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With the cutting-edge advancements in computer vision, facial expression recognition (FER) is an active research area due to its broad practical applications. It has been utilized in various fields, including education, advertising and marketing, entertainment and gaming, health, and transportation. The facial expression recognition-based systems are rapidly evolving due to new challenges, and significant research studies have been conducted on both basic and compound facial expressions of emotions; however, measuring emotions is challenging. Fueled by the recent advancements and challenges to the FER systems, in this article, we have discussed the basics of FER and architectural elements, FER applications and use-cases, FER-based global leading companies, interconnection between FER, Internet of Things (IoT) and Cloud computing, summarize open challenges in-depth to FER technologies, and future directions through utilizing Preferred Reporting Items for Systematic reviews and Meta Analyses Method (PRISMA). In the end, the conclusion and future thoughts are discussed. By overcoming the identified challenges and future directions in this research study, researchers will revolutionize the discipline of facial expression recognition in the future.
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In autonomous driving and Intelligent transportation systems, pedestrian detection is vital in reducing traffic accidents. However, detecting small-scale and occluded pedestrians is challenging due to the ineffective utilization of the low-feature content of small-scale objects. The main reasons behind this are the stochastic nature of weight initialization and the greedy nature of nonmaximum suppression. To overcome the aforesaid issues, this work proposes a multifocus feature extractor module by fusing feature maps extracted from the Gaussian and Xavier mapping function to enhance the effective receptive field. We also employ a focused attention feature selection on a higher layer feature map of the single shot detector (SSD) region proposal module to blend with its low-layer feature to tackle the vanishing of the feature detail due to convolution and pooling operation. In addition, this work proposes a decaying nonmaximum suppression function considering score and Intersection Over Union (IOU) parameters to tackle high miss rates caused by greedy nonmaximum suppression used by SSD. Extensive experiments have been conducted on the Caltech pedestrian dataset with the original annotations and the improved annotations. Experimental results demonstrate the effectiveness of the proposed method, particularly for small and occluded pedestrians.
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Condução de Veículo , Pedestres , Humanos , Acidentes de Trânsito , AtençãoRESUMO
Previous studies have shown that perennial Chan training leads to improvements in brain functioning. However, few studies have investigated the effects of short-term Huatou Chan training. The current study explored the effects of a three-day Huatou Chan training on physical and emotional health, as well as brain state. Seventy healthy subjects were recruited and divided into two groups: the Huatou Chan group and the Control group. The Huatou Chan group received a 3-day Huatou Chan training, while the Control group waited for three days. Both groups completed a 6 min Brain State Index recording, the SCL-90, the brief profile of mood state, the meaning in life questionnaire, and the Index of Well-being, prior to and after the training or waiting period. Results showed that short-term Huatou Chan training had significant benefits on some aspects such as physical and emotional health (obsessive-compulsive, depression, hostility, and psychoticism), negative emotions (tension-anxiety, depression-dejection, anger-hostility, fatigue-inertia, and confusion-bewilderment), well-being, and attitude towards life. In addition, short-term Chan training can significantly improve brain state, as shown by the index of depression, anxiety, alerting and intelligence. This is the first study to provide direct evidence for the benefits of short-term intensive Huatou Chan training on physical and mental health.
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Ansiedade , Depressão , Ira , Ansiedade/prevenção & controle , Emoções , Humanos , Inquéritos e QuestionáriosRESUMO
Apolipoprotein C2 (ApoC2) is a key activator of lipoprotein lipase for plasma triglyceride metabolism. ApoC2-deficient patients present with severe hypertriglyceridemia and recurrent acute pancreatitis, for whom the only effective treatment is the infusion of normal plasma containing ApoC2. However, since ApoC2 has a fast catabolic rate, a repeated infusion is required, which limits its clinical use. To explore a safe and efficient approach for ApoC2 deficiency, we herein established an adeno-associated virus expressing human ApoC2 (AAV-hApoC2) to evaluate the efficacy and safety of gene therapy in ApoC2-deficient hypertriglyceridemic hamsters. Administration of AAV-hApoC2 via jugular or orbital vein in adult and neonatal ApoC2-deficient hamsters, respectively, could prevent the neonatal death and effectively improve severe hypertriglyceridemia of ApoC2-deficient hamsters without side effects in a long-term manner. Our novel findings in the present study demonstrate that AAV-hApoC2-mediated gene therapy will be a promising therapeutic approach for clinical patients with severe hypertriglyceridemia caused by ApoC2 deficiency.
