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1.
Biosci Rep ; 39(6)2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31189745

RESUMO

Apocynum venetum is an eco-economic plant that exhibits high stress resistance. In the present paper, we carried out a whole-genome survey of A. venetum in order to provide a foundation for its whole-genome sequencing. High-throughput sequencing technology (Illumina NovaSep) was first used to measure the genome size of A. venetum, and bioinformatics methods were employed for the evaluation of the genome size, heterozygosity ratio, repeated sequences, and GC content in order to provide a foundation for subsequent whole-genome sequencing. The sequencing analysis results indicated that the preliminary estimated genome size of A. venetum was 254.40 Mbp, and its heterozygosity ratio and percentage of repeated sequences were 0.63 and 40.87%, respectively, indicating that it has a complex genome. We used k-mer = 41 to carry out a preliminary assembly and obtained contig N50, which was 3841 bp with a total length of 223949699 bp. We carried out further assembly to obtain scaffold N50, which was 6196 bp with a total length of 227322054 bp. We performed simple sequence repeat (SSR) molecular marker prediction based on the A. venetum genome data and identified a total of 101918 SSRs. The differences between the different types of nucleotide repeats were large, with mononucleotide repeats being most numerous and hexanucleotide repeats being least numerous. We recommend the use of the '2+3' (Illumina+PacBio) sequencing combination to supplement the Hi-C technique and resequencing technique in future whole-genome research in A. venetum.

2.
J Virol ; 93(13)2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30996087

RESUMO

Enterovirus A71 (EV-A71) is a major pathogen that causes hand-foot-and-mouth disease (HFMD), which occasionally results in severe neurological complications. In this study, we developed four EV-A71 (rgEV-A71) strains by reverse genetics procedures as possible vaccine candidates. The four rgEV-A71 viruses contained various codon-deoptimized VP1 capsid proteins (VP1-CD) and showed replication rates and antigenicity similar to that of the wild-type virus, while a fifth virus, rg4643C4VP-CD, was unable to form plaques but was still able to be examined by median tissue culture infectious dose (TCID50) titers, which were similar to those of the others, indicating the effect of CD on plaque formation. However, the genome stability showed that there were some mutations which appeared during just one passage of the VP1-CD viruses. Thus, we further constructed VP1-CD rgEV-A71 containing high-fidelity determinants in 3D polymerase (CD-HF), and the number of mutations in CD-HF rgEV-A71 was shown to have decreased. The CD-HF viruses showed less virulence than the parental strain in a mouse infection model. After 14 days postimmunization, antibody titers had increased in mice infected with CD-HF viruses. The mouse antisera showed similar neutralizing antibody titers against various CD-HF viruses and different genotypes of EV-A71. The study demonstrates the proof of concept that VP1 codon deoptimization combined with high-fidelity 3D polymerase decreased EV-A71 mutations and virulence in mice but retained their antigenicity, indicating it is a good candidate for next-generation EV-A71 vaccine development.IMPORTANCE EV-A71 can cause severe neurological diseases with fatality in infants and young children, but there are still no effective drugs to date. Here, we developed a novel vaccine strategy with the combination of CD and HF substitutions to generate the genetically stable reverse genetics virus. We found that CD combined with HF polymerase decreased the virulence but maintained the antigenicity of the virus. This work demonstrated the simultaneous introduction of CD genome sequences and HF substitutions as a potential new strategy to develop attenuated vaccine seed virus. Our work provides insight into the development of a low-virulence candidate vaccine virus through a series of genetic editing of virus sequences while maintaining its antigenicity and genome stability, which will provide an additional strategy for next-generation vaccine development of EV-A71.

3.
Neural Regen Res ; 13(11): 1893-1899, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30233061

RESUMO

Myelin-associated glycoprotein (MAG) inhibits the growth of neurites from nerve cells. Extraction and purification of MAG require complex operations; therefore, we attempted to determine whether commercially available MAG-Fc can replace endogenous MAG for research purposes. Immunofluorescence using specific antibodies against MAG, Nogo receptor (NgR) and paired immunoglobulin-like receptor B (PirB) was used to determine whether MAG-Fc can be endocytosed by neuro-2a cells. In addition, neurite outgrowth of neuro-2a cells treated with different doses of MAG-Fc was evaluated. Enzyme linked immunosorbent assays were used to measure RhoA activity. Western blot assays were conducted to assess Rho-associated protein kinase (ROCK) phosphorylation. Neuro-2a cells expressed NgR and PirB, and MAG-Fc could be endocytosed by binding to NgR and PirB. This activated intracellular signaling pathways to increase RhoA activity and ROCK phosphorylation, ultimately inhibiting neurite outgrowth. These findings not only verify that MAG-Fc can inhibit the growth of neural neurites by activating RhoA signaling pathways, similarly to endogenous MAG, but also clearly demonstrate that commercial MAG-Fc is suitable for experimental studies of neurite outgrowth.

