other sterol intermediates. In conclusion, our results suggest that vitamin D3, rather than calcitriol, inhibits growth of patient-derived glioma cell lines via inhibition of the cholesterol homeostasis pathway.
Subject(s)
Cholecalciferol , Glioblastoma , Humans , Cholecalciferol/pharmacology , Calcitriol/pharmacology , Glioblastoma/drug therapy , Vitamin D/pharmacology , Cell Line , Homeostasis , CholesterolABSTRACT
This paper presents a modified unified critical state model to predict the mechanical responses of both clays and sands under over-consolidation and cyclic loading conditions on the basis of clay and sand model (CASM), which is named as CASM-kII. Through the application of subloading surface concept, CASM-kII is able to describe the plastic deformation inside the yield surface and the reverse plastic flow, and is thus expected to capture the over-consolidation and cyclic loading behaviours of soils. CASM-kII is numerical implemented by the using of the forward Euler scheme with automatic substepping and error control. Then, a sensitivity study is carried out to check the influences of the three new parameters of CASM-kII on the mechanical response of soils in over-consolidation and cyclic loading conditions. Through the comparisons of experimental data and simulated results, it is found that CASM-kII is able to satisfactorily describe the mechanical responses of both clays and sands in over-consolidation and cyclic loading conditions.
ABSTRACT
In traditional slope stability analyses, soil is usually approximated as isotropic. However, naturally cohesive soil deposits are inherently anisotropic, primarily due to the directional arrangement of soil particles during their deposition process. In this paper, a generalized anisotropic constitutive model for c-φ soil is introduced to evaluate the influence of varying shear strength on slope stability. In this model, the initial strength anisotropy is defined by the variety of friction angles to the direction of the principle stress. This model is utilized by two approaches to estimate the slope stability. Firstly, the upper bound limit analysis solution for slope stability is developed, and the safety factor of the slopes is studied. Secondly, this model is coupled with the finite element method to get insight of the influence of anisotropy on slope stability. One typical slope case of slope is studied by numerical analyses. It is found that the slope stability is largely overestimated when the strength anisotropy is ignored, and the overestimation, in terms of safety factors, can reach up to 32.9%. The complex interrelations between the degree of anisotropy and evolution of the ensuing safety factor are revealed by a series of parametric studies in terms of different degrees of anisotropy.
Subject(s)
Soil , Anisotropy , Finite Element Analysis , Shear Strength , FrictionABSTRACT
With the increasing penetration of renewable energy, uncertainty has become the main challenge of power systems operation. Fortunately, system operators could deal with the uncertainty by adopting stochastic optimization (SO), robust optimization (RO) and distributionally robust optimization (DRO). However, choosing a good decision takes much experience, which can be difficult when system operators are inexperienced or there are staff shortages. In this paper, a decision-making approach containing robotic assistance is proposed. First, advanced clustering and reduction methods are used to obtain the scenarios of renewable generation, thus constructing a scenario-based ambiguity set of distributionally robust unit commitment (DR-UC). Second, a DR-UC model is built according to the above time-series ambiguity set, which is solved by a hybrid algorithm containing improved particle swarm optimization (IPSO) and mathematical solver. Third, the above model and solution algorithm are imported into robots that assist in decision making. Finally, the validity of this research is demonstrated by a series of experiments on two IEEE test systems.
