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1.
J Diabetes Investig ; 2019 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-31758850

RESUMO

AIMS/INTRODUCTION: To assess the effect of dulaglutide (DU) 1.5/0.75 mg in comparison with glimepiride (GLIM) or insulin glargine (GLAR) on the composite end-point in Chinese type 2 diabetes patients. MATERIALS AND METHODS: Post-hoc analyses of two randomized phase III trials (NCT01644500 and NCT01648582) were carried out using Fisher's exact test. The primary composite end-point was the number of patients reaching glycated hemoglobin (HbA1c) <7.0%, without weight gain and hypoglycemia. Secondary composite end-points included the number of patients reaching HbA1c <7.0% without weight gain and HbA1c <7.0% without hypoglycemia. RESULTS: Data of 1,147 Chinese type 2 diabetes patients were analyzed (NCT01644500 = 556; NCT01648582 = 591). In each analyzed trial, 40-48% of patients received DU (1.5 mg), 30-39% of patients received DU (0.75 mg) and 15-20% of patients on active comparators (GLIM/GLAR) reached the primary composite end-point at week 26 (P < 0.001 for DU vs GLIM/GLAR). At 52 weeks, 26% of patients that received DU (1.5 mg), 23% of patients that received DU (0.75 mg) and 7% of patients that received GLAR attained the primary composite end-point (P < 0.001 for DU vs GLAR). A similar trend of results was found for secondary composite end-points. CONCLUSIONS: Dulaglutide is found to be an effective therapeutic alternative for Chinese type 2 diabetes patients. Compared with GLIM/GLAR, significantly greater proportions of patients on DU attained the HbA1c target of <7.0% without weight gain or hypoglycemia.

2.
J Oral Pathol Med ; 2019 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-31610043

RESUMO

BACKGROUND: Oral submucous fibrosis (OSF) is a chronic progressive oral disease with cancerous tendency. Arecoline plays an important role in the pathogenesis of OSF. Fibroblasts (FBs) are the primary cells involved in the pathogenesis of OSF. There is a change in CD4+ IL-17+ helper T cells (Th17) and CD4+ CD25+ Foxp3+ regulatory T cells (Treg) in OSF patients, but the molecular mechanisms of are not clearly understood. In this work, we studied the molecular mechanisms. METHODS: Enzyme digestion was used to extract primary FBs, and immunofluorescence was used to identify FBs. Cytotoxic experiment was then performed to determine the effect of arecoline on FB activity. Enzyme-linked immunosorbent assay (ELISA) was used to detect changes in the amount of cytokines. In addition, we treated human peripheral blood mononuclear cells (PBMC) with the above cytokines and detected their changes. Flow cytometry was used to detect the changes of Th17 and Treg, and quantitative-polymerase chain reaction (Q-PCR) was used to detect the changes of RORγt and Foxp3. RESULTS: We have found that the stimulation of arecoline on FBs increased interleukin-2, interleukin-6, and interleukin-21 (IL-2, IL-6, and IL-21) while decreased transforming growth cytokine-ß (TGF-ß). After the cytokine-containing supernatant was co-cultured with PBMC, the cytometry results showed that Th17 was increased, while Treg was significantly decreased and Q-PCR results showed that RORγt expression was increased and Foxp3 expression was decreased. CONCLUSION: The arecoline can affect inflammatory cytokines produced by FBs, which then act on immune cells Th17 and Treg, and make them change.