4.
Mol Pain ; 14: 1744806918777614, 2018 Jan-Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29768956

RESUMO

Aims The main objective was to investigate the effects of the transient receptor potential cation channel subfamily V member 1 (TRPV1) on nerve regeneration following sciatic transection injury by functional blockage of TRPV1 using AMG-517, a specific blocker of TRPV1. Methods AMG-517 was injected into the area surrounding ipsilateral lumbar dorsal root ganglia 30 min after unilateral sciatic nerve transection. The number of sciatic axons and the expression of growth-associated protein-43 (GAP-43) and glial fibrillary acidic protein was examined using semithin sections, Western blot, and immunofluorescence analyses. Results Blockage of TRPV1 with AMG-517 markedly promoted axonal regeneration, especially at two weeks after sciatic injury; the number of axons was similar to the uninjured control group. After sciatic nerve transection, expression of glial fibrillary acidic protein was decreased and GAP-43 was increased at the proximal stump. However, the expression of both glial fibrillary acidic protein and GAP-43 increased significantly in AMG-517-treated groups. Conclusions TRPV1 may be an important therapeutic target to promote peripheral nerve regeneration after injury.


Assuntos
Axônios/patologia , Benzotiazóis/farmacologia , Benzotiazóis/uso terapêutico , Regeneração Nervosa/efeitos dos fármacos , Traumatismos dos Nervos Periféricos/tratamento farmacológico , Traumatismos dos Nervos Periféricos/patologia , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Canais de Cátion TRPV/metabolismo , Animais , Axônios/efeitos dos fármacos , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Proteína GAP-43/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Células de Schwann/metabolismo , Células de Schwann/patologia , Nervo Isquiático/patologia , Corno Dorsal da Medula Espinal/metabolismo , Corno Dorsal da Medula Espinal/patologia , Canais de Cátion TRPV/antagonistas & inibidores , Regulação para Cima/efeitos dos fármacos
5.
PLoS One ; 13(5): e0196727, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29746492

RESUMO

It is generally agreed that human influenza virus preferentially binds to α-2,6-linked sialic acid-containing receptors, and mutations that change the binding preference may alter virus infectivity and host tropism. Limited information is available on the glycan-binding specificity of epidemic influenza viruses. In this study, we systemically investigated the glycan-binding preferences of human influenza A(H3N2) viruses isolated from 1999 to 2007 in Taiwan using a high-throughput carbohydrate array. The binding patterns of 37 H3N2 viruses were classified into three groups with significant binding-pattern variations. The results showed that the carbohydrate-binding patterns of H3N2 varied over time. A phylogenetic analysis of the hemagglutinin gene also revealed progressive drift year to year. Of note, the viruses that caused large outbreaks in 1999 and 2003 showed glycan-binding preferences to both α-2,3 and α-2,6 sialylated glycans. Twenty amino acid substitutions were identified primarily at antigenic sites that might contribute to H3N2 virus evolution and the change in the glycan-binding patterns. This study provides not only a systematic analysis of the receptor-binding specificity of influenza clinical isolates but also information that could help to monitor the outbreak potential and virus evolution of influenza viruses.


Assuntos
Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/metabolismo , Polissacarídeos/metabolismo , Substituição de Aminoácidos/genética , Evolução Molecular , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Influenza Humana/virologia , Mutação/genética , Filogenia , Receptores Virais/metabolismo , Estações do Ano , Taiwan , Ligação Viral
6.
Toxins (Basel) ; 10(2)2018 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-29393906

