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1.
Mem Inst Oswaldo Cruz ; 114: e190150, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31432892

RESUMO

BACKGROUND: Zika virus (ZIKV) infections reported in recent epidemics have been linked to clinical complications that had never been associated with ZIKV before. Adaptive mutations could have contributed to the successful emergence of ZIKV as a global health threat to a nonimmune population. However, the causal relationships between the ZIKV genetic determinants, the pathogenesis and the rapid spread in Latin America and in the Caribbean remain widely unknown. OBJECTIVES: The aim of this study was to characterise three ZIKV isolates obtained from patient samples during the 2015/2016 Brazilian epidemics. METHODS: The ZIKV genomes of these strains were completely sequenced and in vitro infection kinetics experiments were carried out in cell lines and human primary cells. FINDINGS: Eight nonsynonymous substitutions throughout the viral genome of the three Brazilian isolates were identified. Infection kinetics experiments were carried out with mammalian cell lines A549, Huh7.5, Vero E6 and human monocyte-derived dendritic cells (mdDCs) and insect cells (Aag2, C6/36 and AP61) and suggest that some of these mutations might be associated with distinct viral fitness. The clinical isolates also presented differences in their infectivity rates when compared to the well-established ZIKV strains (MR766 and PE243), especially in their abilities to infect mammalian cells. MAIN CONCLUSIONS: Genomic analysis of three recent ZIKV isolates revealed some nonsynonymous substitutions, which could have an impact on the viral fitness in mammalian and insect cells.


Assuntos
Aedes/virologia , Replicação Viral , Infecção por Zika virus/virologia , Zika virus/genética , Animais , Brasil , Cercopithecus aethiops , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Filogenia , Células Vero , Carga Viral , Cultura de Vírus
2.
Exp Parasitol ; 204: 107731, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31374185

RESUMO

Neospora caninum is an obligate intracellular parasite related to cases of abortion and fertility impairment in cattle. The control of the parasite still lacks an effective protective strategy and the understanding of key mechanisms for host infection might be crucial for identification of specific targets. There are many proteins related to important mechanisms in the host cell infection cycle such as adhesion, invasion, proliferation and immune evasion. The surface proteins, especially SRS (Surface Antigen Glycoprotein - Related Sequences), have been demonstrated to have a pivotal role in the adhesion and invasion processes, making them potential anti-parasite targets. However, several predicted surface proteins were not described concerning their function and importance in the parasite life cycle. As such, a novel SRS protein, NcSRS57, was described. NcSRS57 antiserum was used to detect SRS proteins by immunofluorescence in parasites treated or not with phosphatidylinositol-specific phospholipase C (PI-PLC). The treatment with PI-PLC also allowed the identification of NcSRS29B and NcSRS29C, which were the most abundant SRS proteins in the soluble fraction. Our data indicated that SRS proteins in N. caninum shared a high level of sequence similarity and were susceptible to PI-PLC. In addition, the description of the SRS members, regarding abundance, function and immunogenicity will be useful in guiding specific methods to control the mechanism of adhesion and invasion mediated by these surface proteins.


Assuntos
Antígenos de Protozoários/metabolismo , Antígenos de Superfície/metabolismo , Neospora/química , Fosfoinositídeo Fosfolipase C/farmacologia , Proteínas de Protozoários/metabolismo , Animais , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Antígenos de Superfície/genética , Antígenos de Superfície/imunologia , Cercopithecus aethiops , Clonagem Molecular , DNA de Protozoário/isolamento & purificação , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Soros Imunes/imunologia , Soros Imunes/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Confocal , Neospora/efeitos dos fármacos , Neospora/genética , Neospora/imunologia , Fosfoinositídeo Fosfolipase C/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Espectrometria de Massas em Tandem , Fosfolipases Tipo C/metabolismo , Fosfolipases Tipo C/farmacologia , Células Vero
3.
J Water Health ; 17(4): 597-608, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31313997

RESUMO

Water-borne diseases like diarrheagenic Escherichia coli (DEC)-induced gastroenteritis are major public health problems in developing countries. In this study, the microbiological quality of water from mines and shallow wells was analyzed for human consumption. Genotypic and phenotypic characterization of DEC strains was performed. A total of 210 water samples was analyzed, of which 153 (72.9%) contained total coliforms and 96 (45.7%) E. coli. Of the E. coli isolates, 27 (28.1%) contained DEC genes. The DEC isolates included 48.1% Shiga toxin-producing E. coli (STEC), 29.6% enteroaggregative E. coli (EAEC), 14.9% enteropathogenic E. coli (EPEC), 3.7% enterotoxigenic E. coli (ETEC), and 3.7% enteroinvasive E. coli (EIEC). All the STECs had cytotoxic effects on Vero cells and 14.8% of the DEC isolates were resistant to at least one of the antibiotics tested. All DEC formed biofilms and 92.6% adhered to HEp-2 cells with a prevalence of aggregative adhesion (74%). We identified 25 different serotypes. One EPEC isolate was serotype O44037:H7, reported for the first time in Brazil. Phylogenetically, 63% of the strains belonged to group B1. The analyzed waters were potential reservoirs for DEC and could act as a source for infection of humans. Preventive measures are needed to avoid such contamination.