ABSTRACT
BACKGROUND: Acetaminophen is a widely clinically used analgesic. However, the clinical effect of the route of administration on postoperative analgesia as well as on postoperative nausea and vomiting in patients undergoing general anesthesia remains unclear. This study aimed to explore whether the route of administration of acetaminophen affects postoperative analgesia, nausea, and vomiting in patients undergoing general anesthesia. METHODS: We included all randomized controlled trials investigating the effects of the route of administration of acetaminophen on postoperative pain, nausea, and vomiting in patients undergoing general anesthesia. Independent examiners reviewed the literature and extracted data, with disagreements resolved through negotiation or the involvement of a third party. The Cochrane risk assessment tool was used to evaluate the quality of the included randomized controlled trials. A narrative synthesis was conducted to summarize the qualitative information from the included studies. A meta-integration of quantitative data was performed using RevMan 5.4. RESULTS: Ten studies met the inclusion criteria. Eight studies assessed postoperative pain, whereas two assessed postoperative nausea and vomiting. Data from the eight studies assessing postoperative pain confirmed that there was no difference between intravenously and orally administered acetaminophen in adults (OR = -0.13; 95% CI, -0.36 to 0.11; p = 0.3). Data from the two studies assessing postoperative nausea and vomiting revealed no difference between intravenously and orally administered acetaminophen in adults (OR = 0.89; 95% CI, 0.64-1.25; p = 0.51). The included studies were of poor quality, with a heterogeneity of 68%. CONCLUSIONS: No differences in postoperative analgesia or postoperative nausea and vomiting were observed between the routes of administration (intravenous vs. oral) of acetaminophen in adult patients undergoing general anesthesia. There is a need for future large sample studies to increase the reliability of the results.
Subject(s)
Acetaminophen , Postoperative Nausea and Vomiting , Acetaminophen/therapeutic use , Administration, Intravenous , Administration, Oral , Adult , Analgesics, Opioid/therapeutic use , Anesthesia, General/methods , Humans , Pain, Postoperative/drug therapy , Postoperative Nausea and Vomiting/drug therapy , Postoperative Nausea and Vomiting/epidemiology , Randomized Controlled Trials as Topic , Reproducibility of ResultsABSTRACT
Estimation of postmortem interval (PMI) has always been one of the difficult problems for forensic scientists. It is especially hard to estimate the PMI of highly decomposed corpses in the wild or in secluded houses with conventional methods. Therefore, application of insect evidence at the scene is usually required for estimation. Sarcosaprophagous flies of different species have totally different developmental rates. In actual cases, direct measurement of the body length of the larvae, calculation of accumulated temperature and succession stages without species identification, or calculation based on incorrect species identification would often lead to a large deviation between the calculated results and the real PMI. This mistake would also mislead the case investigation. Therefore, accurate species identification should be implemented before any PMI estimation of decomposed corpses with forensic entomological methods. This article reviews the general and ultramicroscopic species identification and molecular biological species identification methods of different stages of sarcosaprophagous flies, in order to provide new ideas and methods for related research and practice, and provide reference for the application and promotion of forensic entomology in the front line of public security.
Subject(s)
Animals , Autopsy , Cadaver , Diptera , Entomology , Larva , Postmortem ChangesABSTRACT
Monascus spp. are fungi that have been applied in food production for centuries and produce a range of bioactive metabolites. The objective of this study was to investigate the characteristics of proteolysis, lipolysis, textural and sensory properties of internal mold-ripened cheese ripened by Monascus fumeus x08. The contents of pH 4.6-soluble nitrogen, 12% trichloroacetic acid-soluble nitrogen and 5% phosphotungstic acid-soluble nitrogen changed significantly during the ripening of the Monascus-ripened cheese. The peptides in the Monascus-ripened cheese were evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reversed-phase high-performance liquid chromatography. During ripening, many low-molecular-weight peptides (MW < 14400) were found to increase significantly and the ratio of hydrophobic to hydrophilic peptides decreased to 0.81 and 0.34 in the Red-O (outer) and Red-I (interior) parts of the cheese, respectively. The content of total free fatty acids (15 acids, C4:0-C18:3) was significantly increased and the content of unsaturated fatty acids reached 47.89 mg/g fat on day 42 of ripening. The sensory attributes of the Monascus-ripened cheese was assessed using quantitative descriptive analysis. Compared with blue cheese, purchased from a local market, the odor and taste intensity of the Monascus-ripened cheese were significantly lower, while the color intensity and overall receptivity were significantly higher. M. fumeus x08 can be used as a secondary starter to make Monascus-ripened cheese and may be particularly acceptable to consumers in China.