RESUMO

(1) Background: The botulinum toxin A (BoNT-A) heavy chain (HC) can stimulate the growth of primary motor neurites. (2) Methods: A recombinant BoNT/A HC was injected locally plus interval intrathecal catheter of BoNT/A HC to rats with ipsilateral semi-dissociated lumbar spinal cord injuries (SCIs). First, 2D gel with a silver nitrate stain was applied to detect the general pattern of protein expression. Growth associated protein 43 (GAP-43) and superior cervical ganglion 10 (SCG10) were chosen to represent the altered proteins, based on their molecular weight and pI, and were used to further detect their expression. Meanwhile, the neuronal processes were measured. The measurements of thermal hyperalgesia and grasp power at the ipsilateral hindlimb were used to evaluate spinal sensory and motor function, respectively. (3) Results: The local injection of BoNT/A HC followed by its intrathecal catheter intervally altered the spinal protein expression pattern after an SCI; protein expression was similar to normal levels or displayed a remarkable increase. The changes in the expression and distribution of phosphorylated growth associated protein 43(p-GAP 43) and superior cervical ganglion 10 (SCG 10) indicated that the administration of BoNT/A HC to the SCI significantly amplified the expression of p-GAP43 and SCG10 (p < 0.05). Meanwhile, the positive immunofluorescent staining for both p-GAP43 and SCG10 was mainly present near the rostral aspect of the injury, both in the cytoplasm and the neuronal processes. Moreover, the outgrowth of neurites was stimulated by the BoNT/A HC treatment; this was evident from the increase in neurite length, number of branches and the percentage of cells with neuronal processes. The results from the spinal function tests suggested that the BoNT/A HC did not affect sensation, but had a large role in improving the ipsilateral hindlimb grasp power (p < 0.05). (4) Conclusions: The local injection with the intermittent intrathecal administration of BoNT/A heavy chain to rats with SCI increased the local expression of GAP-43 and SCG 10, which might be affiliated with the regeneration of neuronal processes surrounding the injury, and might also be favorable to the relief of spinal motor dysfunction.

7.
Mol Med Rep ; 16(6): 8392-8398, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28944883

RESUMO

Baicalein (BA), one of the major bioactive flavonoids isolated from Scutellariae Radix, possesses various pharmacological activities. The present study aimed to investigate the protective effects of BA on tert­butyl hydroperoxide (t­BHP)­induced hepatotoxicity, and to investigate the potential mechanisms in LO2 cells. BA was demonstrated to possess protective properties against t­BHP injury in LO2 cells, as evidenced by MTT and lactate dehydrogenase assays. BA significantly prevented t­BHP­induced depolarization of mitochondrial membrane potential (MMP), decreased the percentage of apoptotic cells caused by t­BHP, and prevented intracellular reactive oxygen species (ROS) generation in LO2 cells. Furthermore, BA slightly triggered autophagy in LO2 cells, as evidenced by the elevation of LC3­II expression, while BA combined treatment with an autophagy inhibitor (chloroquine) or activator (rapamycin) did not alter the hepatoprotective properties. In conclusion, BA may possess a hepatoprotective effect against t­BHP­induced liver cell injury, dependent on ROS removal. Therefore, BA may represent a potential drug candidate in protecting hepatotoxicity.


Assuntos
Flavanonas/farmacologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Substâncias Protetoras/farmacologia , terc-Butil Hidroperóxido/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Biomarcadores , Linhagem Celular , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
8.
Nat Commun ; 8(1): 274, 2017 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-28819251

RESUMO

G9a, a H3K9 methyltransferase, shows elevated expression in many types of human cancers, particularly breast cancer. However, the tumorigenic mechanism of G9a is still far from clear. Here we report that G9a exerts its oncogenic function in breast cancer by repressing hephaestin and destruction cellular iron homeostasis. In the case of pharmacological inhibition or short hairpin RNA interference-mediated suppression of G9a, the expression and activity of hephaestin increases, leading to the observed decrease of intracellular labile iron content and the disturbance of breast cancer cell growth in vitro and in vivo. We also provide evidence that G9a interacts with HDAC1 and YY1 to form a multi-molecular complex that contributes to hephaestin silencing. Furthermore, high G9a expression and low hephaestin expression correlate with poor survival of breast cancer are investigated. All these suggest a G9a-dependent epigenetic program in the control of iron homeostasis and tumor growth in breast cancer.G9a is a histone methyltransferase highly expressed in several cancers including breast cancer. Here the authors propose a mechanism through which G9a promotes breast cancer by regulating iron metabolism through the repression of ferroxidase hephaestin.


Assuntos
Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica , Antígenos de Histocompatibilidade/genética , Histona-Lisina N-Metiltransferase/genética , Ferro/metabolismo , Proteínas de Membrana/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Antígenos de Histocompatibilidade/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Homeostase , Humanos , Proteínas de Membrana/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
9.
Am J Chin Med ; 45(1): 123-136, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28081631

RESUMO

Baicalein (BA), one of the major compounds isolated from the root of Scutellaria baicalensis Gerogi, exhibits various pharmacological effects, such as anti-oxidant, anti-inflammatory, and anticancer effects. In this study, we found that BA reduced cell viability and increased apoptosis in ovarian cancer cells. Treatment of cells with BA enhanced microtubule-associated protein light chain 3-II (LC3-II) expression, acidic vesicular organelle and GFP-LC3 fluorescence dot accumulation. Combined treatment with chloroquine and BA apparently reduced cell viability and increased the cleavage of poly (ADPribose) polymerase (PARP) in both HEY and A2780 ovarian cancer cell lines, indicating that BA induces a protective autophagy in these cells. Knockdown of Beclin 1 by siRNA remarkably decreased BA-induced LC3-II lipidation. In addition, we found an increase in the phosphorylation of extracellular signal-regulated kinase (ERK, Thr202/Thr204) and AKT (Ser473) after BA treatment, and inhibition of ERK activation by the pharmacological inhibitor U0126 or ERK siRNA blocked BA-induced autophagy. Taken together, these results suggest that BA induces Beclin 1- and ERK-dependent autophagy in ovarian cancer cells.


