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1.
J Cell Mol Med ; 2020 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-32337831

RESUMO

The purpose of this study was to alleviate liver disturbance by applying polysaccharides from Dicliptera chinensis (DCP) to act on the adenosine monophosphate-activated protein kinase/ nuclear factor erythroid 2-related factor 2 (AMPK/ Nrf2) oxidative stress pathway and the Toll-like receptor 4 (TLR-4)/ nuclear factor kappa-B (NF-κB) inflammatory pathway and to establish an in vivo liver disturbance model using male C57BL/6J and TLR-4 knockout (-/- ) mice. For this, we evaluated the expression levels of SREBP-1 and Nrf2 after silencing the expression of AMPK using siRNA technology. Our results show that with regard to the TLR-4/ NF-κB inflammatory pathway, DCP inhibits TLR-4, up-regulates the expression of peroxisome proliferator-activated receptor-γ (PPAR-γ), reduces the expression of phospho(p)-NF-κB and leads to the reduction of downstream inflammatory factors, such as tumour necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-1ß, thereby inhibiting the inflammatory response. Regarding the AMPK/ Nrf2 oxidative stress pathway, DCP up-regulates the expression of p-AMPK and Nrf2, in addition to regulating glucose and lipid metabolism, oxidative stress and ameliorating liver disturbance symptoms. In summary, our study shows that DCP alleviates liver disturbances by inhibiting mechanisms used during liver inflammation and oxidative stress depression, which provides a new strategy for the clinical treatment of liver disturbance.

2.
Medicine (Baltimore) ; 99(8): e19044, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32080079

RESUMO

INTRODUCTION: The purpose of this study is to evaluate the efficacy and safety of acupuncture on relieving abdominal pain and distension in acute pancreatitis. METHODS AND ANALYSIS: We will electronically search PubMed, MEDLINE, Embase, Web of Science, the Cochrane Central Register of Controlled Trial, China National Knowledge Infrastructure, China Biomedical Literature Database, China Science Journal Database, and Wanfang Database from their inception. Furthermore, we will manually retrieve other resources, including reference lists of identified publications, conference articles, and gray literature. The clinical randomized controlled trials or quasi-randomized controlled trials related to acupuncture treating acute pancreatitis will be included in the study. The language is limited to Chinese and English. Research selection, data extraction, and research quality assessment will be independently completed by 2 researchers. Data will be synthesized using a fixed effects model or random effects model depending on the heterogeneity test. The overall response rate and the visual analog scale score will be the primary outcomes. The time of first bowel sound, the time of first defecation, the length of hospitalization, acute physiology and chronic health evaluation II score, and the adverse events will also be assessed as secondary outcomes. RevMan 5 (version 5.3) statistical software will be used for meta-analysis, and the level of evidence will be assessed by Grading of Recommendations Assessment, Development, and Evaluation. Continuous data will be expressed in the form of weighted mean difference or standardized mean difference with 95% confidence intervals, whereas dichotomous data will be expressed in the form of risk ratios with 95% confidence intervals. ETHICS AND DISSEMINATION: The protocol of this systematic review does not require ethical approval because it does not involve humans. We will publish this article in peer-reviewed journals and present at relevant conferences. PROSPERO REGISTRATION NUMBER: CRD42019147503.


Assuntos
Dor Abdominal/terapia , Terapia por Acupuntura/métodos , Pancreatite/terapia , Terapia por Acupuntura/efeitos adversos , China/epidemiologia , Defecação/efeitos dos fármacos , Defecação/fisiologia , Humanos , Tempo de Internação/estatística & dados numéricos , Metanálise como Assunto , Pancreatite/complicações , Ensaios Clínicos Controlados Aleatórios como Assunto , Índice de Gravidade de Doença , Escala Visual Analógica
4.
Psychooncology ; 29(3): 532-538, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31742784

