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1.
Chaos ; 30(1): 013126, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32013481

RESUMO

Networks of coupled systems may exhibit a form of incomplete synchronization called partial synchronization or cluster synchronization, which refers to the situation where only some, but not all, systems exhibit synchronous behavior. Moreover, due to perturbations or uncertainties in the network, exact partial synchronization in the sense that the states of the systems within each cluster become identical, cannot be achieved. Instead, an approximate synchronization may be observed, where the states of the systems within each cluster converge up to some bound, and this bound tends to zero if (the size of) the perturbations tends to zero. In order to derive sufficient conditions for this robustified notion of synchronization, which we refer to as practical partial synchronization, first, we separate the synchronization error dynamics from the network dynamics and interpret them in terms of a nonautonomous system of delay differential equations with a bounded additive perturbation. Second, by assessing the practical stability of this error system, conditions for practical partial synchronization are derived and formulated in terms of linear matrix inequalities. In addition, an explicit relation between the size of perturbation and the bound of the synchronization error is provided.

2.
Cell Death Differ ; 2020 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-32015502

RESUMO

Angiogenesis plays crucial roles in maintaining the complex operation of central nervous system (CNS) development. The architecture of communication between neurogenesis and angiogenesis is essential to maintain normal brain development and function. Hence, any disruption of neuron-vascular communications may lead to the pathophysiology of cerebrovascular diseases and blood-brain barrier (BBB) dysfunction. Here we demonstrate that neural differentiation and communication are required for vascular development. Regarding the cellular and molecular mechanism, our results show that PRDM16 activity determines the production of mature neurons and their specific positions in the neocortex. In the cortical plate (CP), aberrant neurons fail to secrete modular calcium-binding protein 1 (SMOC1), an important neuronal signal that participates in neurovascular communication to regulate CNS angiogenesis. Neuronal SMOC1 interacts with TGFBR1 by activating the transcription factors phospho-Smad2/3 to convey intercellular signals to endothelial cells (ECs) in the TGF-ß-Smad signaling pathway. Together, our results highlight a crucial coordinated neurovascular development process orchestrated by PRDM16 and reveal the importance of intimate communication for building the neurovascular network during brain development.

3.
Onco Targets Ther ; 12: 7823-7831, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31576140

RESUMO

Background: Hepatocellular carcinoma is a common malignant cancer and the second most common cause of cancer-related deaths worldwide. Collagen triple helix repeat containing 1 (CTHRC1) has been increasingly reported to be involved in tumorigenesis and/or tumor progression. However, limited data are available regarding the role of CTHRC1 in hepatocellular carcinoma. Methods: Paraffin-embedded specimens from a total of 29 patients with HCC were collected in our study. The expression of CTHRC1 in hepatocellular carcinoma was evaluated using immunohistochemistry and bioinformatics analysis. Furthermore, Spearman analysis was performed to identify factors of correlation between CTHRC1 and clinicopathological features. Survival curves for hepatocellular carcinoma were produced using the Kaplan-Meier method and the log rank test. Results: In this study, we confirmed that CTHRC1 is highly expressed in tissues and hepatoma cell lines. The statistical analysis revealed that the levels of CTHRC1 were significantly correlated with cirrhosis (P=0.024), tumor size (P=0.006), vascular invasion (P<0.001), TNM stage (P<0.001), and BCLC stage (P<0.001). High expression of CTHRC1 in hepatocellular carcinoma tissues is significantly associated with poor survival. Conclusion: CTHRC1 may serve as a prognostic biomarker for hepatocellular carcinoma.

4.
Nucleic Acids Res ; 46(17): 8817-8831, 2018 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-29982651

RESUMO

Astrocytes play crucial roles in the central nervous system, and defects in astrocyte function are closely related to many neurological disorders. Studying the mechanism of gliogenesis has important implications for understanding and treating brain diseases. Epigenetic regulations have essential roles during mammalian brain development. Here, we demonstrate that histone H2A.Z.1 is necessary for the specification of multiple neural precursor cells (NPCs) and has specialized functions that regulate gliogenesis. Depletion of H2A.Z.1 suppresses gliogenesis and results in reduced astrocyte differentiation. Additionally, H2A.Z.1 regulates the acetylation of H3K56 (H3K56ac) by cooperating with the chaperone of ASF1a. Furthermore, RNA-seq data indicate that folate receptor 1 (FOLR1) participates in gliogenesis through the JAK-STAT signaling pathway. Taken together, our results demonstrate that H2A.Z.1 is a key regulator of gliogenesis because it interacts with ASF1a to regulate H3K56ac and then directly affects the expression of FOLR1, which acts as a signal-transducing component of the JAK-STAT signaling pathway.