Assuntos
Autofagia/efeitos dos fármacos , Proteína Beclina-1/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Flavanonas/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Associadas aos Microtúbulos/efeitos dos fármacos , Antimaláricos/farmacologia , Proteína Beclina-1/metabolismo , Western Blotting , Linhagem Celular Tumoral , Cloroquina/farmacologia , Feminino , Técnicas de Silenciamento de Genes , Humanos , Técnicas In Vitro , Microscopia Confocal , Microscopia de Fluorescência , Proteínas Associadas aos Microtúbulos/metabolismo , Neoplasias Ovarianas , Poli(ADP-Ribose) Polimerases/efeitos dos fármacos , Poli(ADP-Ribose) Polimerases/metabolismo , RNA Interferente Pequeno
10.
Medicine (Baltimore) ; 95(18): e3577, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-27149483

RESUMO

Human adenoviruses (HAdVs) are important causes of respiratory infections in children. They usually cause mild upper respiratory symptoms, but they can also produce severe pneumonia and other complications. The aims of this retrospective study were to better define the molecular epidemiology of respiratory adenoviruses circulating in Taiwanese children during 2002 and 2013, detect reinfections and co-infections, and characterize the clinical features and laboratory findings according to the causative genotypes.We collected a representative sample of 182 isolates of adenoviruses from 175 children during the 12-year study period. The most prevalent species was HAdV-B genotype 3 (HAdV-3) (92/182, 50.5%) followed by HAdV-C (HAdV-2) (38/182, 20.9%). A single outbreak of HAdV-E (6/182, 3.3%) was noted in 2007. The mean age of children with adenovirus infections was 3.7 ±â€Š2.0 years, with a slight predominance of males (53.1%). Children with HAdV-B tended to be older, had more lower respiratory tract infections, gastrointestinal symptoms, and a higher rate of hospitalization than those with HAdV-C (P < 0.05). Adenovirus co-infections were noted in 25/175 (14.3%) of the children. The most frequent co-infections were with species B (HAdV-3) and C (HAdV-2) (14/25, 56.0%). Additional infections were noted in 23/175 (13.1%) of the children. Of these repeated infections, the initial isolates were always genotypes of HAdV-C. The second isolates were genotypes of HAdV-B or HAdV-E. The clinical features of the first HAdV-B infection and the reinfection of HAdV-B followed the HAdV-C were similar.In conclusion, HAdV-B, C, and E were the only adenovirus species that were isolated from children who were sufficiently ill with respiratory infections to require a visit to the hospital. Human adenovirus B (HAdV-3) accounted for half of these species. HAdV-B was more likely than other species to produce severe disease. The high incidence of adenovirus co-infection and reinfections with different HAdV species supports the need for continued surveillance and has major implications for development of vaccines.


Assuntos
Infecções por Adenovirus Humanos/epidemiologia , Infecções por Adenovirus Humanos/virologia , Adenovírus Humanos/genética , Infecções Respiratórias/epidemiologia , Infecções Respiratórias/virologia , Fatores Etários , Pré-Escolar , Coinfecção/epidemiologia , Coinfecção/virologia , Feminino , Genótipo , Hospitalização/estatística & dados numéricos , Humanos , Incidência , Lactente , Masculino , Recidiva , Estudos Retrospectivos , Taiwan/epidemiologia
11.
Sci Rep ; 6: 26241, 2016 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-27184816

