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Changes in the oxidative (redox) environment accompany idiopathic pulmonary fibrosis (IPF). S-glutathionylation of reactive protein cysteines is a post-translational event that transduces oxidant signals into biological responses. We recently demonstrated that increases in S-glutathionylation promote pulmonary fibrosis, which was mitigated by the deglutathionylating enzyme glutaredoxin (GLRX). However, the protein targets of S-glutathionylation that promote fibrogenesis remain unknown. In the present study we addressed whether the extracellular matrix is a target for S-glutathionylation. We discovered increases in collagen 1A1 S-glutathionylation (COL1A1-SSG) in lung tissues from IPF subjects compared to control subjects in association with increases in ER oxidoreductin 1 (ERO1A) and enhanced oxidation of ER-localized peroxiredoxin 4 (PRDX4) reflecting an increased oxidative environment of the endoplasmic reticulum (ER). Human lung fibroblasts exposed to transforming growth factor beta 1 (TGFB1) show increased secretion of COL1A1-SSG. Pharmacologic inhibition of ERO1A diminished oxidation of PRDX4, attenuated COL1A1-SSG and total COL1A1 levels and dampened fibroblast activation. Absence of Glrx enhanced COL1A1-SSG and overall COL1A1 secretion and promoted activation of mechanosensing pathways. Remarkably, COL1A1-SSG resulted in marked resistance to collagenase degradation. Compared to COL1, lung fibroblasts plated on COL1-SSG proliferated more rapidly, and increased expression of genes encoding extracellular matrix crosslinking enzymes and genes linked to mechanosensing pathways. Overall, these findings suggest that glutathione-dependent oxidation of COL1A1 occurs in settings of IPF in association with enhanced ER oxidative stress and may promote fibrotic remodeling due to increased resistance to collagenase-mediated degradation and fibroblast activation.
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A simple and cost-effective fabrication method of gold nanorods (AuNRs) nanoparticles hybridized with polyvinyl alcohol hydrogel (AuNR/PVA) for SERS substrate is described. The AuNR/PVA achieves the control of inter-particle nanogap by modulating the density of gold nanorods, and inter-particle nanogap by the spatial deformation of the hydrogel, and the reduction of the gap between the AuNRs deposited on hydrogel makes the SERS enhancement. In addition, the AuNR/PVA substrate maintains high SERS activity after more than 100 cycles of bending and storage in air for 30 days, and the substrate possesses high sensitivity and high reproducibility. Combining a flexible and transparent surface-enhanced Raman spectroscopy (SERS) substrate for in situ detection with a small portable Raman can be applied to scenarios such as environmental detection and hazardous materials detection. The substrate showed excellent SERS activity against malachite green (MG) and crystal violet (CV) with limits of detection of 1.18 × 10-13 M and 7.17 × 10-12 M, respectively. The usability of the proposed SERS substrate was demonstrated by detecting the above contaminants in aquatic water. This work not only utilizes a cost-effective method for mass production but also provides a reliable and convenient platform for the preparation of other noble metal flexible substrates.
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Soft template designing is the most promising strategy for the synthesis of zeolite nanosheets. MFI nanosheets directed by soft templates (containing long-chain alkyl groups or aromatic groups as hydrophobic component) can be found frequently; however, so far, MFI nanosheets synthesized by soft templates with aromatic heterocycle groups (e. g., s-triazine groups) are rare. Herein, a nanosheet-stacked hierarchical MFI zeolite (NSHM) has been synthesized by using a triply branched s-triazine-based surfactant as a bifunctional organic structure-directing agent. On the basis of a geometrical match relationship, a formation model has been proposed. Synthesized NSHM had abundant mesopores stacked by nanosheets and exhibited a high surface area (430â m2 â g-1 ). The 1â wt% Pd/NSHM attained a significant increase in yield of cyclohexanol/cyclohexanone mixture (from 66 to 85 %) in the oxidation of cyclohexane compared with Silicalite-1 and SBA-15 as supports.