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RATIONALE: ApoC2 is an important activator for lipoprotein lipase-mediated hydrolysis of triglyceride-rich plasma lipoproteins. ApoC2-deficient patients display severe hypertriglyceridemia (sHTG) and recurrent acute pancreatitis. However, due to embryonic lethality in ApoC2 deleted mouse extensive understanding of ApoC2 function is limited in mammalian species. OBJECTIVE: We sought to generate an animal model with ApoC2 deficiency in a rodent with some human-like features and then study the precise effects of ApoC2 on lipid and glucose homeostasis. METHODS AND RESULTS: Using CRISPR/Cas9, we deleted Apoc2 gene from golden Syrian hamster and the homozygous (-/-) pups can be born in matured term but exhibited neonatal lethality. By continuous iv administration of normal hamster serum the ApoC2-/- pups could survive till weaning and displayed severe HTG in adulthood on chow diet. A single iv injection of AAV-hApoC2 at birth can also rescue the neonatal death of ApoC2-/- pups. Adult ApoC2-/-hamsters exhibited a unique phenotype of sHTG with hypoglycemia, hypoinsulinemia and spontaneous atherosclerosis. The sHTG in ApoC2-/- adult hamsters could not be corrected by various lipid-lowering medications, but partially ameliorated by medium chain triglyceride diet and completely corrected by AAV-hApoC2. CONCLUSIONS: Our study provides a novel ApoC2-deleted mammalian model with severe hypertriglyceridemia that was fully characterized and highlights a potential therapeutic approach for the treatment of ApoC2 deficient patients.
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Apolipoproteína C-II/deficiência , Aterosclerose/etiologia , Hipertrigliceridemia/etiologia , Animais , Animais Recém-Nascidos/genética , Apolipoproteína C-II/uso terapêutico , Glicemia , Cricetinae , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Homeostase , Humanos , Hipertrigliceridemia/tratamento farmacológico , Lipídeos , MesocricetusRESUMO
Glaucoma is characterized by retinal ganglion cell (RGC) death and axonal loss. Therefore, neuroprotection is important in treating glaucoma. In this study, we explored whether exoenzyme C3 transferase (C3)-based gene therapy could protect retinas in an ischemia/reperfusion (I/R) injury rat model. Self-complementary adeno-associated virus 2 (scAAV2) vectors encoding either C3 protein (scAAV2-C3) or enhanced green fluorescence protein (scAAV2-EGFP) were intravitreally delivered into both eyes of rats, and I/R models (acute ocular hypertension) were made in one eye of each rat at day 7 after the injection. The rats were divided into six groups: scAAV2-C3, scAAV2-C3 with I/R, scAAV2-EGFP, scAAV2-EGFP with I/R, blank control, and blank control with I/R. TUNEL (terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling), immunohistochemistry of cleaved caspase-3, NeuN and Brn-3a, and H&E staining were used to detect apoptotic cells and other changes in the retina. The results showed that scAAV2-C3 significantly reduced the number of apoptotic RGCs and decreased cell loss in the ganglion cell layer after I/R injury, and the I/R-injured retinas treated with scAAV2-C3 were the thickest in all I/R groups. These results suggest that scAAV2-mediated C3 gene therapy is able to protect the rat retina from I/R injury and has potential in the treatment of glaucoma in the future.