3.
Clin Ther ; 41(10): 2057-2065, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31514971

RESUMO

PURPOSE: In China, although insulin has been prescribed for decades, glucagon-like peptide 1 receptor agonists (GLP-1-RAs) have been available as an injectable treatment for patients with type 2 diabetes mellitus (T2DM) since 2009. GLP-1 RAs are listed as second-line treatment in the 2017 Chinese Guideline for patients with T2DM in whom prior oral antidiabetic therapy has failed. This study compares the baseline characteristics of Chinese patients with T2DM taking different prescriptions of first injectable therapy (GLP-1-RA or insulin). METHODS: The IQVIA Patient Diary Study database, which captures data from a patient medical record-based physician online survey, was the data source used in this study. Cross-sectional patient data were collected from hospitals in 15 major Chinese cities from June 1, 2016, to June 30, 2018. Adults with T2DM commencing either GLP-1-RA or insulin use as their first injectable antidiabetic therapy were included. Baseline demographic and clinical characteristics were compared between the GLP-1-RA and insulin treatment groups, using t tests and χ2 or Fisher exact tests. FINDINGS: Overall, 563 patients using GLP-1-RAs and 2387 using insulin were identified. In general, patients using GLP-1-RA were younger (mean [SD], 49.6 [10.8] years vs 59.3 [10.9] years), had lower mean (SD) glycosylated hemoglobin levels (8.5% [1.2%] vs 9.6 [1.7%]), had lower mean (SD) fasting plasma glucose levels (9.0 [1.9] mmol/L vs 10.8 [2.6] mmol/L), higher mean (SD) body mass indexes (29.4 [3.9] kg/m2 vs 24.6 [3.1] kg/m2), had higher comorbidity of obesity (75% vs 15%), had a higher occurrence of hyperlipidemia (63% vs 44%), and had lower occurrence of neuropathy (13% vs 34%) when compared with those using insulin (P < 0.0001 for all). The results of multivariate logistic regression model indicate that when controlling other variables in the multivariate logistic regression model, a higher fasting plasma glucose level and a longer diagnosis duration are associated with higher odds of insulin therapy commencement, but higher body mass index and some comorbidities, such as obesity and hyperlipidemia, are associated with higher odds of being a GLP-1-RA user. IMPLICATIONS: Significant differences were identified between selected baseline characteristics of patients initiating GLP-1-RA and insulin therapy, suggesting that these medicines are more likely to be prescribed to different types of patients with T2DM in China. These findings may help to inform Chinese physicians regarding the characteristics of those patients with T2DM who are initiating treatment with a GLP-1-RA or insulin. Because the Patient Diary Study data were collected from hospitals in 15 major cities in China, one noteworthy limitation is that the results may not represent the overall treatment pattern in rural areas of China.

4.
Toxicology ; 419: 32-39, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30910432

RESUMO

Arecoline, the major active ingredient of the betel nut, is involved in the pathogenesis of oral submucous fibrosis. However, the underlying mechanism of this pathogenesis remains unclear. In this study, we found that arecoline suppresses the cell proliferation of the HaCaT epithelial cell and induces cell cycle arrest at the G1/S phase with an IC50 of 50 µg/mL. Furthermore, we found that arecoline reduces the protein level of cyclin D1, but it has no effect on its mRNA level and protein stability, implying that arecoline may modulate the translation of cyclin D1. We also observed the downregulation of the Akt/mTOR signaling pathway after treatment with arecoline, which may be related to the translation of cyclin D1. RNA-seq analysis identified that PHLPP2, the direct upstream target of Akt, is significantly upregulated after arecoline treatment. siRNA-mediated knockdown of PHLPP2 recovered the phosphorylation state of Akt, as well as attenuated the effect of arecoline on cell viability. Thus, our study revealed the crucial role of PHLPP2 in arecoline-induced cell viability suppression.

