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Coccidiosis, caused by a protozoan parasite of the genus Eimeria, is one of the most severe contagious parasite diseases affecting the poultry industry worldwide. Using phytogenics to prevent chicken coccidiosis is a strategy aimed at combating the increasing issue of drug-resistant strains of Eimeria spp. This study demonstrates the anticoccidial activities of a medicinal herb, Trifolium pratense (TP) powder, and its ethanolic extract (designated TPE) against Eimeria spp. TPE exhibited significant suppressive activity against E. maxima oocyst sporulation and E. tenella sporozoite invasion and reproduction in Madin-Darby bovine kidney cells. Furthermore, administration of basal chicken diets containing TP powder or TPE to Eimeria-infected chickens significantly reduced the output of oocysts and severity of intestinal lesions. Dietary supplementation with TP significantly improved relative weight gain in E. tenella- and E. acervulina-infected chickens, while there was no significant improvement in E. maxima-infected chickens. The anticoccidial activities of TP and TPE on E. acervulina, E. tenella and E. maxima were further supported by anticoccidial index scores, which showed greater efficacy than those of amprolium, a commercial coccidiostat used in poultry. TP supplementation positively impacted the primary metabolism of chickens challenged with E. tenella or E. acervulina. The chemical fingerprints of TPE were established using liquid column chromatography; TPE contained 4 major compounds: ononin, sissotrin, formononetin, and biochanin A. In addition, various spectrometric methods were used to ensure the batch-to-batch consistency of TP/TPE. In conclusion, T. pratense is demonstrated to be a novel phytogenic supplement that can be used to control Eimeria-induced coccidiosis in chickens.
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Marek's disease (MD) is an important neoplastic disease caused by serotype 1 Marek's disease virus (MDV-1), which results in severe economic losses worldwide. Despite vaccination practices that have controlled the MD epidemic, current increasing MD-suspected cases indicate the persistent viral infections circulating among vaccinated chicken farms in many countries. However, the lack of available information about phylogeny and molecular characterization of circulating MDV-1 field strains in Taiwan reveals a potential risk in MD outbreaks. This study investigated the genetic characteristics of 18 MDV-1 strains obtained from 17 vaccinated chicken flocks in Taiwan between 2018 and 2020. Based on the sequences of the meq oncogene, the phylogenetic analysis demonstrated that the circulating Taiwanese MDV-1 field strains were predominantly in a single cluster that showed high similarity with strains from countries of the East Asian region. Because the strains were obtained from CVI988/Rispens vaccinated chicken flocks and the molecular characteristics of the Meq oncoprotein showed features like vvMDV and vv+MDV strains, the circulating Taiwanese MDV-1 field strains may have higher virulence compared with vvMDV pathotype. In conclusion, the data presented demonstrates the circulation of hypervirulent MDV-1 strains in Taiwan and highlights the importance of routine surveillance and precaution strategies in response to the emergence of enhanced virulent MDV-1.
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Pollos , Herpesvirus Gallináceo 2 , Enfermedad de Marek , Proteínas Oncogénicas Virales , Animales , Pollos/virología , Herpesvirus Gallináceo 2/clasificación , Herpesvirus Gallináceo 2/genética , Herpesvirus Gallináceo 2/patogenicidad , Enfermedad de Marek/virología , Enfermedad de Marek/prevención & control , Vacunas contra la Enfermedad de Marek/genética , Vacunas contra la Enfermedad de Marek/inmunología , Proteínas Oncogénicas Virales/genética , Filogenia , Enfermedades de las Aves de Corral/virología , Enfermedades de las Aves de Corral/epidemiología , Enfermedades de las Aves de Corral/prevención & control , Taiwán/epidemiología , Vacunación/veterinaria , Virulencia/genéticaRESUMEN
Quantifying hemoglobin is vital yet invasive through blood draws. We developed a wearable diffuse reflectance spectroscopy device comprising control and sensor boards with photodiodes and light-emitting diodes to noninvasively determine hemoglobin. Neural networks enabled recovery of optical parameters for chromophore fitting to calculate hemoglobin. Testing healthy and elderly subjects revealed strong correlation (r=0.9) between our system and invasive methods after data conversion. Bland-Altman analysis demonstrated tight 95% limits of agreement from -1.98 to 1.98â g/dL between the DRS and invasive hemoglobin concentrations. By spectroscopically isolating hemoglobin absorption, interference from melanin was overcome. Our device has the potential for future integration into wearable technology, enabling hemoglobin level tracking.