Subject(s)
Cheese , Monascus , Cheese/analysis , China , Food Handling , Lipolysis , ProteolysisABSTRACT
A core-satellite-structured surface molecularly imprinted polymer has been synthesized for the enrichment of 3-phenoxybenzaldehyde by pipette tip solid-phase extraction (SPE). In a typical sol-gel process, two silane reagents as functional monomers and 3-phenoxybenzoic acid as the dummy template, the surface imprinting layer was coated on the core-satellite silica microsphere, which formed the core-satellite-structured molecularly imprinted polymer (CSMIP). Compared to the silica-based core-shell ones, this CS-MIP exhibits a stunning surface area (142 m2 g-1) in micrometer size and also overcomes the aggregation trends of core-shell structure in nanoscale. Taking potassium permanganate solution as oxidizer and indicator, the adsorbed 3-phenoxybenzaldehyde can be a quantitatively determined through redox reaction after elution. The value of maximum adsorption capacity and imprinting factor of CS-MIP were calculated to be 87.5 µg mg-1 and 2.13, respectively. These CS-MIPs were packed into commercial pipette tip as the sorbent to concentrate 3-phenoxybenzaldehyde. Under the optimum condition, a liner relationship was achieved in the range 0.200 to 1.00 µg mL-1 and the limit of detection was 81 ng mL-1. Moreover, this customized SPE device exhibits good adsorption capability after six sequential adsorption-desorption cycles, and the high recovery range of 92.2~99.7% of spiked tap water assay demonstrated its potential application for real sample analysis. Graphical abstract Schematic presentation of core-satellite molecularly imprinted polymer preparation strategy and customized pipette tip solid-phase extraction device.
Subject(s)
Benzaldehydes/analysis , Molecularly Imprinted Polymers/chemistry , Solid Phase Extraction/methods , Adsorption , Benzaldehydes/chemistry , Benzaldehydes/isolation & purification , Benzoates/chemistry , Colorimetry/methods , Drinking Water/analysis , Molecular Imprinting , Potassium Permanganate/chemistry , Solid Phase Extraction/instrumentation , Water Pollutants, Chemical/analysis , Water Pollutants, Chemical/chemistry , Water Pollutants, Chemical/isolation & purificationABSTRACT
PURPOSE: Topical anesthesia analgesic therapy has diverse applicability in solving the barrier properties of skin and unfavorable physicochemical properties of drugs. Lidocaine (LID) combined with prilocaine (PRI) has been used as a topical preparation for dermal anesthesia for treatment of conditions such as paresthesia. MATERIALS AND METHODS: In this study, for combination anesthesia and overcoming the drawbacks of LID and PRI, respectively, LID- and PRI-loaded solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs) were prepared and characterized by determination of their particle size, drug loading capacity, stability, in vitro drug release behavior and in vitro cellular viability. Ex vivo skin permeation and in vivo anesthesia analgesic efficiency of these two systems were also evaluated and compared. RESULTS: Results revealed that combination delivery of the dual drugs exhibited more remarkable efficiency than signal drug-loaded systems. SLN systems have better ex vivo skin permeation ability than NLCs. NLC systems revealed a stronger in vivo anesthesia analgesic effect than SLN systems. CONCLUSION: It can be concluded that SLNs and NLCs have different advantages, and that both carriers are promising dual drug delivery systems for topical anesthetic analgesic therapy.