RESUMO

BACKGROUND: Both demoralization and depression are common forms of psychological distress in cancer patients. This study aimed to investigate the severity of demoralization in Chinese cancer patients and to explore the factors influencing depression and demoralization and their effects on quality of life to better understand the differences and the relationship between demoralization and depression. METHODS: Cross-sectional study design, in-patients sequentially recruited from a tertiary-level cancer hospital in Beijing between January 2016 and April 2016 completed Mandarin version of Demoralization Scale (DS-MV), Patient Health Questionnaire-9 (PHQ-9), Revised Life Orientation Test (CLOT-R), Beck Hopelessness Scale (BHS), and the 12-items Short Form Health Survey, version 2 (SF-12 V2) and provided socio-demographic and clinical information. RESULTS: 296/424 (70.0%) of patients completed questionnaires. The mean score of DS is 30.4 (SD = 13.0). There are 28% (83/296) patients who experienced a low level of depression but a high level of demoralization. Resignation medical coping method (b = 0.279, P < .001), hopelessness (b = 0.492, P < .001), positive life orientation (b = -0.170, P < .001), and education level (b = -0.132, P < .001) were found to be predictors of demoralization, while only resignation medical coping method (b = 0.373, P < .001) and hopelessness (b = 0.350, P < .001) were found to be predictors of depression. Depression is a mediator between demoralization and physical aspects of quality of life (γ = -0.1604, LLCI = -0.244, ULCI = -0.080). CONCLUSION: Demoralization is a prevalent psychiatric problem in Chinese cancer patients, and a large proportion of patients had low depression but high demoralization. Therefore, screening for demoralization in Chinese oncology practice is essential. A positive life orientation was found to be protective against demoralization.

6.
BMJ Open ; 9(9): e026328, 2019 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-31519667

RESUMO

OBJECTIVES: The aim of this study was to explore perspectives and reasoning of medical staff from Class A tertiary hospitals about the factors hindering and facilitating the uptake and use of clinical practice guidelines (CPGs) during medical procedures. DESIGN: Mixed-method research study to collect and analyse both quantitative and qualitative data. SETTING: Class A tertiary hospitals in China. PARTICIPANTS: The inclusion criteria for the questionnaire survey and qualitative research were (1) medical practitioners and (2) years of practice: above 5 years in a tertiary hospital. METHODS: Questionnaires were distributed to medical staff in 11 cities to collect quantitative data. Frequency and ranking of barriers and enablers were analysed. Spearman correlations were computed to explore the correlation between years of practice, professional title ranking and educational background with self-reported guideline adherence. Using a constructivist grounded theory method, qualitative data were generated via in-depth face-to-face interviews with Chinese medical practitioners. RESULTS: A total of 359 medical practitioners were surveyed and 32 medical practitioners interviewed in 11 cities. Higher frequency and higher ranking of barriers all converged on 'lack of access', 'less convenient', 'lack of applicability' and 'lack of evidence from Chinese sample'. Higher frequency and higher ranking of enablers converged on 'Short formats presentation', 'Utilisation of various media', 'Information visualisation' and 'Linking to patient electronic medical records'. There were no relationships between characteristics of respondents with self-reported adherence. This research produced a theoretical understanding of the experience of medical practitioners when using guidelines. Themes identified were as follows: existing intrinsic flaws in guidelines, deficient or incomplete system mechanism and being ambiguous. CONCLUSION: Our findings provide a comprehensive and culturally sensitive perspective in understanding guideline implementation in China. Strategies addressing those barriers should be further discussed and researched in the future.

