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1.
Cell Prolif ; 53(1): e12722, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31737959

RESUMO

OBJECTIVES: The mechanisms underlying the effects of Toll-like receptor 9 (TLR9) and autophagy on rheumatoid arthritis (RA)-aggravated periodontitis are unclear. We aimed to explore a novel target, cathepsin K (Ctsk)-mediated TLR9-related autophagy, during the progress of periodontitis with RA. MATERIALS AND METHODS: DBA/J1 mouse model of periodontitis with RA was created by local colonization of Porphyromonas gingivalis (Pg) and injection of collagen. The expression of Ctsk was inhibited by adeno-associated virus (AAV). Micro-CT, immunohistochemistry (IHC), Western blot and quantitative real-time polymerase chain reaction (qRT-PCR) were used to detect the expression of TLR9-related autophagy in periodontitis with RA. Small interfering RNA (siRNA) and CpG oligodeoxynucleotides (CpG ODN) were applied in macrophages. Western blot, immunofluorescence (IF) and qRT-PCR were used to verify the in vivo results. RESULTS: RA can promote periodontitis bone destruction in the lesion area, while inhibiting Ctsk could effectively alleviate this effect. The infiltration of macrophages, TLR9, autophagy proteins (TFEB and LC3) and inflammatory cytokines increased in the periodontitis-with-RA group and was reduced by the inhibition of Ctsk in the periodontal region. Macrophage stimulation confirmed the in vivo results. With the activation of TLR9 by CpG ODN, inhibition of Ctsk could suppress both TLR9 downstream signalling proteins and autophagy-related proteins. CONCLUSIONS: This study advanced a novel role for Ctsk in TLR9 and autophagy to explain the interaction between periodontitis and RA.


Assuntos
Artrite Reumatoide/tratamento farmacológico , Catepsina K/antagonistas & inibidores , Regulação para Baixo/efeitos dos fármacos , Oligodesoxirribonucleotídeos/farmacologia , Periodontite/tratamento farmacológico , Receptor Toll-Like 9/imunologia , Animais , Artrite Reumatoide/genética , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/imunologia , Catepsina K/genética , Catepsina K/imunologia , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos DBA , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/imunologia , Oligodesoxirribonucleotídeos/genética , Periodontite/genética , Periodontite/imunologia , Periodontite/patologia , Receptor Toll-Like 9/genética
2.
J Microbiol ; 58(2): 153-162, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31872374

RESUMO

Oligodeoxynucleotides containing unmethylated CpG dinucleotides (CpG-ODN) can be specifically recognized by Toll-like receptor 9 (TLR9), provoking innate immune responses. Designed according to this structural feature, many synthetic phosphorothioate CpG-ODNs successfully activate macrophages. However, it is difficult to find potent stimulatory CpG-DNA fragments in microbial genomes. Therefore, whether microbial CpG-DNA substantially contributes to infectious and immune diseases remains controversial. In this study, high-throughput scanning was carried out for thousands of bacterial genomes with bioinformatics tools to comprehensively evaluate the distribution of CpG-DNA fragments. A random sampling test was then performed to verify their immunostimulatory properties by experiments in vitro and in vivo. Natural TLR9-dependent and potent stimulatory CpG-DNA fragments were found in microbial genomes. Interestingly, highly conserved stimulatory CpG-DNA fragments were found in 16S and 23S rDNA sequences with multiple copies, while others were species-specific. Additionally, we found that the reported active motifs were mostly non-stimulatory in natural CpG fragments. This evidence indicates that the previous structural descriptions of functional CpG-ODNs are incomplete. Our study has assessed the distribution of microbial CpG-DNA fragments, and identified natural stimulatory CpG-DNA fragments. These findings provide a deeper understanding of CpG-ODN structures and new evidence for microbial DNA inflammatory function and pathogenicity.