Assuntos
Receptor 1 de Folato/genética , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Neurogênese/genética , Neuroglia/fisiologia , Acetilação , Animais , Astrócitos/fisiologia , Proteínas de Ciclo Celular , Células Cultivadas , Proteínas Cromossômicas não Histona/metabolismo , Embrião de Mamíferos , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Histonas/fisiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Chaperonas Moleculares , Células-Tronco Neurais , Gravidez , Transdução de Sinais/genética , Transcrição Genética
5.
J Cell Biol ; 217(10): 3464-3479, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30037926

RESUMO

In mammals, a constant body temperature is an important basis for maintaining life activities. Here, we show that when pregnant mice are subjected to cold stress, the expression of RBM3, a cold-induced protein, is increased in the embryonic brain. When RBM3 is knocked down or knocked out in cold stress, embryonic brain development is more seriously affected, exhibiting abnormal neuronal differentiation. By detecting the change in mRNA expression during maternal cold stress, we demonstrate that Yap and its downstream molecules are altered at the RNA level. By analyzing RNA-binding motif of RBM3, we find that there are seven binding sites in 3'UTR region of Yap1 mRNA. Mechanistically, RBM3 binds to Yap1-3'UTR, regulates its stability, and affects the expression of YAP1. RBM3 and YAP1 overexpression can partially rescue the brain development defect caused by RBM3 knockout in cold stress. Collectively, our data demonstrate that cold temperature affects brain development, and RBM3 acts as a key protective regulator in cold stress.


Assuntos
Regiões 3' não Traduzidas , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Encéfalo/embriologia , Resposta ao Choque Frio , Embrião de Mamíferos/embriologia , Neurogênese , Fosfoproteínas/biossíntese , Estabilidade de RNA , Proteínas de Ligação a RNA/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas de Ciclo Celular , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Endogâmicos ICR , Camundongos Knockout , Motivos de Nucleotídeos , Fosfoproteínas/genética , Proteínas de Ligação a RNA/genética
6.
J Genet ; 95(4): 751-760, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27994173

RESUMO

The B-cell activating factor (BAFF) is a member of tumour necrosis factor (TNF) superfamily that specifically regulates B lymphocyte proliferation and survival. Excess BAFF leads to overproduction of antibodies for secretion, anti-dsDNA antibodies and a lupus-like syndrome in mice. To investigate whether transgenic overexpression of the zebrafish BAFF leads to immunoglobulin changes and/or early maturing of the immune system, a Tol2-GFP-2A-BAFF/His recombinant plasmid was constructed by inserting a 2A peptide between the green fluorescent protein (GFP) and BAFF sequences. Functional GFP and BAFF proteins were expressed separately and confirmed in HeLa cells. The relative expression of immune-related genes (IgLC-1, IgLC-2, IgLC-3, IgD, IgM and IL-4), early lymphoid markers (Ikaros, Rag-1 and TCRAC), and the protooncogene Bcl-2 were evaluated by quantitative polymerase chain reaction (PCR) in F0 founder of transgenic zebrafish juveniles and adults. Ectopic expression of BAFF in adults was confirmed using Western blots and was shown to upregulate IgLC-1, IgLC-2, IgD, IgM, IgZ/T, Ikaros, Rag-1, TCRAC, IL-4 and Bcl-2 expression in juveniles on day 21 and IgLC-1, IgLC-2, IgD, IgM,IgZ/T, Rag-1, TCRAC and Bcl-2 expression in zebrafish three months postfertilization. The relative titers of specific IgM against Edwardsiella tarda WED were assessed using modified enzyme-linked immunosorbent assay (ELISA) with the whole body homogenate of zebrafish and demonstrated a significant increase in BAFF-transgenic group. Therefore, our findings provided novel insight into further exploration of modulating adaptive immunity and studying autoimmune diseases caused by regulating BAFF.


