RESUMO
Several studies have examined the functions of nucleic acids in small extracellular vesicles (sEVs). However, much less is known about the protein cargos of sEVs and their functions in recipient cells. This study demonstrates the presence of lysine-specific demethylase 1 (LSD1), which is the first identified histone demethylase, in the culture medium of gastric cancer cells. We show that sEVs derived from gastric cancer cells and the plasma of patients with gastric cancer harbor LSD1. The shuttling of LSD1-containing sEVs from donor cells to recipient gastric cancer cells promotes cancer cell stemness by positively regulating the expression of Nanog, OCT4, SOX2, and CD44. Additionally, sEV-delivered LSD1 suppresses oxaliplatin response of recipient cells in vitro and in vivo, whereas LSD1-depleted sEVs do not. Taken together, we demonstrate that LSD1-loaded sEVs can promote stemness and chemoresistance to oxaliplatin. These findings suggest that the LSD1 content of sEV could serve as a biomarker to predict oxaliplatin response in gastric cancer patients.
Assuntos
Vesículas Extracelulares , Neoplasias Gástricas , Histona Desmetilases/genética , Humanos , Lisina , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/genéticaRESUMO
In the present study, a specific and reliable duplex SYBR green I-based quantitative real-time polymerase chain reaction assay was established to detect pseudorabies virus (PRV) and porcine circovirus 3 (PCV3) simultaneously. Viral genomes of PRV and PCV3 in one specimen were identified by their different melting temperatures with melting peaks at 87 °C and 81 °C for PRV and PCV3 respectively, whilst other non-targeted swine pathogens exhibited no fluorescent signals. The assay displayed a high degree of linearity (R2 > 0.997), and the limits of detection were 37.8 copies/µL, 30.6 copies/µL and 60 copies/µL for PRV, PCV3 and the mixture of two recombinant plasmids, respectively. It had good repeatability and reproducibility, and the coefficients of variation in intra-batch and inter-batch assays were all less than 2.0%. In this research, the duplex assay was further evaluated using 117 clinical tissue specimens from diseased pigs in the field. The results revealed the infection rates of PRV and PCV3 were 23.08% (27/117) and 55.56% (65/117) respectively, and PRV and PCV3 co-infection rate was 14.53% (17/117). The assay could be utilized as a diagnostic tool with specificity, sensitivity, and reliability for molecular epidemiological surveillance of PRV and PCV3.
Assuntos
Benzotiazóis/química , Infecções por Circoviridae/diagnóstico , Circovirus/isolamento & purificação , Coinfecção/diagnóstico , Diaminas/química , Herpesvirus Suídeo 1/isolamento & purificação , Pseudorraiva/diagnóstico , Quinolinas/química , Doenças dos Suínos/virologia , Animais , China/epidemiologia , Infecções por Circoviridae/epidemiologia , Infecções por Circoviridae/veterinária , Circovirus/genética , Coinfecção/epidemiologia , Coinfecção/veterinária , Genoma Viral , Herpesvirus Suídeo 1/genética , Limite de Detecção , Reação em Cadeia da Polimerase Multiplex , Pseudorraiva/epidemiologia , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Especificidade da Espécie , Suínos , Temperatura de TransiçãoRESUMO
The SYBR Green Ð-based duplex real-time PCR assay was developed for simultaneous detection of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus 3 (PCV-3) genomes. PRRSV and PCV-3 were distinguished in the same sample by their distinctive melting temperature (Tm) which was 84⯰C for PRRSV and 81.5⯰C for PCV-3, and other non-targeted swine viruses showed no specific melting peaks. The detection limits of this assay were 46.1copies/µL for PRRSV and 49.3copies/µL for PCV-3, respectively. Thirty-three lung samples of porcine with respiratory and reproductive failure symptoms were collected and confirmed by the SYBR Green Ð-based real-time PCR assay and conventional PCR assay. The real-time PCR detection results showed that the PRRSV positive rate was 45.45%, the PCV-3 positive rate was 63.63%, the PRRSV and PCV-3 co-infection positive rate was 36.36%, which were more sensitive than conventional PCR detection. This duplex real-time PCR assay could be a rapid, sensitive and reliable method for the detection of PRRSV and PCV-3 co-infection.
