RESUMO
The whole-genome sequencing of coxsackievirus (CV)-A10 does not follow a conventional experimental protocol. To fully understand the genetic variation and evolution of CV-A10, complete genome amplification is necessary. Most previous studies have concentrated on partial sequences of the CV-A10 genome, such as the VP1 gene. The few studies that have investigated CV-A10 at the genomic level have reported only two complete genome sequences to GenBank. The basic fault may be attributed to the regional nature of the genetics and evolution of CV-A10 and to the lack of laboratory procedures for obtaining the genomes. In this study, we present a robust "three-step" protocol performed with A105UF/A820, EVP4/A6141, and A4879/A1005R for the full-length genome amplification of CV-A10. The results revealed that the method is able to accurately and reproducibly amplify three fragments with overlaps of the full-length genome of eight CV-A10 strains. Compared with other methods, this assay is both quick and specific. In addition, the three-step protocol could be capable of amplifying the full-length genomes of CV-A10 strains isolated from different countries and regions. The specific three-step protocol may be particularly useful for investigating samples co-infected with CV-A10 and other viruses.
Assuntos
Enterovirus Humano A/genética , Genoma Viral , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Mapeamento Cromossômico , Primers do DNA/síntese química , Primers do DNA/genética , GenótipoRESUMO
We investigated the application of liquid chromatography-tandem mass spectrometry (LC-MS/MS) for pan-drug-resistant Acinetobacter baumannii (PDR-AB) with carbapenem resistance. Eight strains were randomly selected from 84 clinical isolates of PDR-AB strains obtained by the Kirby-Bauer and agar dilution methods. An efflux pump inhibition test was used to screen for the efflux pump phenotype. An ethylenediaminetetraacetic acid (EDTA) synergy test was used to screen for the ß-lactamase phenotype, and a three-dimensional test was used to detect extended spectrum ß-lactamase (ESBL) and ampicillin C, KPC, and carbapenemase. ESBL genes were amplified by polymerase chain reaction and sequenced. Outer membrane proteins were extracted from a sensitive strain and the PDR-AB strains by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and subjected to LC-MS/MS. Peptide mass fingerprinting data were retrieved, and proteins with differential expression were identified. Results of the efflux pump inhibition tests showed that the minimum inhibitory concentrations for meropenem were decreased in 4 of the 8 strains by at least 25% of the original value. The results of the EDTA synergy test were negative, and the modified Hodge's tests were positive for all strains. PCR and sequencing confirmed that seven, five, and all eight of the PDR-AB strains contained blaOXA-23, blaTEM-1, and KPC-2, respectively. OXA-23 and CsuC proteins were differentially expressed between the drug-resistant and -sensitive strains. Production of blaOXA-23 enzyme and pilus molecular chaperone to guide synthesis of CsuC protein may be involved in the resistance of A. baumannii to carbapenems. LC-MS/ MS provides a quick and easy method for carbapenemase detection.
Assuntos
Proteínas de Bactérias/isolamento & purificação , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , beta-Lactamases/isolamento & purificação , Acinetobacter baumannii/química , Acinetobacter baumannii/efeitos dos fármacos , Proteínas de Bactérias/química , Farmacorresistência Bacteriana/genética , Humanos , beta-Lactamases/químicaRESUMO
TERMINAL FLOWER1 (TFL1) homologous genes play major roles in maintaining vegetative growth and inflorescence meristem characteristics in various plant species; however, to date, the function of the bamboo TFL1 homologous gene has not been described. In this study, a TFL1 homologous gene was isolated from Bambusa oldhamii and designated as BoTFL1-like. Phylogenetic analysis of TFL1 homologous genes revealed that BoTFL1-like shared more than 90% identity with the TFL1 genes of other Gramineae. RT-PCR analysis showed that the expression level of BoTFL1-like in floral buds was almost 3.5 times higher than in vegetative buds. In 35S::BoTFL1-like transgenic Arabidopsis thaliana plants, the time of flowering was significantly delayed by 5 to 9 days, and development of floral buds and sepals was severely affected compared to wild type Arabidopsis plants. This suggests that the BoTFL1-like gene may play roles in flowering time and flower morphological structure in B. oldhamii. The BoTFL1-like gene driven by the 35S promoter almost fully rescued the phenotype of the tfl1 mutant apart from the number of rosette inflorescences, indicating that the function of BoTFL1-like was similar to TFL1 in Arabidopsis. We conclude the TFL1 gene function has been conserved between B. oldhamii and A. thaliana.