Assuntos
Adjuvantes Imunológicos/genética , Genoma Bacteriano/imunologia , Oligodesoxirribonucleotídeos/genética , Animais , Biologia Computacional , Escherichia coli/genética , Imunidade Inata , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/microbiologia , Camundongos , Streptococcus/genética , Receptor Toll-Like 9/imunologia
3.
Anal Chim Acta ; 1094: 130-135, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31761039

RESUMO

Quantification of plasma membrane proteins (PMPs) is crucial for understanding the fundamentals of cellular signaling systems and their related diseases. In this work, a super-quadruplex scaffold was designed to regulate assembly of oligonucleotide-grafted AIEgens for detection of PMPs. The nonfluorescence oligonucleotide-grafted AIEgen (Oligo-AIEgen) was firstly synthesized by attaching the AIEgen to 3'-terminus of the oligonucleotide through click chemistry. Meanwhile, the tetramolecular hairpin-conjugated super-quadruplex (THP-G4) as cleavage element and signal enhancement scaffold composited of three elements: a substrate sequence of DNAzyme in the loop region, partial hybridization region in the stem, and six guanine nucleotides to form G-quadruplex. Once the DNAzyme was anchored on the specific PMPs through aptamer-protein recognition, the substrate sequence on the loop of THP-G4 was cleaved by DNAzyme with the aid of cofactor MnII, resulting in the conformation switch of THP-G4 to the activated G-quadruplex scaffold. The latter could assemble Oligo-AIEgens to generate aggregation-induced emission (AIE) enhancement, resulting in a simple and sensitive strategy for detection of membrane proteins. Moreover, the DNAzyme continuously cut the next THP-G4 to achieve recycling amplification. Under the optimized conditions, this AIE-based strategy exhibited good linear relationship with the logarithm of MUC1 concentration from 0.01 to 10 µg mL-1 with the limit of detection down to 4.3 ng mL-1. The G4-assembled AIEgens provides a universal platform for detecting various biomolecules and a proof-of concept for AIE biosensing.


Assuntos
Acrilonitrila/análogos & derivados , Técnicas Biossensoriais/métodos , Corantes Fluorescentes/química , Quadruplex G , Mucina-1/análise , Estilbenos/química , Acrilonitrila/síntese química , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Linhagem Celular Tumoral , DNA Catalítico/química , DNA Catalítico/genética , Corantes Fluorescentes/síntese química , Humanos , Limite de Detecção , Mucina-1/química , Hibridização de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/genética , Estudo de Prova de Conceito , Estilbenos/síntese química
4.
Life Sci ; 239: 117067, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31738882

RESUMO

AIMS: Both CpG oligodeoxynucleotide (CpG-ODN) and melatonin have been reported to induce Th1 response and contribute to allergic asthma resistance. Here, we aimed to reveal how they confer such effect as well as whether they crosstalk with each other. MAIN METHODS: Six-week-old Female C57BL/6 mice were challenged by OVA to induce allergic airway inflammation, and were treated with CpG-ODN, CpG-ODN plus Luzindole or melatonin respectively. Bronchoalveolar lavage fluid (BALF) cellularity was classified and counted by Wright's-Giemsa staining. HE and PAS staining were used to analyze airway inflammation. The levels of IL-4, IL-5, IL-13,GM-CSF and IFN-γ, as well as IL-1ß and IL-18 were analyzed by ELISA. Protein expressions of ASMT, AANAT, NLRP3, IL-1ß and caspase-1 in lung tissue were detected by Western blotting, expression of ASMT and AANAT were further observed by immunohistochemistry. KEY FINDINGS: We found that CpG-ODN considerably suppressed OVA-induced airway leukocytes infiltration, goblet cell hyperplasia and Th2 cytokines production. Furthermore, the resolution effect of CpG-ODN on OVA-induced allergic airway inflammation occurred in parallel with decreased-activation of NLRP3 inflammasome and increased biosynthesis of melatonin. Blocking the effect of endogenous melatonin by Luzindole abolished the suppressive effect of CpG-ODN on OVA-induced airway inflammation and activation of NLRP3 inflammasome, suggesting such effect was mediated by endogenous melatonin. Moreover, exogenous melatonin pronouncedly ameliorated airway inflammation and decreased the activation of NLRP3 inflammasome. SIGNIFICANCE: These results proven that CpG-ODN protects against allergic airway inflammation via suppressing the activation of NLRP3 inflammasome, and such effect may be resulted from the restored-production of melatonin.