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Chicken infectious anemia caused by chicken anemia virus (CAV) is a very important immunosuppressive disease in chickens. The horizontal spread of CAV in field chickens has been confirmed mainly through oral infection in our published article. Anemia is the main symptom of this disease. Studies by other scientists have shown that infection of CAV in 1-day-old chicks can cause anemia, and the degree of anemia is directly proportional to the dose of infectious virus. However, the pathogenesis of oral inoculation of CAV in older chickens is still not well understood. The purpose of this study was to determine whether 3-weeks-old specific-pathogen-free (SPF) chickens infected with different viral doses in oral route would cause anemia, as well as other signs associated with age-resistance. The experimental design was divided into a high-dose inoculated group (106 1050), low-dose inoculated group (103 TCID50), and non-virus inoculated control group, and 12 birds in each group at the beginning of the trial. The packed cell volumes (PCVs), CAV genome copies in tissues, CAV titer in peripheral blood fractions, and serology were evaluated at 7, 14, and 21 days post-infection (dpi). Virus replication and spread were estimated using quantitative polymerase chain reaction (qPCR) and viral titration in cell culture, respectively. The results showed that the average PCVs value of the high-dose inoculated group was significantly lower than that of the control group at 14 dpi (p < 0.05), and 44.4% (4/9) of the chickens reached the anemia level (PCVs < 27%). At 21 dpi, the average PCV value rebounded but remained lower than the control group without significant differences. In the low-dose inoculated group, all birds did not reach anemia during the entire trial period. Peripheral blood analysis showed that the virus titer in all erythrocyte, granulocyte and mononuclear cell reached the peak at 14 dpi regardless of the high-dose or low-dose inoculated group, and the highest virus titer appeared in the high-dose inoculated group of mononuclear cell. In the low-dose inoculated group, CAV was detected only at 14 dpi in erythrocyte. Taken together, our results indicate that the older birds require a higher dose of infectious CAV to cause anemia after about 14 days of infection, which is related to apoptosis caused by viral infection of erythrocytes. In both inoculated groups, the viral genome copies did not increase in the bone marrow, which indicated that minimal cell susceptibility to CAV was found in older chickens. In the low-dose inoculated group, only mononuclear cells can still be detected with CAV at 21 dpi in seropositive chickens, indicating that the mononuclear cell is the target cell for persistent infection. Therefore, complete elimination of the CAV may still require the aid of a cell-mediated immune response (CMI), although it has previously been reported to be inhibited by CAV infection. Prevention of early exposure to CAV could be possible by improved hygiene procedures.
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BACKGROUND: VP1 of the chicken anemia virus (CAV) is a structural protein that is required for virus encapsulation. VP1 proteins are present both in the nucleus and cytoplasm; however, the functional nuclear localization signal (NLS) and nuclear export signal (NES) of VP1 are still unknown. This study aimed to characterize the NLS and NES motifs of VP1 using bioinformatics methods and multiple-site fragment deletions, and investigate shuttling of VP2 from nucleus to cytoplasm by co-transfection with VP1. METHODS: Two putative NLS motifs were predicted by the WoLF PSORT and NLStradamus programs from the amino acid sequence of VP1. Three NES motifs of VP1 were predicted by the NetNES 1.1 Server and ELM server programs. All mutants were created by multiple-site fragment deletion mutagenesis. VP1 and VP2 were co-expressed in cells using plasmid transfection. RESULTS: A functional NLS motif was identified at amino acid residues 3 to 10 (RRARRPRG) of VP1. Critical amino acids 3 to 10 were significantly involved in nuclear import in cells and were evaluated using systematic deletion mutagenesis. Three NES motifs of VP1 were predicted by the NetNES 1.1 Server and ELM server programs. A functional NES was identified at amino acid residues 375 to 388 (ELDTNFFTLYVAQ). Leptomycin B (LMB) treatment demonstrated that VP1 export from nucleus to cytoplasm occurred through a chromosome region maintenance 1 (CRM1)-dependent pathway. With co-expression of VP1 and VP2 in cells, we observed that VP1 may transport VP2 from nucleus to cytoplasm. CONCLUSION: Our data showed that VP1 of CAV contained functional NLS and NES motifs that modulated nuclear import and export through a CRM1-dependent pathway. Further, VP1 may play a role in the transport of VP2 from nucleus to cytoplasm.