Subject(s)
Anesthetics, Local/administration & dosage , Drug Delivery Systems , Lidocaine/administration & dosage , Prilocaine/administration & dosage , Administration, Cutaneous , Anesthetics, Local/pharmacokinetics , Anesthetics, Local/pharmacology , Animals , BALB 3T3 Cells , Chemistry, Pharmaceutical/methods , Drug Carriers/chemistry , Drug Liberation , Lidocaine/pharmacokinetics , Lidocaine/pharmacology , Lipids/chemistry , Mice , Nanoparticles , Particle Size , Prilocaine/pharmacokinetics , Prilocaine/pharmacology , Rats , Rats, Wistar , Skin/metabolism , Skin AbsorptionABSTRACT
Brown adipose tissue (BAT) has attracted considerable research interest because of its therapeutic potential to treat obesity and associated metabolic diseases. Augmentation of brown fat mass and/or its function may represent an attractive strategy to enhance energy expenditure. Using high-throughput phenotypic screening to induce brown adipocyte reprogramming in committed myoblasts, we identified a retinoid X receptor (RXR) agonist, bexarotene (Bex), that efficiently converted myoblasts into brown adipocyte-like cells. Bex-treated mice exhibited enlarged BAT mass, enhanced BAT function, and a modest browning effect in subcutaneous white adipose tissue (WAT). Expression analysis showed that Bex initiated several "browning" pathways at an early stage during brown adipocyte reprogramming. Our findings suggest RXRs as new master regulators that control brown and beige fat development and activation, unlike the common adipogenic regulator PPARγ. Moreover, we demonstrated that selective RXR activation may potentially offer a therapeutic approach to manipulate brown/beige fat function in vivo.
Subject(s)
Adipose Tissue, Brown/metabolism , Cellular Reprogramming/genetics , Adipogenesis/drug effects , Adipose Tissue, Brown/cytology , Adipose Tissue, White/metabolism , Animals , Bexarotene , Body Weight/drug effects , Cells, Cultured , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Mice , Mice, Inbred C57BL , Myoblasts/cytology , Myoblasts/drug effects , Myoblasts/metabolism , Oxygen Consumption/drug effects , PPAR gamma/metabolism , RNA Interference , Retinoid X Receptor alpha/antagonists & inhibitors , Retinoid X Receptor alpha/genetics , Retinoid X Receptor alpha/metabolism , Retinoid X Receptor beta/antagonists & inhibitors , Retinoid X Receptor beta/genetics , Retinoid X Receptor beta/metabolism , Retinoid X Receptor gamma/antagonists & inhibitors , Retinoid X Receptor gamma/genetics , Retinoid X Receptor gamma/metabolism , Tetrahydronaphthalenes/pharmacology , Thermogenesis/drug effects , Transcription Factors/antagonists & inhibitors , Transcription Factors/genetics , Transcription Factors/metabolism , Uncoupling Protein 1/metabolismABSTRACT
Both mammals and adult humans possess classic brown adipocytes and beige adipocytes, and the amount and activity of these adipocytes are considered key factors in combating obesity and its associated metabolic diseases. Uncoupling protein 1 (Ucp1) is the functional marker of both brown and beige adipocytes. To facilitate a reliable, easy, and sensitive measurement of Ucp1 expression both in vivo and in vitro, we generated a Ucp1-2A-luciferase knock-in mouse by deleting the stop codon for the mouse Ucp1 gene and replacing it with a 2A peptide. This peptide was followed by the luciferase coding sequence to recapitulate the expression of the Ucp1 gene at the transcriptional and translational levels. With this mouse, we discovered a cold-sensitive brown/beige adipose depot underneath the skin of the ears, which we named uBAT. Because of the sensitivity and high dynamic range of luciferase activity, the Ucp1-2A-luciferase mouse is useful for both in vitro quantitative determination and in vivo visualization of nonshivering thermogenesis. With the use of this model, we identified and characterized axitinib, an oral small-molecule tyrosine kinase inhibitor, as an effective browning agent.
