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1.
Biochem Biophys Res Commun ; 728: 150340, 2024 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-38968770

RESUMO

BACKGROUND: Colorectal cancer (CRC) is a prevalent malignancy and poses a significant clinical challenge. Piperine, an alkaloid molecule extracted from Piper nigrum and Piper longum, has emerged as a promising anticancer agent. However, the molecular mechanisms of piperine' antitumor effects in CRC need to be further elucidated. METHODS: Human colorectal cancer cells were treated with piperine in vitro. CCK-8 and clone formation assays were adopted to detect cell viability. The accumulation of autophagosomes was assessed by Western blotting and immunofluorescence. Apoptosis and reactive oxygen species (ROS) levels were analyzed by flow. In vivo, a xenograft tumor mouse model was constructed using CT26 cells. RESULTS: Piperine inhibited CRC cell viability and suppressed tumor weight and volume in a mouse model. Additionally, piperine treatment induced the accumulation of autophagosomes in CRC cells. This effect was attributed to the inhibition of the AKT/mTOR pathway and the accumulation of ROS. activation of AKT or clearance of ROS attenuated piperine-mediated tumor suppression. CONCLUSION: This study shows that piperine induces autophagy-dependent cell death in CRC cells by increasing ROS production and inhibiting Akt/mTOR signaling.

2.
Chemistry ; 30(30): e202401010, 2024 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-38517333

RESUMO

Electrocatalytic nitrogen reduction reaction (NRR) has attracted much attention as a sustainable ammonia production technology, but it needs further exploration due to its slow kinetics and the existence of competitive side reactions. In this research, xAu/MIL-101(Fe) catalysts were obtained by loading gold nanoparticles (Au NPs) onto MIL-101(Fe) using a one-step reduction strategy. Herein, MIL-101(Fe), with high specific surface area and strong N2 adsorption capacity, is used as a support to disperse Au NPs to increase the electrochemical active surface area. Au NPs, with a high NRR activity, is introduced as the active site to promote charge transfer and intermediate formation rates. More importantly, the strong interaction between Au NPs and MIL-101(Fe) enhances the electron transfer between Au NPs and MIL-101(Fe), thereby enhancing the activation of N2 and achieving efficient NRR. Among the prepared catalysts, 15 %Au/MIL-101(Fe) has the highest NH3 yield of 46.37 µg h-1 mg-1 cat and a Faraday efficiency of 39.38 % at -0.4 V (vs. RHE). In-situ FTIR reveals that the NRR mechanism of 15 %Au/MIL-101(Fe) follows the binding alternating pathway and also indicates that the interaction between Au NPs and MIL-101(Fe) strengthens the activation of the N≡N bond in the rate-limiting process, thereby accelerating the NRR process.

3.
Bioorg Chem ; 142: 106958, 2024 01.
Artigo em Inglês | MEDLINE | ID: mdl-37979322

RESUMO

UPLC-TOF-MS/MDF directed phytochemical research of Chloranthus japonicus led to the isolation of 46 lindenane sesquiterpenoid dimers, which included 13 new analogs. Their structures with absolute configurations were elucidated by analysis of spectroscopic data. Fourteen compounds with ester chains significantly decreased PCSK9 protein level in medium of HepG2 cells, especially for compounds 14 and 29 (5 µM) with inhibition rates of 69.0% and 72.8%, respectively. Compound 14 in HepG2 cells was evaluated via DiI-LDL uptake assays and found to increase LDL uptake by upregulating LDLR mRNA and protein level. Meanwhile, 14 decreased the secretion of PCSK9 protein in medium and downregulated intracellular PCSK9 protein and mRNA level. The discovery of these natural small molecule compounds provides a novel structure basis for design PCSK9 regulators, making them a promising lead for development of new lipid-lowering agents.