7.
Front Genet ; 10: 531, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31275351

RESUMO

Background: Diabetic retinopathy (DR) is a common diabetes complication and was considered as the major cause of blindness among young adults. MiRNAs are a group of small non-coding RNAs regulating the expression of target genes and have been reported to be associated with the development of DR in a variety of molecular mechanisms. In this study, we aimed to identify miRNAs that are differentially expressed (DE) in the serum of DR patients. Methods: We recruited 21 type 2 diabetes mellitus (T2DM) inpatients of Chinese Han ancestry, consisting of 10 non-proliferative DR patients (DR group) and 11 non-DR T2DM patients (NDR group). MiRNA was extracted from fasting peripheral serum and quantified by RNA-seq. The expression levels of miRNA were evaluated and compared between the two groups, with adjustments made for age differences. The validated target genes of miRNAs were subjected to a pathway analysis. We also constructed a weighted polygenic risk score using the DE miRNA and evaluated its predictive power. Results: Five miRNAs were DE between DR and NDR groups (p-Value ≤ 0.01, LFC ≥ 2 or LFC ≤-2). These included miR-4448, miR-338-3p, miR-190a-5p, miR-485-5p, and miR-9-5p. In total, these miRNAs were validated to regulate 55 target genes. Four target genes were found to overlap with the NAD metabolism, sirtuin, and aging pathway, which was thought to control the vascular growth and morphogenesis. The predictive power of our polygenic risk score was apparently high (AUC = 0.909). However, it needs to be interpreted with caution. Conclusion: In this study, we discovered novel DR-specific miRNAs in human serum samples. These circulating miRNAs may represent the pathological changes in the retina in response to diabetes and may serve as non-invasive biomarkers for early DR risk prediction.

8.
Biosens Bioelectron ; 137: 52-57, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31078840

RESUMO

Previously reported photoelectrochemical (PEC) cytosensing commonly relied on the use of photoelectrodes as both signaling sources and platforms to accommodate the biorecognition events. In such a design, the side reaction between photoelectrodes and potential reductive species in the biological media as well as the interplay between the photoelectrodes and biomolecules will inevitably impair the performance of the corresponding PEC cytosensors. Herein, we presented a facile and efficient PEC cytosensor by separating capture probe from the photoelectrode. The system is operated upon by using a ternary heterostructured photoanode to provide an evident and stable photocurrent signal, an aptamer-based biocathode for recognizing and capturing the target cells, and the corresponding signal reduction from the cell-induced steric hindrance effect. Exemplified by human breast cancer cells (MCF-7), the proposed system realizes the separation of the signaling photoanode and the sensing biocathode toward a sensitive and selective self-powered PEC MCF-7 cytosensor. This work reports a new PEC cytosensing protocol, and it is expected to attract more interest in the research of high-performance PEC cytosensing.


Assuntos
Técnicas Biossensoriais , Técnicas Eletroquímicas , Processos Fotoquímicos , Eletrodos , Ouro/química , Humanos , Células MCF-7
9.
Acta Otolaryngol ; 139(5): 444-450, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30806131

RESUMO

BACKGROUND: Intraparotid facial nerve schwannoma (IFNS) is rare and its definite preoperative diagnosis is challenging. OBJECTIVE: To improve available knowledge regarding the diagnosis of IFNS and to suggest an appropriate treatment plan. MATERIAL AND METHODS: We retrospectively analyzed medical records of IFNS patients at our hospital. Inclusion criteria were surgery (from January 2000, to December 2016) for a parotid mass, pathologically diagnosed as a schwannoma. RESULTS: The study included 42 eligible patients who had undergone tumor resection from 5977 parotid tumor patients. Mostly presented hard-textured (18/39) or medium-textured (15/39), with limited mobility (21/39) mass (three tumors were not palpable). Their facial nerve function outcomes were House-Brackmann Grade I (n = 14), Grade II (n = 7), Grade III (n = 11), Grade IV (n = 5), Grade V (n = 3), and Grade VI (n = 2). Significant differences were noted in results based on different surgical methods used (p = .000) and tumor involvement (p = .002). CONCLUSIONS AND SIGNIFICANCE: A hard-textured tumor with limited mobility mass in the parotid gland should prompt the diagnosis of a schwannoma. Tumors involving main trunk usually lead to unsatisfactory facial nerve outcomes. Facial nerve preservation should always be essential, and stripping surgery or intracapsular enucleation could be the preferred surgical methods of choice.