Assuntos
Fator Ativador de Células B/genética , Expressão Gênica , Imunidade/genética , Peixe-Zebra/genética , Peixe-Zebra/imunologia , Animais , Animais Geneticamente Modificados , Linhagem Celular , Clonagem Molecular , Regulação da Expressão Gênica , Ordem dos Genes , Genes Reporter , Vetores Genéticos/genética , Humanos , Transfecção
7.
Infect Immun ; 82(9): 3855-66, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24980971

RESUMO

Salmonella enterica serovar Typhimurium is a Gram-negative food-borne pathogen that is a major cause of acute gastroenteritis in humans. The ability of the host to control such bacterial pathogens may be influenced by host immune status and by concurrent infections. Helminth parasites are of particular interest in this context because of their ability to modulate host immune responses and because their geographic distribution coincides with those parts of the world where infectious gastroenteritis is most problematic. To test the hypothesis that helminth infection may negatively regulate host mucosal innate immunity against bacterial enteropathogens, a murine coinfection model was established by using the intestinal nematode Heligmosomoides polygyrus and S. Typhimurium. We found that mice coinfected with S. Typhimurium and H. polygyrus developed more severe intestinal inflammation than animals infected with S. Typhimurium alone. The enhanced susceptibility to Salmonella-induced intestinal injury in coinfected mice was found to be associated with diminished neutrophil recruitment to the site of bacterial infection that correlated with decreased expression of the chemoattractants CXCL2/macrophage inflammatory protein 2 (MIP-2) and CXCL1/keratinocyte-derived chemokine (KC), poor control of bacterial replication, and exacerbated intestinal inflammation. The mechanism of helminth-induced inhibition of MIP-2 and KC expression involved interleukin-10 (IL-10) and, to a lesser extent, IL-4 and IL-13. Ly6G antibody-mediated depletion of neutrophils reproduced the adverse effects of H. polygyrus on Salmonella infection. Our results suggest that impaired neutrophil recruitment is an important contributor to the enhanced severity of Salmonella enterocolitis associated with helminth coinfection.


Assuntos
Coinfecção/imunologia , Imunidade Inata/imunologia , Inflamação/imunologia , Mucosa Intestinal/imunologia , Salmonelose Animal/imunologia , Salmonella typhimurium/imunologia , Animais , Quimiocina CXCL1/imunologia , Quimiocina CXCL2/imunologia , Coinfecção/microbiologia , Modelos Animais de Doenças , Feminino , Inflamação/microbiologia , Interleucinas/imunologia , Mucosa Intestinal/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Nematospiroides dubius/imunologia , Neutrófilos/imunologia , Neutrófilos/microbiologia , Salmonelose Animal/microbiologia , Infecções por Strongylida/imunologia , Infecções por Strongylida/microbiologia
8.
PLoS Negl Trop Dis ; 8(7): e2987, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25010669

RESUMO

Infections with intestinal helminth and bacterial pathogens, such as enteropathogenic Escherichia coli, continue to be a major global health threat for children. To determine whether and how an intestinal helminth parasite, Heligomosomoides polygyrus, might impact the TLR signaling pathway during the response to a bacterial enteropathogen, MyD88 knockout and wild-type C57BL/6 mice were infected with H. polygyrus, the bacterial enteropathogen Citrobacter rodentium, or both. We found that MyD88 knockout mice co-infected with H. polygyrus and C. rodentium developed more severe intestinal inflammation and elevated mortality compared to the wild-type mice. The enhanced susceptibility to C. rodentium, intestinal injury and mortality of the co-infected MyD88 knockout mice were found to be associated with markedly reduced intestinal phagocyte recruitment, decreased expression of the chemoattractant KC, and a significant increase in bacterial translocation. Moreover, the increase in bacterial infection and disease severity were found to be correlated with a significant downregulation of antimicrobial peptide expression in the intestinal tissue in co-infected MyD88 knockout mice. Our results suggest that the MyD88 signaling pathway plays a critical role for host defense and survival during helminth and enteric bacterial co-infection.