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Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Virus de la Anemia del Pollo/genética , Señales de Exportación Nuclear , Señales de Localización Nuclear/genética , Transporte Activo de Núcleo Celular , Secuencias de Aminoácidos , Animales , Células CHO , Núcleo Celular/metabolismo , Virus de la Anemia del Pollo/efectos de los fármacos , Biología Computacional , Cricetulus , Citoplasma/metabolismo , Ácidos Grasos Insaturados/farmacología , Carioferinas/metabolismo , Mutagénesis , Señales de Localización Nuclear/química , Unión Proteica , Transporte de Proteínas , Receptores Citoplasmáticos y Nucleares/metabolismo , Transfección , Proteína Exportina 1RESUMEN
BACKGROUND: Chicken anaemia virus (CAV) is commonly found in poultry. VP1 is the sole structural protein of CAV, which is the major component responsible for capsid assembly. The CAV virion consists of the VP1 protein and a viral genome. However, there is currently no information on the protein-nucleic acid interactions between VP1 and DNA molecules. RESULTS: In this study, the recombinant VP1 protein of CAV was expressed and purified to characterize its DNA binding activity. When VP1 protein was incubated with a DNA molecule, the DNA molecule exhibited retarded migration on an agarose gel. Regardless of whether the sequence of the viral genome was involved in the DNA molecule, DNA retardation was not significantly influenced. This outcome indicated VP1 is a DNA binding protein with no sequence specificity. Various DNA molecules with different conformations, such as circular dsDNA, linear dsDNA, linear ssDNA and circular ssDNA, interacted with VP1 proteins according to the results of a DNA retardation assay. Further quantification of the amount of VP1 protein required for DNA binding, the circular ssDNA demonstrated a high affinity for the VP1 protein. The preferences arranged in the order of affinity for the VP1 protein with DNA are circular ssDNA, linear ssDNA, supercoiled circular dsDNA, open circular DNA and linear dsDNA. CONCLUSIONS: The results of this study demonstrated that the interaction between VP1 and DNA molecules exhibited various binding preferences that were dependent on the structural conformation of DNA. Taken together, the results of this report are the first to demonstrate that VP1 has no sequence-specific DNA binding activity. The particular binding preferences of VP1 might play multiple roles in DNA replication or encapsidation during the viral life cycle.
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Proteínas de la Cápside/metabolismo , Virus de la Anemia del Pollo/metabolismo , ADN Viral/metabolismo , Proteínas de Unión al ADN/metabolismo , Virus de la Anemia del Pollo/genética , Proteínas de Unión al ADN/genética , Unión Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Chicken anaemia virus (CAV) is an important contagious agent that causes immunosuppressive disease in chickens. CAV Apoptin is a nucleoplasmic shuffling protein that induces apoptosis in chicken lymphoblastoid cells. In the present study, confocal microscopy revealed co-localisation of expressed CAV non-structural protein VP2 with Apoptin in the nucleus of MDCC-MSB1 cells and the nucleoplasmic compartment of CHO-K1 cells. In vitro pull-down and ex vivo biomolecular fluorescent complementation (BiFC) assays further showed that the VP2 protein directly interacts with Apoptin. Transient co-expression of VP2 and Apoptin in MDCC-MSB1 cells significantly decreased the rate of apoptosis compared with that in cells transfected with the Apoptin gene alone. In addition, the phosphorylation status of threonine 108 (Thr108) of Apoptin was found to decrease upon interaction with VP2. Although dephosphorylated Thr108 did not alter the subcellular distribution of Apoptin in the nucleus of MDCC-MSB1 cells, it did suppress apoptosis. These findings provide the first evidence that VP2 directly interacts with Apoptin in the nucleus to down-regulate apoptosis through alterations in the phosphorylation status of the latter. This information will be useful to further elucidate the underlying mechanism of viral replication in the CAV life cycle.
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Apoptosis , Proteínas de la Cápside/metabolismo , Virus de la Anemia del Pollo/fisiología , Regulación hacia Abajo , Regulación Viral de la Expresión Génica , Replicación Viral , Animales , Células CHO , Proteínas de la Cápside/genética , Pollos , Cricetulus , Fosforilación , TreoninaRESUMEN
In this study, loop-mediated isothermal amplification (LAMP) and real-time LAMP assays were developed to detect the dioxin-degrading bacterium Ochrobactrum anthropi strain BD-1 in soil. Four primers were designed to use ITS gene amplification for the strain O. anthropi BD-1. The real-time LAMP assay was found to accomplish the reaction by 1 pg of genomic DNA load when used for nucleic acid amplification. This assay was then applied to detect O. anthropi BD-1 in eight soil samples collected from a dioxin-contaminated site. The results demonstrated that these newly developed LAMP and real-time LAMP assays will not only be useful and efficient tools for detecting the target gene, but also be used as molecular tools for monitoring the growth of dioxin-degrading O. anthropi in the soil. This is the first report to demonstrate the use of LAMP assays to monitor the presence of O. anthropi in dioxin-contaminated soil. The application of this method should improve the biomonitoring of dioxin contamination.