Assuntos
Infecções por Escherichia coli , Água Subterrânea/microbiologia , Animais , Brasil , Cercopithecus aethiops , Diarreia , Humanos , Células Vero
4.
Pol J Microbiol ; 68(2): 165-171, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31257789

RESUMO

Hand, foot, and mouth disease (HFMD) is caused by various serotypes of Enterovirus genus. Coxsackievirus A16 (CV-A16) and enterovirus A71 (EV-A71) were known to be the only responsible agents for these epidemics; however, this opinion was challenged after the detection that coxsackievirus A6 (CV-A6) was the responsible species for the outbreak in Finland in 2008. HFMD is frequently seen in Turkey, and no detailed study on its clinical and microbiological epidemiology has previously been reported. The present study addresses this question. Twenty-seven patient samples collected between 2015 and 2017 were included in the study. Typing was conducted by RT-PCR and the sequencing applied directly to patient's samples and as well as to the viral cultures with pan-enterovirus and serotype-specific primers. The presence of Enterovirus in 12 of 27 HFMD samples was shown with RT-PCR. The causative agent for three of these 12 samples was CV-A16, one of the most frequent two serotypes around the world, and the remaining nine samples was CV-A6. The findings of the study are relevant since it pertains to the molecular epidemiology of HFMD in Turkey, a gateway country where different serotypes might be circulating and transmitted. The findings also support the notion that CV-A6 cases are rising in number, which has caused more severe clinical features and widespread rashes in recent outbreaks.Hand, foot, and mouth disease (HFMD) is caused by various serotypes of Enterovirus genus. Coxsackievirus A16 (CV-A16) and enterovirus A71 (EV-A71) were known to be the only responsible agents for these epidemics; however, this opinion was challenged after the detection that coxsackievirus A6 (CV-A6) was the responsible species for the outbreak in Finland in 2008. HFMD is frequently seen in Turkey, and no detailed study on its clinical and microbiological epidemiology has previously been reported. The present study addresses this question. Twenty-seven patient samples collected between 2015 and 2017 were included in the study. Typing was conducted by RT-PCR and the sequencing applied directly to patient's samples and as well as to the viral cultures with pan-enterovirus and serotype-specific primers. The presence of Enterovirus in 12 of 27 HFMD samples was shown with RT-PCR. The causative agent for three of these 12 samples was CV-A16, one of the most frequent two serotypes around the world, and the remaining nine samples was CV-A6. The findings of the study are relevant since it pertains to the molecular epidemiology of HFMD in Turkey, a gateway country where different serotypes might be circulating and transmitted. The findings also support the notion that CV-A6 cases are rising in number, which has caused more severe clinical features and widespread rashes in recent outbreaks.


Assuntos
Enterovirus Humano B/isolamento & purificação , Doença de Mão, Pé e Boca/epidemiologia , Doença de Mão, Pé e Boca/virologia , Epidemiologia Molecular/métodos , Animais , Linhagem Celular , Cercopithecus aethiops , Criança , Pré-Escolar , Enterovirus Humano B/classificação , Enterovirus Humano B/genética , Feminino , Humanos , Lactente , Masculino , Tipagem Molecular , Turquia/epidemiologia , Células Vero
5.
Pol J Microbiol ; 68(2): 185-191, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31257791

RESUMO

We observed the death of insect caterpillars of Spodoptera exigua in the laboratory culture line and identified Serratia marcescens as the bacterial causative agent of the insect death. We confirmed that S. marcescens had insecticidal activity against S. exigua and caused high mortality of larvae. The LC50 values of S. marcescens CFU per 1 cm2 of insect diet surface were similar for all isolates. Our research reports novel strains with high pesticidal activity as candidates for future research on a new bioinsecticide. As bioinsecticides cannot be harmful to non-target organisms, we determined the pathogenic properties of S. marcescens to humans. We proved the ability of S. marcescens to damage mammalian epithelial cells. All strains had cytopathic effects to Vero cells with a cytotoxic index ranging from 51.2% ± 3.8% to 79.2% ± 4.1%. We found that all of the strains excreted catecholate siderophore - enterobactin. All isolates were resistant to sulfamethoxazole, tobramycin, gentamicin, cefepime, and aztreonam. We did not observe the ESBL phenotype and the integrons' integrase genes. Resistance to sulfamethoxazole was due to the presence of the sul1 or sul2 gene. The use of resistant S. marcescens strains that are pathogenic to humans in plant protection may cause infections difficult to cure and lead to the spread of resistance genes. The results of our study emphasize the necessity of determination of the safety to vertebrates of the bacteria that are proposed to serve as biocontrol agents. The novelty of our study lies in the demonstration of the indispensability of the bacteria verification towards the lack of hazardous properties to humans.We observed the death of insect caterpillars of Spodoptera exigua in the laboratory culture line and identified Serratia marcescens as the bacterial causative agent of the insect death. We confirmed that S. marcescens had insecticidal activity against S. exigua and caused high mortality of larvae. The LC50 values of S. marcescens CFU per 1 cm2 of insect diet surface were similar for all isolates. Our research reports novel strains with high pesticidal activity as candidates for future research on a new bioinsecticide. As bioinsecticides cannot be harmful to non-target organisms, we determined the pathogenic properties of S. marcescens to humans. We proved the ability of S. marcescens to damage mammalian epithelial cells. All strains had cytopathic effects to Vero cells with a cytotoxic index ranging from 51.2% ± 3.8% to 79.2% ± 4.1%. We found that all of the strains excreted catecholate siderophore ­ enterobactin. All isolates were resistant to sulfamethoxazole, tobramycin, gentamicin, cefepime, and aztreonam. We did not observe the ESBL phenotype and the integrons' integrase genes. Resistance to sulfamethoxazole was due to the presence of the sul1 or sul2 gene. The use of resistant S. marcescens strains that are pathogenic to humans in plant protection may cause infections difficult to cure and lead to the spread of resistance genes. The results of our study emphasize the necessity of determination of the safety to vertebrates of the bacteria that are proposed to serve as biocontrol agents. The novelty of our study lies in the demonstration of the indispensability of the bacteria verification towards the lack of hazardous properties to humans.