Assuntos
Pró-Proteína Convertase 9 , Sesquiterpenos , Humanos , Pró-Proteína Convertase 9/genética , Pró-Proteína Convertase 9/metabolismo , Células Hep G2 , Sesquiterpenos/química , RNA Mensageiro
4.
Nature ; 556(7700): 255-258, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29618817

RESUMO

Cross-species transmission of viruses from wildlife animal reservoirs poses a marked threat to human and animal health 1 . Bats have been recognized as one of the most important reservoirs for emerging viruses and the transmission of a coronavirus that originated in bats to humans via intermediate hosts was responsible for the high-impact emerging zoonosis, severe acute respiratory syndrome (SARS) 2-10 . Here we provide virological, epidemiological, evolutionary and experimental evidence that a novel HKU2-related bat coronavirus, swine acute diarrhoea syndrome coronavirus (SADS-CoV), is the aetiological agent that was responsible for a large-scale outbreak of fatal disease in pigs in China that has caused the death of 24,693 piglets across four farms. Notably, the outbreak began in Guangdong province in the vicinity of the origin of the SARS pandemic. Furthermore, we identified SADS-related CoVs with 96-98% sequence identity in 9.8% (58 out of 591) of anal swabs collected from bats in Guangdong province during 2013-2016, predominantly in horseshoe bats (Rhinolophus spp.) that are known reservoirs of SARS-related CoVs. We found that there were striking similarities between the SADS and SARS outbreaks in geographical, temporal, ecological and aetiological settings. This study highlights the importance of identifying coronavirus diversity and distribution in bats to mitigate future outbreaks that could threaten livestock, public health and economic growth.


Assuntos
Alphacoronavirus/isolamento & purificação , Alphacoronavirus/patogenicidade , Doenças dos Animais/epidemiologia , Doenças dos Animais/virologia , Quirópteros/virologia , Infecções por Coronavirus/veterinária , Diarreia/veterinária , Suínos/virologia , Alphacoronavirus/classificação , Alphacoronavirus/genética , Doenças dos Animais/transmissão , Animais , Biodiversidade , China/epidemiologia , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/transmissão , Diarreia/patologia , Diarreia/virologia , Reservatórios de Doenças/veterinária , Reservatórios de Doenças/virologia , Genoma Viral/genética , Humanos , Jejuno/patologia , Jejuno/virologia , Filogenia , Síndrome Respiratória Aguda Grave/epidemiologia , Síndrome Respiratória Aguda Grave/veterinária , Síndrome Respiratória Aguda Grave/virologia , Análise Espaço-Temporal , Zoonoses/epidemiologia , Zoonoses/transmissão , Zoonoses/virologia
5.
J Nat Prod ; 85(5): 1294-1303, 2022 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-35561431

RESUMO

Seven new hexasaccharide resin glycosides, named calysepins I-VII (1-7), with 27-membered rings, were obtained from the aerial parts of Calystegia sepium. Their structures with absolute configuration were established on the basis of spectroscopic data interpretation analysis and the use of chemical methods. They were defined as hexasaccharides composed of one d-quinovose, four d-glucose, and one l-rhamnose unit, and their sugar moieties were partially acylated by (2S)-methylbutanoic acid in 1-7 and (2R,3R)-nilic acid in 1-5 and 7, which mainly differed at the positions of acylation. Additionally, calysepin IV (4) exhibited cytotoxicity against A549 cells with an IC50 value of 5.2 µM.


Assuntos
Antineoplásicos , Calystegia , Convolvulus , Calystegia/química , Glicosídeos/química , Glicosídeos/farmacologia , Estrutura Molecular , Resinas Vegetais/química
6.
Mol Cell Probes ; 50: 101494, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31863825

RESUMO

Spring viremia of carp virus (SVCV) is a significant pathogenic agent that can cause large-scale outbreaks of spring viremia of carp (SVC) in many types of fish and bring huge economic losses to the aquaculture industry. A simple and convenient detection method is imperative for SVCV diagnosis. In this study, the real-time reverse transcription recombinase polymerase amplification (RT-RPA) assay was developed and validated. Primers and probe targeting the conserved region of M gene were designed and applied to the real-time RT-RPA assay that performed at 39 °C for 20 min. The specificity analysis showed that no cross-reaction with other pathogenic viruses of fish was found, indicating appropriate specificity of the assay. In vitro transcribed RNA standards were used to estimate the sensitivity of the assay and the detection limit was 102copies/reaction. To further evaluate the assay, 65 clinical samples were tested using both real-time RT-RPA assay and real-time RT-PCR method. The same detection results were observed, suggesting the potential application of real-time RT-RPA assay in clinical sample detection. This is the first report on RPA assay for SVCV detection and this new developed assay would be useful in both laboratory and in the field for diagnosis of SVCV.