RESUMO

Licochalcone A (LCA), a flavonoid isolated from the famous Chinese medicinal herb Glycyrrhiza uralensis Fisch, presents obvious anti-cancer effects. In this study, the anti-cancer effects and potential mechanisms of LCA in non-small cell lung cancer (NSCLC) cells were studied. LCA decreased cell viability, increased lactate dehydrogenase release, and induced apoptosis in a concentration-dependent manner in NSCLC cells while not in human embryonic lung fibroblast cells. The expression of phosphatidylethanolamine-modified microtubule-associated protein light-chain 3 (LC3-II) and formation of GFP-LC3 punta, two autophagic markers, were increased after treatment with LCA. LCA-induced LC3-II expression was increased when combined with chloroquine (CQ), while knock-down of autophagy related protein (ATG) 7 or ATG5 reversed LCA-induced LC3-II expression and GFP-LC3 punta formation, suggesting that LCA induced autophagy in NSCLC cells. Inhibition of autophagy could not reverse the LCA-induced cell viability decrease and apoptosis. In addition, LCA increased the expression of endoplasmic reticulum stress related proteins, such as binding immunoglobulin protein and C/EBP homologous protein (CHOP). Knock-down of CHOP reversed LCA-induced cell viability decrease, apoptosis, and autophagy. Taken together, LCA-induced autophagic effect is an accompanied phenomenon in NSCLC cells, and CHOP is critical for LCA-induced cell viability decrease, apoptosis, and autophagy.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Chalconas/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Fator de Transcrição CHOP/metabolismo , Células A549 , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Técnicas de Silenciamento de Genes , Glycyrrhiza uralensis/química , Humanos , L-Lactato Desidrogenase/metabolismo , Neoplasias Pulmonares/patologia , Proteínas Associadas aos Microtúbulos/metabolismo , Fator de Transcrição CHOP/antagonistas & inibidores , Fator de Transcrição CHOP/genética
12.
Acta Neurol Belg ; 116(3): 287-93, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26615535

RESUMO

Vertebral artery dominance (VAD), which is a common congenital variation of vertebral artery, may be associated with an increased risk of cerebral posterior circulation infarction (PCI). The aims of this study were to investigate the correlation of VAD with incidence and laterality of PCI, and oblige the correlation of VAD and basilar artery (BA) curvature. Incidence of separate territory infarction in posterior circulation and incidence of BA curvature were compared between 78 VAD patients and 68 controls. VA dominance, laterality of BA curvature and separate territory infarction, and their directional relationships were observed in VAD group. The incidence of BA curvature in VAD group was significantly higher than that in controls (P = 0.000). 89.7 % (35/39) of patients had an opposite directional relationship between dominant VA and BA curvature. The total incidence of PCI in VAD group was significantly higher than that in controls (P = 0.001). The incidences of posterior inferior cerebellar artery (PICA) and BA territory infarction were both significantly higher than those in controls [11.5 % (9/78) vs. 1.5 % (1/68), P = 0.016; 20.5 % (16/78) vs. 7.4 % (5/68), P = 0.024]. No differences were found in superior cerebellar artery and posterior cerebral artery territory infarction between two groups. 77.8 % (7/9) of PICA infarction were on the opposite side of dominant VA. 75.0 % (12/16) of BA infarction were on the side of dominant VA. The incidence of PCI in BA curvature patients was significantly higher than that in BA straight patients. The incidence of BA curvature is higher in VAD patients, and BA usually bends to the opposite side of dominant VA. The incidence of PCI is higher in VAD patients, especially in PICA infarction and BA infarction patients.


Assuntos
Artéria Basilar/diagnóstico por imagem , Infarto Encefálico/etiologia , Angiografia Cerebral , Circulação Cerebrovascular/fisiologia , Imagem por Ressonância Magnética , Artéria Vertebral/diagnóstico por imagem , Adulto , Idoso , Infarto Encefálico/fisiopatologia , Cerebelo/irrigação sanguínea , Angiografia Cerebral/métodos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
13.
PLoS One ; 10(11): e0142745, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26555962

RESUMO

Wild poliovirus (WPV) persists in diverse locales worldwide, spreading outward from endemic areas. In response to the international threat of WPV transmission and changes in the national vaccination policy, we established an environmental surveillance system to monitor the circulation of wild and vaccine-related poliovirus in Taiwan. From July 2012 to December 2013, we collected sewage specimens every month from 10 sewage treatment plants located throughout Taiwan. The specimens were concentrated by the two-phase separation method and then inoculated into L20B, RD, and A549 cells for virus isolation. Viral isolates were identified and serotyped by immunofluorescence assay or molecular analysis. A total of 300 sewage samples were collected, and the results showed 163 samples (54.3%) were positive for virus, and 268 isolates were identified. Among these, 75 samples (25%) were positive for enterovirus (EV), but no poliovirus was found. In addition, 92 isolates were identified as enteroviruses and the most common serotypes were coxsackievirus B4, coxsackievirus B3, and coxsackievirus B2. Interestingly, 102 (34%) and 82 (27.3%) specimens were positive for mammalian orthoreovirus (MRV) and adenovirus, respectively. This study confirmed that sewage surveillance can be a useful additional modality for monitoring the possible presence of wild-type or vaccine-derived poliovirus in wastewater, and can indicate the current types of viruses circulating in the population. Furthermore, since MRV was found in children with acute necrotizing encephalopathy and meningitis, the high incidence of MRV detected by environmental surveillance warrants further investigation.