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Dioxinas/química , Técnicas de Amplificación de Ácido Nucleico/métodos , Ochrobactrum anthropi/genética , Microbiología del Suelo , Cartilla de ADN , ADN Bacteriano/análisis , Humanos , Reproducibilidad de los ResultadosRESUMEN
Taraxacum formosanum (TF) is a medicinal plant used as an important component of health drinks in Taiwan. In this study, a rapid, sensitive and specific loop-mediated isothermal amplification (LAMP) assay for authenticating TF was established. A set of four specific LAMP primers was designed based on the nucleotide sequence of the internal transcribed spacer 2 (ITS2) nuclear ribosomal DNA (nrDNA) of TF. LAMP amplicons were successfully amplified and detected when purified genomic DNA of TF was added in the LAMP reaction under isothermal condition (65 °C) within 45 min. These specific LAMP primers have high specificity and can accurately discriminate Taraxacum formosanum from other adulterant plants; 1 pg of genomic DNA was determined to be the detection limit of the LAMP assay. In conclusion, using this novel approach, TF and its misused plant samples obtained from herbal tea markets were easily identified and discriminated by LAMP assay for quality control.
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Genes de Plantas , Taraxacum/metabolismo , Secuencia de Bases , Cartilla de ADN/metabolismo , ADN Ribosómico/genética , ADN Ribosómico/metabolismo , Análisis Discriminante , Datos de Secuencia Molecular , Técnicas de Amplificación de Ácido Nucleico , Alineación de Secuencia , TemperaturaRESUMEN
BACKGROUND: Pigeon circovirus (PiCV) is considered to be a viral agent central to the development of young pigeon disease syndrome (YPDS). The Cap protein, a structural protein encoded by the cap (or C1) gene of PiCV, has been shown to be responsible for not only capsid assembly, but also has been used as antigen for detecting antibody when the host is infected with PiCV. The antigenic characteristics of the Cap protein potentially may allow the development of a detection kit that could be applied to control PiCV infection. However, poor expression and poor protein solubility have hampered the production of recombinant Cap protein in the bacteria. This study was undertaken to develop the optimal expression of recombinant full-length Cap protein of PiCV using an E. coli expression system. RESULTS: The PiCV cap gene was cloned and fused with different fusion partners including a His-tag, a GST-tag (glutathioine-S-transferase tag) and a Trx-His-tag (thioredoxin-His tag). The resulting constructs were then expressed after transformation into a number of different E. coli strains; these then had their protein expression evaluated. The expression of the recombinant Cap protein in E. coli was significantly increased when Cap protein was fused with either a GST-tag or a Trx-His tag rather than a His-tag. After various rare amino acid codons presented in the Cap protein were optimized to give the sequence rCapopt, the expression level of the GST-rCapopt in E. coli BL21(DE3) was further increased to a significant degree. The highest protein expression level of GST-rCapopt obtained was 394.27 ± 26.1 mg/L per liter using the E. coli strain BL21(DE3)-pLysS. Moreover, approximately 74.5% of the expressed GST-rCapopt was in soluble form, which is higher than the soluble Trx-His-rCapopt expressed using the BL21(DE3)-pLysS strain. After purification using a GST affinity column combined with ion-exchange chromatography, the purified recombinant GST-rCapopt protein was found to have good antigenic activity when tested against PiCV-infected pigeon sera. CONCLUSIONS: These findings shows that the E. coli-expressed full-length PiCV Cap protein has great potential in terms of large-scaled production and this should allow in the future the development of a serodiagnostic kit that is able to clinically detect PiCV infection in pigeons.