Assuntos
Infecções por Enterobacteriaceae , Helmintíase , Inflamação , Enteropatias , Fator 88 de Diferenciação Mieloide/genética , Animais , Citrobacter rodentium , Infecções por Enterobacteriaceae/genética , Infecções por Enterobacteriaceae/microbiologia , Infecções por Enterobacteriaceae/parasitologia , Feminino , Helmintíase/genética , Helmintíase/microbiologia , Helmintíase/parasitologia , Inflamação/genética , Inflamação/microbiologia , Inflamação/parasitologia , Enteropatias/genética , Enteropatias/microbiologia , Enteropatias/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
9.
Vet Parasitol ; 194(2-4): 183-5, 2013 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-23465747

RESUMO

To obtain novel antigen genes for use as an anti-tumor vaccine, a Trichinella spiralis cDNA expression library was constructed from muscle larvae RNA and screened with sera from Balb/C mice injected with Sp2/0 myeloma cells. One positive clone was obtained after three rounds of immunoscreening of the cDNA expression library and was subsequently excised in vivo using the ExAssist helper phage with SOLR strain. A full-length gene was amplified using 5'-RACE technology and analyzed by BLAST, Protein Analysis System of ELM, and DNAStar Software. The sequencing results showed that the fragment was 569 bp in length and contained an open reading frame. It was predicted that the full-length gene encoded 136 amino acids. This gene, TS2, contained four putative N-Arg dibasic convertase (nardilysine) cleavage sites, one peptide C-terminal amidation site, and one glycosaminoglycan attachment site. Six antibody epitopes were predicted by bioinformatic analysis.


Assuntos
Antígenos de Helmintos/genética , Vacinas Anticâncer/genética , Soros Imunes/imunologia , Trichinella spiralis/genética , Proteínas Supressoras de Tumor/genética , Animais , Antígenos de Helmintos/imunologia , Sítios de Ligação de Anticorpos/genética , Sítios de Ligação de Anticorpos/imunologia , Linhagem Celular Tumoral , Biologia Computacional , Mapeamento de Epitopos , Epitopos/genética , Epitopos/imunologia , Biblioteca Gênica , Proteínas de Helminto/genética , Proteínas de Helminto/imunologia , Larva/genética , Camundongos , Camundongos Endogâmicos BALB C , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/patologia , RNA de Helmintos/genética , Trichinella spiralis/imunologia , Proteínas Supressoras de Tumor/imunologia
10.
J Immunol ; 189(3): 1459-66, 2012 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-22732589

RESUMO

Autophagy is an important mechanism used by macrophages to kill intracellular pathogens. The results reported in this study demonstrate that autophagy is also involved in the macrophage killing of the extracellular enteropathogen Citrobacter rodentium after phagocytosis. The process was significantly impaired in macrophages isolated from mice chronically infected with the helminth parasite Heligmosomoides polygyrus. The H. polygyrus-mediated inhibition of autophagy was Th2 dependent because it was not observed in macrophages isolated from helminth-infected STAT6-deficient mice. Moreover, autophagy of Citrobacter was inhibited by treating macrophages with IL-4 and IL-13. The effect of H. polygyrus on autophagy was associated with decreased expression and processing of L chain protein 3 (LC3), a key component of the autophagic machinery. The helminth-induced inhibition of LC3 expression and processing was STAT6 dependent and could be recapitulated by treatment of macrophages with IL-4 and IL-13. Knockdown of LC3 significantly inhibited autophagic killing of Citrobacter, attesting to the functional importance of the H. polygyrus-mediated downregulation of this process. These observations reveal a new aspect of the immunosuppressive effects of helminth infection and provide mechanistic insights into our earlier finding that H. polygyrus significantly worsens the in vivo course of Citrobacter infection.


Assuntos
Autofagia/imunologia , Citrobacter rodentium/imunologia , Infecções por Enterobacteriaceae/imunologia , Macrófagos Peritoneais/imunologia , Nematospiroides dubius/imunologia , Infecções por Strongylida/imunologia , Animais , Citrobacter rodentium/crescimento & desenvolvimento , Citrobacter rodentium/patogenicidade , Regulação para Baixo/imunologia , Infecções por Enterobacteriaceae/parasitologia , Infecções por Enterobacteriaceae/patologia , Feminino , Macrófagos Peritoneais/microbiologia , Macrófagos Peritoneais/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/biossíntese , Proteínas Associadas aos Microtúbulos/genética , Nematospiroides dubius/crescimento & desenvolvimento , Nematospiroides dubius/patogenicidade , Processamento de Proteína Pós-Traducional/imunologia , Infecções por Strongylida/microbiologia , Infecções por Strongylida/patologia
11.
Artigo em Chinês | MEDLINE | ID: mdl-21823320