Assuntos
Circovirus/isolamento & purificação , Compostos Orgânicos/metabolismo , Vírus da Síndrome Respiratória e Reprodutiva Suína/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Suínos/virologia , Animais , Benzotiazóis , Linhagem Celular , Circovirus/genética , Diaminas , Desnaturação de Ácido Nucleico , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Quinolinas , Padrões de Referência , Reprodutibilidade dos TestesRESUMO
Pseudorabies (PR), also called Aujeszky's disease (AD), is a highly infectious viral disease which is caused by pseudorabies virus (PRV). It has been nearly 200 years since the first PR case occurred. Currently, the virus can infect human beings and various mammals, including pigs, sheep, dogs, rabbits, rodents, cattle and cats, and among them, pigs are the only natural host of PRV infection. PRV is characterized by reproductive failure in pregnant sows, nervous disorders in newborn piglets, and respiratory distress in growing pigs, resulting in serious economic losses to the pig industry worldwide. Due to the extensive application of the attenuated vaccine containing the Bartha-K61 strain, PR was well controlled. With the variation of PRV strain, PR re-emerged and rapidly spread in some countries, especially China. Although researchers have been committed to the design of diagnostic methods and the development of vaccines in recent years, PR is still an important infectious disease and is widely prevalent in the global pig industry. In this review, we introduce the structural composition and life cycle of PRV virions and then discuss the latest findings on PRV pathogenesis, following the molecular characteristic of PRV and the summary of existing diagnosis methods. Subsequently, we also focus on the latest clinical progress in the prevention and control of PRV infection via the development of vaccines, traditional herbal medicines and novel small RNAs. Lastly, we provide an outlook on PRV eradication.
Assuntos
Herpesvirus Suídeo 1 , Pseudorraiva , Doenças dos Suínos , Animais , Bovinos , Cães , Feminino , Herpesvirus Suídeo 1/genética , Humanos , Mamíferos , Pseudorraiva/prevenção & controle , Vacinas contra Pseudorraiva , Coelhos , Ovinos , Suínos , Vacinas AtenuadasRESUMO
To understand the biological characteristics of the reemerging pseudorabies virus (PRV) strains, a total of 392 tissue samples were collected from diseased pigs during reemerging PR outbreaks between 2012 and 2019 on farms in central China where swine had been immunized with Bartha-K61 and 51 (13. 01%) were positive for the gE gene by PCR. Sixteen PRV strains were isolated and caused clinical symptoms and death in mice. Subsequently, gE, gC, gB, and gD complete genes were amplified from the 16 PRV isolates and sequenced. Phylogenetic analysis based on these four gene sequences shows that the 16 PRV isolates were more closely related to the Chinese PRV variants (after 2012) but genetically differed from early Chinese PRV isolates (before 2012). Sequence analysis reveals that PRV isolates exhibited amino acid insertions, substitutions, or deletions compared with early Chinese PRV isolates and European-American PRV strains. In addition, this is the first report that eight isolates (8/16) in this study harbor a unique amino acid substitution at position 280 (F to L) of the gC protein, and six isolates have an amino acid substitution at position 338 (A to V) of the gD protein compared with the Chinese PRV variants. The emulsion containing inactivated PRV NY isolate could provide complete protection against the NY isolate. This study might enrich our understanding of the evolution of reemerging PRV strains as well as pave the way for finding a model virus to develop a novel vaccine based on reemerging PRV strains.
RESUMO
In late 2011, the outbreak of pseudorabies (PR) occurred in Bartha-K61-vaccinated pig farms and spread rapidly to many provinces of China, causing substantial economic losses to the swine industry. A total of 4708 pig serum samples from Henan province during 2018-2019 were collected to screen for the presence of pseudorabies virus (PRV) gE-specific antibodies, and phylogenetic analysis based on the gE gene of PRV was performed. Of the 4708 serum samples tested, 30.14% (1419/4708) were seropositive for PRV antibodies, based on PRV gE-coated enzyme-linked immunosorbent assay (ELISA), with slaughterhouses having the highest seroprevalence. The seropositive rates of PRV also varied with the region and the season. Phylogenetic analysis showed that three PRV isolates from this study were clustered in an independent branch together with the Chinese variant PRV strains (after 2012), and had a closer genetic relationship with the Chinese variant PRV strains, but differed genetically from the 4 early Chinese PRV strains and 4 European-American strains. This study suggests that three PRV isolates may belong to PRV variants, and the development of a novel vaccine against PRV variants is particularly urgent.