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BACKGROUND/AIMS: Recent studies indicate that therapies targeting the vasculature can significantly sensitize tumors to radiation. Ultrasound-stimulated microbubbles (USMBs) are regarded as a promising radiosensitizer. In this study, we investigated the effect of USMBs on the sensitivity of nasopharyngeal carcinoma (NPC) to radiation. METHODS: Human NPC (CNE-2) cells and human umbilical vein endothelial cells (HUVECs) were exposed to radiation (0, 2, and 8 Gy) alone or in combination with USMBs. Cell viability and apoptosis were measured with the MTT assay and flow cytometry, respectively. The angiogenic activity of HUVECs was detected using matrigel tubule formation. The in vitro effects induced by these treatments were confirmed in vivo with xenograft models of CNE-2 cells in nude mice by examining vascular integrity using color Doppler flow imaging and cell survival using immunohistochemistry. Additionally, the in vivo and in vitro expressions of angiotensin II (ANG II) and its receptor (AT1R) were detected by immunohistochemistry and western blotting, respectively. With CNE-2 cells and HUVECs transfected with control, ANG II, or AT1R, perindopril (an inhibitor of angiotensin-converting enzyme) and candesartan (an inhibitor of AT1R) were used to verify the role of ANG II and AT1R in the radiosensitivity of tumor and endothelial cells by USMBs, by determining cell viability and apoptosis and angiogenic activity. RESULTS: In the NPC xenografts, USMBs slightly reduced blood flow and CD34 expression, increased tumor cell death and ANG II and AT1R expression, and significantly enhanced the effects of radiation. With CNE-2 cells and HUVECs, the USMBs further enhanced the inhibition of tumor cell viability and endothelial tubule formation and further enhanced the increase in ANG II and AT1R due to radiation. Furthermore, perindopril and candesartan significantly enhanced the inhibitory effect of radiation and USMBs on tumor cell growth and angiogenesis in vitro. CONCLUSIONS: We have demonstrated for the first time that USMB exposure can significantly enhance the destructive effect on NPC of radiation, and this effect might be further increased by ANG II and AT1R inhibition. Our findings suggest that USMBs can be used as a promising sensitizer of radiotherapy to treat NPC, and the clinical effect might be increased by ANG II and AT1R inhibition.
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Meios de Contraste/química , Microbolhas/uso terapêutico , Angiotensina II/metabolismo , Animais , Antígenos CD34/metabolismo , Benzimidazóis/metabolismo , Compostos de Bifenilo , Carcinoma/metabolismo , Carcinoma/patologia , Carcinoma/radioterapia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Meios de Contraste/farmacologia , Meios de Contraste/uso terapêutico , Feminino , Raios gama , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Camundongos Nus , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/patologia , Neoplasias Nasofaríngeas/radioterapia , Perindopril/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Sonicação , Tetrazóis/metabolismoRESUMO
PURPOSE: This study is aimed to explore the combined use of goiter dispersion formula and antithyroid drugs in the treatment of patients with neurologic manifestations of Graves' disease by examining its modulating effects on patients' cytokines. METHODS: A total of 80 patients with Graves' disease were randomly divided into treatment and control groups. Patients of the treatment group received goiter dispersion formula and antithyroid drugs (methimazole or propylthiouracil), whereas those of the control group received antithyroid drug alone. FT3, FT4, and TSH contents were detected by chemiluminescence immunoassay at pre- and post-treatment; interleukin (IL)-2, IL-8, and IL-17 serum levels before and after the treatment were detected by radioimmunoassay; thyroid B-mode ultrasound and liver and renal function tests were performed in all patients of both groups. An additional cohort of 40 healthy subjects was recruited for baseline measurement. RESULTS: All the enrolled patients completed the trial. The effective treatment rate was higher in the treatment group than in the control group, of which the difference was statistically significant (treatment group, 95%; control group, 75%, p < 0.01). For blood cytokine, before treatment, IL-2 was reduced whereas IL-8 and IL-17 were increased significantly in both groups of patients with Graves' disease comparing with those in healthy subjects (p < 0.01). For patients of the treatment group, after treatment, their IL-2 levels were increased (p < 0.01) with concomitant decreases in IL-8 and IL-17 levels (p < 0.05). There were no significant changes in blood cytokine levels before and after treatment in the control group. CONCLUSIONS: Goiter dispersion formula significantly improved the treatment outcomes of antithyroid drug in hyperthyroidism patients with neurologic manifestations of Graves' disease by modulating IL-2, IL-8, and IL-17. The data supported the rationale for the use of goiter dispersion formula in Graves' disease treatment.