Assuntos
Arabidopsis/genética , Bambusa/genética , Expressão Ectópica do Gene , Flores/genética , Mutação , Fenótipo , Proteínas de Plantas/genética , Sequência de Aminoácidos , Bambusa/classificação , Sequência de Bases , Clonagem Molecular , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Proteínas de Plantas/química , Conformação Proteica , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
We investigated the relationship between peripheral blood T lymphocyte subsets and hepatitis C virus (HCV) RNA levels in patients with hepatitis C. Samples from 69 chronic hepatitis C (CHC) patients and 20 healthy controls were analyzed using quantitative polymerase chain reaction (PCR) to detect HCV RNA and flow cytometry to determine the expression levels of CD3, CD4, and CD8 in lymphocytes. The percentage of CD4+ T cells (42.87 ± 6.11%) and the ratio of CD4+/CD8+ (1.34 ± 0.25) in these patients were significantly lower than those in the healthy control group (49.55 ± 6.68%, 1.82 ± 0.11, respectively) (P < 0.01, P < 0.01), while the percentage of CD8+ T cells (32.78 ± 5.48%) was higher than that in the control group (27.35 ± 4.32%) (P < 0.01). There was no significant difference in the percentage of CD3+ T cells between the two groups (P > 0.05). With the increase in HCV RNA replication, the percentage of CD8+ T cells increased gradually, while the CD4+ T cell percentage and CD4/CD8 ratio decreased. The change in the percentage of T lymphocyte subsets may be one of the reasons for persistent HCV infection, and the high expression levels of HCV RNA might be the reason for the low frequency of CD4+ T lymphocytes in patients with chronic HCV.
Assuntos
Hepacivirus/imunologia , Hepatite C Crônica/imunologia , Hepatite C Crônica/virologia , Subpopulações de Linfócitos T/imunologia , Carga Viral , Adolescente , Adulto , Idoso , Antígenos CD/metabolismo , Contagem de Linfócito CD4 , Relação CD4-CD8 , Estudos de Casos e Controles , Feminino , Hepatite C Crônica/sangue , Humanos , Imunofenotipagem , Masculino , Pessoa de Meia-Idade , Subpopulações de Linfócitos T/metabolismo , Adulto JovemRESUMO
To compare the efficacy of dendritic and cytokine-induced killer cells (DC-CIK) therapy combined with concurrent radiochemotherapy on stage IIIB non-small cell lung cancer. Sixty-three patients with stage IIIB non-small cell lung cancer were randomly divided into the study and control groups. The study group, comprising 30 patients, was treated with DC-CIK combined with docetaxel-cisplatin chemotherapy and synchronization conformal radiotherapy. The control group including 33 patients was only treated with docetaxel-cisplatin chemotherapy and synchronization conformal radiotherapy. The efficacy, Karnofsky performance score (KPS), tumor markers, 6-month and 12-month survival rate, T cell subsets, and adverse reactions of the two groups were compared. The response rate of the study group was 83.3% (25/30), and that of the control group was only 54.5% (18/33). Furthermore, the KPS, T cell subsets, and 12-month survival rate was significantly higher in the study group, and there were significant differences between the two groups. The two groups had no significant difference in adverse reactions. The combined DC-CIK therapy, with synchronous radiotherapy and chemotherapy to treat stage IIIB non-small cell lung cancer was superior to single synchronous radiotherapy and chemotherapy. The combined therapy can improve the life quality and prolong the survival time of the patients.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/terapia , Quimiorradioterapia , Células Matadoras Induzidas por Citocinas/imunologia , Células Dendríticas/imunologia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/terapia , Idoso , Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Carcinoma Pulmonar de Células não Pequenas/patologia , Quimiorradioterapia/efeitos adversos , Feminino , Seguimentos , Humanos , Avaliação de Estado de Karnofsky , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Subpopulações de Linfócitos/imunologia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Taxa de Sobrevida , Fatores de Tempo , Resultado do TratamentoRESUMO