Assuntos
Herpesvirus Suídeo 1/genética , Pseudorraiva/epidemiologia , Pseudorraiva/virologia , Doenças dos Suínos/epidemiologia , Doenças dos Suínos/virologia , Suínos/virologia , Animais , Anticorpos Antivirais/imunologia , China/epidemiologia , Surtos de Doenças/prevenção & controle , Surtos de Doenças/veterinária , Herpesvirus Suídeo 1/imunologia , Filogenia , Pseudorraiva/imunologia , Estudos Soroepidemiológicos , Suínos/imunologia , Doenças dos Suínos/imunologia , Vacinas Virais/imunologiaRESUMO
BACKGROUND: Some patients with knee osteoarthritis (KOA) show pain, stiffness and limited flexion and extension at the back of the knee, leading to dysfunction and affecting life. This may be related to changes in the biomechanical properties of skeletal muscles. Shear wave elastography (SWE) can detect these changes by measuring muscle shear modulus. AIMS: To investigate hamstring muscle shear modulus of healthy people and patients was studied using SWE method, and the correlation analysis between the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) score of patients' subjective feeling and shear modulus of objective quantification was conducted. METHODS: The hamstring shear modulus was measured by SWE in 50 patients and 50 healthy individuals. Pearson correlation coefficient was used to evaluate the correlation between hamstring stiffness and shear modulus in patients. RESULTS: The hamstring shear modulus were significantly higher in the KOA group [the semimembranosus (SM) 15.23 ± 7.23, the semitendinosus (ST) 15.94 ± 5.40, the biceps femoris long tendinitis (BFL) 14.21 ± 6.55] than in the control group (the SM 10.95 ± 2.41, the ST 11.25 ± 2.23, the BFL 9.98 ± 2.81) (p = 0.000, p = 0.000, p = 0.001). The hamstring shear modulus in the KOA group was moderately positively correlated with pain, shear modulus, and physical function score. CONCLUSION: Preliminary results show that the shear modulus of the hamstring of KOA patients is higher than that of healthy people, the WOMAC score and the shear modulus of patients are moderately correlated. These preliminary results show that ultrasonic shear wave elastography measurement of shear modulus may be enough to sensitive, can detect these effects, more targeted in order to assist the doctor's diagnosis and treatment.
RESUMO
Porcine circovirus 4 (PCV4), a new circovirus with a distinct relationship to other circoviruses, was identified in 2019 in several pigs with severe clinical disease in Hunan Province, China. To investigate the epidemic profile and genetic diversity of the virus, 63 clinical samples were collected from 24 different pig farms in 14 cities in Henan and Shanxi Provinces, China, between February 2018 and December 2019, and the partial Cap gene of PCV4 was amplified by PCR. Among the 63 samples, 16 (25.40%) were positive for PCV4, and 50% (12/24) of the pig farms were positive for PCV4. PCV4 was detected in samples from pigs with different clinical presentations. One PCV4 strain (Henan-LY1-2019) was sequenced in this study, and shared 98.4% genomic nucleotide identity with PCV4 strain HNU-AHG1-2019 (accession no. MK986820) detected on a pig farm in Hunan Province in 2019. A phylogenetic analysis based on the genomes of Henan-LY1-2019 and 31 reference strains showed that the Henan-LY1-2019 strain together with PCV4 strain HNU-AHG1-2019 was grouped in a relatively independent sub-branch, and separated from other viruses in the genus Circovirus. The results of this study extend our understanding of the molecular epidemiology of PCV4.