Assuntos
Carpas/virologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Recombinases/metabolismo , Transcrição Reversa/genética , Rhabdoviridae/genética , Viremia/diagnóstico , Viremia/veterinária , Animais , Primers do DNA/genética , Sensibilidade e Especificidade , Viremia/genética , Viremia/virologia
7.
Arch Virol ; 164(6): 1639-1646, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30982935

RESUMO

Rabbits are widely used as models in biological research, and the pathogen status of rabbits used in studies can directly affect the results of experiments. Serological surveillance is the common monitoring method used in laboratory animals. A rapid, sensitive, and cost-effective high-throughput Luminex xMAP assay could be an attractive alternative to labor-intensive enzyme-linked immunosorbent assay (ELISA) methods. In this study, recombinant proteins from rabbit hemorrhagic disease virus and rabbit rotavirus and whole viral lysates of Sendai virus were used as coating antigens in an xMAP assay for the simultaneous detection of antibodies against these pathogens. The xMAP assay showed high specificity, with no cross-reaction with other pathogens. The coefficient of variation for intra-assay and inter-assay comparisons was less than 3% and 4%, respectively, indicating good repeatability and stability of the assay. The xMAP assay exhibited similar limits of detection for rabbit hemorrhagic virus and Sendai virus and was less sensitive for the detection of rabbit rotavirus when compared with commercial ELISA kits. A total of 52 clinical samples were tested simultaneously using both the xMAP assay and ELISA kits. The results obtained using these two methods were 100% coincident. In summary, the novel xMAP assay offers an alternative choice for rapid and sensitive high-throughput detection of antibodies in rabbit serum and can be used as a daily monitoring tool for laboratory animals.


Assuntos
Anticorpos Antivirais/sangue , Vírus da Doença Hemorrágica de Coelhos/imunologia , Rotavirus/imunologia , Vírus Sendai/imunologia , Animais , Especificidade de Anticorpos , Reações Cruzadas , Imunoensaio/veterinária , Coelhos , Kit de Reagentes para Diagnóstico
8.
BMC Vet Res ; 15(1): 305, 2019 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-31443656

RESUMO

BACKGROUND: Porcine circovirus type 3 (PCV3) is an emerging circovirus species, that has been reported in major pig-raising countries including the United States, China, South Korea, Brazil, Spain, and Poland. RESULTS: A real-time loop-mediated isothermal amplification (LAMP) assay was developed for rapid detection of porcine circovirus 3 (PCV3). The method had a detection limit of 1 × 101 copies/µL with no cross-reactions with classical swine fever virus (CSFV) C strain, foot-and-mouth disease virus (FMDV), porcine circovirus 2 (PCV2) LG vaccine strain, porcine epidemic diarrhoea virus (PEDV), porcine respiratory and reproductive syndrome virus (PRRSV), or pseudorabies virus (PRV). The PCV3 positive detection rate of 203 clinical samples for the real-time LAMP assay was 89.66% (182/203). CONCLUSIONS: The real-time LAMP assay is highly sensitive, and specific for use in epidemiological investigations of PCV3.


Assuntos
Circovirus/genética , Circovirus/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/veterinária , Suínos/virologia , Animais , Técnicas de Amplificação de Ácido Nucleico/métodos , Sensibilidade e Especificidade
9.
Virol J ; 15(1): 146, 2018 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-30241540

RESUMO

BACKGROUND: A serological method to simultaneously detect antibodies against infectious laryngotracheitis virus (ILTV) and infectious bronchitis virus (IBV) is imperative for the differential diagnosis and evaluation of antibodies titers after vaccination. METHOD: The microspheres coated with purified recombinant glycoprotein D (gD) of ILTV or nucleocapsid (N) protein of IBV were incubated with serum samples. The simultaneous quantification of ILTV and IBV antibodies were achieved through the interrogation of microspheres by Luminex 200 detection system. . RESULTS: This xMAP detection demonstrated no nonspecific reactions with avian influenza virus (AIV), avian leukosis virus (ALV), newcastle disease virus (NDV), and Marek's disease virus (MDV). The results also demonstrated that the xMAP assay was four times more sensitive than the enzyme-linked immunosorbent assay (ELISA) for ILTV detection and two times more sensitive for IBV detection. A total of 90 chicken serum samples from a chicken farm were tested by xMAP and ELISA assays. The results showed that the coincidence rates were 84.44 and 100% for ILTV and IBV detection, respectively. CONCLUSION: This study exhibited an opportunity for the differential diagnosis through simultaneous detection of multiplex antibodies in serum and can be used for the multiplex antibodies evaluation after vaccination.