Assuntos
Monitoramento Ambiental/métodos , Orthoreovirus de Mamíferos/isolamento & purificação , Esgotos/virologia , Adenovírus Humanos/isolamento & purificação , Criança , Enterovirus Humano B/isolamento & purificação , Genes Virais , Humanos , Tipagem Molecular , Orthoreovirus de Mamíferos/classificação , Orthoreovirus de Mamíferos/genética , Filogenia , Poliovirus/isolamento & purificação , Taiwan
14.
Acta Pharmacol Sin ; 36(12): 1503-13, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26592509

RESUMO

AIM: Platycodin D, the main saponin isolated from Chinese herb Platycodonis Radix, exhibits anticancer activities against various cancer cell lines. Here we evaluated its anticancer action against human hepatocellular carcinoma cells in vitro and in vivo, and elucidated the relationship between platycodin D-induced apoptosis and autophagy. METHODS: The viability of human hepatocellular carcinoma BEL-7402 cells was evaluated with MTT assay, and the apoptosis was examined using Annexin V/PI and Hoechst 33342 staining assays. Monodansylcadaverine (MDC) staining was used to label autophagic vacuoles. The proteins were detected using Western blot analysis. For studying its anticancer action in vivo, platycodin D (5 and 10 mg· kg(-1)·d(-1)) was intraperitoneally injected to BEL-7402-bearing mice for 21 days. RESULTS: Platycodin D (5-40 µmol/L) inhibited the cell proliferation in vitro with IC50 values of 37.70±3.99, 24.30±2.30 and 19.70±2.36 µmol/L at 24, 48 and 72 h, respectively. Platycodin D (5-20 µmol/L) dose-dependently increased BEL-7402 cell apoptosis, increased the Bax/Bcl-2 ratio and the levels of cleaved PARP and cleaved caspase-3, and decreased the level of Bcl-2. Furthermore, platycodin D (5-20 µmol/L) induced autophagy in BEL-7402 cells, as evidenced by formation of cytoplasmic vacuoles, increased amounts of LC3-II, and increased numbers of MDC-positive cells. Pretreatment with the autophagy inhibitor chloroquine (5 µmol/L) or BAF (50 nmol/L) significantly enhanced platycodin D-induced proliferation inhibition and apoptosis. Moreover, platycodin D (20 µmol/L) activated the ERK and JNK pathways in BEL-7402 cells, and simultaneous blockage of the two pathways effectively suppressed platycodin D-induced autophagy and enhanced platycodin D-induced apoptosis. In BEL-7402-bearing mice, platycodin D (10 mg·kg(-1)•d(-1)) significantly reduced relative tumor volume with decreased body weight. CONCLUSION: Platycodin D not only inhibits the proliferation of BEL-7402 cells but also suppresses BEL-7402 xenograft tumor growth. Platycodin D-induced cell proliferation inhibition and apoptosis are amplified by co-treatment with autophagy inhibitors.


Assuntos
Antineoplásicos Fitogênicos/uso terapêutico , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Saponinas/uso terapêutico , Triterpenos/uso terapêutico , Animais , Antineoplásicos Fitogênicos/farmacologia , Autofagia/efeitos dos fármacos , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Camundongos Endogâmicos BALB C , Camundongos Nus , Platycodon/química , Saponinas/farmacologia , Triterpenos/farmacologia
15.
PLoS One ; 10(3): e0120793, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25799397

RESUMO

Avian-origin influenza A (H7N9) viruses emerged as human pathogens in China in early 2013 and have killed >100 persons. Influenza vaccines are mainly manufactured using egg-based technology which could not meet the surging demand during influenza pandemics. In this study, we evaluated cell-based influenza H7N9 vaccines in ferrets. An egg-derived influenza H7N9 reassortant vaccine virus was adapted in MDCK cells. Influenza H7N9 whole virus vaccine antigen was manufactured using a microcarrier-based culture system. Immunogenicity and protection of the vaccine candidates with three different formulations (300 µg aluminum hydroxide, 1.5 µg HA, and 1.5 µg HA plus 300 µg aluminum hydroxide) were evaluated in ferrets. In ferrets receiving two doses of vaccination, geometric mean titers of hemagglutination (HA) inhibition and neutralizing antibodies were <10 and <40 for the control group (adjuvant only), 17 and 80 for the unadjuvanted (HA only) group, and 190 and 640 for the adjuvanted group (HA plus adjuvant), respectively. After challenge with wild-type influenza H7N9 viruses, virus titers in respiratory tracts of the adjuvanted group were significantly lower than that in the control, and unadjuvanted groups. MDCK cell-derived influenza H7N9 whole virus vaccine candidate is immunogenic and protective in ferrets and clinical development is highly warranted.