5.
Diabetes Obes Metab ; 21(2): 234-243, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30129089

RESUMO

AIM: To compare the efficacy and safety of once-weekly dulaglutide with that of insulin glargine in combination with metformin and/or a sulphonylurea in mainly Asian patients with type 2 diabetes mellitus (T2DM). MATERIALS AND METHODS: In this 52-week, randomized, parallel-arm open-label study, we enrolled patients aged ≥18 years with T2DM for at least 6 months and a glycated haemoglobin (HbA1c) concentration ≥53.0 mmol/mol (7.0%) and ≤96.7 mmol/mol (11.0%). The primary outcome was change in HbA1c from baseline to week 26 to determine non-inferiority of dulaglutide 1.5 mg versus glargine. RESULTS: A total of 774 patients from China, South Korea, Mexico and Russia were randomly assigned (1:1:1) to dulaglutide 1.5 mg, dulaglutide 0.75 mg or glargine treatment groups. The patients' mean age was 55 years and the average T2DM duration was ~8 years. The least squares mean (SE) changes from baseline in HbA1c at 26 weeks were - 18.9 (0.73) mmol/mol (-1.73 [0.067]%) for dulaglutide 1.5 mg and -14.5 (0.73) mmol/mol (-1.33 [0.067]%) for dulaglutide 0.75 mg, compared with -12.7 (0.73) mmol/mol (-1.16 [0.067]%) for glargine. Statistical criteria for superiority were met with both dulaglutide 1.5 mg and dulaglutide 0.75 mg. More patients in the dulaglutide 1.5 and 0.75 mg groups achieved HbA1c target <53.0 mmol/mol (<7.0%) than in the glargine group at week 26 (P < 0.001 and P = 0.004, respectively). Body weight decreased with dulaglutide and increased with glargine. The incidence and rate of total hypoglycaemia were lower with dulaglutide versus glargine. Gastrointestinal adverse events, including diarrhoea and nausea, were the most frequently reported for patients taking dulaglutide. CONCLUSIONS: Once-weekly dulaglutide provides greater improvement in HbA1c, with weight loss and less hypoglycaemia, than once-daily insulin glargine in a population of mainly Asian patients with T2DM who had failed to achieve optimal glycaemic control on metformin and/or a sulphonylurea.


Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Peptídeos Semelhantes ao Glucagon/análogos & derivados , Fragmentos Fc das Imunoglobulinas/administração & dosagem , Insulina Glargina/administração & dosagem , Metformina/administração & dosagem , Proteínas Recombinantes de Fusão/administração & dosagem , Compostos de Sulfonilureia/administração & dosagem , Adulto , Idoso , Grupo com Ancestrais do Continente Asiático/estatística & dados numéricos , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , China/epidemiologia , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/etnologia , Esquema de Medicação , Quimioterapia Combinada , Feminino , Peptídeos Semelhantes ao Glucagon/administração & dosagem , Peptídeos Semelhantes ao Glucagon/efeitos adversos , Hemoglobina A Glicada/análise , Hemoglobina A Glicada/metabolismo , Humanos , Fragmentos Fc das Imunoglobulinas/efeitos adversos , Insulina Glargina/efeitos adversos , Masculino , Metformina/efeitos adversos , México/epidemiologia , Pessoa de Meia-Idade , Proteínas Recombinantes de Fusão/efeitos adversos , República da Coreia/epidemiologia , Federação Russa/epidemiologia , Compostos de Sulfonilureia/efeitos adversos , Resultado do Tratamento
6.
Conf Proc IEEE Eng Med Biol Soc ; 2018: 4492-4495, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-30441349

RESUMO

Rapid and accurate detection of single-nucleotide polymorphism (SNP) in pathogenic mutants is crucial for broad fields from food safety monitoring to disease diagnostics and prognosis. Here, we developed a nanopore single-molecule sensor, coupled with the locked nucleic acid (LNA) technique, to accurately discriminate SNPs for detection of Shiga toxin producing Escherichia coli (STEC) O157:H7 pathogen serotype, and cancer-derived driver mutations EGFR L858R and KRAS G12D. This sensitive method, with a simplified, low cost, easy-to-operate LNA design, can be applied in food science and medical detection that need rapid and accurate determination of genetic variations.