Assuntos
Anticorpos Antivirais/sangue , Infecções por Coronavirus/veterinária , Infecções por Herpesviridae/veterinária , Herpesvirus Galináceo 1/imunologia , Imunoensaio/métodos , Vírus da Bronquite Infecciosa/imunologia , Doenças das Aves Domésticas/diagnóstico , Animais , Galinhas , Infecções por Coronavirus/diagnóstico , Diagnóstico Diferencial , Infecções por Herpesviridae/diagnóstico , Sensibilidade e Especificidade
10.
Mol Cell Probes ; 37: 1-5, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29054443

RESUMO

Acute respiratory tract infections are of paramount importance in the poultry industry. We developed an xTAG bead assay for the simultaneous detection and discrimination of avian influenza virus (AIV), Newcastle disease virus (NDV), infectious bronchitis virus (IBV) and infectious laryngotracheitis virus (ILTV). The assay lacked nonspecific reactions with other common avian viruses and the limit of detection was 6.75 × 102- 3.52 × 103copies/µL. We examined 60 clinical specimens and found 18 positive for respiratory viruses. Our result demonstrated that xTAG-multiplex PCR method is a high-throughput, rapid, specific and sensitive assay for use in epidemiological studies and clinical detection of avian respiratory pathogens.


Assuntos
Herpesvirus Galináceo 1/isolamento & purificação , Vírus da Bronquite Infecciosa/isolamento & purificação , Vírus da Influenza A/isolamento & purificação , Reação em Cadeia da Polimerase Multiplex/métodos , Vírus da Doença de Newcastle/isolamento & purificação , Doenças das Aves Domésticas/diagnóstico , Animais , Infecções por Vírus de DNA/diagnóstico , Infecções por Vírus de DNA/veterinária , Infecções por Vírus de DNA/virologia , Herpesvirus Galináceo 1/genética , Vírus da Bronquite Infecciosa/genética , Vírus da Influenza A/genética , Limite de Detecção , Vírus da Doença de Newcastle/genética , Aves Domésticas , Doenças das Aves Domésticas/virologia , Infecções por Vírus de RNA/diagnóstico , Infecções por Vírus de RNA/veterinária , Infecções por Vírus de RNA/virologia , Sensibilidade e Especificidade
11.
Mol Cell Probes ; 39: 25-32, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29609038

RESUMO

Differentiation of classical strains and highly pathogenic strains of porcine reproductive and respiratory syndrome virus is crucial for effective vaccination programs and epidemiological studies. We used nested PCR and high resolution melting curve analysis with unlabeled probe to distinguish between the classical and the highly pathogenic strains of this virus. Two sets of primers and a 20 bp unlabeled probe were designed from the NSP3 gene. The unlabeled probe included two mutations specific for the classical and highly pathogenic strains of the virus. An additional primer set from the NSP2 gene of the highly pathogenic vaccine strain JXA1-R was used to detect its exclusive single nucleotide polymorphism. We tested 107 clinical samples, 21 clinical samples were positive for PRRSV (consistent with conventional PCR assay), among them four were positive for the classical strain with the remainder 17 for the highly pathogenic strain. Around 10 °C difference between probe melting temperatures showed the high discriminatory power of this method. Among highly pathogenic positive samples, three samples were determined as positive for JXA1-R vaccine-related strain with a 95% genotype confidence percentage. All these genotyping results using the high resolution melting curve assay were confirmed with DNA sequencing. This unlabeled probe method provides an alternative means to differentiate the classical strains from the highly pathogenic porcine reproductive and respiratory syndrome virus strains rapidly and accurately.