Subject(s)
Adipocytes, Beige/metabolism , Adipocytes, Brown/metabolism , Subcutaneous Fat/metabolism , Adipocytes, Beige/drug effects , Adipocytes, Brown/drug effects , Animals , Axitinib , Blotting, Western , Ear , Gene Knock-In Techniques , Glucose Tolerance Test , Imidazoles/pharmacology , Immunohistochemistry , In Vitro Techniques , Indazoles/pharmacology , Luciferases/genetics , Luminescent Measurements , Male , Mice , Models, Animal , Oxygen Consumption , Protein Kinase Inhibitors/pharmacology , Reverse Transcriptase Polymerase Chain Reaction , Subcutaneous Fat/drug effects , Thermogenesis/drug effects , Uncoupling Protein 1/geneticsABSTRACT
As a member of the tissue inhibitor of metallo-proteinases (TIMP) family, it has been reported that TIMP-3 is involved in human cancer development. However, the function of TIMP-3 in hepatocellular carcinoma (HCC) development is unclear. We aimed to determine the biological role of TIMP-3 in HCC by evaluating the effects of its methylation status and expression on HCC cell function. TIMP-3 expression in HCC tissues was visibly analyzed by immunohistochemistry. Methylation of the TIMP-3 promoter was evaluated by methylation-specific PCR. Effects of TIMP-3 on HCC cell growth, apoptosis, migration, and invasion were examined by transfecting the TIMP-3-expressing plasmid, pCMV6. TIMP-3 was expressed in non-tumorous live tissue, but silenced or downregulated in 60% of HCC cases (P<0.05). Reduced protein expression of TIMP-3 was associated with reduced tumor differentiation (P=0.003) and increased metastatic activity (P=0.005) in HCC cell lines. Promoter methylation contributed to the TIMP-3 inactivation. Overexpression of TIMP-3 in HCC cell lines suppressed cell proliferation, induced apoptosis, and inhibited migration and invasion in vitro. TIMP-3 expression is suppressed by promoter methylation in HCC. This inhibitory protein acts as a functional tumor suppressor by inhibiting HCC cell proliferation, invasion, and migration and by inducing apoptosis and cell cycle arrest at the G2/M phase.
Subject(s)
Carcinoma, Hepatocellular/genetics , Gene Expression Regulation, Neoplastic/genetics , Liver Neoplasms/genetics , Tissue Inhibitor of Metalloproteinase-3/genetics , Apoptosis/genetics , Carcinoma, Hepatocellular/pathology , Cell Cycle Checkpoints/genetics , Cell Differentiation/genetics , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , DNA Methylation/genetics , Down-Regulation/genetics , Female , G2 Phase Cell Cycle Checkpoints/genetics , Hep G2 Cells , Humans , Liver Neoplasms/pathology , Male , Middle Aged , Neoplasm Invasiveness/genetics , Neoplasm Invasiveness/pathology , Promoter Regions, Genetic/genetics , Tumor Suppressor Proteins/geneticsABSTRACT
Obesity is accompanied by an increase in the size and the number of adipocytes. As adipocytes are thought to differentiate from pre-adipocytes, we postulate that non-adipogenic fibroblasts contribute to adipocyte formation under certain conditions such as obesity. We report for the first time that NIH-3T3 fibroblasts, which are generally considered to be non-adipogenic, can be converted into mature adipocytes by treatment with a defined hormone mixture comprising EGF (epidermal growth factor), HGF (hepatocyte growth factor), Dex (dexamethasone) and insulin. Furthermore, NIH-3T3 cells transplanted into obese immunodeficient NOD/SCID (non-obese diabetic/severe combined immunodeficient) mice formed adipocytes in vivo. Interestingly, the mixture elicited conversion of NIH-3T3 cells directly into adipocytes without a preceding pre-adipocyte stage. Functional analysis revealed that each component of the mixture was necessary for the induction of adipogenesis, including Dex which inhibited the cell proliferation stimulated by EGF. Upon profiling the signalling pathways employed by EGF and HGF, we found STAT5 (signal transducer and activator of transcription 5) signalling to be activated, predominantly at the levels of both transcription and post-translational modification. Inhibition of the STAT5 pathway, either by genetic knockdown or a specific pharmacological agent, blocked adipogenesis in NIH-3T3 cells. Taken together, these data not only establish a newly recognized grouping of factors that can induce trans-differentiation of non-adipogenic fibroblasts into adipocytes, but also give us a greater understanding of obesity.