Assuntos
Agentes de Controle Biológico/efeitos adversos , Inseticidas/efeitos adversos , Inseticidas/farmacologia , Serratia marcescens/patogenicidade , Spodoptera/microbiologia , Animais , Antibacterianos/farmacologia , Aztreonam/farmacologia , Cefepima/farmacologia , Cercopithecus aethiops , Enterobactina/metabolismo , Gentamicinas/farmacologia , Humanos , Testes de Sensibilidade Microbiana , Serratia marcescens/efeitos dos fármacos , Serratia marcescens/isolamento & purificação , Sulfametoxazol/farmacologia , Tobramicina/farmacologia , Células Vero
6.
Anticancer Res ; 39(7): 3727-3737, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31262899

RESUMO

BACKGROUND/AIM: This study aims to investigate whether the combination of oncolytic viruses with chemoradiotherapy or other therapies is a promising strategy for cancer treatment. MATERIALS AND METHODS: The anticancer effects of measles virus (MeV) in combination with nimotuzumab in the treatment of laryngeal cancer were evaluated in vitro and in nude mice inoculated with Hep2 tumors. MTT assay and flow cytometry were used to examine cell death. RESULTS: Laryngeal cancer cells treated with MeV+nimotuzumab combination had a significantly lower survival rate compared to those treated with MeV or nimotuzumab alone (p<0.0001). In an animal model bearing human laryngeal tumor, the treated group had a higher survival rate (60%) compared to a untreated group (20%) (p<0.05), and the survival rate of the group treated with MeV+nimotuzumab combination was higher compared to the groups received single treatment. CONCLUSION: The MeV+nimotuzumab combination has greater anticancer activities in both laryngeal cancer cells and an animal model.


Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Antineoplásicos/uso terapêutico , Neoplasias Laríngeas/terapia , Vírus do Sarampo , Terapia Viral Oncolítica , Vírus Oncolíticos , Animais , Linhagem Celular Tumoral , Cercopithecus aethiops , Terapia Combinada , Humanos , Vacina contra Sarampo , Camundongos Nus , Células Vero
7.
Chem Commun (Camb) ; 55(57): 8227-8230, 2019 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-31268107

RESUMO

An original family of multivalent vectors encompassing gemini and facial amphiphilicity, namely cationic Siamese twin surfactants, has been prepared from the disaccharide trehalose; molecular engineering lets us modulate the self-assembling properties and the topology of the nanocomplexes with plasmid DNA for efficient gene delivery in vitro and in vivo.


Assuntos
Nanoestruturas/química , Plasmídeos/química , Tensoativos/química , Transfecção/métodos , Trealose/química , Animais , Linhagem Celular , Cercopithecus aethiops , Humanos , Camundongos , Plasmídeos/metabolismo
8.
BMC Complement Altern Med ; 19(1): 149, 2019 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-31238921

RESUMO

BACKGROUND: The kidney is an essential organ required by the body to perform several important functions. Nephrotoxicity is one of the most prevailing kidney complications that result from exposure to an extrinsic or intrinsic toxicant, which increase the need for the acquisition of proper remedies. Recently, natural remedies are gaining great attention owed to the fact that they have fewer side effects than most conventional drugs. METHODS: The current study recorded a new therapeutic role of the well-known medicinal plants for kidney stones [Ammi visnaga (AVE), Petroselinum crispum (PCE), Hordeum vulgare (HVE), and Cymbopogon schoenanthus (CSE)]. Hence, the aqueous extracts of these plants examined against CCl4-induced toxicity in mammalian kidney (Vero) cells. RESULTS: These extracts showed the presence of varying amounts of phenolic and triterpenoid compounds, as well as vitamin C. Owing to the antioxidant potential of these constituents, the extracts suppressed the CCl4-induced oxidative stress significantly (p < 0.05) by scavenging the reactive oxygen species and enhancing the cellular antioxidant indices. In addition, these extracts significantly (p < 0.05) reduced the CCl4-induced inflammation by inhibiting the gene expression of NF-кB, iNOS, and in turn the level of nitric oxide. Consequently, the morphological appearance of Vero cells, cellular necrosis, and the gene expression of kidney injury molecule-1 (a marker of renal injury) after these treatments were improved. The AVE improved CCl4-induced oxidative and inflammatory stress in Vero cells and showed a more potent effect than the commonly used alpha-Ketoanalogue drug (ketosteril) in most of the studied assays. CONCLUSION: Thus, the studied plant extracts, especially AVE can be considered as promising extracts in the management of nephrotoxicity and other chronic diseases associated with oxidative stress and inflammation.