Assuntos
Desnaturação de Ácido Nucleico/genética , Reação em Cadeia da Polimerase/métodos , Vírus da Síndrome Respiratória e Reprodutiva Suína/isolamento & purificação , Animais , Primers do DNA/metabolismo , Sondas de DNA/metabolismo , Filogenia , Síndrome Respiratória e Reprodutiva Suína/diagnóstico , Síndrome Respiratória e Reprodutiva Suína/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Análise de Sequência de DNA , Suínos
12.
Mol Cell Probes ; 41: 27-31, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30157451

RESUMO

Theiler's murine encephalomyelitis virus (TMEV) is one of the most common viral pathogens that circulate widely in captive mouse colonies. A molecular biology detection method would be a useful tool to use in an integrated program to monitor and prevent TMEV infection and transmission. Thus, a reverse transcription recombinase polymerase amplification (RT-RPA) assay was developed to detect TMEV infection. The sensitivity of the RT-RPA assay approached 8 copies per reaction, which is equivalent to the sensitivity of RT-qPCR reactions. This assay did not detect RNA extracts from other murine pathogens included in this study or TMEV negative samples. Brain tissues and contaminated biological materials were used to assess the clinical performance of the RT-RPA. The detection results of RT-RPA and RT-qPCR were very similar, except that a contaminated biological material sample which was positive by RT-qPCR, with a CT value of 38, was negative by RT-RPA. In summary, the developed RT-RPA assay offers a rapid, sensitive and specific alternative method for monitoring of TMEV, especially in resource-limited conditions.


Assuntos
Reação em Cadeia da Polimerase em Tempo Real/métodos , Recombinases/metabolismo , Transcrição Reversa/genética , Theilovirus/isolamento & purificação , Animais , Primers do DNA/metabolismo , Sondas de DNA/metabolismo , Camundongos , Sensibilidade e Especificidade , Theilovirus/genética
13.
BMC Vet Res ; 14(1): 399, 2018 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-30547776

RESUMO

BACKGROUND: Murine norovirus (MNV) is recognized as the most prevalent viral pathogen in captive mouse colonies. The rapid detection assay for MNV would be a useful tool for monitoring and preventing MNV infection. A recombinase polymerase amplification (RPA) assay was established in this study to provide a solution for rapid and sensitive detection of MNV. RESULTS: The detection limit of the RT-RPA assay for the detection of MNV was 1 × 102 copies of RNA molecules per reaction. The assay was specific since there was no cross-reaction with other common murine viruses. In addition, the broad reactivity of the RT-RPA assay was validated using the synthesized template carrying seven point mutations among several MNV strains. The MNV RT-RPA assay could detect as few as 1 × 102 copies of the mutant per reaction, suggesting the assay could be broadly reactive against a large diversity of MNV strains. Forty eight clinical samples including 16 gastric tissue specimens, 16 cecal tissue specimens and 16 fecal specimens were tested for the validation of the new developed RT-RPA assay. The detection results of RT-RPA and RT-qPCR for clinical samples were very similar, except that a gastric tissue sample which was positive by RT-qPCR, with a RNA titer of 27 copies, was negative by RT-RPA. CONCLUSIONS: A broadly reactive RT-RPA assay was successfully established for MNV detection.


Assuntos
Infecções por Caliciviridae/veterinária , Norovirus/genética , Doenças dos Roedores/diagnóstico , Animais , Animais de Zoológico , Infecções por Caliciviridae/diagnóstico , Infecções por Caliciviridae/virologia , Camundongos , Técnicas de Amplificação de Ácido Nucleico , Doenças dos Roedores/virologia , Sensibilidade e Especificidade
14.
BMC Vet Res ; 14(1): 347, 2018 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-30442149

RESUMO

BACKGROUND: Chicken anemia virus (CAV), avian reovirus (ARV), infectious bursal disease virus (IBDV), Marek's disease virus (MDV) and reticuloendotheliosis virus (REV) all cause immunosuppressive disease in birds through vertical or horizontal transmission. Mixed infections with these immunosuppressive pathogens lead to atypical clinical signs and obstruct accurate diagnoses and epidemiological investigations. Therefore, it is essential to develop a high-throughput assay for the simultaneous detection of these immunosuppressive viruses with high specificity and sensitivity. The aim of this study was to establish a novel method using a RT-PCR assay combined with fluorescence labeled polystyrene bead microarray (multiplex xTAG assay) to detect single or mixed viral infections. RESULTS: The results showed that the established xTAG assay had no nonspecific reactions with avian influenza virus (AIV), infectious bronchitis virus (IBV), newcastle disease virus (NDV), infectious laryngotracheitis virus (ILTV), Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS). The limit of detection was 1.0 × 103 copies/µL for IBDV and 1.0 × 102copies/µL for the other four viruses. Ninety field samples were tested and the results were confirmed using conventional RT-PCR methods. The detection results of these two methods were 100% consistent. The established multiplex xTAG assay allows a high throughput and simultaneous detection of five chicken immunosuppressive viruses. CONCLUSION: The multiplex xTAG assay has been showed to be an additional tool for molecular epidemiology studies of five chicken immunosuppressive viruses in the poultry industry.