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Proteínas de la Cápside/metabolismo , Circovirus/clasificación , Escherichia coli/fisiología , Regulación Viral de la Expresión Génica/fisiología , Replicación Viral/fisiología , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas de la Cápside/genética , Cromatografía , Circovirus/fisiología , Clonación Molecular , Datos de Secuencia Molecular , Proteínas RecombinantesRESUMEN
BACKGROUND: Chicken anemia virus (CAV) is an important viral pathogen that causes anemia and severe immunodeficiency syndrome in chickens worldwide. Generally, CAV infection occurs via vertical transmission in young chicks that are less than two weeks old, which are very susceptible to the disease. Therefore, epidemiological investigations of CAV infection and/or the evaluation of the immunization status of chickens is necessary for disease control. Up to the present, systematically assessing viral protein antigenicity and/or determining the immunorelevant domain(s) of viral proteins during serological testing for CAV infection has never been performed. The expression, production and antigenic characterization of CAV viral proteins such as VP1, VP2 and VP3, and their use in the development of diagnostic kit would be useful for CAV infection prevention. RESULTS: Three CAV viral proteins VP1, VP2 and VP3 was separately cloned and expressed in recombinant E. coli. The purified recombinant CAV VP1, VP2 and VP3 proteins were then used as antigens in order to evaluate their reactivity against chicken sera using indirect ELISA. The results indicated that VP2 and VP3 show good immunoreactivity with CAV-positive chicken sera, whereas VP1 was found to show less immunoreactivity than VP2 and VP3. To carry out the further antigenic characterization of the immunorelevant domains of the VP2 and VP3 proteins, five recombinant VP2 subunit proteins (VP2-435N, VP2-396N, VP2-345N, VP2-171C and VP2-318C) and three recombinant VP3 subunit proteins (VP3-123N, VP3-246M, VP3-366C), spanning the defined regions of VP2 and VP3 were separately produced by an E. coli expression system. These peptides were then used as antigens in indirect ELISAs against chicken sera. The results of these ELISAs using truncated recombinant VP2 and VP3 subunit proteins as coating antigen showed that VP2-345N, VP2-396N and VP3-246M gave good immunoreactivity with CAV-positive chicken sera compared to the other subunit proteins. Moreover, the VP2-396N and VP2-345 based ELISAs had better sensitivity (97.5%) and excellent specificity (100%) during serodiagnosis testing using a mean plus three standard deviations cut-off. The VP3-246M based ELISA showed a sensitivity of 85% and a specificity of 100% at the same cut-off value. CONCLUSIONS: This is the first report to systematically assess the antigenic characteristics of CAV viral proteins for sero-diagnosis purposes. Purified recombinant VP2-396N and VP2-345N subunit proteins, which span defined regions of VP2, were demonstrated to have good antigenicity and higher sensitivities than VP3-246M and were able to recognize CAV-positive chicken serum using an ELISA assay. The defined antigenicity potential of these chimeric subunit proteins produced by expression in E. coli seem to have potential and could be useful in the future for the development of the CAV diagnostic tests based on a subunit protein ELISA system.
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Proteínas de la Cápside/inmunología , Virus de la Anemia del Pollo/inmunología , Enfermedades de las Aves de Corral/inmunología , Animales , Antígenos Virales/biosíntesis , Antígenos Virales/inmunología , Proteínas de la Cápside/biosíntesis , Virus de la Anemia del Pollo/metabolismo , Pollos/virología , Infecciones por Circoviridae/diagnóstico , Infecciones por Circoviridae/inmunología , Infecciones por Circoviridae/veterinaria , Infecciones por Circoviridae/virología , Clonación Molecular , Ensayo de Inmunoadsorción Enzimática/veterinaria , Escherichia coli/metabolismo , Enfermedades de las Aves de Corral/diagnóstico , Enfermedades de las Aves de Corral/metabolismo , Enfermedades de las Aves de Corral/virología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Pruebas Serológicas/métodos , Pruebas Serológicas/veterinariaRESUMEN
BACKGROUND: Apoptin, a nonstructural protein encoded by the VP3 gene of chicken anemia virus (CAV), has been shown to not only induce apoptosis when introduced into the precursors of chicken thymocytes, but has been found to specifically kill human cancer cells, tumor cell and transformed cells without affecting the proliferation of normal cells. This tumor-specific apoptotic characteristic of the protein potentially may allow the development of a protein drug that has applications in tumor therapy. However, several major problems, which include poor expression and poor protein solubility, have hampered the production of apoptin in bacteria. RESULTS: Significantly increased expression of recombinant full-length apoptin that originated from chicken anemia virus was demonstrated using an E. coli expression system. The CAV VP3 gene was fused with a synthetic sequence containing a trans-acting activator of transcription (TAT) protein transduction domain (PTD). The resulting construct was cloned into various different expression vectors and these were then expressed in various E. coli strains. The expression of the TAT-Apoptin in E. coli was significantly increased when TAT-Apoptin was fused with GST-tag rather than a His-tag. When the various rare amino acid codons of apoptin were optimized, the expression level of the GST-TAT-Apoptin(opt) in E. coli BL21(DE3) was significantly further increased. The highest protein expression level obtained was 8.33 g/L per liter of bacterial culture after induction with 0.1 mM IPTG for 4 h at 25 °C. Moreover, approximately 90% of the expressed GST-TAT-Apoptin(opt) under these conditions was soluble. After purification by GST affinity chromatography, the purified recombinant TAT-Apoptin(opt) protein was used to evaluate the recombinant protein's apoptotic activity on tumor cells. The results demonstrated that the E. coli-expressed GST-TAT-apoptin(opt) showed apoptotic activity and was able to induce human premyelocytic leukemia HL-60 cells to enter apoptosis. CONCLUSIONS: On expression in E. coli, purified recombinant TAT-Apoptin(opt) that has been fused to a GST tag and had its codons optimized, was found to have great potential. This protein may in the future allow the development of a therapeutic protein that is able to specifically kill tumor cells.