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A manganese-catalyzed N-alkylation reaction of amines with alcohols via hydrogen autotransfer strategy has been demonstrated. The developed practical catalytic system including an inexpensive, nontoxic, commercially available MnCl2 or MnBr(CO)5 as the metal salt and triphenylphosphine as a ligand provides access to diverse aromatic, heteroaromatic, and aliphatic secondary amines in moderate-to-high yields. In addition, this operationally simple protocol is scalable to the gram level and suitable for synthesizing heterocycles such as indole and resveratrol-derived amines known to be active for Alzheimer's disease.
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Álcoois , Aminas , Alquilação , Catálise , Cloretos , Manganês , Estrutura MolecularRESUMO
The mammalian runt-related factor 1 (RUNX1) is a master transcription factor that regulates lineage specification of hematopoietic stem cells. RUNX1 translocations result in the development of myeloid leukemias. Recently, RUNX1 has been implicated as a tumor suppressor in other cancers. We postulated RUNX1 expression may be associated with lung adenocarcinoma etiology and/or progression. We evaluated the association of RUNX1 mRNA expression with overall survival data from The Cancer Genome Atlas (TCGA), a publically available database. Compared to high expression levels, Low RUNX1 levels from lung adenocarcinomas were associated with a worse overall survival (Hazard Ratio = 2.014 (1.042-3.730 95% confidence interval), log-rank p = 0.035) compared to those that expressed high RUNX1 levels. Further immunohistochemical examination of 85 surgical specimens resected at the University of Vermont Medical Center identified that low RUNX1 protein expression was associated with larger tumors (p = 0.038). Gene expression network analysis was performed on the same subset of TCGA cases that demonstrated differential survival by RUNX1 expression. This analysis, which reveals regulatory relationships, showed that reduced RUNX1 levels were closely linked to upregulation of the transcription factor E2F1. To interrogate this relationship, RUNX1 was depleted in a lung cancer cell line that expresses high levels of RUNX1. Loss of RUNX1 resulted in enhanced proliferation, migration, and invasion. RUNX1 depletion also resulted in increased mRNA expression of E2F1 and multiple E2F1 target genes. Our data implicate loss of RUNX1 as driver of lung adenocarcinoma aggression, potentially through deregulation of the E2F1 pathway.
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Adenocarcinoma/metabolismo , Agressão/fisiologia , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Regulação da Expressão Gênica/fisiologia , Neoplasias Pulmonares/metabolismo , Adenocarcinoma de Pulmão/metabolismo , Proliferação de Células/fisiologia , Perfilação da Expressão Gênica/métodos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Ativação Transcricional/fisiologiaRESUMO
Autophagy has emerged as a mechanism critical to both tumorigenesis and development of resistance to multiple lines of anti-cancer therapy. Therefore, targeting autophagy and alternative cell death pathways has arisen as a viable strategy for refractory tumors. The anti-malarial 4-aminoquinoline compounds chloroquine and hydroxychloroquine are currently being considered for re-purposing as anti-cancer therapies intended to sensitize different tumors by targeting the lysosomal cell death pathway. Here, we describe a novel organometallic chloroquine derivative, cymanquine, that exhibits enhanced bioactivity compared to chloroquine in both normal, and reduced pH tumor microenvironments, thus overcoming a defined limitation of traditional 4-aminoquinolines. In vitro, cymanquine exhibits greater potency than CQ in a diverse panel of human cancer cell lines, including melanoma, in both normal pH and in reduced pH conditions that mimic the tumor microenvironment. Cymanquine treatment results in greater lysosomal accumulation than chloroquine and induces lysosomal dysfunction leading to autophagy blockade. Using a mouse model of vemurafenib-resistant melanoma, cymanquine slowed tumor growth greater than hydroxychloroquine, and when used in combination with vemurafenib, cymanquine partially restored sensitivity to vemurafenib. Overall, we show that cymanquine exhibits superior lysosomal accumulation and autophagy blockade than either chloroquine or hydroxychloroquine in vitro; and in addition to its high level of tolerability in mice, exhibits superior in vivo efficacy in a model of human melanoma.
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Proliferação de Células/efeitos dos fármacos , Cloroquina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Melanoma/tratamento farmacológico , Compostos Organometálicos/farmacologia , Animais , Antimaláricos/farmacologia , Apoptose/efeitos dos fármacos , Feminino , Humanos , Melanoma/patologia , Camundongos , Camundongos Nus , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A novel C(sp3)-H bond arylation of benzyl ethers with Knochel-type arylzinc reagents has been developed. This transition-metal-catalyst-free reaction proceeds well under mild conditions in a simple and effective manner and enables the synthesis of a wide range of potentially biologically active benzyl ethers by using highly functionalized organozinc reagents as a carbon nucleophile.