RESUMO

OBJECTIVE: To clone and express S-dsRNA gene of Cryptosporidium parvum virus, and investigate the reactogenicity of the recombinant. METHODS: Total RNA was extracted from Cryptosporidium parvum and S-dsRNA gene was amplified by RT-PCR. The PCR product was cloned into pET-28a(+) expression vector. The recombinant plasmid pET-28a(+)-S was transformed into E. coli BL21 (DE3) and induced with IPTG. The expression situation of recombinant protein was analyzed by SDS-PAGE. Its reactogenicity was examined by Western blotting analysis. RESULTS: pET-28a (+)-S was identified by PCR and double endonuclease digestion. SDS-PAGE result showed that the recombinant protein (M, 37,000) was expressed in the form of inclusion body. High level expression of recombinant protein was found at 1 mmol/L IPTG condition after incubation at 37 degrees C for 4 h and reached up to 72.6% of the total protein. The protein was recognized by the antisera from mice immunized with antigens from Cryptosporidium parvum oocysts. CONCLUSION: The S-dsRNA gene of Cryptosporidium parvum virus has been expressed with adequate reactogenicity.


Assuntos
Proteínas do Capsídeo/genética , Proteínas do Capsídeo/isolamento & purificação , Cryptosporidium parvum/virologia , Vírus de RNA/genética , RNA Viral/genética , Animais , Feminino , Expressão Gênica , Vetores Genéticos , Camundongos , Camundongos Endogâmicos BALB C , Plasmídeos , RNA de Cadeia Dupla
12.
Protein Expr Purif ; 77(2): 207-13, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21300155

RESUMO

The alpha and epsilon toxins are 2 of the 4 major lethal toxins of the pathogen Clostridium perfringens. In this study, the expression of the epsilon toxin (etx) gene of C. perfringens was optimized by replacing rare codons with high-frequency codons, and the optimized gene was synthesized using overlapping PCR. Then, the etx gene or the alpha-toxin gene (cpa) was individually inserted into the pTIG-Trx expression vector with a hexahistidine tag and a thioredoxin (Trx) to facilitate their purification and induce the expression of soluble proteins. The recombinant alpha toxin (rCPA) and epsilon toxin (rETX) were highly expressed as soluble forms in the recipient Escherichia coli BL21 strain, respectively. The rCPA and rETX were purified using Ni(2+)-chelating chromatography and size-exclusion chromatography. And the entire purification process recovered about 40% of each target protein from the starting materials. The purified target toxins formed single band at about 42kDa (rCPA) or 31kDa (rETX) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their functional activity was confirmed by bioactivity assays. We have shown that the production of large amounts of soluble and functional proteins by using the pTIG-Trx vector in E. coli is a good alternative for the production of native alpha and epsilon toxins and could also be useful for the production of other toxic proteins with soluble forms.


Assuntos
Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Fosfolipases Tipo C/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Toxinas Bacterianas/genética , Toxinas Bacterianas/isolamento & purificação , Sequência de Bases , Bioensaio , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/isolamento & purificação , Linhagem Celular , Cromatografia de Afinidade , Cromatografia em Gel , Clonagem Molecular , Clostridium perfringens/química , Cães , Eletroforese em Gel de Poliacrilamida , Eritrócitos , Escherichia coli , Vetores Genéticos/metabolismo , Hemólise , Histidina/metabolismo , Camundongos , Dados de Sequência Molecular , Oligopeptídeos/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Solubilidade , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/isolamento & purificação
13.
Exp Parasitol ; 127(4): 784-8, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21232537

RESUMO

To investigate the presence of myeloma-associated antigens in Trichinella spiralis and their anti-tumor effect, cross-immune responses between antigens of the myeloma cell SP2/0 versus positive sera to T. spiralis, and antigens of T. spiralis versus positive sera to myeloma cell SP2/0 were determined using T. spiralis and myeloma specific enzyme-linked immunosorbent assays (ELISA). The myeloma-associated antigens in T. spiralis were separated by ultrafiltration and 2-D electrophoresis, and the amino acid sequences and molecular weights were determined by spectrometry. An obvious reaction was found between a 33 kDa antigen and positive sera, and the major component of the antigen was tropomyosin (TM), which is an surface acidic protein with 284 amino acids. Mice were immunized with TM to determine the anti-tumor effect in vivo. The results showed that CD4(+), CD8(+) T lymphocyte, and CD19(+) B lymphocyte were significantly increased (P<0.05). The anti-tumor effects were significantly different between mice immunized with the antigens or adjuvant alone (P<0.05), while the difference between mice immunized with antigens and whole T. spiralis was not significant (P>0.05). The results indicated that TM identified in this study may play a role in eliciting cross-protective immunity.