Assuntos
Infecções por Birnaviridae/veterinária , Vírus da Anemia da Galinha , Infecções por Circoviridae/veterinária , Coinfecção/veterinária , Vírus da Doença Infecciosa da Bursa , Mardivirus , Doença de Marek/diagnóstico , Análise em Microsséries/veterinária , Reação em Cadeia da Polimerase Multiplex/veterinária , Orthoreovirus Aviário , Doenças das Aves Domésticas/diagnóstico , Infecções por Reoviridae/veterinária , Vírus da Reticuloendoteliose Aviária , Infecções por Retroviridae/veterinária , Infecções Tumorais por Vírus/veterinária , Animais , Infecções por Birnaviridae/diagnóstico , Infecções por Birnaviridae/virologia , Galinhas/virologia , Infecções por Circoviridae/diagnóstico , Infecções por Circoviridae/virologia , Coinfecção/diagnóstico , Coinfecção/virologia , Doença de Marek/virologia , Análise em Microsséries/métodos , Reação em Cadeia da Polimerase Multiplex/métodos , Doenças das Aves Domésticas/virologia , Infecções por Reoviridae/diagnóstico , Infecções por Reoviridae/virologia , Reprodutibilidade dos Testes , Infecções por Retroviridae/diagnóstico , Infecções por Retroviridae/virologia , Sensibilidade e Especificidade , Infecções Tumorais por Vírus/diagnóstico , Infecções Tumorais por Vírus/virologia
15.
BMC Vet Res ; 14(1): 127, 2018 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-29625588

RESUMO

BACKGROUND: Domestic rabbits especially New Zealand white rabbits play an important role in biological research. The disease surveillance and quality control are essential to guarantee the results of animal experiments performed on rabbits. Rabbit hemorrhagic disease virus, rabbit rotavirus and Sendai virus are the important pathogens that needed to be eliminated. Rapid and sensitive method focus on these three viruses should be established for routine monitoring. The Luminex x-TAG assay based on multiplex PCR and fluorescent microsphere is a fast developing technology applied in high throughput detection. Specific primers modified with oligonucleotide sequence/biotin were used to amplify target fragments. The conjugation between oligonucleotide sequence of the PCR products and the MagPlex-TAG microspheres was specific without any cross-reaction, and the hybridization products could be analyzed using the Luminex 200 analyzer instrument. Recombinant plasmids were constructed to estimate the detection limit of the viruses. Furthermore, 40 clinical samples were used to evaluate the efficiency of this multiplex PCR based Luminex x-TAG assay. RESULTS: According to the results, this new method showed high specificity and good stability. Assessed by the recombinant plasmids, the detection limit of three viruses was 100copies/µl. Among 40 clinical specimens, 18 specimens were found positive, which was completely concordant with the conventional PCR method. CONCLUSIONS: The new developed Luminex x-TAG assay is an accurate, high throughput method for rapid detection of three important viruses of rabbits.


Assuntos
Infecções por Caliciviridae/veterinária , Reação em Cadeia da Polimerase Multiplex/veterinária , Coelhos/virologia , Infecções por Respirovirus/veterinária , Infecções por Rotavirus/veterinária , Animais , Animais de Laboratório/virologia , Infecções por Caliciviridae/diagnóstico , Reações Cruzadas , Fluorescência , Vírus da Doença Hemorrágica de Coelhos , Microesferas , Reação em Cadeia da Polimerase Multiplex/métodos , Infecções por Respirovirus/diagnóstico , Rotavirus , Infecções por Rotavirus/diagnóstico , Vírus Sendai , Sensibilidade e Especificidade
16.
J Nanosci Nanotechnol ; 18(3): 1811-1817, 2018 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-29448664