We explored the expression and clinical significance of circulating tumor cells (CTCs) in patients with advanced non-small cell lung cancer (NSCLC). Sixty-six patients with advanced NSCLC at the Oncology Department of Jinzhou Hospital were selected as an observation group between February and December 2013. Healthy volunteers and 20 benign lung disease patients were taken as a control group. Peripheral blood CTCs in the observation and control groups were detected using the CellSearch(®). CTC detection and analysis system, and the relationship between the expression and clinical effect of CTCs and disease progression was analyzed. Peripheral blood CTCs were observed in 47 of the 66 observation group cases (71.21%), but none were found in the control group (P < 0.05). The CTC-positive rate was independent of NSCLC patients' age, gender, smoking habits, histological features, and degree of differentiation (P > 0.05). The CTC-positive rate correlated with pathological staging (P < 0.05). After two courses of chemotherapy, the number of cases with CTCs ≥3 decreased significantly, compared with pre-chemotherapy cases (P < 0.05), and the disease did not progress in 37 cases (34 cases with <3 CTCs and three cases with ≥3 CTCs). Eight cases displayed disease progression, of which five cases had <3 CTCs and three cases had ≥3CTCs. There was a statistically significant correlation between CTC changes and disease progression (P < 0.05). The CTC-positive rate correlated with the pathological staging and changes in the number of CTCs were associated with chemotherapy efficacy and disease progression.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/patologia , Células Neoplásicas Circulantes/patologia , Adulto , Idoso , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Progressão da Doença , Feminino , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Masculino , Pessoa de Meia-IdadeRESUMO
We investigated the expression differences of the TEL-AML1 fusion gene in a leukemia glucocorticoid (GC)-sensitive cell line (CEM) and a GC-resistant cell line (Jurkat). Changes in TEL-AML1 expression before and after GC exposure were analyzed. Expression of GC-sensitive and GC-resistant leukemia cells following initial diagnosis and during treatment was simulated. Leukemia cells were divided into a GC-unexposed or a GC-exposed group. A methyl thiazolyl tetrazolium assay was used to detect cell proliferation inhibition, flow cytometry was used to observe cell apoptosis, reverse transcription-polymerase chain reaction was used to detect the mRNA expression of TEL-AML1 before and after exposure, and western blotting was used to analyze protein levels of TEL-AML1 before and after exposure. Inhibitory concentrations of 50% of cells in the Jurkat and CEM cells at 24 h were 382 and 9 mM, respectively, and at 48 h they were 216 and 2 mM. The proliferation inhibition effect of dexamethasone sodium phosphate on Jurkat cells was much lower than that on CEM cells. Jurkat cells showed obvious apoptosis after exposure to 100 mM dexamethasone sodium phosphate for 48 h. In the exposed group, Jurkat cells showed higher TEL-AML1 expression than did CEM cells (P < 0.05). In the unexposed group, TEL-AML1 gene expression in Jurkat cells was not affected by GC exposure (P > 0.05), while the CEM cells presented significant differences before and after exposure (P < 0.05). Sustained high expression of TEL-AML1 participated in and maintained the occurrence of GC resistance. Inhibition of TEL-AML1 may provide a new therapeutic approach to reverse GC resistance.