Assuntos
Inflamassomos/efeitos dos fármacos , Melatonina/farmacologia , Oligodesoxirribonucleotídeos/farmacologia , Animais , Asma , Líquido da Lavagem Broncoalveolar , Citocinas/metabolismo , Feminino , Hipersensibilidade/metabolismo , Inflamassomos/metabolismo , Inflamassomos/fisiologia , Inflamação/metabolismo , Inflamação/prevenção & controle , Pulmão/metabolismo , Melatonina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/fisiologia , Oligodesoxirribonucleotídeos/genética , Oligodesoxirribonucleotídeos/metabolismo , Células Th2 , Triptaminas/farmacologia
5.
Int J Mol Sci ; 20(17)2019 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-31470511

RESUMO

Approximately 30% of pancreatic cancer patients harbor targetable mutations. However, there has been no therapy targeting these molecules clinically. Nucleic acid medicines show high specificity and can target RNAs. Nucleic acid medicine is expected to be the next-generation treatment next to small molecules and antibodies. There are several kinds of nucleic acid drugs, including antisense oligonucleotides, small interfering RNAs, microRNAs, aptamers, decoys, and CpG oligodeoxynucleotides. In this review, we provide an update on current research of nucleic acid-based therapies. Despite the challenging obstacles, we hope that nucleic acid drugs will have a significant impact on the treatment of pancreatic cancer. The combination of genetic diagnosis using next generation sequencing and targeted therapy may provide effective precision medicine for pancreatic cancer patients.


Assuntos
Aptâmeros de Nucleotídeos/uso terapêutico , Ensaios Clínicos como Assunto/métodos , Oligodesoxirribonucleotídeos/uso terapêutico , Oligonucleotídeos Antissenso/uso terapêutico , Neoplasias Pancreáticas/tratamento farmacológico , RNA Interferente Pequeno/uso terapêutico , Animais , Aptâmeros de Nucleotídeos/genética , Humanos , Oligodesoxirribonucleotídeos/genética , Oligonucleotídeos Antissenso/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , RNA Neoplásico/antagonistas & inibidores , RNA Neoplásico/genética , RNA Interferente Pequeno/genética
6.
Int J Mol Sci ; 20(16)2019 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-31426274

RESUMO

Over two decades ago, short oligodeoxynucleotides (ODNs) were proven to be an effective and rapid technique for analysis of gene function without interference in the plant genome. Our previous research has shown the successful regulation of chalcone synthase (CHS) gene expression in flax by ODN technology. The CHS gene encodes a pivotal enzyme in flavonoid biosynthesis. The manipulation of its transcript level was the result of the specific methylation status developed after treatment with ODNs. In further analysis of the application of oligodeoxynucleotides in plants, we will focus on maintaining the methylation status induced originally by ODNs homologous to the regulatory regions of the CHS gene in flax. This article reports the latest investigation applied to stabilization and inheritance of the epigenetic marks induced by plants' treatment with ODNs. The methylation status was analyzed in the particular CCGG motifs located in the CHS gene sequence. Individual plants were able to maintain alterations induced by ODNs. In order to confirm the impact of methylation marks on the nucleosome rearrangement, chromatin accessibility assay was performed. The perpetuation of targeted plant modulation induced by ODNs exhibits strong potential for improving crops and intensified application for medicine, nutrition and industry.


Assuntos
Aciltransferases/genética , Metilação de DNA , Linho/genética , Engenharia Genética/métodos , Proteínas de Plantas/genética , Sequência de Bases , Epigênese Genética , Regulação da Expressão Gênica de Plantas , Oligodesoxirribonucleotídeos/genética , Plantas Geneticamente Modificadas/genética
7.
Anal Chim Acta ; 1081: 176-183, 2019 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-31446955

RESUMO

Precise description of temperature at the microscale level is essential in many biological applications. In this study, we prepared a DNA-based thermometer that reports low and high temperatures by providing two distinct optical signals. The system is a molecular beacon that carries a loop and a stem, whose conformation is subject to change from a hairpin to a random coil when the temperature changes from low to high. A fluorophore, Cy5, and a quencher, BHQ3, are terminally labeled at the stem ends. Moreover, perylene is included in the middle of the 3'-end stem. The signaling state of Cy5 relies on the relative distance to BHQ3. However, the perylene emission is regulated by its microenvironment (i.e., the oligonucleotide or duplex state). With a temperature variation, the designed thermometer undergoes a change in conformation that leads to two signal patterns with Cy5/off and perylene/on at low temperature and Cy5/on and perylene/off at high temperature. The reversibility and biocompatibility of the thermometer design were examined for potential applications in biological systems.