Assuntos
Ammi/química , Cymbopogon/química , Hordeum/química , Rim/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Petroselinum/química , Extratos Vegetais/farmacologia , Animais , Antioxidantes/química , Antioxidantes/isolamento & purificação , Antioxidantes/farmacologia , Cercopithecus aethiops , Rim/imunologia , NF-kappa B/genética , NF-kappa B/imunologia , Óxido Nítrico/imunologia , Fenóis/química , Fenóis/isolamento & purificação , Fenóis/farmacologia , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Folhas de Planta/química , Sementes/química , Células Vero
9.
Virol J ; 16(1): 81, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31221185

RESUMO

BACKGROUND: Pseudorabies virus (PRV) protein UL56 (pUL56) has been implicated in viral dissemination and virulence in vivo. However, the properties of PRV pUL56 remain largely unknown. In the present study, we aim to investigate the subcellular localization of pUL56 and the underlying molecular basis in transfected cells. METHODS: Constructs of N-terminal green fluorescent protein (GFP) fused pUL56 and its truncations were employed for investigating subcellular localization and further identifying amino acids crucial for pUL56 localization in transfected Vero cells. Finally, the identified amino acids were replaced with alanine for confirming if these mutations could impair the specific localization of pUL56. RESULTS: The pUL56 predominantly localized at the Golgi and trans-Golgi network (TGN) through its predicted C-terminal transmembrane helix in transfected Vero cells. A Golgi-associated protein Rab6a, independent of interaction with pUL56, was significantly downregulated by pUL56. Further, we found three truncated pUL56 C-terminal fragments (174-184, 175-185 and 191-195) could restrict GFP in the perinuclear region, respectively. Within these truncations, the 174proline (P), 181leucine (L), 185L and 191L were essential for maintaining perinuclear accumulation, thus suggesting an important role of leucine. Alanine (A) mutagenesis assays were employed to generate a series of pUL56 C-terminal mutants on the basis of leucine. Finally, a pUL56 mutant M10 (174P/A-177L/A-181L/A-185L/A-191L/A-194L/A-195I/A-196-197L/A-200L/A) lost Golgi-TGN localization. Thus, our data revealed that the leucine-rich transmembrane helix was responsible for pUL56 Golgi-TGN localization and retention, probably through specific intracellular membrane insertion. CONCLUSION: Our data indicated that the C-terminal transmembrane helix was responsible for the Golgi-TGN localization of pUL56. In addition, the leucines within C-terminal transmembrane helix were essential for maintaining pUL56 Golgi-TGN retention in cells. Further, the pUL56 can induce downregulation of Golgi-associated protein Rab6a.


Assuntos
Complexo de Golgi/fisiologia , Leucina/química , Pseudorraiva , Proteínas Estruturais Virais/metabolismo , Rede trans-Golgi/fisiologia , Animais , Cercopithecus aethiops , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Transporte Proteico , Transfecção , Células Vero , Proteínas Estruturais Virais/genética
10.
Biomed Environ Sci ; 32(5): 324-333, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31217049

RESUMO

OBJECTIVE: To investigate the mechanisms underlying ozone-induced inactivation of poliovirus type 1 (PV1). METHODS: We used cell culture, long-overlapping RT-PCR, and spot hybridization assays to verify and accurately locate the sites of action of ozone that cause PV1 inactivation. We also employed recombinant viral genome RNA infection models to confirm our observations. RESULTS: Our results indicated that ozone inactivated PV1 primarily by disrupting the 5'-non-coding region (5'-NCR) of the PV1 genome. Further study revealed that ozone specifically damaged the 80-124 nucleotide (nt) region in the 5'-NCR. Recombinant viral genome RNA infection models confirmed that PV1 lacking this region was non-infectious. CONCLUSION: In this study, we not only elucidated the mechanisms by which ozone induces PV1 inactivation but also determined that the 80-124 nt region in the 5'-NCR is targeted by ozone to achieve this inactivation.


Assuntos
Genoma Viral/efeitos dos fármacos , Oxidantes Fotoquímicos/farmacologia , Ozônio/farmacologia , Poliovirus/efeitos dos fármacos , Inativação de Vírus , Regiões 5' não Traduzidas , Animais , Cercopithecus aethiops , Células Vero
11.
Int J Nanomedicine ; 14: 4105-4121, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31239670

