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1.
Arch Microbiol ; 202(2): 275-282, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31641798

RESUMO

Candida species cause a wide range of opportunistic infections in humans and animals. The detection of Candida species by conventional diagnosis methods is costly and time consuming. This study was conducted for the first time to evaluate and compare a relatively new molecular assay and the loop-mediated isothermal amplification (LAMP) technique with conventional methods for detection of Candida albicans. In this study, 70 different species of Candida identified by conventional methods were cultured on Sabouraud chloramphenicol agar medium and then the genomic DNA was extracted. The LAMP technique was performed using specific primers targeting the ITS2 gene of C. albicans. The analytical sensitivity and specificity of LAMP were measured using a tenfold serial dilution prepared from extracted DNA from standard C. albicans strain from 1 ng to 1 fg and the DNA samples of other clinical Candida species and three non-Candida yeast. Out of 70 yeast samples analyzed by LAMP technique, 24 samples (34.3%) were positive for C. albicans. Comparison of the results showed that the CHROMagar Candida and germ tube production methods are quite consistent with the LAMP technique, while the agreement amount between the results of carbohydrate assimilation and chlamydoconidia generation assays and LAMP technique was 98.5% and 72.8%, respectively. The detection limits of the LAMP assay were 10 fg of the DNA from the standard C. albicans strain. No amplification was observed in the DNA samples of other yeast species and only the DNA sample of standard C. albicans strain was amplified. Based on the results, it can be concluded that the LAMP method is as specific and precise as common diagnostic methods, but is faster, easier deployable or more sensitive. Therefore, this method can be used as a suitable complementary assay for Candida diagnosis in medical diagnostic laboratories and field conditions.


Assuntos
Candida albicans/genética , Candidíase/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Animais , Candida albicans/classificação , Candida albicans/isolamento & purificação , Primers do DNA/genética , DNA Fúngico/genética , Humanos , Sensibilidade e Especificidade
2.
Biochemistry ; 58(26): 2906-2920, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31145594

RESUMO

NF-κB essential modulator (NEMO) regulates NF-κB signaling by acting as a scaffold for the kinase IKKß to direct its activity toward the NF-κB inhibitor, IκBα. Here, we show that a highly conserved central region of NEMO termed the intervening domain (IVD, amino acids 112-195) plays a key role in NEMO function. We determined a structural model of full-length NEMO by small-angle X-ray scattering and show that full-length, wild-type NEMO becomes more compact upon binding of a peptide comprising the NEMO binding domain of IKKß (amino acids 701-745). Mutation of conserved IVD residues (9SG-NEMO) disrupts this conformational change in NEMO and abolishes the ability of NEMO to propagate NF-κB signaling in cells, although the affinity of 9SG-NEMO for IKKß compared to that of the wild type is unchanged. On the basis of these results, we propose a model in which the IVD is required for a conformational change in NEMO that is necessary for its ability to direct phosphorylation of IκBα by IKKß. Our findings suggest a molecular explanation for certain disease-associated mutations within the IVD and provide insight into the role of conformational change in signaling scaffold proteins.


Assuntos
Quinase I-kappa B/metabolismo , Sequência de Aminoácidos , Animais , Células HEK293 , Humanos , Quinase I-kappa B/química , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Multimerização Proteica , Espalhamento a Baixo Ângulo , Alinhamento de Sequência , Transdução de Sinais , Difração de Raios X
3.
Dev Comp Immunol ; 104: 103559, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31751628

RESUMO

Herein, we characterize transcription factor NF-κB from the demosponge Amphimedon queenslandica (Aq). Aq-NF-κB is most similar to NF-κB p100/p105 among vertebrate proteins, with an N-terminal DNA-binding domain, a C-terminal Ankyrin (ANK) repeat domain, and a DNA binding-site profile akin to human NF-κB proteins. Like mammalian NF-κB p100, C-terminal truncation allows nuclear translocation of Aq-NF-κB and increases its transcriptional activation activity. Expression of IκB kinases (IKKs) induces proteasome-dependent C-terminal processing of Aq-NF-κB in human cells, and processing requires C-terminal serines in Aq-NF-κB. Unlike NF-κB p100, C-terminal sequences of Aq-NF-κB do not inhibit its DNA-binding activity. Tissue of a black encrusting demosponge contains NF-κB site DNA-binding activity, as well as nuclear and processed NF-κB. Treatment of sponge tissue with LPS increases both DNA-binding activity and processing of NF-κB. A. queenslandica transcriptomes contain homologs to upstream NF-κB pathway components. This is first functional characterization of NF-κB in sponge, the most basal multicellular animal.


Assuntos
Sequência Conservada/genética , Proteínas de Ligação a DNA/genética , NF-kappa B/genética , Poríferos/imunologia , Domínios Proteicos/genética , Animais , Proteínas de Ligação a DNA/metabolismo , Evolução Molecular , Regulação da Expressão Gênica , NF-kappa B/metabolismo , Transdução de Sinais , Transcrição Gênica
4.
PLoS One ; 14(9): e0222588, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31553754

RESUMO

The use of alternative promoters for the cell type-specific expression of a given mRNA/protein is a common cell strategy. NEMO is a scaffold protein required for canonical NF-κB signaling. Transcription of the NEMO gene is primarily controlled by two promoters: one (promoter B) drives NEMO transcription in most cell types and the second (promoter D) is largely responsible for NEMO transcription in liver cells. Herein, we have used a CRISPR/Cas9-based approach to disrupt a core sequence element of promoter B, and this genetic editing essentially eliminates expression of NEMO mRNA and protein in 293T human kidney cells. By cell subcloning, we have isolated targeted 293T cell lines that express no detectable NEMO protein, have defined genomic alterations at promoter B, and do not support activation of canonical NF-κB signaling in response to treatment with tumor necrosis factor. Nevertheless, non-canonical NF-κB signaling is intact in these NEMO-deficient cells. Expression of ectopic wild-type NEMO, but not certain human NEMO disease mutants, in the edited cells restores downstream NF-κB signaling in response to tumor necrosis factor. Targeting of the promoter B element does not substantially reduce NEMO expression (from promoter D) in the human SNU-423 liver cancer cell line. Thus, we have created a strategy for selectively eliminating cell type-specific expression from an alternative promoter and have generated 293T cell lines with a functional knockout of NEMO. The implications of these findings for further studies and for therapeutic approaches to target canonical NF-κB signaling are discussed.


Assuntos
Edição de Genes/métodos , Técnicas de Silenciamento de Genes/métodos , Quinase I-kappa B/genética , Elementos Reguladores de Transcrição/genética , Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Células HEK293 , Humanos , NF-kappa B/metabolismo , Transdução de Sinais
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