Assuntos
Temperatura Baixa , DNA/química , Temperatura Alta , Termômetros , Carbocianinas/química , DNA/genética , Fluorescência , Corantes Fluorescentes/química , Células Hep G2 , Humanos , Sequências Repetidas Invertidas , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/genética , Concentração Osmolar , Perileno/química
8.
Immunology ; 158(2): 85-93, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31335975

RESUMO

Bacterial DNA contains CpG oligonucleotide (ODN) motifs to trigger innate immune responses through the endosomal receptor Toll-like receptor 9 (TLR9). One of the cell surface receptors to capture and deliver microbial DNA to intracellular TLR9 is the C-type lectin molecule DEC-205 through its N-terminal C-type lectin-like domain (CTLD). CD93 is a cell surface protein and member of the lectin group XIV with a CTLD. We hypothesized that CD93 could interact with CpG motifs, and possibly serve as a novel receptor to deliver bacterial DNA to endosomal TLR9. Using ELISA and tryptophan fluorescence binding studies we observed that the soluble histidine-tagged CD93-CTLD was specifically binding to CpG ODN and bacterial DNA. Moreover, we found that CpG ODN could bind to CD93-expressing IMR32 neuroblastoma cells and induced more robust interleukin-6 secretion when compared with mock-transfected IMR32 control cells. Our data argue for a possible contribution of CD93 to control cell responsiveness to bacterial DNA in a manner reminiscent of DEC-205. We postulate that CD93 may act as a receptor at plasma membrane for DNA or CpG ODN and to grant delivery to endosomal TLR9.


Assuntos
DNA Bacteriano/imunologia , Regulação da Expressão Gênica/imunologia , Glicoproteínas de Membrana/imunologia , Oligodesoxirribonucleotídeos/imunologia , Receptores de Complemento/imunologia , Receptor Toll-Like 9/imunologia , Antígenos CD/genética , Antígenos CD/imunologia , Transporte Biológico/genética , Transporte Biológico/imunologia , Linhagem Celular Tumoral , Clonagem Molecular , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Endossomos/imunologia , Endossomos/metabolismo , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Inflamação , Interleucina-6/genética , Interleucina-6/imunologia , Lectinas Tipo C/genética , Lectinas Tipo C/imunologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/imunologia , Modelos Biológicos , Neurônios/imunologia , Neurônios/metabolismo , Neurônios/patologia , Oligodesoxirribonucleotídeos/genética , Oligodesoxirribonucleotídeos/metabolismo , Ligação Proteica , Domínios Proteicos , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/imunologia , Receptores de Complemento/genética , Receptores de Complemento/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Receptor Toll-Like 9/genética
9.
Nucleic Acids Res ; 47(14): 7213-7222, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31265072

RESUMO

Recent studies suggest noncoding RNAs interact with genomic DNA, forming an RNA•DNA-DNA triple helix that regulates gene expression. However, base triplet composition of pyrimidine motif RNA•DNA-DNA triple helices is not well understood beyond the canonical U•A-T and C•G-C base triplets. Using native gel-shift assays, the relative stability of 16 different base triplets at a single position, Z•X-Y (where Z = C, U, A, G and X-Y = A-T, G-C, T-A, C-G), in an RNA•DNA-DNA triple helix was determined. The canonical U•A-T and C•G-C base triplets were the most stable, while three non-canonical base triplets completely disrupted triple-helix formation. We further show that our RNA•DNA-DNA triple helix can tolerate up to two consecutive non-canonical A•G-C base triplets. Additionally, the RNA third strand must be at least 19 nucleotides to form an RNA•DNA-DNA triple helix but increasing the length to 27 nucleotides does not increase stability. The relative stability of 16 different base triplets in DNA•DNA-DNA and RNA•RNA-RNA triple helices was distinctly different from those in RNA•DNA-DNA triple helices, showing that base triplet stability depends on strand composition being DNA and/or RNA. Multiple factors influence the stability of triple helices, emphasizing the importance of experimentally validating formation of computationally predicted triple helices.