RESUMO

In the present work, the nanocomposite of the silver nanoparticles decorated partially unzipped carbon nanotubes (PUCNTs) (PUCNTs/Ag) was fabricated by in situ method, and its application as a sensitive non-enzymatic hydrogen peroxide (H2O2) sensor was then explored correspondingly. The measurements of transmission electron microscopy (TEM), fourier transform infrared (FTIR) spectroscopy and ultraviolet visible (UV-vis) absorption spectrum proved that the PUCNTs/Ag composite has been successfully prepared and the Ag nanoparticles dispersed on the surface of the PUCNTs and entered the inner of unzipped MWCNTs. Cyclic voltammetric (CV) measurement indicated that the PUCNTs/Ag nanocomposite showed the well-defined redox characteristics in 0.1 M phosphate buffer solution (PBS, pH = 7.5), and under the optimized experimental conditions, the current response of the as-obtained sensor towards electrochemical oxidation of H2O2 was linear with the concentration of H2O2 in the range of 0.0419 mM to 87 mM (R = 0.997) in the solution of 0.1 M PBS (pH = 7.5) at the applied potential of 0 V. The detection limit was 1 µM with the sensitivity calculated as 1.115 × 103 µA · M-1 · cm-2 and the fast response achieved within 3 s. The constructed non-enzymatic sensor is one of the promising candidates due to it's good sensitivity, selectivity, and reproducibility.

17.
Helicobacter ; 22(2)2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27687595

RESUMO

BACKGROUND: Enterohepatic Helicobacter species (EHS) are widespread in rodent species around the world. Several studies have demonstrated that infection with EHS can interfere with the outcomes of animal experiments in cancer research and significantly influence the study results. Therefore, it is essential to establish a rapid detection and identification of EHS for biomedical research using laboratory rodents. Our study aimed to develop a rapid and sensitive method to detect and distinguish five enterohepatic Helicobacter species. MATERIALS AND METHODS: Nested PCR followed by high-resolution melting curve analysis (HRM) was developed for identification of H. bilis, H. rodentium, H. muridarum, H. typhlonius, as well as H. hepaticus. To validate the accuracy of nested PCR-HRM analysis, quantitative real-time PCR methods for five different enterohepatic Helicobacter species were developed. A total of 50 cecal samples were tested using both nested PCR-HRM analysis and qPCR method. RESULTS: The nested PCR-HRM method could distinguish five enterohepatic Helicobacter species by different melting temperatures. The melting curve were characterized by peaks of 78.7 ± 0.12°C for H. rodentium, 80.51 ± 0.09°C for H. bilis, 81.6 ± 0.1°C for H. typhlonius, 82.11 ± 0.18°C for H. muridarum, and 82.95 ± 0.09°C for H. hepaticus. CONCLUSIONS: The nested PCR-HRM assay is a simple, rapid, and cost-effective assay. This assay could be a useful tool for molecular epidemiology study of enterohepatic Helicobacter infection and an attractive alternative for genotyping of enterohepatic Helicobacter species.


Assuntos
DNA Bacteriano/química , DNA Bacteriano/genética , Helicobacter/classificação , Helicobacter/genética , Técnicas Microbiológicas/métodos , Reação em Cadeia da Polimerase/métodos , Temperatura de Transição , Animais , Ceco/microbiologia , Custos e Análise de Custo , Técnicas de Genotipagem/métodos , Helicobacter/isolamento & purificação , Camundongos , Fatores de Tempo
18.
Virus Genes ; 52(6): 877-882, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27470155

RESUMO

A new family of IFNs called type III IFN or IFN-λ has been described, and shown to induce antiviral activity against several viruses in the cell culture. In this study, the molecular cloning, expression, and antiporcine epidemic diarrhea virus (PEDV) activity of porcine IFN-λ3 (poIFN-λ3) were reported. The full-length poIFN-λ3 cDNA sequence encoded 196 amino acids with a 23 amino acid signal peptide. Sequence alignments showed that poIFN-λ3 had an amino acid sequence similarity to Ovis aries (78.1 %), Bos taurus (76.0 %), Tupaia belangeri (71.3 %), Equus caballus (69.9 %), and Homo sapiens (69.9 %). The phylogenetic analysis based on the genomic sequences indicated that poIFN-λ3 is located in the same branch as B. taurus and O. aries IFN-λ3. The poIFN-λ3 without a signal anchor sequence was efficiently expressed in Escherichia coli, and the purified recombinant poIFN-λ3 exhibited significant antiviral effects against PEDV in a dose- and time-dependent manner. This inhibitory effect of poIFN-λ3 on PEDV was observed under three different treatment conditions. The highest inhibition of PEDV was observed in Vero E6 cell cultures pretreated with poIFN-λ3 (prior to PEDV infection). In addition, poIFN-λ3 was able to induce the expression of IFN-stimulated genes, including ISG15, OAS1, and Mx1 in Vero E6 cells. These data demonstrate that poIFN-λ3 has antiviral activity against PEDV and may serve as a useful biotherapeutic candidate to inhibit PEDV or other viruses in swine.