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Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Virus de la Anemia del Pollo/genética , Neoplasias/tratamiento farmacológico , Ingeniería de Proteínas , Apoptosis/efectos de los fármacos , Secuencia de Bases , Proteínas de la Cápside/aislamiento & purificación , Proteínas de la Cápside/uso terapéutico , Línea Celular Tumoral , Virus de la Anemia del Pollo/metabolismo , Codón , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Humanos , Datos de Secuencia Molecular , Neoplasias/fisiopatología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/uso terapéuticoRESUMEN
Chalcones have been described to represent cancer chemopreventive food components that are rich in fruits and vegetables. In this study, we examined the anti-oral cancer effect of flavokawain B (FKB), a naturally occurring chalcone isolated from Alpinia pricei (shell gingers), and revealed its molecular mechanism of action. Treatment of human oral carcinoma (HSC-3) cells with FKB (1.25-10 µg/mL; 4.4-35.2 µM) inhibited cell viability and caused G(2)/M arrest through reductions in cyclin A/B1, Cdc2, and Cdc25C levels. Moreover, FKB treatment resulted in the induction of apoptosis, which was associated with DNA fragmentation, mitochondria dysfunction, cytochrome c and AIF release, caspase-3 and caspase-9 activation, and Bcl-2/Bax dysregulation. Furthermore, increased Fas activity and procaspase-8, procaspase-4, and procaspase-12 cleavages were accompanied by death receptor and ER-stress, indicating the involvement of mitochondria, death-receptor, and ER-stress signaling pathways. FKB induces apoptosis through ROS generation as evidenced by the upregulation of oxidative-stress markers HO-1/Nrf2. This mechanism was further confirmed by the finding that the antioxidant N-acetylcysteine (NAC) significantly blocked ROS generation and consequently inhibited FKB-induced apoptosis. Moreover, FKB downregulated the phosphorylation of Akt and p38 MAPK, while their inhibitors LY294002 and SB203580, respectively, induced G(2)/M arrest and apoptosis. The profound reduction in cell number was observed in combination treatment with FKB and Akt/p38 MAPK inhibitors, indicating that the disruption of Akt and p38 MAPK cascades plays a functional role in FKB-induced G(2)/M arrest and apoptosis in HSC-3 cells.
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Apoptosis/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Flavonoides/farmacología , Sistema de Señalización de MAP Quinasas/fisiología , Neoplasias de la Boca/patología , Especies Reactivas de Oxígeno/metabolismo , Anticarcinógenos , Línea Celular Tumoral , Chalconas/farmacología , Regulación hacia Abajo , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Neoplasias de la Boca/prevención & controlRESUMEN
A fast, sensitive, and specific reverse-transcription (RT) loop-mediated isothermal amplification (RT-LAMP) assay was developed that involved a single tube and a 1-step reaction for detecting infectious bursal disease virus (IBDV). Four specific primers were used for amplification of the VP2 gene of IBDV. The amplified LAMP products were detected by DNA electrophoresis and by direct observation with the naked eye in the presence of SYBR Green I. The sensitivity of RT-LAMP was determined to be 0.01 fg of IBDV viral RNA. This assay for IBDV is more sensitive than the conventional RT-polymerase chain reaction assay, which has a detection limit of 1 ng. The LAMP assay was also assessed for specificity and was found to precisely discriminate between positive and negative test samples. This newly established LAMP assay, combined with RT, is a practical diagnostic tool because IBDV-infected and uninfected clinical samples collected from an experimental farm could be discriminated. Full verification of a sample's IBDV status was obtained within 40 min of extraction of the viral RNA, which could then be directly added to the RT-LAMP reaction mixture.
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Infecciones por Birnaviridae/veterinaria , Pollos/virología , Virus de la Enfermedad Infecciosa de la Bolsa/aislamiento & purificación , Enfermedades de las Aves de Corral/diagnóstico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria , Virología/métodos , Animales , Infecciones por Birnaviridae/diagnóstico , Infecciones por Birnaviridae/virología , Western Blotting/veterinaria , Virus de la Enfermedad Infecciosa de la Bolsa/genética , Enfermedades de las Aves de Corral/virología , ARN Viral/análisis , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: Chicken anemia virus (CAV), the causative agent chicken anemia, is the only member of the genus Gyrovirus of the Circoviridae family. CAV is an immune suppressive virus and causes anemia, lymph organ atrophy and immunodeficiency. The production and biochemical characterization of VP1 protein and its use in a subunit vaccine or as part of a diagnostic kit would be useful to CAV infection prevention. RESULTS: Significantly increased expression of the recombinant full-length VP1 capsid protein from chicken anemia virus was demonstrated using an E. coli expression system. The VP1 gene was cloned into various different expression vectors and then these were expressed in a number of different E. coli strains. The expression of CAV VP1 in E. coli was significantly increased when VP1 was fused with GST protein rather than a His-tag. By optimizing the various rare amino acid codons within the N-terminus of the VP1 protein, the expression level of the VP1 protein in E. coli BL21(DE3)-pLysS was further increased significantly. The highest protein expression level obtained was 17.5 g/L per liter of bacterial culture after induction with 0.1 mM IPTG for 2 h. After purification by GST affinity chromatography, the purified full-length VP1 protein produced in this way was demonstrated to have good antigenicity and was able to be recognized by CAV-positive chicken serum in an ELISA assay. CONCLUSIONS: Purified recombinant VP1 protein with the gene's codons optimized in the N-terminal region has potential as chimeric protein that, when expressed in E. coli, may be useful in the future for the development of subunit vaccines and diagnostic tests.