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Organomanganese reagents were prepared by the insertion of magnesium into aryl halides in the presence of MnCl2·2LiCl. These organomanganese reagents smoothly undergo 1,2-addition, acylation, and Pd-catalyzed cross-coupling with various electrophiles. Especially, the oxidative homocoupling of organomanganese reagents was completed in one pot without an additional transition-metal catalyst.
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Background: From the end of 2019 to December 2023, the world grappled with the COVID-19 pandemic. The scope and ultimate repercussions of the pandemic on global health and well-being remained uncertain, ushering in a wave of fear, anxiety, and worry. This resulted in many individuals succumbing to fear and despair. Acupoint massage emerged as a safe and effective alternative therapy for anxiety relief. However, its efficacy was yet to be extensively backed by evidence-based medicine. This study aimed to enhance the clinical effectiveness of acupoint massage and extend its benefits to a wider population. It undertakes a systematic review of the existing randomized controlled trials (RCTs) assessing the impact of acupoint massage on anxiety treatment, discussing its potential benefits and implications. This research aims to furnish robust evidence supporting anxiety treatment strategies for patients afflicted with COVID-19 disease and spark new approaches to anxiety management. Objectives: This study evaluates the evidence derived from randomised controlled trials (RCTs), quantifies the impact of acupressure on anxiety manifestations within the general population, and proposes viable supplementary intervention strategies for managing COVID-19 related anxiety. Materials and methods: This review included RCTs published between February 2014 and July 2023, that compared the effects of acupressure with sham control in alleviating anxiety symptomatology as the outcome measure. The studies were sourced from the multiple databases, including CINAHL, EBM Reviews, Embase, Medline, PsycINFO, Scopus and Web of Science. A meta-analysis was performed on the eligible studies, and an overall effect size was computed specifically for the anxiety outcome. The Cochrane Collaboration Bias Risk Assessment Tool (RevMan V5.4) was employed to assess bias risk, data integration, meta-analysis, and subgroup analysis. The mean difference, standard mean deviation, and binary data were used to represent continuous outcomes. Results: Of 1,110 studies of potential relevance, 39 met the criteria for inclusion in the meta-analysis. The majority of the studies reported a positive effect of acupressure in assuaging anticipatory anxiety about treatment. Eighteen studies were evaluated using the STAI scale. The acupressure procedures were thoroughly documented, and studies exhibited a low risk of bias. The cumulative results of the 18 trials showcased a more substantial reduction in anxiety in the acupressure group compared to controls (SMD = -5.39, 95% CI -5.61 to -5.17, p < 0.01). A subsequent subgroup analysis, based on different interventions in the control group, demonstrated improvement in anxiety levels with sham acupressure in improving changes in anxiety levels (SMD -1.61, 95% CI: -2.34 to -0.87, p < 0.0001), and blank controls (SMD -0.92, 95% CI: -2.37 to 0.53, p = 0.22). Conclusion: In the clinical research of traditional Chinese medicine treatment of anxiety, acupressure demonstrated effectiveness in providing instant relief from anxiety related to multiple diseases with a medium effect size. Considering the increasing incidence of anxiety caused by long COVID, the widespread application of acupressure appears feasible. However, the results were inconsistent regarding improvements on physiological indicators, calling for more stringent reporting procedures, including allocation concealment, to solidify the findings.