RESUMO

Purpose: Doxorubicin (DOX) encapsulated O-succinyl chitosan graft Pluronic® F127 (OCP) copolymer nanoparticles conjugated with an anti-HER2 monoclonal antibody were developed as targeted drug delivery vehicles for the treatment of HER2-overexpressing breast cancer. Methods: Five percent and 10% (w/w) of O-succinyl chitosan was grafted onto Pluronic® F127 using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) as mediated cross-linking agents. DOX was added to the copolymer solution to form DOX-nanoparticles before conjugation with anti-HER2 on the surface of the nanoparticles. Results: DOX was encapsulated within the NP matrices at an encapsulation efficiency of 73.69 ± 0.53% to 74.65 ± 0.44% (the initial DOX concentration was 5 µg/mL). Anti-HER2 was successfully conjugated onto the surface of the nanoparticles at a moderately high conjugation efficiency of approximately 57.23 ± 0.38% to 61.20 ± 4.42%. In the in vitro DOX dissolution study, the nanoparticle formulations exhibited a biphasic drug release with an initial burst release followed by a sustained release profile at both pH 5.0 and pH 7.4. The drug was rapidly and completely released from the nanoparticles at pH 5.0. In the in vitro cytotoxicity, the anti-HER2 conjugated OCP copolymer nanoparticles showed the lowest IC50, which indicated an increase in the therapeutic efficacy of DOX to treat human breast cancer cells with the HER2 overexpression. Conclusion: Our study shows that anti-HER2 conjugated OCP copolymer nanoparticles have the potential for the development of anticancer drug carriers.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Quitosana/análogos & derivados , Doxorrubicina/uso terapêutico , Nanopartículas/química , Receptor ErbB-2/metabolismo , Animais , Neoplasias da Mama/patologia , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cercopithecus aethiops , Quitosana/química , Doxorrubicina/farmacologia , Portadores de Fármacos/química , Liberação Controlada de Fármacos , Feminino , Humanos , Ligantes , Células MCF-7 , Micelas , Tamanho da Partícula , Células Vero
12.
Ecotoxicol Environ Saf ; 181: 214-223, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31195230

RESUMO

In the aftermath of the Great East Japan Earthquake of March 11, 2011, marine fish in Kesennuma Bay, Japan, have been contaminated with heavy oil containing polycyclic aromatic hydrocarbons (PAHs). To estimate the risk of six PAHs (benzo[α]pyrene, dibenzothiophene, phenanthrene, 2,3,5-trimethylnaphthalene, acenaphthene, and 1-methylphenanthrene), which have been detected at high levels in the tissues of fish from Kesennuma Bay, we attempted to evaluate the effects of these PAHs on the fish aryl hydrocarbon receptor (AHR) signaling pathway. We initially measured PAH concentrations and cytochrome P4501A catalytic activities (EROD: ethoxyresorufin-O-deethylase and MROD: methoxyresorufin-O-demethylase) as markers of AHR activation in greenlings (Hexagrammos otakii) collected from Kesennuma Bay in 2014. The results showed that alkylated PAH concentrations and EROD/MROD activities were higher in sites close to the oil-spilled sites than in the control site, suggesting AHR activation by spilled alkylated PAHs. We then investigated AHR-mediated responses to these PAHs in the in vitro reporter gene assay system where red seabream (Pagrus major) AHR1 (rsAHR1) or rsAHR2 expression plasmids were transiently transfected into COS-7 cells. The in vitro assay showed rsAHR isoform-, PAH-, and dose-dependent transactivation potencies. The relative effective concentrations of benzo[α]pyrene, dibenzothiophene, phenanthrene, 2,3,5-trimethylnaphthalene, acenaphthene, and 1-methylphenanthrene that induce 20% of the maximum benzo[α]pyrene response (REC20-BaP) for rsAHR1 activation were 0.052, 38, 79, 88, 270 nM, and no response, respectively, and those for rsAHR2 activation were 0.0049, 32, 53, 88, 60 nM, and no response, respectively. The results showed that the REC20-BaP values of benzo[α]pyrene for both the rsAHR1 and rsAHR2 isoforms were lower than the concentrations (0.041-0.20 nM) detected in the muscle tissue of fish from Kesennuma Bay, while the REC20-BaP values of other PAHs were higher than their tissue concentrations. In silico rsAHR homology modeling and subsequent ligand docking simulation analyses indicated that the rsAHR activation potencies of PAHs could be predicted from a rsAHR2 model. This study shows that in vitro and in silico rsAHR analyses may be a useful tool for assessing the risks to fish contaminated with PAHs.


Assuntos
Peixes/metabolismo , Poluição por Petróleo , Hidrocarbonetos Policíclicos Aromáticos/análise , Receptores de Hidrocarboneto Arílico/metabolismo , Animais , Células COS , Cercopithecus aethiops , Simulação por Computador , Citocromo P-450 CYP1A1/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Genes Reporter , Japão , Perciformes/metabolismo , Petróleo , Hidrocarbonetos Policíclicos Aromáticos/química , Hidrocarbonetos Policíclicos Aromáticos/metabolismo , Hidrocarbonetos Policíclicos Aromáticos/toxicidade , Receptores de Hidrocarboneto Arílico/química , Receptores de Hidrocarboneto Arílico/genética , Medição de Risco , Dourada/genética
13.
J Med Microbiol ; 68(7): 1109-1117, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31199226