Assuntos
Furões , Subtipo H7N9 do Vírus da Influenza A/imunologia , Vacinas contra Influenza/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Adaptação Biológica , Animais , Antígenos Virais/imunologia , Cães , Feminino , Imunização , Subtipo H7N9 do Vírus da Influenza A/ultraestrutura , Pulmão/imunologia , Pulmão/patologia , Pulmão/virologia , Células Madin Darby de Rim Canino , Vírus Reordenados
16.
PLoS One ; 10(2): e0116278, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25706563

RESUMO

Enterovirus 71 (EV71) infection causes a myriad of diseases from mild hand-foot-and-mouth disease or herpangina to fatal brain stem encephalitis complicated with pulmonary edema. Several severe EV71 endemics have occurred in Asia-Pacific region, including Taiwan, and have become a serious threat to children's health. EV71 infection is initiated by the attachment of the virion to the target cell surface. Although this process relies primarily upon interaction between viruses and cell surface receptors, soluble factors may also influence the binding of EV71 to host cells. Galectin-1 has been reported to participate in several virus infections, but is not addressed in EV71. In this study, we found that the serum levels of galectin-1 in EV71-infected children were higher than those in non-infected people. In EV71 infected cells, galectin-1 was found to be associated with the EV71 VP1 and VP3 via carbohydrate residues and subsequently released and bound to another cell surface along with the virus. EV71 propagated from galectin-1 knockdown SK-N-SH cells exhibited lower infectivity in cultured cells and less pathogenicity in mice than the virus propagated from parental cells. In addition, this galectin-1-free EV71 virus was sensitive to high temperature and lost its viability after long-term storage, which could be restored following supplement of recombinant galectin-1. Taken together, our findings uncover a new role of galectin-1 in facilitating EV71 virus infection.


Assuntos
Enterovirus Humano A/metabolismo , Infecções por Enterovirus/sangue , Galectina 1/metabolismo , Vírion/metabolismo , Replicação Viral/fisiologia , Criança , Enterovirus Humano A/patogenicidade , Infecções por Enterovirus/virologia , Galectina 1/sangue , Galectina 1/genética , Humanos
17.
Phytother Res ; 29(5): 674-9, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25641124

RESUMO

Baicalein (BA), isolated from the Chinese medicinal herb Scutellariae radix (Huangqin in Chinese), is a flavonoid with various pharmacological effects. Herein, we found that BA only slightly reduced the cell viability on HepG2 cells after 24-h treatment as determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. However, BA (50 µM) effectively blocked the colony formation. Meanwhile, BA remarkably induced the formation of autophagosomes after 24-h treatment as determined by immunofluorescence with monodansylcadaverine staining as well as transmission electron microscopy, respectively. Moreover, BA obviously up-regulated the expression of microtubule-associated protein 1A/1B-light chain 3-II in concentration-dependent and time-dependent manners in HepG2 cells. When combined with the autophagy inhibitor chloroquine and BA, the cell viability and colony formation were significantly decreased, indicating that BA triggered protective autophagy, which prevented cell death. Further study showed that BA concentration-dependently and time-dependently decreased the expression of p-AKT (S473), p-ULK1 (S757) and p-4EBP1 (T37 and S65), suggesting the involvement of protein kinase B (AKT)/mammalian target of rapamycin (mTOR) in BA-triggered autophagy.


Assuntos
Autofagia/efeitos dos fármacos , Carcinoma Hepatocelular/patologia , Flavanonas/farmacologia , Neoplasias Hepáticas/patologia , Transdução de Sinais/efeitos dos fármacos , Células Hep G2/efeitos dos fármacos , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo
18.
J Virol ; 89(8): 4527-38, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25673703

RESUMO

UNLABELLED: Because the pathogenesis of enterovirus 71 (EV71) remains mostly ambiguous, identifying the factors that mediate viral binding and entry to host cells is indispensable to ultimately uncover the mechanisms that underlie virus infection and pathogenesis. Despite the identification of several receptors/attachment molecules for EV71, the binding, entry, and infection mechanisms of EV71 remain unclear. Herein, we employed glycoproteomic approaches to identify human nucleolin as a novel binding receptor for EV71. Glycoproteins purified by lectin chromatography from the membrane extraction of human cells were treated with sialidase, followed by immunoprecipitation with EV71 particles. Among the 16 proteins identified by tandem mass spectrometry analysis, cell surface nucleolin attracted our attention. We found that EV71 interacted directly with nucleolin via the VP1 capsid protein and that an antinucleolin antibody reduced the binding of EV71 to human cells. In addition, the knockdown of cell surface nucleolin decreased EV71 binding, infection, and production in human cells. Furthermore, the expression of human nucleolin on the cell surface of a mouse cell line increased EV71 binding and conferred EV71 infection and production in the cells. These results strongly indicate that human nucleolin can mediate EV71 binding to and infection of cells. Our findings also demonstrate that the use of glycoproteomic approaches is a reliable methodology to discover novel receptors for pathogens. IMPORTANCE: Outbreaks of EV71 have been reported in Asia-Pacific countries and have caused thousands of deaths in young children during the last 2 decades. The discovery of new EV71-interacting molecules to understand the infection mechanism has become an emergent issue. Hence, this study uses glycoproteomic approaches to comprehensively investigate the EV71-interacting glycoproteins. Several EV71-interacting glycoproteins are identified, and the role of cell surface nucleolin in mediating the attachment and entry of EV71 is characterized and validated. Our findings not only indicate a novel target for uncovering the EV71 infection mechanism and anti-EV71 drug discovery but also provide a new strategy for virus receptor identification.