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Our previous study revealed that acupuncture may exhibit therapeutic effects on Alzheimer's disease (AD) through the activation of metabolism in memory-related brain regions. However, the underlying functional mechanism remains poorly understood and warrants further investigation. In this study, we used resting-state functional magnetic resonance imaging (rsfMRI) to explore the potential effect of electroacupuncture (EA) on the 5xFAD mouse model of AD. We found that the EA group exhibited significant improvements in the number of platforms crossed and the time spent in the target quadrant when compared with the Model group (p < 0.05). The functional connectivity (FC) of left hippocampus (Hip) was enhanced significantly among 12 regions of interest (ROIs) in the EA group (p < 0.05). Based on the left Hip as the seed point, the rsfMRI analysis of the entire brain revealed increased FC between the limbic system and the neocortex in the 5xFAD mice after EA treatment. Additionally, the expression of amyloid-ß(Aß) protein and deposition in the Hip showed a downward trend in the EA group compared to the Model group (p < 0.05). In conclusion, our findings indicate that EA treatment can improve the learning and memory abilities and inhibit the expression of Aß protein and deposition of 5xFAD mice. This improvement may be attributed to the enhancement of the resting-state functional activity and connectivity within the limbic-neocortical neural circuit, which are crucial for cognition, motor function, as well as spatial learning and memory abilities in AD mice.
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Doença de Alzheimer , Eletroacupuntura , Neocórtex , Camundongos , Animais , Doença de Alzheimer/diagnóstico por imagem , Doença de Alzheimer/terapia , Doença de Alzheimer/metabolismo , Eletroacupuntura/métodos , Peptídeos beta-Amiloides/metabolismo , Hipocampo/metabolismo , Neocórtex/diagnóstico por imagem , Neocórtex/metabolismo , Aprendizagem Espacial , Modelos Animais de Doenças , Camundongos TransgênicosRESUMO
BACKGROUND: During the coronavirus disease 2019 (COVID-19) epidemic, the fever clinic is an important link for screening and diagnosing whether a patient is infected with the novel coronavirus. Blood collection from children's fingertips is a commonly used detection method; however, in children, the blood collection process may cause discomfort and resistance. To address this problem, the use of heating gloves combined with hand swinging can be considered for fingertip blood collection in children. AIM: To explore the application of fever gloves with the handshaking method for fingertip blood collection from children in fever clinics during the COVID-19 epidemic. METHODS: A total of 100 children were selected for fingertip blood collection at the fever clinic of our hospital from June 2022 to June 2023 and were divided into two groups using a randomized numerical table method, with 50 cases in each group, including the control and observation groups. The patients in the control group followed the doctor's instructions to cooperate with the routine fingertip blood collection method, and the patients in the observation group followed the doctor's instructions to cooperate with the static fever gloves with the shaking hands method of children's fingertip blood collection. The level of the six blood routine and collection indexes, and the satisfaction of the examination of the patients in the peripheral blood group and the fever gloves with the shaking hands method of the children's fingertip blood collection group were compared. RESULTS: The red and white blood cell count, hemoglobin, and red blood cell pressure volume in the observation group were higher than those in the control group (P < 0.05); the platelet count in the control group was lower than that in the observation group (P < 0.05); the number of times of squeezing the fingertip, the average time of blood collection, and the score of puncture pain in the observation group were significantly better than those in the control group (P < 0.05); and satisfaction with the routine blood examination in the observation group was greater than that in the control group. CONCLUSION: The application value of the fever gloves with shaking hands method for children's fingertip blood collection was better, the accuracy of examination indexes was higher, and patient satisfaction with the examination was greater.
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In this work, we demonstrated a sensor array with multiple fluorescence channels using dendritic mesoporous silica nanoparticles embedded with three quantum dots for the determination of four heavy metal ions (Hg2+, Cu2+, Cr3+, and Ag+). Carboxyl-modified CdTe QDs with three different fluorescence emission wavelengths were loaded onto a dendritic mesoporous supporter by an amidation reaction. The fluorescence sensor array exhibited excellent analytical performance for discrimination and semi-quantification of heavy metal ions from a single test, which simplified detection procedures. The four heavy metal ions exhibited different degrees of quenching of the fluorescence emission intensities of the three quantum dots and resulted in a variant data matrix for linear discriminant analysis. Under optimized conditions, the fluorescence sensor array discriminated the four heavy metal ions in a concentration range of 0.05-5 µmol/L, and semi-quantified Hg2+, Cu2+, Cr3+, and Ag+ with a limit of detection of 2.51 nmol/L, 5.15 nmol/L, 3.81 nmol/L, and 5.74 nmol/L, respectively. The fluorescence sensor array integrated the sensing units into a single nanoparticle instead of the complex multiple detection steps used in traditional sensor arrays, providing an alternative strategy for constructing a single-well sensing platform. Furthermore, the fluorescence sensor array showed great practical potential for distinguishing heavy metal ions in raw water and crayfish samples.