Assuntos
Doenças do Nervo Facial/cirurgia , Traumatismos do Nervo Facial/etiologia , Recidiva Local de Neoplasia , Neurilemoma/cirurgia , Glândula Parótida/patologia , Adolescente , Adulto , Idoso , Doenças do Nervo Facial/patologia , Feminino , Humanos , Doença Iatrogênica , Masculino , Pessoa de Meia-Idade , Neurilemoma/patologia , Estudos Retrospectivos , Adulto Jovem
10.
Org Lett ; 21(4): 1082-1086, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30694071

RESUMO

An efficient synthesis of N-fused polycyclic indoles by a palladium-catalyzed annulation/acyl migration cascade reaction is described. The reaction is ligand-free, scalable, and provides access to a diverse range of useful indole scaffolds from readily available starting materials. Supporting mechanistic studies indicate that the reaction likely proceeds via an intramolecular α-arylation mechanism. The synthetic utility of this protocol is demonstrated by a gram-scale reaction and syntheses toward indole alkaloids and a HSP90 inhibitor.

11.
J Biol Chem ; 294(9): 3312-3320, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30626735

RESUMO

The Ctp1 protein in Schizosaccharomyces pombe is essential for DNA double-strand break (DSB) repair by homologous recombination. Fission yeast Ctp1 and its budding yeast (Sae2) and human (CtIP) homologs control Mre11-Rad50-Nbs1 nuclease complex activity and harbor DNA-binding and -bridging activities. However, the molecular basis for Ctp1-DNA transactions remains undefined. Here, we report atomic force microscopy (AFM) imaging of S. pombe Ctp1-DNA complexes revealing that Ctp1 polymerizes on dsDNA molecules and forms synaptic filaments that bridge two dsDNA strands. We observed that Ctp1 DNA filaments are typified by an average filament length of ∼180 bp of dsDNA and a Ctp1 tetramer footprint of ∼15 bp. Biochemical results characterizing Ctp1 variants with impaired DNA-binding or -bridging properties were consistent with Ctp1-mediated DNA bridging requiring the intact and correctly folded Ctp1 tetramer. Furthermore, mutations altering Ctp1 oligomerization and DNA bridging in vitro conferred cell sensitivity to DSB-producing agents. Together, these results support an important role for Ctp1-regulated DNA strand coordination required for DNA DSB repair in S. pombe.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , DNA Fúngico/genética , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Genoma Fúngico/genética , Modelos Moleculares , Mutação , Ligação Proteica , Domínios Proteicos , Dobramento de Proteína , Multimerização Proteica , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética
12.
ACS Appl Mater Interfaces ; 10(44): 38506-38516, 2018 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-30360113

RESUMO

Natural fibers with functionalities have attracted considerable attention. However, developing facile and versatile strategies to modify natural fibers is still a challenge. In this study, cotton fibers, the most widely used natural fibers, were partially oxidized by sodium periodate in aqueous solution, to give oxidized cotton fibers containing multiple aldehyde groups on their surface. Then poly(hexamethylene guanidine) was chemically grafted onto the oxidized cotton fibers forming Schiff bases between the terminal amines of poly(hexamethylene guanidine) and the aldehyde groups of oxidized cotton fibers. Finally, carbon-nitrogen double bonds were reduced by sodium cyanoborohydride, to bound poly(hexamethylene guanidine) covalently to the surface of cotton fibers. These functionalized fibers show strong and persistent antibacterial activity: complete inhibition against Escherichia coli and Staphylococcus aureus was maintained even after 1000 consecutive washing in distilled water. On the other hand, cotton fibers with only physically adsorbed poly(hexamethylene guanidine) lost their antibacterial activity entirely after a few washes. According to Cell Counting Kit-8 assay and hemolytic analysis, toxicity did not significantly increase after chemical modification. Attributing to the hydrophilicity of poly(hexamethylene guanidine) coatings, the modified cotton fibers were also more hygroscopic compared to untreated cotton fibers, which can improve the comfort of the fabrics made of modified cotton fibers. This study provides a facile and versatile strategy to prepare modified polysaccharide natural fibers with durable antibacterial activity, biosecurity, and comfortable touch.