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Proteínas de la Cápside/metabolismo , Escherichia coli/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas de la Cápside/genética , Proteínas de la Cápside/inmunología , Pollos/virología , Codón , Escherichia coli/crecimiento & desarrollo , Glutatión Transferasa/biosíntesis , Glutatión Transferasa/genética , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Vacunas Sintéticas/biosíntesis , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Vacunas Virales/biosíntesis , Vacunas Virales/genética , Vacunas Virales/inmunologíaRESUMEN
Cymbidium mosaic virus (CymMV) is the most prevalent orchid virus. A single-tube one-step betaine-free reverse transcription (RT) loop-mediated isothermal amplification (LAMP) assay was developed for the rapid and easy detection of orchid-infecting CymMV. Five sets of primers were designed based on the conserved regions among various virus isolates. The specificity and the sensitivity of the assay were then evaluated using the RT-LAMP reaction. Within 1h under isothermal conditions at 60°C the target viral gene was amplified successfully. This RT-LAMP assay was found to be quick, specific, sensitive and easy to perform assay that involved only one step and was simpler to carry out than alternative approaches. Thus this assay is an alternative for the rapid and easy detection of CymMV in orchids. This is first time that a RT-LAMP method for the detection of an orchid virus has been described.
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Técnicas de Amplificación de Ácido Nucleico/métodos , Orchidaceae/virología , Enfermedades de las Plantas/virología , Potexvirus/aislamiento & purificación , Virología/métodos , Secuencia Conservada , Cartilla de ADN/genética , ARN Viral/genética , Sensibilidad y Especificidad , TemperaturaRESUMEN
UNLABELLED: Intrahepatic cholangiocarcinoma typically presents in an advanced stage in which treatment options are limited. In an effort to recapitulate key biological and clinical features of the progressive disease, we established a novel rat model based on bile duct inoculation of rat cholangiocyte cell lines in different stages of tumor progression. Our BDEneu cell line, which is highly tumorigenic, originated from an immortalized rat cholangiocyte cell line (BDE1 cells) that was stably transfected to constitutively overexpress mutationally activated rat neu oncogene. Our less aggressive tumorigenic BDEsp cholangiocyte cell line was derived from the spontaneous in vitro neoplastic transformation of the same parent BDE1 cell line. Unlike BDEneu cells, BDEsp cells expressed wild-type c-neu and exhibited in vitro growth rates intermediate between those of BDEneu and BDE1 cholangiocytes. Cyclooxygenase-2 and activated Akt were significantly overexpressed in BDEsp cells over those of BDE1 cells, and at higher levels than those expressed in BDEneu cells. Only BDEneu cells overexpressed activated p185(neu), which was associated with a significant increase in phospho-p44/42 mitogen-activated protein kinase (MAPK). Mucin 1 (MUC1) messenger RNA (mRNA), an indicator of cholangiocarcinoma cell progression, was also significantly overexpressed in BDEneu cells over that of BDEsp cells. BDEneu cells inoculated into the bile duct of isogenic rats resulted over a 21- to 26-day period in rapid exponential cholangiocarcinoma tumor growth within liver, paralleled by increases in bile duct obstruction and gross peritoneal metastases. Under comparable conditions, BDEsp cells yielded only small nonmetastatic intrahepatic cholangiocarcinomas without bile duct obstruction. CONCLUSIONS: A novel model of cholangiocarcinoma progression mimicking progressive development of the advanced human disease has been established, which may serve as a powerful preclinical platform to study cholangiocarcinoma progression and for rapidly testing treatment approaches.