Assuntos
Nanoporos , Neoplasias/genética , Oligonucleotídeos/química , Escherichia coli Shiga Toxigênica/isolamento & purificação , Receptores ErbB/genética , Humanos , Mutação , Polimorfismo de Nucleotídeo Único , Proteínas Proto-Oncogênicas p21(ras)/genética , Sorogrupo , Escherichia coli Shiga Toxigênica/genética
7.
Bioconjug Chem ; 29(11): 3810-3816, 2018 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-30350578

RESUMO

Many strategies for the detection of nucleic acid sequence rely upon Watson-Crick hybridization of a probe strand to the target strand, but the reversible nature of nucleic acid hybridization presents an inherent challenge: short probes that provide high target specificity have relatively low target affinity resulting in signal losses. Sequence-specific covalent cross-linking reactions have the potential to provide both selective target capture and durable signal. We explore a novel approach involving sequence-specific covalent cross-linking of a probe to target DNA combined with single-molecule nanopore detection of the cross-linked DNA. Here, we exploited the selective reaction of mechlorethamine at a C-C mismatch for covalent capture of a target DNA sequence corresponding to a cancer-driving mutation at position 1799 of the human BRAF kinase gene. We then demonstrated that the α-hemolysin protein nanopore can be employed for the unambiguous, single-molecule detection of the cross-linked probe-target complex. Cross-linked DNA generates an unmistakable deep and persistent current block (≥5 s) that is easily distinguished from the microsecond and millisecond blocks generated by translocation of single-stranded DNA and uncross-linked duplexes through the nanopore.


Assuntos
Reagentes para Ligações Cruzadas/química , DNA/química , Mecloretamina/química , Polimorfismo de Nucleotídeo Único , Proteínas Proto-Oncogênicas B-raf/genética , Pareamento Incorreto de Bases , Sequência de Bases , DNA/genética , Proteínas Hemolisinas/química , Humanos , Nanoporos/ultraestrutura , Hibridização de Ácido Nucleico
8.
Nanoscale ; 10(29): 13857-13866, 2018 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-29998253

RESUMO

The aerolysin pore (ARP) is a newly emerging nanopore that has been extensively used for peptide and protein sensing. Recently, several groups have explored the application of ARP in detecting genetic and epigenetic markers. This brief review summarizes the current applications of ARP, progressing from peptidomic to genomic detection; the recently reported site-directed mutagenesis of ARP; and new genomic DNA sensing approaches, and their advantages and disadvantages. This review will also discuss the perspectives and future applications of ARP for nucleic acid sequencing and biomolecule sensing.


Assuntos
Toxinas Bacterianas/química , Genômica , Nanoporos , Proteínas Citotóxicas Formadoras de Poros/química , Mutagênese Sítio-Dirigida , Oligonucleotídeos/análise , Estrutura Terciária de Proteína
9.
Diabetes Obes Metab ; 20(9): 2121-2130, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29708650

RESUMO

AIMS: To compare the efficacy and safety of once-weekly glucagon-like peptide-1 receptor agonist dulaglutide 1.5 and 0.75 mg with glimepiride in East-Asian patients with type 2 diabetes (T2D). MATERIALS AND METHODS: In this phase III, multinational, multicentre, double-blind, randomized, parallel-arm, 26-week study, patients with inadequate glycaemic control were randomized 1:1:1 to once-weekly dulaglutide 1.5 or 0.75 mg or daily glimepiride (1-3 mg/d). The primary endpoint was assessment of the non-inferiority of dulaglutide (1.5 mg), as measured by change in glycated haemoglobin (HbA1c), compared with glimepiride using a 0.4% non-inferiority margin. RESULTS: A total of 737 patients were randomized (dulaglutide 1.5 mg, n = 244; dulaglutide 0.75 mg, n = 248; glimepiride, n = 245). At week 26, both doses of dulaglutide were non-inferior and also superior to glimepiride for HbA1c reduction from baseline with a least squares mean difference of -6.34 mmol/mol (95% confidence interval [CI] -8.31, -4.26) or -0.58% (95% CI -0.76, -0.39) for dulaglutide 1.5 mg and -3.50 mmol/mol (95% CI -5.47, -1.42) or -0.32% (95% CI -0.50, -0.13) for dulaglutide 0.75 mg (P < .001). A greater proportion of patients in the dulaglutide 1.5 mg group achieved the HbA1c target of <53 mmol/mol (<7.0%) compared with the glimepiride group (74.1% vs 57.4%; P < .001). The mean body weight decreased (P < .005) and total hypoglycaemia rates were lower (P < .001) in the dulaglutide groups compared with the glimepiride group. The most common drug-related adverse events in both dulaglutide groups (≥5% of patients) included diarrhoea, nausea, increased lipase, decreased appetite, abdominal distension and vomiting. CONCLUSIONS: Dulaglutide (both doses) demonstrated superior glycaemic control vs glimepiride, with a favourable tolerability and safety profile in East-Asian patients with T2D.


Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Peptídeos Semelhantes ao Glucagon/análogos & derivados , Hipoglicemiantes/administração & dosagem , Fragmentos Fc das Imunoglobulinas/administração & dosagem , Proteínas Recombinantes de Fusão/administração & dosagem , Compostos de Sulfonilureia/administração & dosagem , Idoso , Glicemia/efeitos dos fármacos , Diabetes Mellitus Tipo 2/sangue , Método Duplo-Cego , Extremo Oriente , Feminino , Peptídeos Semelhantes ao Glucagon/administração & dosagem , Peptídeos Semelhantes ao Glucagon/efeitos adversos , Hemoglobina A Glicada/efeitos dos fármacos , Humanos , Hipoglicemia/induzido quimicamente , Hipoglicemiantes/efeitos adversos , Fragmentos Fc das Imunoglobulinas/efeitos adversos , Masculino , Pessoa de Meia-Idade , Proteínas Recombinantes de Fusão/efeitos adversos , Compostos de Sulfonilureia/efeitos adversos , Resultado do Tratamento
10.
ACS Nano ; 12(5): 4194-4205, 2018 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-29664612

RESUMO

Accurate and rapid detection of single-nucleotide polymorphism (SNP) in pathogenic mutants is crucial for many fields such as food safety regulation and disease diagnostics. Current detection methods involve laborious sample preparations and expensive characterizations. Here, we investigated a single locked nucleic acid (LNA) approach, facilitated by a nanopore single-molecule sensor, to accurately determine SNPs for detection of Shiga toxin producing Escherichia coli (STEC) serotype O157:H7, and cancer-derived EGFR L858R and KRAS G12D driver mutations. Current LNA applications that require incorporation and optimization of multiple LNA nucleotides. But we found that in the nanopore system, a single LNA introduced in the probe is sufficient to enhance the SNP discrimination capability by over 10-fold, allowing accurate detection of the pathogenic mutant DNA mixed in a large amount of the wild-type DNA. Importantly, the molecular mechanistic study suggests that such a significant improvement is due to the effect of the single-LNA that both stabilizes the fully matched base-pair and destabilizes the mismatched base-pair. This sensitive method, with a simplified, low cost, easy-to-operate LNA design, could be generalized for various applications that need rapid and accurate identification of single-nucleotide variations.


Assuntos
Mutação , Nanoporos , Neoplasias/genética , Oligonucleotídeos/química , Polimorfismo de Nucleotídeo Único/genética , Escherichia coli/química , Escherichia coli/imunologia , Humanos , Simulação de Dinâmica Molecular , Neoplasias/imunologia , Sorogrupo , Toxina Shiga/biossíntese , Toxina Shiga/imunologia
11.
Oncol Lett ; 15(2): 1379-1388, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29434828

RESUMO

The mitogen-activated protein kinase (MAPK) signaling pathway is associated with tumor cell proliferation, differentiation, apoptosis, angiogenesis, invasion and metastasis. The present review assesses the involvement of the MAPK signaling pathway in oral cancer progression and invasion based on analysis of individual sub-pathways and their mechanisms of action. The regulation of this pathway for targeted oral cancer therapy is explored and the challenges confronting this, as well as corresponding potential solutions, are discussed. Exploring this pathway with an emphasis on its components, subfamilies, sub-pathways, interactions with other pathways and clinical practice modes may improve oral cancer treatment.