Assuntos
DNA/química , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , RNA não Traduzido/química , RNA/química , Algoritmos , Composição de Bases , Sequência de Bases , Códon/genética , DNA/genética , Código Genético , Concentração de Íons de Hidrogênio , Cinética , Motivos de Nucleotídeos , Oligodesoxirribonucleotídeos/genética , RNA/genética , RNA não Traduzido/genética
10.
Protein Eng Des Sel ; 32(1): 41-45, 2019 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-31297523

RESUMO

User-defined mutagenic libraries are fundamental for applied protein engineering workflows. Here we show that unamplified oligo pools can be used to prepare site saturation mutagenesis libraries from plasmid DNA with near-complete coverage of desired mutations and few off-target mutations. We find that oligo pools yield higher quality libraries when compared to individually synthesized degenerate oligos. We also show that multiple libraries can be multiplexed into a single oligo pool, making preparation of multiple libraries less expensive and more convenient. We provide software for automatic oligo pool design that can generate mutagenic oligos for saturating or focused libraries.


Assuntos
Biblioteca Gênica , Mutagênese Sítio-Dirigida/métodos , Oligodesoxirribonucleotídeos , Engenharia de Proteínas/métodos , Oligodesoxirribonucleotídeos/síntese química , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/genética , Plasmídeos/química , Plasmídeos/genética
11.
Anal Chim Acta ; 1078: 176-181, 2019 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-31358217

RESUMO

Intracellular microRNA (miRNA) analysis in single cell is highly informative and offers valuable insights to its physiological and pathological state, but it must confront the pivotal challenge of gene probe delivery and conditional release. Herein, we report an assembled DNA mini-hexahedron (DMH) that can selectively package and protect miRNA probe, target-cell-specific delivery and release it based on the target sequence recognition for intracellular miRNA detection. In brief, the DMH is self-assembled from six single-stranded oligonucleotide strands through rational design, one of which containing AS1411 sequence for specific uptake. Two fluorescent dye labeled recognition strands are inserted into two DMH edges with quencher groups through partially complementary hybridization. We find that this DMH possesses great biocompatibility, good trans-membrane ability and are able to protect the gene cargo against enzymatic degradation and protein binding. Fluorescence restoration caused by the target-mediated competitive chain replacement reaction allows to simultaneous detection of two cancer-related intracellular miRNAs with little false-positive signal, providing a powerful tool to discriminate healthy normal cell and cancerous cell. Thus, the construct opens a new avenue to circumvent the challenges in gene delivery, specific delivery and intrinsic interferences resistance.


Assuntos
DNA de Cadeia Simples/química , MicroRNAs/análise , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Sequência de Bases , Linhagem Celular Tumoral , DNA de Cadeia Simples/genética , Portadores de Fármacos/química , Fluoresceínas/química , Fluorescência , Corantes Fluorescentes/química , Humanos , MicroRNAs/genética , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Hibridização de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/genética
12.
Anal Chim Acta ; 1078: 24-31, 2019 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-31358225

RESUMO

A novel electrochemical DNA biosensor was developed and MON89788 of soybean transgenic gene sequence was detected based on a strategy of rolling circle amplification (RCA) and gold nanoparticle cube (AuNPC)-labeled multiple probes. First, the mercapto-modified capture DNA was immobilized on the surface of the Fe3O4@Au magnetic nanoparticles via an Au-S bond, and the capture DNA was opened and complementarily hybridized with the target DNA to form a double-stranded DNA. In the 10 × reaction buffer, Exonuclease III (ExoIII) specifically recognized and sheared the double-stranded DNA to release the target DNA, which led to the next round of reaction. Afterward, AuNP cube-loaded ssDNA (AuNPC/DNA) was added with the rolling circle reaction with the help of Phi29 DNA polymerase and T4 ligase. Finally, [Ru(NH3)6]3+ was attracted directly by the anionic phosphate of ssDNA via electrostatic interaction. The determination was carried out by using chronocoulometry (CC), and the CC signal was recorded. The mass amount of DNA strands extended infinitely on the AuNPs cube and numerous [Ru(NH3)6]3+ were absorbed, thus the detected signal was highly amplified. The corresponding CC signal showed a good linear relationship with the logarithm of the target DNA concentration in the range of 1 × 10-16 to 1 × 10-7 mol L-1, with a detection limit of 4.5 × 10-17 mol L-1. Specific gene sequence of MON89788 in soybean samples was determined, and the recoveries ranged from 97.3% to 102.0%. This sensor is one of the most sensitive sensors for genetic sequence assessment at present. Moreover, it demonstrates good selectivity, stability, and reproducibility.