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Línea Celular/metabolismo , Colangiocarcinoma/patología , Colestasis Intrahepática/patología , Modelos Animales de Enfermedad , Neoplasias Hepáticas/patología , Hígado/patología , Ratas , Animales , Pruebas de Carcinogenicidad , Proliferación Celular , Colangiocarcinoma/complicaciones , Colestasis Intrahepática/etiología , Ciclooxigenasa 2/metabolismo , Progresión de la Enfermedad , Glicoproteínas/metabolismo , Humanos , Hígado/metabolismo , Neoplasias Hepáticas/complicaciones , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Mucina-1/metabolismo , Invasividad Neoplásica , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor ErbB-2 , Factores de TiempoRESUMEN
BACKGROUND & AIMS: Cholangiocarcinomas appear to arise from the malignant transformation of cholangiocytes lining the biliary tract. Because the development of an in vitro model of malignant transformation can provide a powerful new tool for establishing critical events governing the molecular pathogenesis of cholangiocarcinoma, we investigated the potential of achieving malignant transformation of cultured rat cholangiocytes in relation to aberrant overexpression of mutationally activated erbB-2/neu. METHODS: Malignant neoplastic transformation was achieved after infection of the rat cholangiocyte cell line, designated BDE1, with the retrovirus Glu664-neu, containing the transforming rat erbB-2/neu oncogene. RESULTS: Compared with untransformed control cells, malignant transformants carrying the activating erbB-2/neu mutation prominently overexpressed p185neu receptor protein, which was phosphorylated strongly at its major autophosphorylation site at tyrosine 1248. Moreover, erbB-2/neu transformation of BDE1 cells resulted in increased telomerase activity, up-regulation of cyclooxygenase-2 with overproduction of prostaglandin E(2), enhanced phosphorylation of mitogen-activated protein kinase and of serine/threonine kinase Akt/PKB, overexpression of vascular endothelial growth factor, and increased mucin 1 messenger RNA expression. Only erbB-2/neu transformants were tumorigenic when transplanted into isogeneic rats, yielding a 100% incidence of tumors closely resembling human desmoplastic ductal cholangiocarcinomas in their morphology. Malignant cholangiocytes in the tumors were strongly immunoreactive for biliary cytokeratin 19, p185neu, and cyclooxygenase-2. CONCLUSIONS: This unique malignant transformation model recapitulates key molecular features of the human disease and appears to be well suited for testing novel molecular therapeutic strategies against cholangiocarcinoma.
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Neoplasias de los Conductos Biliares , Conductos Biliares/citología , Transformación Celular Neoplásica , Receptor ErbB-2/metabolismo , Animales , Neoplasias de los Conductos Biliares/metabolismo , Neoplasias de los Conductos Biliares/patología , Células Cultivadas , Modelos Animales de Enfermedad , Humanos , Hígado/patología , Ratas , Ratas Endogámicas F344 , Receptor ErbB-2/genética , Análisis de Secuencia de ADN , Telomerasa/metabolismoRESUMEN
Recently, we demonstrated that the cyclooxygenase-2 (COX-2) inhibitor celecoxib acts to significantly suppress the growth of rat C611B cholangiocarcinoma (ChC) cells in vitro. To establish a molecular mechanism for this growth suppression, we investigated the effects of celecoxib on apoptotic signaling pathways in cultured rat C611B ChC cells. Celecoxib and another COX-2 inhibitor, rofecoxib, at 5 microM were almost equally effective in inhibiting prostaglandin E(2) (PGE(2)) production by these cells, but at this low concentration, neither inhibitor suppressed growth or induced apoptosis. Celecoxib at 50 microM induced prominent apoptosis in these cells, whereas rofecoxib at 50 microM was without effect in either suppressing growth or inducing apoptosis. Celecoxib (50 microM) did not alter Bcl-2, Bcl-x(L), or COX-2 protein levels, nor did it inhibit p42/44 mitogen-activated protein kinase (MAPK) phosphorylation; however, it significantly suppressed serine/threonine kinase Akt/PKB (Akt) phosphorylation and kinase activity in cultured C611B cells. This effect, in turn, directly correlated with Bax translocation to mitochondria, cytochrome c release into cytosol, activation of caspase-9 and caspase-3, and cleavage of poly (ADP-ribose) polymerase (PARP). Addition of 25 microM PGE(2) to C611B cell cultures blocked the apoptotic actions of celecoxib. Rofecoxib (50 microM) was without effect in suppressing Akt phosphorylation and caspase-3 activation. In vivo, celecoxib partially suppressed tumorigenic growth of C611B ChC cells. In conclusion, our results indicate that celecoxib preferentially acts in vitro to induce apoptosis in ChC cells through a mechanism involving Akt inactivation, Bax translocation, and cytochrome c release. Our in vivo results further suggest celecoxib might have potential therapeutic or chemopreventive value against ChC.