Assuntos
Enterovirus Humano D/metabolismo , Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ligação Viral , Internalização do Vírus , Cromatografia , Enterovirus Humano D/fisiologia , Ensaio de Imunoadsorção Enzimática , Técnicas de Silenciamento de Genes , Humanos , Imunoprecipitação , Proteínas de Membrana/genética , Microscopia Imunoeletrônica , Neuraminidase , Fosfoproteínas/genética , Proteômica , Proteínas de Ligação a RNA/genética , Espectrometria de Massas em Tandem
19.
Eur J Pharmacol ; 749: 81-8, 2015 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-25592318

RESUMO

Platycodin D (PD), isolated from the Chinese medicinal herb named Platycodonis Radix, is a triterpenoid saponin with well-known anti-tumor effects. In this study, we provided reliable evidence that PD triggered autophagy in a number of cell lines in vitro. PD-triggered autophagy was identified by observation of cytoplasmic vacuole, up-regulation of microtubule-associated protein 1 light chain 3 II (LC3-II), and accumulation of autophagosomes. The Akt/mammalian target of rapamycin (mTOR) pathway may be not involved in PD-triggered autophagy, as evidenced by the increased phosphorylation of Akt (Thr308), mTOR (Ser2448), ribosomal protein S6 kinase (Ser371), and ULK1 (Ser757). However, the extracellular signal-regulated kinase (ERK) was activated after PD treatment. The decreased ERK phosphorylation caused by pretreatment with U0126, an inhibitor of MEK, suppressed the expression of LC3-II compared with PD treatment alone, suggesting that ERK pathway may have a critical function in PD-triggered autophagy. In addition, the PD-induced proliferative inhibition and apoptosis were enhanced when pretreatment with autophagy inhibitor chloroquine (CQ) or bafilomycin A1 (BAF), indicating that PD may trigger a protective autophagy in HepG2 cells. To the best of our knowledge, this paper is the first to report that PD triggers autophagy in a series of cell lines and ERK activation is important for PD-triggered autophagy in hepatocellular carcinoma HepG2 cells. The combined treatment with PD and CQ or BAF may be a promising regimen for hepatocellular carcinoma treatment.


Assuntos
Autofagia/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Saponinas/farmacologia , Triterpenos/farmacologia , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Cloroquina/farmacologia , Humanos , Neoplasias Hepáticas/metabolismo , Macrolídeos/farmacologia
20.
Fa Yi Xue Za Zhi ; 31(5): 337-40, 2015 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-26821471

RESUMO

OBJECTIVE: To investigate the relationship between the expression of secreted frizzled-related protein 5 (SFRP5) mRNA and the time interval after skeletal muscle injury in rats by real-time PCR. METHODS: A total of ninety SD rats were randomly divided into the contusion groups at different times including 4h, 8h, 12h, 16h, 20h, 24h, 28h, 32h, 36h, 40h, 44h, 48h after contusion, incision groups at different times including 4h and 8h after incision and the control group. The samples were taken from the contused zone at different time points. The total RNA was isolated from the samples and reversely transcribed to analyze the expression levels of SFRP5 mRNA. RESULTS: Compared to the control group, the expression of SFRP5 mRNA in contusion groups were down-regulated within 48 h after contusion and reached the lowest level at 20 h, and the expression of SFRP5 mRNA gradually increased from 20 h to 48 h after contusion. The expression of SFRP5 mRNA in the incised groups were significantly lower than that of the contusion groups at 4 h after injury. At the time of 8 h, the expression levels between the contusion and incision groups showed no statistically significant difference. CONCLUSION: It is suggested that SFRP5 mRNA analysis may show regular expression and can be a marker for estimation of skeletal muscle injury age.


Assuntos
Contusões/metabolismo , Proteínas de Membrana/metabolismo , Músculo Esquelético/lesões , Músculo Esquelético/metabolismo , Animais , Biomarcadores/metabolismo , RNA Mensageiro , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real
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