12.
Surg Radiol Anat ; 40(6): 609-614, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29079941

RESUMO

PURPOSE: The positional relationship between the mandibular canal and corresponding third molars is a key anatomic factor of inferior alveolar nerve (IAN) injury. The aim of the present study is to classify the anatomic three-dimensional relationship between the mandibular third molar and the mandibular canal on cone-beam computed tomography (CBCT) images. METHODS: This study used CBCT images to classify the positional relationship between the mandibular canal and corresponding third molars. CBCT images of 749 patients (1296 mandibular third molars) were analyzed to draw up a classification. RESULTS: On a total of 1296 third molars, the mandibular canal relative to the roots of the mandibular third molar was on the apical side (88.1%), followed by the buccal side (7.9%), the lingual side (3.5%), and then between the roots (0.5%). Ninety-five (7.1%) third molars had a close relation with the mandibular canal, while 1201 (92.7%) third molars had no direct contact. The percentage of the mandibular canal contacts with the mandibular third molar was higher when the mandibular canal was lingually positioned. CONCLUSIONS: The anatomic structures of the mandibular third molar and the mandibular canal may be helpful to make adequate surgical planning to avoid or reduce nerve involvement.


Assuntos
Tomografia Computadorizada de Feixe Cônico , Mandíbula/anatomia & histologia , Dente Serotino/anatomia & histologia , Adulto , Idoso , Humanos , Imagem Tridimensional , Mandíbula/diagnóstico por imagem , Nervo Mandibular/anatomia & histologia , Nervo Mandibular/diagnóstico por imagem , Pessoa de Meia-Idade , Dente Serotino/diagnóstico por imagem , Adulto Jovem
14.
Virulence ; 9(1): 555-562, 2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-28795862

RESUMO

In vitro interaction of osthol (Ost) and fluconazole (FLC) was investigated against 11 fluconazole-resistant clinical isolates of Candida albicans. Synergistic activities were determined using the checkerboard microdilution assay. The results of agar diffusion test confirmed the synergistic interaction. We used an enteric material Eudragit S100 for preparation of Ost nanoparticle (Ost-NP) to improve the oral bioavailability, biological activity of Ost. The physicochemical characteristics of Ost-S100-NP revealed Ost-S100-NP with mean particle size of 55.4±0.4 nm, encapsulation efficiency of 98.95±0.06%, drug loading efficiency of 23.89±0.25%, yield of 98.5±0.1% and a polydispersity index (PDI) of 0.165. As the Ost concentration-time curve showed, Ost-S100-NP can increase the plasma concentration and relative bioavailability of Ost compared with Ost-suspension by oral administration. In vivo, Ost-S100-NP enhanced the therapeutic efficacy of Ost against FLC-resistant C. albicans in immunosuppressed candidiasis mice model. The available information strongly suggests that Ost-S100-NP may be used as a promising compound against drug-resistant fungi.


Assuntos
Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Cumarínicos/farmacologia , Portadores de Fármacos/metabolismo , Sinergismo Farmacológico , Ácidos Polimetacrílicos/metabolismo , Administração Oral , Animais , Antifúngicos/administração & dosagem , Antifúngicos/farmacocinética , Candidíase/tratamento farmacológico , Candidíase/microbiologia , Modelos Animais de Doenças , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/farmacocinética , Fluconazol/farmacologia , Camundongos , Plasma/química , Ácidos Polimetacrílicos/administração & dosagem , Ácidos Polimetacrílicos/farmacocinética , Resultado do Tratamento
15.
Anal Chem ; 89(24): 13039-13043, 2017 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-29183111