Assuntos
Infecções por Circoviridae/veterinária , Circovirus/classificação , Circovirus/genética , Doenças dos Suínos/epidemiologia , Animais , China/epidemiologia , Infecções por Circoviridae/epidemiologia , Infecções por Circoviridae/virologia , Filogenia , Prevalência , Sus scrofa , Suínos , Doenças dos Suínos/virologiaRESUMO
Differentiation therapy has been successfully applied clinically in cases of acute promyelocytic leukemia (APL), but few differentiation-induction agents other than all-trans retinoic acid (ATRA) have been discovered clinically. Based on our previously reported neuritogenic differentiation activity of synthetic dimeric derivatives of securinine, we explored the leukemia differentiation-induction activity of such as compound, SN3-L6. It was found that SN3-L6 induces transdifferentiation of both acute myeloid leukemia (AML) and chronic myelogenous leukemia (CML) cells but unexpectedly, a new transdifferentiation pathway from APL cells to morphologically and immunologically normal megakaryocytes and platelets were discovered. SN3-L6 fails to induce transdifferentiation of ATRA-produced mature granulocytes into megakaryocytes, indicating its selectivity between mature and immature cells. SN3-L6 induces CML K562 cells to transdifferentiate into apoptotic megakaryocytes but without platelet formation, indicating a desirable selectivity between different leukemia cells. Our data illuminate a differentiation gap between AML cells and platelets, and promises applications in leukemia differentiation therapy strategy.
RESUMO
A real-time polymerase chain reaction (PCR) using a TaqMan probe was developed to detect porcine parvovirus (PPV). Real-time PCR was optimized to quantify PPV using a detection system (Rotor Gene 2000 detector) and a dual-labeled fluorogenic probe. The gene-specific labeled fluorogenic probe for the VP2 gene of PPV was used to detect PPV. Quantitation of PPV was accomplished by a standard curve plotting cycle threshold values (Ct) against each dilution of standard plasmids. When the specificity of the assay using specific PPV primers was evaluated by testing the PPV standard strain and other viruses, no cross-reactions were detected with non-PPV reference viruses. The detection limit of real-time PCR for PPV was 2.08log10 genome copy equivalent (gce). In this study, a real-time PCR assay was performed on 80 clinical samples and compared with a conventional PCR assay. In 48 of 80 samples, PPV DNA was detected by the conventional PCR assay. All samples positive for PPV DNA by the conventional PCR assay were also positive by the real-time PCR assay, and 12 of 32 samples that tested negative for PPV DNA by the conventional method tested positive by the real-time PCR assay. Using the real-time PCR assay, the number of samples in which PPV was detected increased by 15%. Therefore, it is considered to be a useful tool for the detection of PPV.
Assuntos
Parvovirus Suíno/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Suínos/virologia , Animais , Sondas de DNA , DNA Viral/análise , Feto/virologia , Infecções por Parvoviridae/diagnóstico , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Doenças dos Suínos/diagnósticoRESUMO
Multidrug resistance (MDR) is a main cause of chemotherapy failure and patient death. This situation usually involves a glycoprotein (P-gp) mediated drug efflux, resulting in a low cellular drug concentration and insensitivity. Here we report the design, synthesis and evaluation of novel (+/-)-securinine bivalents as P-gp inhibitors in vitro and in vivo. MTT assays reflected that bivalent mimetics of securinine particularly the virosecurinine bivalent mimetic 8C showed promissing MDR reversal potential in both P-gp highly expressed cell line HepG2/DOX and MCF-7/ADM. At a 10⯵M concentration, 8C can entirely reverse the resistance of HepG2/DOX to doxorubicin (DOX), and is more effective than the positive control verapamil (VRP). Fluorescence, flow cytometry, and DOX efflux assays demonstrated that 8C can facilitate the accumulation and diminish the efflux of intracellular DOX. Molecular docking analysis and western blot assays indicated that 8C accomplished this by competitively inhibiting the activity of P-gp rather than by affecting its expression. Compound 8C was also observed to reverse drug resistance effectively in xenograft models when combined with DOX. This study lays a foundation for the discovery of (+/-)-securinine ramifications as P-gp inhibitors and provides a promising lead compound 8C as a P-gp mediated MDR reversal agent.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Alcaloides , Antineoplásicos , Azepinas , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Lactonas , Piperidinas , Alcaloides/química , Alcaloides/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Azepinas/química , Azepinas/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/farmacologia , Compostos Heterocíclicos de Anel em Ponte/química , Compostos Heterocíclicos de Anel em Ponte/farmacologia , Humanos , Lactonas/química , Lactonas/farmacologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Piperidinas/química , Piperidinas/farmacologia , Verapamil/farmacologiaRESUMO
Hydroxyl radical (.OH)-initiated photooxidation reaction of toluene was carried out in a self-made smog chamber. Four individual seed aerosols such as ammonium sulfate, ammonium nitrate, sodium silicate and calcium chloride, were introduced into the chamber to assess their influence on the growth of secondary organic aerosols (SOA). It was found that the low concentration of seed aerosols might lead to high concentration of SOA particles. Seed aerosols would promote rates of SOA formation at the start of the reaction and inhibit its formation rate with prolonging the reaction time. In the case of ca. 9000 pt/cm3 seed aerosol load, the addition of sodium silicate induced a same effect on the SOA formation as ammonium nitrate. The influence of the four individual seed aerosols on the generation of SOA decreased in the order of calcium chloride>sodium silicate and ammonium nitrate>ammonium sulfate.