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Compostos de Cádmio , Mercúrio , Metais Pesados , Nanopartículas , Pontos Quânticos , Fluorescência , Telúrio , Dióxido de Silício , Metais Pesados/análise , Mercúrio/análise , ÍonsRESUMO
Although previous studies have suggested that meteorological factors are associated with Bell's palsy, articles on this topic are rare and the results are inconsistent. We aim to reveal the relationship between exposure to different meteorological factors and the onset of severe Bell's palsy (SBP) with daily data. A case-crossover study based on time-series data was applied, and the minimum risk value of each climatic factor was set as the reference value. We fitted a distributed lag non-linear model (DLNM) which applied quasi-Poisson regression to evaluate the exposure-response association and the lag-response association of meteorological factors on the occurrence of SBP. The mode value and per-decile interval value of each meteorological factor were all included in the analysis. Sensitivity analyses were conducted to test the robustness of results. A total of 863 SBP patients (474 males and 389 females) from 7 hospitals in the Shenzhen Futian District were selected from January 2009 to February 2020. The highest relations effect was tested in the cumulative exposure-response result shown as follows; mean temperature at the minimum value 15.3°C with RR of 10.370 (1.557-69.077) over lag 0 to 13; relative humidity at the 30th value 71% with RR of 8.041 (1.016-63.616) over lag 0 to 14; wind speed at the 90th value 31 (0.1 m/s) with RR of 1.286 (1.038-1.593) over lag 0; mean air pressure at the 30th value 1001.4 (pa) with RR of 9.052 (1.039-78.858) over lag 0 to 5; visibility at the 80th value 26.5 (km) with RR of 1.961 (1.005-1.423) over lag 0 to 2; average total cloud cover at the max value 100 (%) with RR 1.787 (1.014-3.148) over lag 0 to 2; sunshine duration at the 10th value 0.1 (h) with RR of 4.772 (1.018-22.361); daily evaporation shows no relationship in the cumulative result; daily average solar radiation at the minimum value 0 (W/m2) with RR of 5.588 (1.184-26.382). There is a relationship between wind speed and the onset of SBP, while mean air pressure, visibility, and average total cloud cover, especially sunshine duration and solar radiation which showed a strong effect, may be associated with severe clinical symptoms of SBP. Mean temperature and relative humidity may affect the course of SBP.
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Paralisia de Bell , Paralisia Facial , Masculino , Feminino , Humanos , Paralisia de Bell/epidemiologia , Paralisia de Bell/etiologia , Estudos Cross-Over , Fatores de Tempo , Dinâmica não Linear , Temperatura , China/epidemiologiaRESUMO
Glutathione (GSH) is a critical component of the cellular redox system that combats oxidative stress. The glutamate-cystine antiporter, system xC-, is a key player in GSH synthesis that allows for the uptake of cystine, the rate-limiting building block of GSH. It is unclear whether GSH or GSH-dependent protein oxidation [protein S-glutathionylation (PSSG)] regulates the activity of system xC-. We demonstrate that an environment of enhanced PSSG promotes GSH increases via a system xC--dependent mechanism. Absence of the deglutathionylase, glutaredoxin (GLRX), augmented SLC7A11 protein and led to significant increases of GSH content. S-glutathionylation of C23 or C204 of the deubiquitinase OTUB1 promoted interaction with the E2-conjugating enzyme UBCH5A, leading to diminished ubiquitination and proteasomal degradation of SLC7A11 and augmentation of GSH, effects that were reversed by GLRX. These findings demonstrate an intricate link between GLRX and GSH via S-glutathionylation of OTUB1 and system xC- and illuminate a previously unknown feed-forward regulatory mechanism whereby enhanced GSH protein oxidation augments cellular GSH.