Assuntos
Antivirais/farmacologia , Interferon gama/farmacologia , Vírus da Diarreia Epidêmica Suína/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Linhagem Celular , Células Cultivadas , Clonagem Molecular , Interferon gama/química , Interferon gama/genética , Interferon gama/isolamento & purificação , Filogenia , Vírus da Diarreia Epidêmica Suína/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Análise de Sequência de DNA , Suínos , Células Vero , Replicação Viral/efeitos dos fármacos
19.
Virus Genes ; 51(2): 252-9, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26329934

RESUMO

Porcine epidemic diarrhea virus (PEDV) is a member of the coronaviridae family, which can cause acute and highly contagious enteric disease of swine characterized by severe entero-pathogenic diarrhea in piglets. Currently, the vaccines of PEDV are only partially effective and there is no specific drug available for treatment of PEDV infection. To exploit the possibility of using RNA interference (RNAi) as a strategy against PEDV infection, five shRNA-expressing plasmids targeting the N, M, and S genes of PEDV were constructed and transfected into Vero cells. The cytopathic effect and MTS assays demonstrated that two shRNAs (pSilencer4.1-M1 and pSilencer4.1-N) were capable of protecting cells against PEDV invasion with very high specificity and efficiency. The two shRNA expression plasmids were also able to inhibit the PEDV replication significantly, as shown by detection of virus titers (TCID50/mL). A real-time quantitative RT-PCR further confirmed that the amounts of viral RNAs in cell cultures pre-transfected with these two plasmids were reduced by 95.0 %. Our results suggest that RNAi might be a promising new strategy against PEDV infection.


Assuntos
Vírus da Diarreia Epidêmica Suína/crescimento & desenvolvimento , Vírus da Diarreia Epidêmica Suína/genética , Interferência de RNA , Animais , Sobrevivência Celular , Chlorocebus aethiops , Efeito Citopatogênico Viral , Plasmídeos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , RNA Viral/análise , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real , Células Vero , Carga Viral
20.
Virus Genes ; 51(3): 361-6, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26573282

RESUMO

Since 2013, the second outbreak of peste des petits ruminants (PPR) caused by Peste des petits ruminants virus (PPRV) has spread over more than 20 provinces, municipalities, and autonomous regions in China, resulting in major economic losses for livestock industry. In 2014, we encountered a clinical PPR case on a goat farm in Guangdong province, southern China. The complete genome of this PPRV strain, named CH/GDDG/2014, was sequenced to determine its similarities and differences with other strains. The CH/GDDG/2014 genome comprised 15,954 nucleotides (six nucleotides more than classical PPRVs identified before 2013, but complying with the rule of six) with six open reading frames encoding nucleocapsid protein, phosphoprotein, matrix protein, fusion protein, hemagglutinin, and large polymerase protein, respectively. The whole-genome-based alignment analysis indicated that CH/GDDG/2014 had the most proximate consensus (99.8 %) to China/XJYL/2013 and the least consensus (87.2 %) to KN5/2011. The phylogenetic analysis showed that CH/GDDG/2014 was clustered in one branch (lineage IV) with other emerging strains during the second outbreak. This study is the first report describing the whole-genome sequence of PPRV in Guangdong province, southern China and also suggests the PPR outbreak may be closely related to illegal cross-regional importation of goats.


Assuntos
Doenças das Cabras/virologia , Peste dos Pequenos Ruminantes/genética , Peste dos Pequenos Ruminantes/virologia , Vírus da Peste dos Pequenos Ruminantes/genética , Filogenia , Animais , Sequência de Bases , China/epidemiologia , Análise por Conglomerados , Surtos de Doenças , Genes Virais , Doenças das Cabras/epidemiologia , Cabras , Proteínas do Nucleocapsídeo/genética , Peste dos Pequenos Ruminantes/mortalidade , Vírus da Peste dos Pequenos Ruminantes/isolamento & purificação , Alinhamento de Sequência , Análise de Sequência de DNA
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