RESUMO

PURPOSE: Eugenol, the main component of clove bud essential oil (Eugenia caryophyllus), has been linked to antimicrobial, anti-inflammatory, insecticidal and immunomodulatory properties. The purpose of this study was to evaluate the antifungal and cytotoxic activity of eugenol, the essential oil of Eugenia caryophyllus, and some semisynthetic derivatives of eugenol against dermatophytes of the genus Trichophyton. METHODOLOGY: We evaluated the antifungal effect of the compounds, determining the minimum inhibitory concentrations (MICs) by the microdilution method and the minimum fungicidal concentrations by cultures from the inhibitions. Additionally, the inhibition of the radial growth of the mycelium of the dermatophyte fungi was tested by poisoned substrate. Cytotoxicity was measured by the colorimetric method on Vero cells. RESULTS: All of the eugenol compounds tested exhibited antifungal properties, showing MICs of 62.5-500 µg ml-1 , determined within three dermatophyte species: Trichophyton rubrum, Trichophyton mentagrophytes and Trichophyton tonsurans. Among these derivatives, methyl isoeugenol, at concentrations of 300 and 100 µg ml-1, was found to completely inhibit (100 %) radial growth of the mycelium of all three species after 20 days of treatment. Additionally, phenotypic variations related to the decrease in pigment production of T. rubrum were observed after treatment with O-ethyl and O-butyl isoeugenol derivatives. Meanwhile, all of the tested (iso)eugenol molecules exhibited moderate toxicity in Vero cells [50 % cytotoxic concentration (the concentration required for a 50 % reduction in cell viability; CC50): 54.06-265.18 µg ml-1 ). CONCLUSION: The results suggest that the semisynthetic eugenol derivatives (SEDs) show promising antifungal activity and selectivity against dermatophyte fungi.


Assuntos
Antifúngicos/farmacologia , Eugenol/análogos & derivados , Eugenol/farmacologia , Trichophyton/efeitos dos fármacos , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cercopithecus aethiops , Eugenol/toxicidade , Testes de Sensibilidade Microbiana
14.
Vet Microbiol ; 233: 140-146, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31176400

RESUMO

Porcine reproductive and respiratory syndrome (PRRS) is caused by PRRS virus (PRRSV), and is characterized by respiratory diseases in piglet and reproductive disorders in sow. Identification of sustainable and effective measures to mitigate PRRSV transmission is a pressing problem. The nucleocapsid (N) protein of PRRSV plays a crucial role in inhibiting host innate immunity during PRRSV infection. In the current study, a new host-restricted factor, tripartite motif protein 25 (TRIM25), was identified as an inhibitor of PRRSV replication. Co-immunoprecipitation assay indicated that the PRRSV N protein interferes with TRIM25-RIG-I interactions by competitively interacting with TRIM25. Furthermore, N protein inhibits the expression of TRIM25 and TRIM25-mediated RIG-I ubiquitination to suppress interferon ß production. Furthermore, with increasing TRIM25 expression, the inhibitory effect of N protein on the ubiquitination of RIG-I diminished. These results indicate for the first time that TRIM25 inhibits PRRSV replication and that the N protein antagonizes the antiviral activity by interfering with TRIM25-mediated RIG-I ubiquitination. This not only provides a theoretical basis for the development of drugs to control PRRSV replication, but also better explains the mechanism through which the PRRSV N protein inhibits innate immune responses of the host.


Assuntos
Proteína DEAD-box 58/metabolismo , Proteínas do Nucleocapsídeo/metabolismo , Vírus da Síndrome Respiratória e Reprodutiva Suína/metabolismo , Proteínas com Motivo Tripartido/antagonistas & inibidores , Proteínas com Motivo Tripartido/genética , Ubiquitinação , Motivos de Aminoácidos , Animais , Linhagem Celular , Cercopithecus aethiops , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Proteínas do Nucleocapsídeo/genética , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Ligação Proteica , RNA Interferente Pequeno , Transdução de Sinais/imunologia , Suínos , Transfecção , Replicação Viral
15.
Molecules ; 24(9)2019 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-31052590

RESUMO

Type 2 diabetic patients possess a two to four fold-increased risk for Cardiovascular Diseases (CVD). Hyperglycemia, oxidative stress associated with endothelial dysfunction and dyslipidemia are regarded as pro-atherogenic mechanisms of CVD. In this study, high-fat diet-induced diabetic and non-diabetic vervet monkeys were treated with 90 mg/kg of aspalathin-rich green rooibos extract (Afriplex GRT) for 28 days, followed by a 1-month wash-out period. Supplementation showed improvements in both the intravenous glucose tolerance test (IVGTT) glycemic area under curve (AUC) and total cholesterol (due to a decrease of the low-density lipoprotein [LDL]) values in diabetics, while non-diabetic monkeys benefited from an increase in high-density lipoprotein (HDL) levels. No variation of plasma coenzyme Q10 (CoQ10) were found, suggesting that the LDL-lowering effect of Afriplex GRT could be related to its ability to modulate the mevalonate pathway differently from statins. Concerning the plasma oxidative status, a decrease in percentage of oxidized CoQ10 and circulating oxidized LDL (ox-LDL) levels after supplementation was observed in diabetics. Finally, the direct correlation between the amount of oxidized LDL and total LDL concentration, and the inverse correlation between ox-LDL and plasma CoQ10 levels, detected in the diabetic monkeys highlighted the potential cardiovascular protective role of green rooibos extract. Taken together, these findings suggest that Afriplex GRT could counteract hyperglycemia, oxidative stress and dyslipidemia, thereby lowering fundamental cardiovascular risk factors associated with diabetes.