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Cistina , Glutarredoxinas , Transporte Biológico , Ácido Glutâmico , GlutationaRESUMO
Norfloxacin (NOX), a kind of quinolone antibiotic, is widely used in disease treatment and the control of human and livestock products. Due to overuse, norfloxacin has become a common organic pollutant in water. We combine the high specific surface area and high stability of metal-organic frameworks with the high selectivity of molecularly imprinted polymers. By grafting a carbon-carbon double bond on the surface of UiO-66-NH2, a molecularly imprinted layer is formed on the surface of UiO-66-NH2 upon free radical polymerization. The saturated adsorption capacity of UiO-66@MIP reaches 58.01 mg g-1. UiO-66@MIP exhibits high adsorption performance in real water samples and its recoveries range from 96.7% to 98.3%, which demonstrates a higher adsorption capacity and recovery than other molecularly imprinted materials and has potential applications in the removal of norfloxacin in real life.
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BACKGROUND: To this day, the molecular mechanism of endotoxin-induced multi-organ failure has not been completely clarified. This study aimed to construct an miRNA-mRNA regulatory network and identify main pathways and key genes in multi-organ of LPS-mediated endotoxemic mice. METHODS: Public datasets from six mRNA and three miRNA microarray datasets were downloaded from the GEO website to screen final differentially expressed genes (FDEGs) and hub genes in the heart, lung, liver, and kidney of LPS-mediated endotoxemic mice. Functional and pathway enrichment analysis of FDEGs was used to identify the main pathways in multi-organ damage of LPS-treated mice. Finally, hub genes of each organ were intersected to obtain the key genes of multi-organ. RESULTS: Firstly, 158, 358, 299, and 91 FDEGs were identified in the heart, lung, liver, and kidney, respectively. The pathway enrichment analysis of the FDEGs then showed that the TNF signaling pathway, Toll-like receptor signaling pathway, and some viral-infection-related pathways (influenza A, measles, and herpes simplex) were the main pathways in multi-organ damage of LPS-mediated endotoxemic mice. Moreover, miRNA-mRNA or PPI regulatory networks were constructed based on FDEGs. According to these networks, 31, 34, 34, and 31 hub genes were identified in the heart, lung, liver, and kidney, respectively. Among them, nine key genes (Cd274, Cxcl1, Cxcl9, Icam1, Ifit2, Isg15, Stat1, Tlr2, and Usp18) were enriched in Toll-like receptor signaling pathway and chemokine signaling pathway. Finally, seven potential drugs were predicted based on these key genes. CONCLUSION: The shared underlying molecular pathways in endotoxin-induced multi-organ damage that have been identified include Toll-like receptor signaling pathway and TNF signaling pathway. Besides, nine key genes (Cd274, Cxcl1, Cxcl9, Icam1, Ifit2, Isg15, Stat1, Tlr2, and Usp18) and seven potential drugs were identified. Our data provide a new sight and potential target for future therapy in endotoxemia-induced multi-organ failure.
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Viral myocarditis (VMC) is a disease characterized as myocardial parenchyma or interstitium inflammation caused by virus infection, especially Coxsackievirus B3 (CVB3) infection, which has no accurate non-invasive examination for diagnosis and specific drugs for treatment. The mechanism of CVB3-induced VMC may be related to direct myocardial damage of virus infection and extensive damage of abnormal immune response after infection. Non-coding RNA (ncRNA) refers to RNA that is not translated into protein and plays a vital role in many biological processes. There is expanding evidence to reveal that ncRNAs regulate the occurrence and development of VMC, which may provide new treatment or diagnosis targets. In this review, we mainly demonstrate an overview of the potential role of ncRNAs in the pathogenesis, diagnosis and treatment of CVB3-induced VMC.