Assuntos
Sulfato de Amônio , Cloreto de Cálcio , Nitratos , Silicatos , Tolueno/química , Tolueno/efeitos da radiação , Aerossóis , Poluentes Atmosféricos/química , Poluentes Atmosféricos/efeitos da radiação , Sulfato de Amônio/química , Sulfato de Amônio/efeitos da radiação , Cloreto de Cálcio/química , Cloreto de Cálcio/efeitos da radiação , Nitratos/química , Nitratos/efeitos da radiação , Óxido Nítrico/química , Óxido Nítrico/efeitos da radiação , Nitritos/química , Nitritos/efeitos da radiação , Oxirredução , Silicatos/química , Silicatos/efeitos da radiação , Raios UltravioletaRESUMO
A series of novel bivalent securinine mimetics incorporating different linkers between C-15 and C-15' were synthesized and their topoisomerase I (Topo I) inhibitory activities evaluated. It was thus revealed that mimetic R2 incorporating a rigid m-substituted benzene linker exhibits Topo I inhibitory activity three times that of parent securinine. Comprehensive structure-activity relationship analyses in combination with docking studies were used to rationalize the potent activity of these bivalent mimetics. Mechanistic studies served to confirm the deductions arising from docking studies that the active bivalent mimetics not only inhibited complexation between Topo I and DNA but also stabilized the Topo I-DNA complex itself.
RESUMO
Photooxidation reaction of toluene in smog chamber systems was initiated by the UV radiation of toluene/CH3ONO/NOx mixtures. The products of the photooxidation reaction of toluene and its subsequent reactions were analyzed directly utilizing Fourier transform infrared spectrometer (FTIR). Detailed assignments to FTIR spectrum of gas-phase products were given. The information of some important functional groups in the products, such as, carbonyl groups (C-O), hydroxyl groups ( -OH), carboxylic acid (-COOH), C-C bonding, N-O bonding and C-H bonding (C-H), was got from this analysis. These results were compared to those analyzed by aerosol time of flight mass spectrometer (ATOFMS). It was found that there are some differences between FTIR analysis of gas-phase products and that of particle-phase, for example, the products with carbonyl groups, which were connected to unsaturated chemical bonds, was relatively higher in the gas phase, while ketones, aldehydes, carboxylic acid and organonitrates were the dominant functional groups in the aerosol-phase reaction products. The possible reaction pathways of some important products in the gas phase were also discussed.