Subject(s)
Adipocytes/cytology , Adipocytes/drug effects , Cell Transdifferentiation/drug effects , Fibroblasts/cytology , Fibroblasts/drug effects , 3T3-L1 Cells , Adipocytes/metabolism , Adipogenesis/drug effects , Animals , Dexamethasone/administration & dosage , Epidermal Growth Factor/administration & dosage , Fibroblasts/metabolism , Gene Knockdown Techniques , Hepatocyte Growth Factor/administration & dosage , Insulin/administration & dosage , Mice , Mice, Inbred NOD , Mice, SCID , NIH 3T3 Cells , Obesity/metabolism , Obesity/pathology , STAT5 Transcription Factor/antagonists & inhibitors , STAT5 Transcription Factor/genetics , STAT5 Transcription Factor/metabolism , Signal Transduction/drug effectsABSTRACT
Hypoxia, a state of low oxygen, is a common feature of solid tumors and is associated with disease progression as well as resistance to radiotherapy and certain chemotherapeutic drugs. Hypoxic regions in tumors, therefore, represent attractive targets for cancer therapy. To date, five distinct classes of bioreactive prodrugs have been developed to target hypoxic cells in solid tumors. These hypoxia-activated prodrugs, including nitro compounds, N-oxides, quinones, and metal complexes, generally share a common mechanism of activation whereby they are reduced by intracellular oxidoreductases in an oxygen-sensitive manner to form cytotoxins. Several examples including PR-104, TH-302, and EO9 are currently undergoing phase II and phase III clinical evaluation. In this review, we discuss the nature of tumor hypoxia as a therapeutic target, focusing on the development of bioreductive prodrugs. We also describe the current knowledge of how each prodrug class is activated and detail the clinical progress of leading examples.
Subject(s)
Antineoplastic Agents/pharmacology , Cell Hypoxia/drug effects , Neoplasms , Prodrugs/pharmacology , Anthraquinones/chemistry , Anthraquinones/pharmacology , Antineoplastic Agents/chemistry , Aziridines/chemistry , Aziridines/pharmacology , Humans , Indolequinones/chemistry , Indolequinones/pharmacology , Molecular Structure , NAD(P)H Dehydrogenase (Quinone)/chemistry , NAD(P)H Dehydrogenase (Quinone)/pharmacology , Neoplasms/drug therapy , Neoplasms/pathology , Nitrogen Mustard Compounds/chemistry , Nitrogen Mustard Compounds/pharmacology , Nitroimidazoles/chemistry , Nitroimidazoles/pharmacology , Phosphoramide Mustards/chemistry , Phosphoramide Mustards/pharmacology , Prodrugs/chemistry , Tirapazamine , Triazines/chemistry , Triazines/pharmacologyABSTRACT
Rolling circle amplification (RCA) is a newly developed experimental technique that can specific ally amplify circular DNA. Since 2008, RCA has been extensively used in hepatitis B virus (HBV) research, such as the amplification of the full-length sequence of the HBV genome, and the analysis of the drug-resistant mutations of HBV covalently closed circular DNA (cccDNA), amongst others. To create an easy assay for the analysis of duck hepatitis B virus (DHBV) cccDNA, this study established an RCA-based method. DHBV cccDNA was amplified from the DHBV DNA samples of duck liver with four pairs of sulfur-modified primers, which were designed according to the highly conserved sequence of DHBV using sera DHBV DNA as the negative control. DHBV cccDNA was detected in the obtained RCA products by the sequencing of RCA amplicons that were amplified with primer pairs on both sides of the gap of DH BV relaxed circular DNA, rather than by digesting RCA products with a restriction enzyme. The liver and sera DHBV DNA samples of 39 ducks infected with DHBV were examined with the RCA-based DHBV cccDNA detection method, and the results showed that while DHBV cccDNA was detected from all 39 liver DHBV DNA samples, no DHBV cccDNA was found in any of the sera DHBV DNA samples. These results suggest that the method established in the study is highly specific and sensitive for the detection of DHBV cccDNA. The establishment of this RCA-based DHBV method for cccDNA detection lays the groundwork for using a DHBV model to study the role of cccDNA in the pathogenesis of hepatitis B and to evaluate the effect of anti-virus therapies.