RESUMO

Aerolysin protein pore has been widely used for sensing peptides and proteins. However, only a few groups explored this nanopore for nucleic acids detection. The challenge is the extremely low capture efficiency for nucleic acids (>10 bases), which severely lowers the sensitivity of an aerolysin-based genetic biosensor. Here we reported a simple and easy-to-operate approach to noncovalently transform aerolysin into a highly nucleic acids-sensitive nanopore. Through a remote pH-modulation mechanism, we simply lower the pH on one side of the pore, then aerolysin is immediately "activated" and enabled to capture target DNA/RNA efficiently from the opposite side of the pore. This mechanism also decelerates DNA translocation, a desired property for sequencing and gene detection, allowing temporal separation of DNAs in different lengths. This method provides insight into the nanopore engineering for biosensing, making aerolysin applicable in genetic and epigenetic detections of long nucleic acids.


Assuntos
Toxinas Bacterianas/genética , Sequenciamento de Nucleotídeos em Larga Escala , Nanoporos , Ácidos Nucleicos/análise , Proteínas Citotóxicas Formadoras de Poros/genética , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Concentração de Íons de Hidrogênio , Pulmão/química , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/metabolismo
16.
Nat Commun ; 8(1): 1458, 2017 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-29133841

RESUMO

The chemical properties and biological mechanisms of RNAs are determined by their tertiary structures. Exploring the tertiary structure folding processes of RNA enables us to understand and control its biological functions. Here, we report a nanopore snapshot approach combined with coarse-grained molecular dynamics simulation and master equation analysis to elucidate the folding of an RNA pseudoknot structure. In this approach, single RNA molecules captured by the nanopore can freely fold from the unstructured state without constraint and can be programmed to terminate their folding process at different intermediates. By identifying the nanopore signatures and measuring their time-dependent populations, we can "visualize" a series of kinetically important intermediates, track the kinetics of their inter-conversions, and derive the RNA pseudoknot folding pathway. This approach can potentially be developed into a single-molecule toolbox to investigate the biophysical mechanisms of RNA folding and unfolding, its interactions with ligands, and its functions.


Assuntos
Bacteriófago T4/genética , Dobramento de RNA/fisiologia , RNA Viral/metabolismo , Sequência de Bases , Simulação de Dinâmica Molecular , Análise de Sequência de RNA/métodos
17.
Methods Mol Biol ; 1632: 255-268, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28730445

RESUMO

MicroRNAs (miRNAs) are a class of noncoding RNAs that are being explored as a new type of disease biomarkers. The nanopore single-molecule sensor offers a potential noninvasive tool to detect miRNAs for diagnostics and prognosis applications. However, one of the challenges that limits its clinical applications is the presence of a large variety of nontarget nucleic acids in the biofluid extracts. Upon interacting with the nanopore, nontarget nucleic acids produce "contaminative" nanopore signals that interfere with target miRNA discrimination, thus severely lowering the accuracy in target miRNA detection. We have reported a novel method that utilizes a designed polycationic peptide-PNA probe to specifically guide the target miRNA migration toward the nanopore, whereas any nontarget nucleic acids without the probe bound is rejected by the nanopore. Consequently, nontarget species are driven away from the nanopore and only the target miRNA can be detected at low concentration. This method is also able to discriminate miRNAs with single-nucleotide difference by using PNA to capture miRNA. Considering the significance and impact of this substantial advance for the future miRNA detection in biofluid samples, we prepared this detailed protocol, by which the readers can view the experimental procedure, data analysis, and resulting explanation.