Assuntos
Antibacterianos/farmacologia , Fibra de Algodão/microbiologia , Têxteis , Aminas/química , Antibacterianos/química , Escherichia coli/efeitos dos fármacos , Escherichia coli/patogenicidade , Guanidina/química , Interações Hidrofóbicas e Hidrofílicas/efeitos dos fármacos , Ácido Periódico/química , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/patogenicidade , Molhabilidade/efeitos dos fármacos
13.
Anal Chem ; 90(18): 10681-10687, 2018 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-30044616

RESUMO

Previous work on photoelectrochemical (PEC) biosensors has demonstrated that the photoanode-based type possesses satisfying sensitivity, because photoanode utilize electrons as the majority charge carriers and a distinct photocurrent can be generated when electron donors are furnished. However, as hole-oxidation reaction occurs at the photoanode interface, the photoanode-based PEC sensor has inferior anti-interference capacity to reductive substances coexisting in the biological sample, leading to a challenged selectivity. Herein, a universal design on selectivity-enhanced PEC enzyme sensor was proposed by integrating a photoanode with a biocathode. Specifically, the CuInS2 sensitization layer and ZnS passivation layer were deposited in sequence on the TiO2 film modified indium-tin oxide (ITO) electrode mainly by successive ionic layer adsorption and reaction (SILAR) means, forming the hybrid ZnS/CuInS2/TiO2/ITO photoanode. A carbon fiber paper (CFP) electrode was modified with biocatalysts of enzymes via the assistance of chitosan (CS) to fabricate the biocathode. Utilizing glucose oxidase (GOx) and horserdish peroxidase (HRP) as biocatalysts, a selectivity-enhanced PEC sensor for glucose was developed. The PEC sensing platform integrating photoanode with biocathode not only inherits distinct photocurrent of the photoanode-based sensor but also possesses enhanced selectivity, because just the biocathode was incubated in the biological sample and there is no interaction between the photoanode and coexisting reductive substances.

14.
Chem Commun (Camb) ; 54(51): 7062-7065, 2018 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-29876572

RESUMO

A general, efficient strategy for a self-powered PEC immunoassay with an evident photocurrent response was proposed by separating the photoanode from recognition events. The immunoassay demonstrates the exciting features of both high sensitivity and anti-interference capabilities.


Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/química , Técnicas Eletroquímicas , Imunoensaio , Nanotubos/química , Nitrilos/química , Polímeros/química , Titânio/química , Condutividade Elétrica , Eletrodos , Tamanho da Partícula , Processos Fotoquímicos , Propriedades de Superfície
15.
Cancer Med ; 7(5): 1578-1603, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29658188

RESUMO

Assessing the CRC subtypes that can predict the outcome of colorectal cancer (CRC) in patients with immunogenicity seems to be a promising strategy to develop new drugs that target the antitumoral immune response. In particular, the disinhibition of the antitumoral T-cell response by immune checkpoint blockade has shown remarkable therapeutic promise for patients with mismatch repair (MMR) deficient CRC. In this review, the authors provide the update of the molecular features and immunogenicity of CRC, discuss the role of possible predictive biomarkers, illustrate the modern immunotherapeutic approaches, and introduce the most relevant ongoing preclinical study and clinical trials such as the use of the combination therapy with immunotherapy. Furthermore, this work is further to understand the complex interactions between the immune surveillance and develop resistance in tumor cells. As expected, if the promise of these developments is fulfilled, it could develop the effective therapeutic strategies and novel combinations to overcome immune resistance and enhance effector responses, which guide clinicians toward a more "personalized" treatment for advanced CRC patients.