Assuntos
Técnicas Biossensoriais/métodos , DNA de Plantas/análise , Técnicas Eletroquímicas/métodos , Plantas Geneticamente Modificadas/genética , Soja/genética , Sequência de Bases , Calibragem , Sondas de DNA/química , Sondas de DNA/genética , DNA de Plantas/química , DNA de Plantas/genética , Exodesoxirribonucleases/química , Ouro/química , Limite de Detecção , Nanopartículas de Magnetita/química , Técnicas de Amplificação de Ácido Nucleico/métodos , Hibridização de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/genética , Reprodutibilidade dos Testes , Compostos de Rutênio/química
13.
Cell Biol Int ; 43(8): 852-862, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31033094

RESUMO

The transcription factor T-cell factor 3 (TCF3), one component of the Wnt pathway, is known as a cell-intrinsic inhibitor of many pluripotency genes in embryonic stem cells (ESCs) that influences the balance between pluripotency and differentiation. In this study, the effects of inhibition of TCF3 transcription factor on the stemness of mouse ESCs (mESCs) were investigated using the decoy oligodeoxynucleotides (ODNs) strategy. The TCF3 decoy and its scramble ODNs were designed and synthesized. The interaction specificity of the TCF3 decoy with the TCF3 transcription factor was evaluated by the electrophoretic mobility shift assay. Subcellular localization was carried out using fluorescence and confocal microscopy. Self-renewal and pluripotency of mESCs were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), cell cycle and apoptosis, alkaline phosphatase (ALP), embryoid body (EB) formation, and real-time assays. All experiments were performed in triplicate. The results showed that knockdown of TCF3 by decoy ODNs transfection in mESCs led to an increase in the cell proliferation, ALP enzyme activity, and master regulatory stemness genes and a decrease in the number and diameter of EBs. These results supported TCF3 as a potential target to maintain the pluripotency and self-renewal capacity of mESCs. Knockdown of the TCF3 transcription factor using decoy ODNs can be a promising method to maintain the stemness of stem cells in regenerative medicine and cell therapy researches.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Proliferação de Células/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Células-Tronco Embrionárias Murinas/metabolismo , Oligodesoxirribonucleotídeos/farmacologia , Medicina Regenerativa , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Oligodesoxirribonucleotídeos/genética , Via de Sinalização Wnt/genética
14.
Nat Commun ; 10(1): 1937, 2019 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-31028261

RESUMO

The development of site-specific recombinases (SSRs) as genome editing agents is limited by the difficulty of altering their native DNA specificities. Here we describe Rec-seq, a method for revealing the DNA specificity determinants and potential off-target substrates of SSRs in a comprehensive and unbiased manner. We applied Rec-seq to characterize the DNA specificity determinants of several natural and evolved SSRs including Cre, evolved variants of Cre, and other SSR family members. Rec-seq profiling of these enzymes and mutants thereof revealed previously uncharacterized SSR interactions, including specificity determinants not evident from SSR:DNA structures. Finally, we used Rec-seq specificity profiles to predict off-target substrates of Tre and Brec1 recombinases, including endogenous human genomic sequences, and confirmed their ability to recombine these off-target sequences in human cells. These findings establish Rec-seq as a high-resolution method for rapidly characterizing the DNA specificity of recombinases with single-nucleotide resolution, and for informing their further development.


Assuntos
DNA Nucleotidiltransferases/genética , DNA/genética , Edição de Genes/métodos , Genoma Humano , Integrases/genética , Sequência de Bases , Clonagem Molecular , DNA/metabolismo , DNA Nucleotidiltransferases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Integrases/metabolismo , Oligodesoxirribonucleotídeos/síntese química , Oligodesoxirribonucleotídeos/genética , Oligodesoxirribonucleotídeos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Recombinação Genética
15.
Mikrochim Acta ; 186(4): 243, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30877395