Assuntos
Chalconas/farmacologia , LDL-Colesterol/sangue , Diabetes Mellitus Experimental/etiologia , Diabetes Mellitus Experimental/metabolismo , Dieta Hiperlipídica/efeitos adversos , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Animais , Biomarcadores , Glicemia/efeitos dos fármacos , Cercopithecus aethiops , Diabetes Mellitus Experimental/tratamento farmacológico , Modelos Animais de Doenças , Feminino , Hipoglicemiantes/química , Hipoglicemiantes/farmacologia , Lipídeos/sangue , Lipoproteínas LDL/sangue , Masculino , Extratos Vegetais/farmacologia , Ubiquinona/análogos & derivados , Ubiquinona/sangue
16.
Fitoterapia ; 136: 104179, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31121252

RESUMO

Essential oils, mixtures of volatile compounds, are targets of research for new antimicrobial drugs. In order to verify the potential from species of the Nectandra genus, the present study evaluated the essential oils from Nectandra amazonum, Nectandra cuspidata, Nectandra gardineri, Nectandra hihua and Nectandra megapotamica to prospect samples with high concentration of a component and its antibacterial, antibiofilm and anti-Trichomonas vaginalis activities. The essential oils from the leaves and barks were extracted by steam distillation and analyzed by gas chromatography coupled to mass spectrometry (GC-MS). The concentrations of 10 and 100 µg/mL of the essential oil were evaluated and the inhibition of bacterial growth and biofilm formation were measured, while for the evaluation of anti-T. vaginalis trophozoite viability, the concentrations from 7.8 to 1000 µg/mL were tested. Seventy-three compounds were identified from essential oils, highlighted bicyclogermacrene (up to 49.9%), elemicin (up to 42.4%), intermedeol (up to 58.2%), (E)-asarone (up to 45.9%) and (+)-α-bisabolol (up to 93.7%). The essential oil from N. megapotamica leaves presented 93.7% of (+)-α-bisabolol and demonstrated the high capacity of inhibition of the biofilm formation, in particular, against Staphylococcus aureus methicillin resistant (MRSA) and Pseudomonas aeruginosa. This sample also had significant activity against T. vaginalis (IC50 of 98.7 µg/mL) and demonstrated cytotoxic and hemolytic effects in Vero cells and human erythrocytes. In general, the Nectandra genus revealed high chemical variability and a N. megapotamica specimen accumulated a compound on high concentration with great potential for biotechnological exploration as a new antibiofilm and anti-T. vaginalis.


Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Lauraceae/química , Óleos Voláteis/farmacologia , Sesquiterpenos/farmacologia , Trichomonas vaginalis/efeitos dos fármacos , Animais , Antiprotozoários/farmacologia , Brasil , Cercopithecus aethiops , Eritrócitos/efeitos dos fármacos , Humanos , Compostos Fitoquímicos/farmacologia , Casca de Planta/química , Folhas de Planta/química , Células Vero
17.
Nat Protoc ; 14(6): 1772-1802, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31101905

RESUMO

Membrane curvatures are involved in essential cellular processes, such as endocytosis and exocytosis, in which they are believed to act as microdomains for protein interactions and intracellular signaling. These membrane curvatures appear and disappear dynamically, and their locations are difficult or impossible to predict. In addition, the size of these curvatures is usually below the diffraction limit of visible light, making it impossible to resolve their values using live-cell imaging. Therefore, precise manipulation of membrane curvature is important to understanding how membrane curvature is involved in intracellular processes. Recent studies show that membrane curvatures can be induced by surface topography when cells are in direct contact with engineered substrates. Here, we present detailed procedures for using nanoscale structures to manipulate membrane curvatures and probe curvature-induced phenomena in live cells. We first describe detailed procedures for the design of nanoscale structures and their fabrication using electron-beam (E-beam) lithography. The fabrication process takes 2 d, but the resultant chips can be cleaned and reused repeatedly over the course of 2 years. Then we describe how to use these nanostructures to manipulate local membrane curvatures and probe intracellular protein responses, discussing surface coating, cell plating, and fluorescence imaging in detail. Finally, we describe a procedure to characterize the nanostructure-cell membrane interface using focused ion beam and scanning electron microscopy (FIB-SEM). Nanotopography-based methods can induce stable membrane curvatures with well-defined curvature values and locations in live cells, which enables the generation of a library of curvatures for probing curvature-related intracellular processes.


Assuntos
Técnicas de Cultura de Células/instrumentação , Membrana Celular/metabolismo , Micromanipulação/instrumentação , Nanoestruturas , Animais , Células COS , Comunicação Celular , Linhagem Celular , Membrana Celular/ultraestrutura , Cercopithecus aethiops , Endocitose , Desenho de Equipamento , Humanos , Camundongos , Microscopia Eletrônica de Varredura , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Imagem Óptica , Proteínas/metabolismo , Propriedades de Superfície
18.
Int J Mol Sci ; 20(9)2019 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-31083458