Assuntos
Técnicas Biossensoriais , MicroRNAs/química , MicroRNAs/genética , Sondas Moleculares , Poliaminas , Humanos , Modelos Moleculares , Conformação Molecular , Ácidos Nucleicos Peptídicos/química , Peptídeos/química , Poliaminas/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química
18.
ACS Sens ; 2(7): 975-981, 2017 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-28750524

RESUMO

Cancer driver mutations are clinically significant biomarkers. In precision medicine, accurate detection of these oncogenic changes in patients would enable early diagnostics of cancer, individually tailored targeted therapy, and precise monitoring of treatment response. Here we investigated a novel nanolock-nanopore method for single-molecule detection of a serine/threonine protein kinase gene BRAF V600E mutation in tumor tissues of thyroid cancer patients. The method lies in a noncovalent, mutation sequence-specific nanolock. We found that the nanolock formed on the mutant allele/probe duplex can separate the duplex dehybridization procedure into two sequential steps in the nanopore. Remarkably, this stepwise unzipping kinetics can produce a unique nanopore electric marker, with which a single DNA molecule of the cancer mutant allele can be unmistakably identified in various backgrounds of the normal wild-type allele. The single-molecule sensitivity for mutant allele enables both binary diagnostics and quantitative analysis of mutation occurrence. In the current configuration, the method can detect the BRAF V600E mutant DNA lower than 1% in the tumor tissues. The nanolock-nanopore method can be adapted to detect a broad spectrum of both transversion and transition DNA mutations, with applications from diagnostics to targeted therapy.

19.
J Food Sci ; 82(7): 1640-1646, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28585714

RESUMO

With growing consumption of aquaculture products, there is increasing demand on rapid and sensitive techniques that can detect prohibited substances in the seafood products. This study aimed to develop a novel surface-enhanced Raman spectroscopy (SERS) method coupled with simplified extraction protocol and novel gold nanorod (AuNR) substrates to detect banned aquaculture substances (malachite green [MG] and crystal violet [CV]) and their mixture (1:1) in aqueous solution and fish samples. Multivariate statistical tools such as principal component analysis (PCA) and partial least squares regression (PLSR) were used in data analysis. PCA results demonstrate that SERS can distinguish MG, CV and their mixture (1:1) in aqueous solution and in fish samples. The detection limit of SERS coupled with standing AuNR substrates is 1 ppb for both MG and CV in fish samples. A good linear relationship between the actual concentration and predicted concentration of analytes based on PLSR models with R2 values from 0.87 to 0.99 were obtained, indicating satisfactory quantification results of this method. These results demonstrate that the SERS method coupled with AuNR substrates can be used for rapid and accurate detection of MG and CV in fish samples.


Assuntos
Violeta Genciana/análise , Ouro/química , Nanotubos/química , Corantes de Rosanilina/análise , Alimentos Marinhos/análise , Animais , Peixes , Análise de Alimentos , Contaminação de Alimentos/análise , Limite de Detecção , Análise de Componente Principal , Análise Espectral Raman
20.
Chembiochem ; 18(14): 1383-1386, 2017 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-28422400

RESUMO

Hybridization-based methods for the detection of nucleic acid sequences are important in research and medicine. Short probes provide sequence specificity, but do not always provide a durable signal. Sequence-specific covalent crosslink formation can anchor probes to target DNA and might also provide an additional layer of target selectivity. Here, we developed a new crosslinking reaction for the covalent capture of specific nucleic acid sequences. This process involved reaction of an abasic (Ap) site in a probe strand with an adenine residue in the target strand and was used for the detection of a disease-relevant T→A mutation at position 1799 of the human BRAF kinase gene sequence. Ap-containing probes were easily prepared and displayed excellent specificity for the mutant sequence under isothermal assay conditions. It was further shown that nanopore technology provides a high contrast-in essence, digital-signal that enables sensitive, single-molecule sensing of the cross-linked duplexes.


Assuntos
Sondas Moleculares/química , Nanoporos , Proteínas Proto-Oncogênicas B-raf/genética , Sequência de Bases , Humanos , Mutação , Proteínas Proto-Oncogênicas B-raf/metabolismo
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