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Citocinas/sangue , Medicamentos de Ervas Chinesas/uso terapêutico , Doença de Graves/complicações , Doença de Graves/fisiopatologia , Hipertireoidismo/complicações , Hipertireoidismo/tratamento farmacológico , Adulto , Idoso , Feminino , Humanos , Hipertireoidismo/sangue , Masculino , Medicina Tradicional Chinesa , Pessoa de Meia-Idade , Adulto JovemRESUMO
Inverted terminal repeats (ITRs) of the adeno-associated virus (AAV) are essential for rescue, replication, packaging, and integration of the viral genome. While ITR mutations have been identified in previous reports, we designed a new truncated ITR lacking the B-B' and C-C' regions named as ITRΔBC and investigated its effects on viral genome replication, packaging, and expression of recombinant AAV (rAAV). The packaging ability was compared between ITRΔBC rAAV and wild-type (wt) ITR rAAV. Our results showed the productivity of ITRΔBC rAAV was reduced 4-fold, which is consistent with the 8-fold decrease in the replication of viral genomic DNA of ITRΔBC rAAV compared with wt ITR rAAV. Surprisingly, transgene expression was significantly higher for ITRΔBC rAAV. A preliminary exploration of the underlying mechanisms was carried out by inhibiting and degrading the ataxia telangiectasia mutated (ATM) protein and the Mre11 complex (MRN), respectively, since the rAAV expression was inhibited by the ATM and/or MRN through cis interaction or binding with wt ITRs. We demonstrated that the inhibitory effects were weakened on ITRΔBC rAAV expression. This study suggests deletion in ITR can affect the transgene expression of AAV, which provides a new way to improve the AAV expression through ITRs modification.
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Dependovirus/genética , Expressão Gênica , Deleção de Sequência , Sequências Repetidas Terminais/genética , Transgenes/genética , Animais , Sequência de Bases , Linhagem Celular Tumoral , Vetores Genéticos/genética , Genoma Viral/genética , Células HEK293 , Humanos , Masculino , Camundongos Endogâmicos C57BL , Homologia de Sequência do Ácido Nucleico , Replicação Viral/genéticaRESUMO
In recent years, Hepatitis B virus (HBV) persistent infection mouse model with recombinant adeno-associated virus 8 carrying 1.3 copies of HBV genome (rAAV8-1.3HBV) is concerned. We studied and compared the efficacy among HBV persistent infection mice models by other serotypes except AAV8. First, we prepared and purified five viruses: rAAV1-1.3HBV, rAAV2-1.3HBV, rAAV5-1.3HBV, rAAV8-1.3HBV and rAAV9-1.3HBV. Then we injected each virus into 3 C57BL/6J mice with the dose of lx 1011 vg (Viral genome, vg) per mouse. We detected HBsAg and HBeAg in sera by enzyme-linked immunosorbent assay (ELISA) at different time points post injection. We killed mice 8 weeks post injection and took blood and livers for assay. We detected copies of HBV DNA by real-time quantitative PCR in sera and livers. Meantime, we detected HBcAg in the livers of mice by immunohistochemistry and further performed pathology analysis of these livers. The five groups of mice, HBeAg and HBsAg expression sustained 8 weeks in serological detection and HBV DNA was both detected in sera and livers at the time of 8 weeks post injection. HBeAg, HBsAg, HBV DNA copies expression levels in descending order were AAV8>AAV9>AAV1>AAV5>AAV2. HBcAg expression was detected in livers as well. Varied degrees of liver damage were shown in five groups of mice. This study provides more alternative AAV vector species to establish a persistent infection with hepatitis B model.