Assuntos
Tolueno/química , Oxirredução , Fotoquímica , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
DNA topoisomerase I (Topo I) has been validated as a target for anticancer agents. In this study, a series of novel securinine derivatives bearing ß'-hydroxy-α,ß-unsaturated ketone moiety were designed and synthesized via a Baylis-Hillman reaction for screening as Topo I inhibitors and antitumor agents. Their topoisomerase I inhibitory activity as well as their cytotoxicity against four human cancer cell lines (A549, HeLa, HepG2, SH-SY5Y) were evaluated, and two pairs of diastereomers 4a-1 and 4a-6 with significant Topo I inhibitory activity and potent anti-proliferative activity against cancer cell lines were identified. The diastereomers were separated, and absolute configurations of five pairs of diastereomers were identified based on X-ray crystallographic analysis and circular dichroism (CD) spectra analysis. Further mechanism studies of the most active compounds 4a-1-R and 4a-1-S indicated that this kind of securinine derivative exhibits a different inhibitory mechanism from that of camptothecin, an established Topo I inhibitor. Unlike camptothecin, compounds 4a-1-R and 4a-1-S specifically inhibits the combination of Topo I and DNA rather than forming the drug-enzyme-DNA covalent ternary complex. In addition, molecular docking and molecular dynamic studies revealed the binding patterns of these compounds with Topo I.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Azepinas/química , Azepinas/farmacologia , DNA Topoisomerases Tipo I/metabolismo , Compostos Heterocíclicos de Anel em Ponte/química , Compostos Heterocíclicos de Anel em Ponte/farmacologia , Lactonas/química , Lactonas/farmacologia , Piperidinas/química , Piperidinas/farmacologia , Inibidores da Topoisomerase I/química , Inibidores da Topoisomerase I/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Clivagem do DNA/efeitos dos fármacos , DNA Topoisomerases Tipo I/química , Desenho de Fármacos , Humanos , Modelos Moleculares , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
Neurite outgrowth is crucial during neuronal development and regeneration, and strategies that aim at promoting neuritogenesis are beneficial for reconstructing synaptic connections after neuronal degeneration and injury. Using a bivalent analogue strategy as a successful approach, the current study identifies a series of novel dimeric securinine analogues as potent neurite outgrowth enhancers. Compounds 13, 14, 17-19, and 21-23, with different lengths of carbon chain of N,N-dialkyl substituting diacid amide linker between two securinine molecules at C-15 position, exhibited notable positive effects on both neuronal differentiation and neurite extension of neuronal cells. Compound 14, one of the most active compounds, was used as a representative compound for mechanistic studies. Its action on neurite outgrowth was through phosphorylation/activation of multiple signaling molecules including Ca2+/calmodulin-dependent protein kinase II (CaMKII), extracellular signal-regulated kinase (ERK) and Akt. These findings collectively identify a new group of beneficial compounds for neuritogenesis, and may provide insights on drug discovery of neural repair and regeneration.
Assuntos
Azepinas/síntese química , Azepinas/farmacologia , Crescimento Celular/efeitos dos fármacos , Compostos Heterocíclicos de Anel em Ponte/síntese química , Compostos Heterocíclicos de Anel em Ponte/farmacologia , Neuritos/efeitos dos fármacos , Fármacos Neuroprotetores/síntese química , Fármacos Neuroprotetores/farmacologia , Animais , Azepinas/química , Western Blotting , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Linhagem Celular Tumoral , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Compostos Heterocíclicos de Anel em Ponte/química , Imuno-Histoquímica , Lactonas/química , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Estrutura Molecular , Neuritos/fisiologia , Fármacos Neuroprotetores/química , Fosforilação/efeitos dos fármacos , Piperidinas/química , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
In a smog chamber, the photooxidation of toluene was initiated by hydroxyl radical (OH*) under different experimental conditions. The size distribution of secondary organic aerosol(SOA) particles from the above reaction was measured using aerodynamic particle sizer spectrometer. It was found from our experimental results that the number of SOA particles increased with increasing the concentration of toluene. As the reaction time prolonged, the sum of SOA particles was also increased. After a reaction time of 130 min, the concentration of secondary organic aerosol particles would be kept constant at 2300 particles/cm3. Increasing illumination power of blacklamps could significantly induce a higher concentration of secondary organic aerosol particle. The density of SOA particles would also be increased with increasing concentration of CH3 ONO, however, it would be decreased as soon as the concentration of CH3 ONO was larger than 225.2 ppm. Nitrogen oxide with initial concentration higher than 30.1 ppm was also found to have little effect on the formation of secondary organic aerosol.
Assuntos
Poluentes Atmosféricos , Fotoquímica , Tolueno/química , Aerossóis/química , Oxirredução , Tamanho da Partícula , Fatores de TempoRESUMO
The development of a SYBR Green-based duplex real-time PCR is described for simultaneous detection of porcine parvovirus (PPV) and porcine circovirus type 2 (PCV-2) genomes. Viral genomes were identified in the same sample by their distinctive melting temperature (T(m)) which is 77.5°C for PPV VP2 313bp amplicon and 82.3°C for PCV-2 ORF2 171bp amplicon, respectively. The detection limit of the method was 0.01TCID(50)/mL for PPV and PCV-2, about 10 times more sensitive than conventional PCR. In addition, PPV and PCV-2 viral load were measured in 126 field samples, confirming the sensitivity and specificity, and the result showed that 70/126 samples were positive for PPV and 92/126 samples were positive for PCV2 by the duplex real-time PCR. This method may be a useful alternative rapid and reliable method for the detection of PPV/PCV-2 co-infection.