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Infecções por Coxsackievirus , Miocardite , Animais , Infecções por Coxsackievirus/diagnóstico , Modelos Animais de Doenças , Enterovirus Humano B/genética , Camundongos , Camundongos Endogâmicos BALB C , Miocárdio , RNA não Traduzido/genéticaRESUMO
BACKGROUND: Atherosclerosis, the underlying cause of the majority of cardiovascular diseases, is a lipid-driven, inflammatory disease of the large arteries. Atherosclerotic cardiovascular disease (ASCVD) threatens human lives due to high morbidity and mortality. Many studies have demonstrated that atherosclerosis is accelerated via activation of the NLRP3 inflammasome. The NLRP3 inflammasome plays a critical role in the development of vascular inflammation and atherosclerosis. In atherosclerotic plaques, excessive generation of reactive oxygen species (ROS) activates the NLRP3 inflammasome. 13-Methylberberine (13-MB) is a newly synthesized compound used in traditional Chinese medicine that has outstanding antibacterial, antitumor, and antiobesity activities, especially anti-inflammatory activity. However, the role of 13-MB in atherosclerosis needs to be explored. METHODS: CCK-8 assays and flow cytometry were conducted to determine the cell viability and apoptotic profiles of human umbilical vein endothelial cells (HUVECs) treated with 13-MB. Carboxy-DCFH-DA and JC-10 assays were used to measure ROS and determine mitochondrial membrane potential. Western blot analysis was performed to investigate proteins that are associated with the NLRP3 inflammasome and autophagy. ELISA was used to detect and quantify inflammatory cytokines related to the NLRP3 inflammasome. Transfection and confocal microscopy were conducted to observe autophagy. RESULTS: Pretreatment with 13-MB markedly reduced cytotoxicity and apoptosis, as well as intracellular ROS production, in H2O2-induced HUVECs. Moreover, 13-MB showed a protective effect in maintaining mitochondrial membrane potential. 13-MB also suppressed NLRP3 inflammasome activation and promoted autophagy induction in HUVECs. CONCLUSION: 13-MB exerts cytoprotective effects in an H2O2-induced cell injury model by inhibiting NLRP3 inflammasome activation via autophagy induction in HUVECs. These anti-inflammatory and autophagy induction activities may provide valuable evidence for further investigating the potential role of 13-MB in atherosclerosis.
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A simple method for stable transfection of Acanthamoeba castellanii using plasmids which confer resistance to neomycin G418 is described. Expression of neomycin phosphotransferase is driven by the Acanthamoeba TBP gene promoter, and can be monitored by cell growth in the presence of neomycin G418 or by Western blot analysis. Transfected cells can be passaged in the same manner as control cells and can be induced to differentiate into cysts, in which form they maintain resistance to neomycin G418 for at least several weeks, although expression of neomycin phosphotransferase is repressed during encystment. Expression of EGFP or an HA-tagged EGFP-TBP fusion can be driven from the same plasmid, using an additional copy of the Acanthamoeba TBP gene promoter or a deletion mutant. The TBP-EGFP fusion is localized to the nucleus, except in a small proportion of presumptive pre-mitotic cells. EGFP expression can also be driven by the cyst-specific CSP21 gene promoter, which is completely repressed in growing cells but strongly induced in differentiating cells. Transfected cells maintain their phenotype for several weeks, even in the absence of neomycin G418, suggesting that transfected genes are stably integrated within the genome. These results demonstrate the utility of the neomycin resistance based plasmids for stable transfection of Acanthamoeba, and may assist a number of investigations.
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Acanthamoeba castellanii/genética , Técnicas Genéticas , Acanthamoeba castellanii/metabolismo , Western Blotting , Diferenciação Celular , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Genoma , Proteínas de Fluorescência Verde/metabolismo , Mitose , Neomicina/farmacologia , Fenótipo , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , RNA Polimerase II/genética , Proteínas Recombinantes/química , Fatores de Tempo , Transcrição Gênica , TransfecçãoRESUMO
Transcription of the TATA box binding protein (TBP) gene in Acanthamoeba castellanii is regulated by TATA box binding protein promoter binding factor (TPBF), which binds to an upstream TBP promoter element to stimulate transcription, and to a TATA proximal element, where it represses transcription. In order to extend these observations to the in vivo chromatin context, the TBP gene was examined by in situ footprinting and chromatin immunoprecipitation (ChIP). Acanthamoeba DNA is nucleosomal with a repeat of approximately 160 bp, and an intranucleosomal DNA periodicity of 10.5 bp. The TBP gene comprises a 220 bp micrococcal nuclease hypersensitive site corresponding to the promoter regulatory elements previously identified, flanked by protected regions of a size consistent with the presence of nucleosomes. ChIP data indicated that TPBF is associated with the TBP, TPBF and MIL gene promoters, but not to the CSP21, MIIHC, 5SrRNA or 39SrRNA promoters, or to the MIL gene C-terminal region. Binding by TPBF to the TPBF and MIL gene promoters was confirmed by in vitro assays. These results validate the in vitro model for TBP gene regulation and further suggest that TPBF may be autoregulated and may participate in the regulation of the MIL gene.