RESUMO

A colorimetric method is presented for the detection of specific nucleotide sequences in plant pathogens. It is based on the use of CRISPR/Cas9-triggered isothermal amplification and gold nanoparticles (AuNPs) as optical probes. The target DNA was recognized and broken up by a given Cas9/sgRNA complex. After isothermal amplification, the product was hybridized with oligonucleotide-functionalized AuNPs. This resulted in the aggregation of AuNPs and a color change from wine red to purple. The visual detection limit is 2 pM of DNA, while a linear relationship exists between the ratio of absorbance at 650 and 525 nm and the DNA concentration in the range from 0.2 pM to 20 nM. In contrast to the previous CRISPR-based amplification platforms, the method has significantly higher specificity with the single-base mismatch and can be visually read out. It was successfully applied to identify the Phytophthora infestans genomic DNA. Graphical abstract Schematic presentation of a colorimetric method for detection of Phytophthora infestans genomic DNA based on CRISPR/Cas9-triggered isothermal amplification. The Cas9 endonuclease cleaves DNA at the design site and the color changes from red to purple with increasing target DNA concentration.


Assuntos
Proteína 9 Associada à CRISPR/química , Sistemas CRISPR-Cas , Colorimetria/métodos , DNA/análise , Sequência de Bases , Técnicas Biossensoriais/métodos , DNA/genética , Ouro/química , Limite de Detecção , Nanopartículas Metálicas/química , Técnicas de Amplificação de Ácido Nucleico/métodos , Hibridização de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/genética , Phytophthora infestans/genética
16.
Analyst ; 144(8): 2704-2715, 2019 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-30864589

RESUMO

Members of the Brucella spp. are facultative intracellular bacteria that can cause global brucellosis, a zoonotic disease. Herein, a novel fluorescence signal amplification (FSA) method for the rapid detection of B. melitensis 16M was developed based on peptide-mediated magnetic separation (PMS) technology and Au nanoparticle (AuNP)-mediated bio-barcode assay technology assembled by quantum dots (QDs). The PMS technology was used to specifically capture and isolate B. melitensis 16M from food. The immunomagnetic bead-B. melitensis 16M bioconjugates (IMBs-B. melitensis 16M) were then identified by IgY on the surface of AuNPs and the oligonucleotide chains on the surface of the gold nanoparticles were hybridized with bio-barcodes assembled by quantum dots (QD-probe2). The IMB/B. melitensis 16M/IgY-AuNP-probe1/QD-probe2 bioconjugates were concentrated by magnetic separation. Therefore, as the concentration of B. melitensis 16M in the sample increased, the unbound QD-probe2 in the supernatant reduced, and the B. melitensis 16M in the sample could be indirectly measured by detecting the fluorescence in the supernatant. This FSA method can detect B. melitensis 16M concentration in the range of 10 to 106 cfu ml-1 without pre-enrichment, and the limit of detection (LOD) is as low as 10 cfu ml-1 with high specificity. Furthermore, the proposed method for the detection of B. melitensis 16M has a LOD of 1.07 × 102 cfu ml-1 and a linear range from 102 to 107 cfu ml-1 in milk, and a LOD of 1.72 × 102 cfu ml-1, and a linear range from 102 to 106 cfu ml-1 in lamb leach. In addition, this method takes less than 3 h to perform. Thus, the assay that was developed in this study shows promise for rapid, sensitive, and specific detection of B. melitensis 16M.


Assuntos
Brucella melitensis/isolamento & purificação , Microbiologia de Alimentos/métodos , Nanopartículas Metálicas/química , Peptídeos/química , Pontos Quânticos/química , Animais , Biotina/química , Brucella melitensis/imunologia , Fluorescência , Ouro/química , Concentração de Íons de Hidrogênio , Isotipos de Imunoglobulinas/imunologia , Limite de Detecção , Fenômenos Magnéticos , Leite/microbiologia , Hibridização de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/genética , Carne Vermelha/microbiologia , Ovinos , Estreptavidina/química
18.
Anal Chim Acta ; 1048: 161-167, 2019 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-30598146

RESUMO

Herein, an original exciton energy transfer-based sensitive fluorescence sensor for the determination of Hg2+ has been designed through DNA aptamer-programmed self-assembly of CdTe quantum dots (QDs). In this work, CdTe QDs were applied as fluorescence signal source. The two pieces of T-rich aptamer played a role as molecular recognition probes which could bind to the target Hg2+ specifically. The extent of Hg2+-triggered self-assembly of QDs depended on the concentration of Hg2+, which resulted in an exciton energy transfer effect between QDs, giving an obvious fluorescence signal decrease and red-shift of the fluorescent peak. Based on this principle, we could detect the Hg2+ in two different signal modes. The limit of detection (LOD) was 3.33 nM. The proposed sensing method exhibited its application in detecting Hg2+ in real water samples with satisfactory performance. The results indicated that this proposed sensor will be of great potential in biological and analytical fields.