Assuntos
Biomarcadores Tumorais/imunologia , Neoplasias Colorretais/tratamento farmacológico , Imunoterapia/métodos , Animais , Ensaios Clínicos como Assunto , Neoplasias Colorretais/genética , Neoplasias Colorretais/imunologia , Reparo de Erro de Pareamento de DNA , Resistencia a Medicamentos Antineoplásicos , Humanos , Medicina de Precisão , Resultado do Tratamento , Microambiente Tumoral
16.
Mikrochim Acta ; 185(5): 249, 2018 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-29623497

RESUMO

An electrochemical sensor for nitrite was fabricated by modifying a glassy carbon electrode (GCE) with the conducting polymer poly(3,4-ethylenedioxythiophene) (PEDOT) that is doped with carbon quantum dots (CQDs). The negatively charged CQDs were doped into PEDOT via electrodeposition to form a conducting polymer nanocomposite on the GCE. The electrode surface has a flake-like nanostructure and a large specific surface area. The elemental mapping analysis revealed that the CQDs are uniformly distributed across the whole nanocomposite. As a result of the superior catalytic activity of CQDs and the good conductivity of PEDOT, the modified GCE displays excellent electrocatalytic activity towards the oxidation of nitrite, and the oxidation peak current is ten times higher than that of a PEDOT modified GCE without CQDs. Under optimum conditions and at a working voltage of 0.80 V (vs. Ag/AgCl), the sensor has a linear response in the 0.5-1110 µM nitrite concentration range, and an 88 nM limit of detection (at S/N = 3). Three different electrodes prepared under the same experimental conditions were applied for the detection of nitrite, and the RSD was 3.1%. The same sensor was employed to quantify nitrite in three replicate measurements, and the RSD was 2.2%. Graphical abstract Poly(3,4-ethylenedioxythiophene) (PEDOT) was doped with carbon quantum dots and deposited on a glassy carbon electrode to obtain an amperometric sensor for nitrite.

17.
J Clin Hypertens (Greenwich) ; 19(12): 1348-1356, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29067768

RESUMO

New-onset diabetes mellitus (NOD) refers to forms of diabetes mellitus that develop during the therapeutic processes of other diseases such as hypertension. This study has been conducted in a network meta-analysis to compare antihypertensive drugs by identifying both the advantages and disadvantages on NOD by focusing on their respective effect rates. Odd ratios and corresponding 95% confidence intervals or credible intervals were calculated within pairwise and network meta-analysis. A total of 38 articles with 224 140 patients were included to evaluate the preventive effect of hypertension drugs on NOD. From the network meta-analysis it was evident that both angiotensin-converting enzyme inhibitor as well as angiotensin receptor blocker treatments are associated with a lower risk of developing NOD compared with placebo, with ranking probabilities of 79.81% and 72.77%, respectively, while ß-blockers and calcium channel blockers may significantly increase the probability of developing NOD (ß-blockers: odds ratio, 2.18 [95% credible intervals: 1.36-3.50]; calcium channel blockers: odds ratio, 1.16 [95% credible intervals, 1.05-1.29]). In conclusion, angiotensin receptor blockers have an advantage over the other treatments regarding the NOD.


Assuntos
Anti-Hipertensivos , Diabetes Mellitus , Hipertensão/tratamento farmacológico , Anti-Hipertensivos/classificação , Anti-Hipertensivos/farmacologia , Diabetes Mellitus/diagnóstico , Diabetes Mellitus/epidemiologia , Diabetes Mellitus/etiologia , Humanos , Medição de Risco , Fatores de Risco
18.
Methods Enzymol ; 592: 187-212, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28668121