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Dependovirus/classificação , Genoma Viral , Vírus da Hepatite B/genética , Hepatite B/virologia , Animais , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Vetores Genéticos , Antígenos do Núcleo do Vírus da Hepatite B/metabolismo , Antígenos de Superfície da Hepatite B/sangue , Antígenos E da Hepatite B/sangue , Camundongos , Camundongos Endogâmicos C57BL , Sorogrupo , Replicação ViralRESUMO
Recently, researchers seeking to understand, modify, and create beneficial traits in organisms have looked for evolutionarily conserved patterns of protein interactions. Their conservation likely means that the proteins of these conserved functional modules are important to the trait's expression. In this paper, we formulate the problem of identifying these conserved patterns as a graph optimization problem, and develop a fast heuristic algorithm for this problem. We compare the performance of our network alignment algorithm to that of the MaWISh algorithm [Koyutürk M, Kim Y, Topkara U, Subramaniam S, Szpankowski W, Grama A, Pairwise alignment of protein interaction networks, J Comput Biol13(2):182-199, 2006.], which bases its search algorithm on a related decision problem formulation. We find that our algorithm discovers conserved modules with a larger number of proteins in an order of magnitude less time. The protein sets found by our algorithm correspond to known conserved functional modules at comparable precision and recall rates as those produced by the MaWISh algorithm.
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Algoritmos , Mapas de Interação de Proteínas , Alinhamento de Sequência/estatística & dados numéricos , Animais , Biologia Computacional , Sequência Conservada , Ontologia Genética/estatística & dados numéricos , Humanos , Mapeamento de Interação de Proteínas/estatística & dados numéricosRESUMO
Activities of 58 miRNAs for BHK21, HEK293, and Vero cell lines were screened using the high-throughput miRNA activity profiling method. miR-206 activity was found specifically high in BHK21. Considering miR-206 was recognized as a muscle-specific miRNA, we further detected the expression and activity level of miR-206 in BHK21 cells, with myoblast cells C2C12 as positive control and HEK293 cells as negative control. Then, we induced BHK21 by culturing with medium containing 2% horse serum (HS) and tested expression level of slow skeletal myosin heavy chain (MHC), activity and expression levels of miR-206, and expression level of Connexin43 (Cx43) which was reported negatively regulated by miR-206. Results demonstrated that activity and expression levels of miR-206 were both higher in BHK21 cells than in C2C12 cells. After induction of HS, MHC expression level was increased in BHK21 cells. The activity and expression levels of miR-206 were further enhanced. The Cx43 expression level was decreased. These results suggested that BHK21 had the characters of myoblast cells. In conclusion, we firstly discovered that miR-206 activity was specifically high in BHK21 cells, suggesting that BHK21 cells were derived from interstitial cells other than parenchyma cells of kidney from miRNA point of view. Our study also indicated that BHK21 cells were able to be used as models in vitro for research of miR-206 function.
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Conexina 43/metabolismo , MicroRNAs/genética , Cadeias Pesadas de Miosina/metabolismo , Animais , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Regulação da Expressão Gênica , Células HEK293 , Humanos , Células VeroRESUMO
There is a lack of methods for high-throughput functional microRNA (miRNA) profiling. In this chapter, we describe a recombinant adeno-associated virus-based miRNA sensor array (miRNA Asensor array), which is able to profile functional miRNAs in cultured cells. The preparation of an miRNA Asensor array and its usage are discussed.
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Técnicas Biossensoriais , Dependovirus/genética , Ensaios de Triagem em Larga Escala , MicroRNAs/química , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Cricetinae , Escherichia coli , Vetores Genéticos , Análise de Sequência com Séries de OligonucleotídeosRESUMO
We evaluated the anti-HBV effects of nucleotide analogues, Entecavir (ETV) and Lamivudine (LAM) targeting mouse model of HBV persistent infection with recombinant adeno-associated virus 8 carrying 1.3 copies of HBV genome (rAAV8-1.3HBV). Ninety percent (27 of 30 mice) of rAAVS-treated mice were chosen as mouse model. Four groups were orally administrated with different doses of ETV (1 mg/(kgd) or 0.1 mg/(kgd)) and LAM (500 mg/(kgd) or 100 mg/(kgd)) once a day for 10 days. The other two groups were set as normal saline treated and untreated control. We detected the levels of HBV DNA, HBeAg and HBsAg in sera at different time. Results indicate that HBV DNA level decreased significantly (P < 0.05) in drug-treated groups compared with normal saline group after drug administration. Fifteen days after the drug withdrawal, HBV DNA level rebounded back obviously (P < 0.05) in groups with low doses of ETV and LAM. However, there was no apparent change of HBeAg and HBsAg in the whole process among all groups. These results showed that our model could reflect the anti-viral effect of nucleotide analogues. This model can be a useful and convenient tool for anti-HBV drug discovery.