Assuntos
Infecções por Circoviridae , Circovirus/genética , Infecções por Parvoviridae , Parvovirus Suíno/genética , Animais , Benzotiazóis , Infecções por Circoviridae/diagnóstico , Infecções por Circoviridae/veterinária , Infecções por Circoviridae/virologia , Coinfecção , Primers do DNA , DNA Viral/análise , Diaminas , Limite de Detecção , Desnaturação de Ácido Nucleico , Compostos Orgânicos , Infecções por Parvoviridae/diagnóstico , Infecções por Parvoviridae/veterinária , Infecções por Parvoviridae/virologia , Quinolinas , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Suínos , Doenças dos Suínos/diagnóstico , Doenças dos Suínos/virologia , Carga ViralRESUMO
A DNA vaccine against infectious laryngotracheitis virus (ILTV) can induce specific humoral and cell-mediated immunity. However, compared to conventional vaccines, DNA vaccines usually induce poor antibody responses. To determine if co-expression of a cytokine can result in a more potent ILTV DNA vaccine, immunogenicity and protective efficacy of a monocistronic vector encoding the glycoprotein B (gB) of ILTV was compared to that of a bicistronic vector separately encoding the gB and chicken interleukin-18. Humoral and cellular responses induced by the DNA vaccines administered to the quadriceps muscle of chickens were evaluated. There were significant differences in antibody levels elicited by either monocistronic or bicistronic DNA vaccines as determined by ELISA. The percentages of CD3(+), CD3(+)CD8(+) and CD3(+)CD4(+) subgroups of peripheral blood T-lymphocytes in chickens immunized with the bicistronic DNA vaccine were higher than those in chickens immunized with monocistronic DNA vaccine. When chickens were challenged with a virulent CG strain of ILTV, the protective efficacy was enhanced significantly after immunization with the bicistronic DNA vaccine. These results demonstrated that co-expression of an adjuvant cytokine from a bicistronic DNA vaccine may be an effective approach to increasing ILTV DNA vaccine immunogenicity.
Assuntos
Adjuvantes Imunológicos/farmacologia , Herpesvirus Galináceo 1/imunologia , Interleucina-18/farmacologia , Vacinas de DNA/imunologia , Proteínas do Envelope Viral/imunologia , Adjuvantes Imunológicos/genética , Animais , Anticorpos Antivirais/sangue , Complexo CD3/análise , Antígenos CD4/análise , Antígenos CD8/análise , Galinhas , Ensaio de Imunoadsorção Enzimática , Herpesvirus Galináceo 1/genética , Injeções Intramusculares , Interleucina-18/genética , Análise de Sobrevida , Subpopulações de Linfócitos T/química , Subpopulações de Linfócitos T/imunologia , Vacinas de DNA/genética , Proteínas do Envelope Viral/genéticaRESUMO
The gene sequence encoding mature porcine interferon-gamma (PoIFN-gamma) fused with a C-terminal 6x histidine tag was cloned into the baculovirus pFastBac Dual vector of the Bac-to-Bac Baculovirus expression system under the control of PH promoter. The authentic signal sequence of porcine interferon-gamma was substituted with the honeybee melittin (HBM) signal sequence, and expressed in insect cells. The recombinant proteins were detected by SDS-PAGE and immunofluorescence assay. The nickel affinity column purified recombinant porcine interferon-gamma with HBM signal peptide (rPoIFN-gammaH) was shown to be a 19kDa protein as confirmed by Western blot analysis. The recombinant PoIFN-gammaH was shown to have cytokine activity, inhibiting the cytopathic effect of vesicular stomatitis virus (VSV) in PK-15 cells at about 1.07x10(6)U/mL. The 2(-7) dilution of the rPoIFN-gammaH in culture supernatant protected the MARC-145 cells from the cytopathic effect caused by 100TCID(50) of porcine reproductive and respiratory syndrome virus.