RESUMO

Atomic force microscopy (AFM) is a scanning probe technique that allows visualization of single biomolecules and complexes deposited on a surface with nanometer resolution. AFM is a powerful tool for characterizing protein-protein and protein-DNA interactions. It can be used to capture snapshots of protein-DNA solution dynamics, which in turn, enables the characterization of the conformational properties of transient protein-protein and protein-DNA interactions. With AFM, it is possible to determine the stoichiometries and binding affinities of protein-protein and protein-DNA associations, the specificity of proteins binding to specific sites on DNA, and the conformations of the complexes. We describe methods to prepare and deposit samples, including surface treatments for optimal depositions, and how to quantitatively analyze images. We also discuss a new electrostatic force imaging technique called DREEM, which allows the visualization of the path of DNA within proteins in protein-DNA complexes. Collectively, these methods facilitate the development of comprehensive models of DNA repair and provide a broader understanding of all protein-protein and protein-nucleic acid interactions. The structural details gleaned from analysis of AFM images coupled with biochemistry provide vital information toward establishing the structure-function relationships that govern DNA repair processes.


Assuntos
Reparo de Erro de Pareamento de DNA , DNA/metabolismo , Microscopia de Força Atômica/métodos , Proteínas MutL/metabolismo , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismo , DNA/química , Desenho de Equipamento , Humanos , Microscopia de Força Atômica/instrumentação , Proteínas MutL/química , Proteína MutS de Ligação de DNA com Erro de Pareamento/química , Ligação Proteica , Conformação Proteica , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Eletricidade Estática , Thermus/química , Thermus/metabolismo
19.
Elife ; 52016 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-27595565

RESUMO

The epigenetic inheritance of DNA methylation requires UHRF1, a histone- and DNA-binding RING E3 ubiquitin ligase that recruits DNMT1 to sites of newly replicated DNA through ubiquitylation of histone H3. UHRF1 binds DNA with selectivity towards hemi-methylated CpGs (HeDNA); however, the contribution of HeDNA sensing to UHRF1 function remains elusive. Here, we reveal that the interaction of UHRF1 with HeDNA is required for DNA methylation but is dispensable for chromatin interaction, which is governed by reciprocal positive cooperativity between the UHRF1 histone- and DNA-binding domains. HeDNA recognition activates UHRF1 ubiquitylation towards multiple lysines on the H3 tail adjacent to the UHRF1 histone-binding site. Collectively, our studies are the first demonstrations of a DNA-protein interaction and an epigenetic modification directly regulating E3 ubiquitin ligase activity. They also define an orchestrated epigenetic control mechanism involving modifications both to histones and DNA that facilitate UHRF1 chromatin targeting, H3 ubiquitylation, and DNA methylation inheritance.


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Epigênese Genética , Histonas/metabolismo , Ubiquitinação , Testamentos , DNA/metabolismo , DNA (Citosina-5-)-Metiltransferase 1 , Células HeLa , Humanos , Ligação Proteica
20.
Mol Cell ; 61(2): 315-23, 2016 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-26774284

RESUMO

Many cellular functions require the assembly of multiprotein-DNA complexes. A growing area of structural biology aims to characterize these dynamic structures by combining atomic-resolution crystal structures with lower-resolution data from techniques that provide distributions of species, such as small-angle X-ray scattering, electron microscopy, and atomic force microscopy (AFM). A significant limitation in these combinatorial methods is localization of the DNA within the multiprotein complex. Here, we combine AFM with an electrostatic force microscopy (EFM) method to develop an exquisitely sensitive dual-resonance-frequency-enhanced EFM (DREEM) capable of resolving DNA within protein-DNA complexes. Imaging of nucleosomes and DNA mismatch repair complexes demonstrates that DREEM can reveal both the path of the DNA wrapping around histones and the path of DNA as it passes through both single proteins and multiprotein complexes. Finally, DREEM imaging requires only minor modifications of many existing commercial AFMs, making the technique readily available.


Assuntos
DNA/química , Imageamento Tridimensional , Microscopia de Força Atômica , Reparo de Erro de Pareamento de DNA , Nucleossomos/metabolismo , Proteínas/metabolismo , Eletricidade Estática
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