Assuntos
Antígenos de Helmintos/análise , Antígenos de Neoplasias/análise , Mieloma Múltiplo/imunologia , Proteínas do Mieloma/análise , Trichinella spiralis/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Helmintos/química , Antígenos de Helmintos/imunologia , Antígenos de Neoplasias/química , Antígenos de Neoplasias/imunologia , Western Blotting , Linhagem Celular Tumoral , Reações Cruzadas , Eletroforese em Gel Bidimensional , Ensaio de Imunoadsorção Enzimática , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Proteínas do Mieloma/química , Proteínas do Mieloma/imunologia , Distribuição Aleatória , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
14.
Vet Parasitol ; 173(1-2): 11-8, 2010 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-20594647

RESUMO

Cryptosporidium parvum and Giardia duodenalis are the most frequently identified enteric parasites associated with diarrhea-causing disease outbreaks, and many non-parvum species of Cryptosporidium also can replicate and cause illness in mammals including humans. In this study, we describe a novel multiplex PCR coupled with Luminex assay for the identification of Cryptosporidium spp., C. parvum and G. duodenalis in a rapid manner. The multiplex PCR for the simultaneous detection of Cryptosporidium and Giardia was developed using three pairs of biotinylated primers which amplify 424, 223 and 267 bp products from the U1 small nuclear ribonucleoprotein (U1 snr) gene, 18S rRNA gene of Cryptosporidium and the beta-giardin gene of Giardia, respectively. The genus and species-specific capture probes linked to carboxylated Luminex microspheres hybridized to the multiplex PCR amplicons to enhance sensitivity and specificity. The conditions of multiplex PCR and Luminex hybridization reaction were optimized to enable the minimum detection limits of 5x10(-6), 5x10(-6), and 5x10(-6) ng DNAs (corresponding approximately to 0.1 oocyst/cyst). The Luminex approach proved to be 100% specific and accurate by testing a total of 240 fecal samples compared with microscopic examination of fecal smears and further modified acid-fast staining or iodine-staining observation. The established assay offers the potential for rapid detection of Cryptosporidium spp., C. parvum and G. duodenalis in fecal and environmental samples.


Assuntos
Cryptosporidium/isolamento & purificação , Giardia/isolamento & purificação , Medições Luminescentes/veterinária , Reação em Cadeia da Polimerase/veterinária , Animais , Criptosporidiose/diagnóstico , Criptosporidiose/veterinária , DNA de Protozoário/isolamento & purificação , Fezes/parasitologia , Giardíase/diagnóstico , Giardíase/veterinária , Reação em Cadeia da Polimerase/métodos , Sensibilidade e Especificidade
15.
Exp Parasitol ; 123(3): 212-7, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19619539

RESUMO

Giardia canis virus (GCV) is a double-stranded RNA (dsRNA) virus of the family Totiviridae. In this study, the full length cDNA of the G. canis virus was constructed in pPoly2/sfinot vector and RNA was transcribed in vitro. Virus-free G. canis trophozoites were transfected with in vitro transcribed GCV RNA by electroporation. Transfected trophozoites were cultured for 12, 24, 36, 48, 60, or 72h post transfection for analysis. The ultrastructures of the transfected trophozoites were determined by transmission electron microscopy. The viral particles were detectable sporadically in the cytoplasm as early as 24h post transfection, but became evident and wide-spread 36h post transfection. The number of viral particles increased dramatically from 48 to 60h. Viral particles were released into the culture medium starting at about 60h and detectable in nuclei 72h post transfection. Severe vacuolization was seen in transfected G. canis trophozoites as early as 36h post transfection and persisted throughout the course of this study. The results of the present study indicate that in vitro transcribed GCV transcripts were capable of infecting Giardia trophozoites, apparently replicated and packaged into mature infectious viral particles which were released from the host.


Assuntos
Giardia/ultraestrutura , Giardia/virologia , Giardiavirus/genética , Animais , DNA Complementar/genética , Eletroporação , Giardiavirus/patogenicidade , Giardiavirus/fisiologia , Giardiavirus/ultraestrutura , Microscopia Eletrônica de Transmissão , Reação em Cadeia da Polimerase , RNA Viral/genética , Transfecção , Vírion/patogenicidade , Vírion/fisiologia , Vírion/ultraestrutura , Replicação Viral
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