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Antivirais/farmacologia , Dependovirus/genética , Hepatite B Crônica/virologia , Nucleotídeos/farmacologia , Animais , Dependovirus/metabolismo , Modelos Animais de Doenças , Vetores Genéticos , Genoma Viral , Guanina/análogos & derivados , Guanina/farmacologia , Anticorpos Anti-Hepatite B/sangue , Vírus da Hepatite B/genética , Vírus da Hepatite B/fisiologia , Lamivudina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Transdução Genética , Replicação ViralRESUMO
A number of tools for the alignment of protein-protein interaction (PPI) networks have laid the foundation for PPI network analysis. Most of alignment tools focus on finding conserved interaction regions across the PPI networks through either local or global mapping of similar sequences. Researchers are still trying to improve the speed, scalability, and accuracy of network alignment. In view of this, we introduce a connected-components based fast algorithm, HopeMap, for network alignment. Observing that the size of true orthologs across species is small comparing to the total number of proteins in all species, we take a different approach based on a precompiled list of homologs identified by KO terms. Applying this approach to S. cerevisiae (yeast) and D. melanogaster (fly), E. coli K12 and S. typhimurium, E. coli K12 and C. crescenttus, we analyze all clusters identified in the alignment. The results are evaluated through up-to-date known gene annotations, gene ontology (GO), and KEGG ortholog groups (KO). Comparing to existing tools, our approach is fast with linear computational cost, highly accurate in terms of KO and GO terms specificity and sensitivity, and can be extended to multiple alignments easily.
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OBJECTIVE: Intratumoral administration of adenoviral vector encoding herpes simplex virus (HSV) thymidine kinase (TK) gene (Ad-TK) followed by systemic ganciclovir (GCV) is an effective approach in treating experimental hepatocellular carcinoma (HCC). However, hepatotoxicity due to unwanted vector spread and suicide gene expression limited the application of this therapy. miR-122 is an abundant, liver-specific microRNA whose expression is decreased in human primary HCC and HCC-derived cell lines. These different expression profiles provide an opportunity to induce tumor-specific gene expression by miR-122 regulation. METHODS: By inserting miR-122 target sequences (miR-122T) in the 3' untranslated region (UTR) of TK gene, we constructed adenovirus (Ad) vectors expressing miR-122-regulated TK (Ad-TK-122T) and report genes. After intratumoral administration of Ad vectors into an orthotopic miR-122-deficient HCC mouse model, we observed the miR-122-regulated transgene expression and assessed the antitumor activity and safety of Ad-TK-122T. RESULTS: Insertion of miR-122T specifically down-regulated transgene expression in vitro and selectively protected the miR-122-positive cells from killing by TK/GCV treatment. Insertion of miR-122T led to significant reduction of tansgene expression in the liver without inhibition of its expression in tumors in vivo, resulting in an 11-fold improvement of tumor-specific transgene expression. Intratumoral injection of Ad vectors mediated TK/GCV system led to a vector dosage-dependent regression of tumor. The insertion of miR-122T does not influence the antitumor effects of suicide gene therapy. Whereas mice administrated with Ad-TK showed severe lethal hepatotoxicity at the effective therapeutic dose, no liver damage was found in Ad-TK-122T group. CONCLUSIONS: miR-122-regulated TK expression achieved effective anti-tumor effects and increased the safety of intratumoral delivery of adenovirus-mediated TK/GCV gene therapy for miR-122-deficient HCC.