Assuntos
Aptâmeros de Nucleotídeos/química , Substâncias Macromoleculares/química , Mercúrio/análise , Pontos Quânticos/química , Espectrometria de Fluorescência/métodos , Aptâmeros de Nucleotídeos/genética , Compostos de Cádmio/química , Transferência de Energia , Fluorescência , Limite de Detecção , Hibridização de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/genética , Telúrio/química
19.
Nucleic Acids Res ; 47(3): 1350-1361, 2019 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-30517685

RESUMO

Nucleic acid-based assemblies that interact with each other and further communicate with the cellular machinery in a controlled manner represent a new class of reconfigurable materials that can overcome limitations of traditional biochemical approaches and improve the potential therapeutic utility of nucleic acids. This notion enables the development of novel biocompatible 'smart' devices and biosensors with precisely controlled physicochemical and biological properties. We extend this novel concept by designing RNA-DNA fibers and polygons that are able to cooperate in different human cell lines and that have defined immunostimulatory properties confirmed by ex vivo experiments. The mutual intracellular interaction of constructs results in the release of a large number of different siRNAs while giving a fluorescent response and activating NF-κB decoy DNA oligonucleotides. This work expands the possibilities of nucleic acid technologies by (i) introducing very simple design principles and assembly protocols; (ii) potentially allowing for a simultaneous release of various siRNAs together with functional DNA sequences and (iii) providing controlled rates of reassociation, stabilities in human blood serum, and immunorecognition.


Assuntos
DNA/genética , NF-kappa B/genética , RNA/genética , Transcrição Genética , DNA/química , Transferência Ressonante de Energia de Fluorescência , Regulação da Expressão Gênica/genética , Humanos , Oligodesoxirribonucleotídeos/genética , Oligonucleotídeos/química , Oligonucleotídeos/genética , RNA/química , RNA Interferente Pequeno/genética
20.
Immunol Invest ; 48(1): 79-95, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30239236

RESUMO

PURPOSE: Toll like receptor (TLR) engagement is primarily a function of the innate immune cells. The purpose of the study was to assess direct uptake of ODN 2216 in T helper cells and effects on cell proliferation and cytokine expression. METHODS: We isolated CD4+ CD25- T helper cells by magnetic sorting and studied the uptake of ODN 2216 using flow cytometry and confocal microscopy. We then studied the effect of ODN 2216 engagement on cell proliferation and cytokine expression using flow cytometry and gene expression of TLR9 signaling genes using real time RT-PCR. RESULTS: We made a chance observation that purified T helper cells from healthy individuals consistently bind to the TLR9 ligand ODN 2216. In PBMCs, on the other hand, 98% of monocytes preferentially bound to ODN 2216 FITC, indicating that they competed with the lymphocytes. We confirmed intracellular localization of ODN 2216 FITC as well as intracellular expression of TLR9 in Thelper cells. Furthermore, ODN 2216 FITC was also co-localized with the lysosomal membrane associated protein 1. The uptake of TLR9 ligand culminated in cellular proliferation, up-regulation of cytokines and increased mRNA expression of TLR9 and IRF7 in T helper cells, in the absence of antigen presenting cells. ODN 2216 uptake was inhibited by promethazine as well as by TLR9 antagonist. CONCLUSIONS: Our results show a direct engagement of TLR9 ligand in T helper cells and suggest involvement of TLR9 signalling in CD4+T cells, which may envisage novel targets for TLR inhibitors.


Assuntos
Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Oligodesoxirribonucleotídeos/genética , Linfócitos T Auxiliares-Indutores/fisiologia , Receptor Toll-Like 9/metabolismo , Proliferação de Células , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Citometria de Fluxo , Humanos , Fator Regulador 7 de Interferon/genética , Fator Regulador 7 de Interferon/metabolismo , Ativação Linfocitária , Microscopia Confocal , Ligação Proteica , Transporte Proteico , Transdução de Sinais/genética , Receptor Toll-Like 9/agonistas , Receptor Toll-Like 9/genética
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