RESUMO

To appraise how evolutionary processes, such as gene duplication and loss, influence an organism's xenobiotic sensitivity is a critical question in toxicology. Of particular importance are gene families involved in the mediation of detoxification responses, such as members of the nuclear receptor subfamily 1 group I (NR1I), the pregnane X receptor (PXR), and the constitutive androstane receptor (CAR). While documented in multiple vertebrate genomes, PXR and CAR display an intriguing gene distribution. PXR is absent in birds and reptiles, while CAR shows a tetrapod-specific occurrence. More elusive is the presence of PXR and CAR gene orthologs in early branching and ecologically-important Chondrichthyes (chimaeras, sharks and rays). Therefore, we investigated various genome projects and use them to provide the first identification and functional characterization of a Chondrichthyan PXR from the chimaera elephant shark (Callorhinchus milii, Holocephali). Additionally, we substantiate the targeted PXR gene loss in Elasmobranchii (sharks and rays). Compared to other vertebrate groups, the chimaera PXR ortholog displays a diverse expression pattern (skin and gills) and a unique activation profile by classical xenobiotic ligands. Our findings provide insights into the molecular landscape of detoxification mechanisms and suggest lineage-specific adaptations in response to xenobiotics in gnathostome evolution.


Assuntos
Elasmobrânquios/classificação , Elasmobrânquios/genética , Evolução Molecular , Redes Reguladoras de Genes , Filogenia , Receptor de Pregnano X/genética , Animais , Células COS , Cercopithecus aethiops , Genes Reporter , Inativação Metabólica/genética , Luciferases/metabolismo , Receptor de Pregnano X/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Sintenia/genética , Ativação Transcricional/genética
19.
Prep Biochem Biotechnol ; 49(7): 686-694, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31035907

RESUMO

In mammalian cell culture technology, viral contamination is one of the main challenges; and, so far, various strategies have been taken to remove or inactivate viruses in the cell-line production process. The suitability and feasibility of each method are determined by different factors including effectiveness in target virus inactivation, maintaining recombinant protein stability, easiness-in terms of the process condition, cost-effectiveness, and eco-friendliness. In this research, Taguchi design-of-experiments (DOE) methodology was used to optimize a non-detergent viral inactivation method via considering four factors of temperature, time, pH, and alcohol concentration in an unbiased (orthogonal) fashion with low influence of nuisance factors. Herpes Simplex Virus-1 (HSV1) and Vero cell-line were used as models for enveloped viruses and cell-line, respectively. Examining the cytopathic effects (CPE) in different dilutions showed that pH (4), alcohol (15%), time (120 min), and temperature (25 °C) were the optimal points for viral inactivation. Evaluating the significance of each parameter in the HSV-1 inactivation using Taguchi and ANOVA analyses, the contributions of pH, alcohol, temperature and time were 56.5%, 19.2%, 12%, and 12%, respectively. Examining the impact of the optimal viral treatment condition on the stability of model recombinant protein-recombinant human erythropoietin, no destabilization was detected.


Assuntos
Técnicas de Cultura de Células/métodos , Herpesvirus Humano 1/fisiologia , Inativação de Vírus , Álcoois/metabolismo , Animais , Técnicas de Cultura de Células/instrumentação , Cercopithecus aethiops , Desenho de Equipamento , Herpesvirus Humano 1/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Projetos de Pesquisa , Temperatura Ambiente , Células Vero , Ensaio de Placa Viral , Inativação de Vírus/efeitos dos fármacos
20.
Microb Pathog ; 132: 215-221, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31075431

RESUMO

Enterovirus 71 (EV71) and coxsackievirus A16 (CA16) are common causative agents of mild and self-limiting symptoms of childhood hand, foot, and mouth disease (HFMD). However, some EV71-infected HFMD patients can develop severe neurological and/or fatal cardiopulmonary complications. In Thailand, HFMD associated with the EV71 subgenotypes C4a and B5 were reported to be associated with diverse outcomes. However, variations in enterovirus subgenotypes and virulence factors have not been fully elucidated; this study elucidated these variations in peripheral blood mononuclear cells (PBMCs) exposed to different subgenotypes of isolated enteroviruses for 24 and 48 h. Following infection, viral titers were determined by plaque assay. Infected cells and intracellular cytokines were quantified using flow cytometry, and multiplex assay was used to examine cytokine release. All isolated subgenotypes showed replication capability in PBMCs; specifically, the replication titer of EV71 C4a tended to be higher than titers of EV71 B5 and CA16. Additionally, the infectivity of EV71 B5 was higher in monocytes than in lymphocytes. Compared with EV71 B5, EV71 C4a and CA16 had greater ability to induce intra- and extracellular cytokine responses. These findings provide new insights into variations in cellular immune responses to different EV71 subgenotypes isolated from Thai patients, which should be considered for the development of vaccines and therapeutic agents.


Assuntos
Citocinas/metabolismo , Enterovirus Humano A/imunologia , Enterovirus Humano A/patogenicidade , Infecções por Enterovirus/imunologia , Adulto , Animais , Anticorpos Monoclonais , Cercopithecus aethiops , Quimopapaína/metabolismo , Enterovirus/imunologia , Enterovirus/isolamento & purificação , Enterovirus Humano A/isolamento & purificação , Infecções por Enterovirus/virologia , Feminino , Humanos , Imunidade Celular/imunologia , Leucócitos Mononucleares , Masculino , Tailândia , Células Vero , Virulência , Adulto Jovem
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