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We recently developed a mouse model of hepatitis B virus (HBV) chronic infection by intravenous (i.v.) injection with rAAV8-1. 3HBV to C57BL/6 mice. To define the responses of different mouse strains after injection with rAAV8-1. 3HBV, we intravenously injected rAAV8-1. 3HBV at doses of 4 x10(9) (Viral genome,vg), 4 x 10(10) vg and 4 x 10(11) vg to C57BL/6 and BALB/c mice,respectively, and determined the levels of serum HBV antigen and antibody by ELISA,serum viral DNA by real-time PCR,and HBcAg expression in liver by immunohistochemical staining. For C57BL/6 mouse strain with injection of rAAV8-1. 3HBV at three doses, 100% of the mice carried HBV for more than 8 months. The levels of serum HBsAg and HBeAg, serum viral DNA and HBcAg-positive hepatocytes increased in a rAAV8-1. 3HBV dose-dependent manner. For C57BL/6 mice injected with rAAV8-1. 3HBV at the dose of 4 x 10(11) vg,over 40% of hepatocytes expressed HBcAg and serum viral DNA reached over 10(5) IU/mL. No HBV antibody was detected in sera of C57BL/6 mice. For BALB/c mice with injection of rAAV8-1. 3HBV at three doses, serum HBeAg, serum viral DNA and HBcAg-positive hepatocytes persisted for more than 8 months, but serum HBsAg declined remarkably at 2 weeks after injection. The levels of serum HBeAg and HBcAg-positive hepatocytes in BALB/c mice increased in a rAAV8-1. 3HBV dose-dependent manner. Injection with rAAV8-1. 3HBV at the dose of 4 x 10(11) vg resulted in over 50% of BALB/c mice hepatocytes expressing HBcAg. Serum anti-HBsAg were detected in BALB/c mice with rAAV8-1. 3HBV injection at the dose of 4 x10 (10) vg. In conclusion, both C57BL/6 and BALB/c strains can be developed to chronic HBV infection mouse models by i. v. injection with rAAV8-1. 3HBV at doses of 4 x10(9) - 4 x 10(11) vg and the levels of HBV replication increase in a rAAV8-1. 3HBV dose-dependent manner. In contrast to C57BL/6 strain, the BALB/c mice carry out humoral immunity to HBsAg, but fail to mediate HBV clearance.
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Dependovirus/genética , Vírus da Hepatite B/fisiologia , Hepatite B/virologia , Transdução Genética , Animais , Dependovirus/metabolismo , Modelos Animais de Doenças , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Hepatite B/imunologia , Anticorpos Anti-Hepatite B/imunologia , Antígenos de Superfície da Hepatite B/imunologia , Antígenos E da Hepatite B/imunologia , Vírus da Hepatite B/genética , Vírus da Hepatite B/imunologia , Hepatócitos/imunologia , Hepatócitos/virologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Replicação ViralRESUMO
In this study, we constructed the plasmid of Sendai virus (SeV) BB1 strain minigenome with Gaussia luciferase (Gluc) as reporter and compared the rescue efficiency of SeV minigenome mediated by T7 promoter with that by CMV promoter. Firstly, the sequence was designed and synthesized which contained hammerhead ribozyme, sequence composed of the trailer, untranslated region of L gene, untranslated region of N gene, and the leader from SeV, and mutant hepatitis delta virus ribozyme sequence. Then, the synthesized sequence was inserted into pVAX1 containing CMV and T7 promoters and the general vector for SeV minigenome pVAX-miniSeV was obtained. Furthermore, pVAX-miniSeV-Gluc (+) and pVAX-miniSeV-Gluc(-) were obtained by inserting Gluc gene into pVAX-miniSeV. From the supernatant of BHK-21 cell transfected with pVAX-miniSeV-Gluc(+), high level of Gluc expression was detection indicating the normal transcription function of CMV promoter. pVAX-SeV-miniGluc(-) and plasmids expressing N,P and L protein of SeV were co-transfected into BST T7/5 cell which derived from BHK-21 and expressed T7 RNA polymerase stably. And high expression of Gluc was found, which indicated that SeV minigenome was efficiently rescued. However, we failed to repeat the result on BHK-21 cell, implying that T7 promoter and CMV promoter may have different effects on the rescue efficiency of SeV minigenome. Therefore, we further constructed SeV minigenome vectors pT7-miniSeV-Gluc (-) and pCMV-miniSeV-Gluc(-) with single promoter of T7 or CMV. Then, these vectors were transfected into BSR T7/ 5 cells respectively accompanied with the N, P, and L protein expression vectors. The result demonstrated that high expression of Gluc was found in the group of pT7-miniSeV-Gluc(-), which failed in the group of pCMV-miniSeV-Gluc(-). It indicated that T7 promoter significantly increased the rescue efficiency of SeV minigenome. We successfully constructed a SeV minigenome vector with secreted luciferase gene as report er and proved T7 promoter can enhance the rescue efficiency of SeV minigenome, which provides basis for construction of infectious clone containing SeV full-length genome.
Assuntos
Citomegalovirus/genética , RNA Polimerases Dirigidas por DNA/genética , Genoma Viral , Regiões Promotoras Genéticas , Infecções por Respirovirus/virologia , Vírus Sendai/genética , Proteínas Virais/genética , Animais , Linhagem Celular , Cricetinae , Humanos , Vírus Sendai/fisiologia , Proteínas Virais/metabolismoRESUMO
BACKGROUND: microRNAs (miRNAs) are small and non-coding RNAs which play critical roles in physiological and pathological processes. A number of methods have been established to detect and quantify miRNA expression. However, method for high-throughput miRNA function detection is still lacking. PRINCIPAL FINDINGS: We describe an adeno-associated virus (AAV) vector-based microRNA (miRNA) sensor (Asensor) array for high-throughput functional miRNA profiling. Each Asensor contains a Gaussia luciferase (Gluc) and a firefly luciferase (Fluc) expression cassette to sense functional miRNA and to serve as an internal control respectively. Using this array, we acquired functional profiles of 115 miRNAs for 12 cell lines and found "functional miRNA signatures" for several specific cell lines. The activities of specific miRNAs including the let-7 family, miR-17-92 cluster, miR-221, and miR-222 in HEK 293 cells were compared with their expression levels determined by quantitative reverse transcriptase polymerase chain reaction (QRT-PCR). We also demonstrate two other practical applications of the array, including a comparison of the miRNA activity between HEK293 and HEK293T cells and the ability to monitor miRNA activity changes in K562 cells treated with 12-O-tetradecanoylphorbol-13-acetate (TPA). CONCLUSIONS/SIGNIFICANCE: Our approach has potential applications in the identification of cell types, the characterization of biological and pathological processes, and the evaluation of responses to interventions.
Assuntos
Técnicas Biossensoriais/instrumentação , Dependovirus/genética , Perfilação da Expressão Gênica/métodos , Ensaios de Triagem em Larga Escala/métodos , MicroRNAs/genética , Análise em Microsséries/instrumentação , Recombinação Genética/genética , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular , Dependovirus/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , MicroRNAs/metabolismo , Recombinação Genética/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
OBJECTIVE: Recombinant adeno-associated virus type 8 (rAAV8) mediating transgene expression in mice was investigated using co-expressed report gene of secreted Gaussia princeps luciferase (Gluc) and non-secreted firefly luciferase(Fluc). METHODS: rAAV8-Gluc/Fluc was prepared and infected HEK293 cells to test its performance in vitro. BALB/c mice were received rAAV8-Gluc/Fluc at different doses by intravenous injection (iv) or intramuscular injection (im). Then Glue activities in blood were measured,the whole-body images for Flue activities were performed and Flue activities of tissue lysate were also detected. RESULTS: rAAV8-Gluc/Fluc was successfully prepared and could infected HEK293 cells. The Glue was mainly detected in the culture media while the Flue was mainly detected within cells. The blood Glue activities of mice received rAAV8-Gluc/Fluc by iv or im peaked at 10-20 d post injection and persisted for at least 120d. The blood Gluc activities increased at the rAAV8-Gluc/Fluc dose-dependent manner. For mice received rAAV8 by iv, Fluc mainly expressed in liver and minor Fluc expression was also found in cardiac muscle and skeletal muscle. For mice received rAAV8-Gluc/Fluc by im, Fluc mainly expressed in local skeletal muscle and secondly in liver. CONCLUSION: rAAV8-Gluc/Fluc has been prepared successfully and its mediating transgene expression in mice has been investigated. This research will facilitate preclinical studies for rAAV8-mediated gene therapy.