RESUMO
The electroencephalogram (EEG) has recently emerged as a pivotal tool in brain imaging analysis, playing a crucial role in accurately interpreting brain functions and states. To address the problem that the presence of ocular artifacts in the EEG signals of patients with obstructive sleep apnea syndrome (OSAS) severely affects the accuracy of sleep staging recognition, we propose a method that integrates a support vector machine (SVM) with genetic algorithm (GA)-optimized variational mode decomposition (VMD) and second-order blind identification (SOBI) for the removal of ocular artifacts from single-channel EEG signals. The SVM is utilized to identify artifact-contaminated segments within preprocessed single-channel EEG signals. Subsequently, these signals are decomposed into variational modal components across different frequency bands using the GA-optimized VMD algorithm. These components undergo further decomposition via the SOBI algorithm, followed by the computation of their approximate entropy. An approximate entropy threshold is set to identify and remove components laden with ocular artifacts. Finally, the signal is reconstructed using the inverse SOBI and VMD algorithms. To validate the efficacy of our proposed method, we conducted experiments utilizing both simulated data and real OSAS sleep EEG data. The experimental results demonstrate that our algorithm not only effectively mitigates the presence of ocular artifacts but also minimizes EEG signal distortion, thereby enhancing the precision of sleep staging recognition based on the EEG signals of OSAS patients.
Assuntos
Artefatos , Apneia Obstrutiva do Sono , Humanos , Máquina de Vetores de Suporte , Processamento de Sinais Assistido por Computador , Eletroencefalografia/métodos , AlgoritmosRESUMO
Breast cancer (BC) is one of the most common malignant tumors in women. CircRNA/miRNA/mRNA regulatory axes have been shown to be involved in the pathogenesis of BC. Here, we sought to analyze the functional mechanism of circ_0104345 in BC. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the levels of circ_0104345, miR-876-3p and zinc finger and BTB domain containing 20 (ZBTB20) mRNA. Cell Counting Kit-8 (CCK8) and 5-ethynyl-2'-deoxyuridine (EdU) assays were used to test cell viability and proliferation, respectively. Cell migration was tested by wound healing assay, and cell invasion was examined by transwell assay. Tube formation ability was tested by angiogenesis assay. Flow cytometry was applied for cell apoptosis. Western blot assay was utilized to measure the protein expression. The relationship between miR-876-3p and circ_0104345 or ZBTB20 was identified by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. Xenografts in mice were conducted to analyze the effect of sh-circ_0104345 on tumor growth in vivo. Circ_0104345 and ZBTB20 were upregulated and miR-876-3p expression was decreased in BC. Circ_0104345 knockdown inhibited cell proliferation, migration, invasion, and enhanced cell apoptosis. MiR-876-3p was targeted by circ_0104345. MiR-876-3p depletion reversed the effects of circ_0104345 downregulation on the progression of BC cells. ZBTB20 was regulated by circ_0104345 through miR-876-3p. The effects of miR-876-3p on BC cell behaviors were restored by ZBTB20 increase. The results of in vivo experiments indicated that silencing of circ_0104345 blocked the growth of xenograft tumors. In this study, we demonstrated, for the first time, the crucial regulation of the new circ_0104345/miR-876-3p/ZBTB20 axis in the biological phenotypes of BC cells.
Assuntos
Apoptose , Neoplasias da Mama , MicroRNAs , RNA Circular , Fatores de Transcrição , RNA Circular/genética , MicroRNAs/genética , Proteínas do Tecido Nervoso/genética , Fatores de Transcrição/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Humanos , Animais , Camundongos , Pessoa de Meia-Idade , Feminino , Linhagem Celular Tumoral , Camundongos Endogâmicos BALB C , Inativação GênicaRESUMO
BACKGROUND: Age-related macular degeneration (AMD) is a multifactorial chronic disease of the eye. Several candidate pathways have been hypothesized to play a role in AMD pathogenesis. Our work and those of others suggests inflammasome activity as a mechanism associated with retinal pigment epithelial (RPE) cell demise. X-linked inhibitor of apoptosis protein (XIAP), an anti-apoptosis factor, has recently been shown to regulate inflammasome activity in non-ocular cells. The purpose of this study is to characterize XIAP's regulatory role in RPE. METHODS: Protein lysates of eye tissues from rats (vinpocetine- or aurin tricarboxylic acid complex-treated, ATAC, vs naïve) and mice (wild type vs Caspase-4-/-) were utilized to analyze XIAP protein levels. Immunohistochemistry was used to detect NLRP3 levels in the RPE layer. In vitro inflammasome activation on RPE cells was achieved with L-leucyl-L-leucine methyl ester (Leu-Leu-OMe) stimulation. Levels of XIAP mRNA and 18S RNA were quantified by RT-PCR. Cell culture supernatants were tested directly for secreted IL-1ß by ELISA or concentrated for the detection of secreted IL-18 by western blot. Protein lysates from RPE in cell culture were collected for the measurement of cleaved caspase-1 p20, XIAP, and GAPDH. Data are presented as Mean ± SD. p < 0.05 is considered statistically significant. RESULTS: The XIAP protein level was significantly increased when the inflammasome was inhibited at the "activation" step by ATAC, but not the "priming" step, in vivo. Concomitantly, NLRP3 immunoreactivity was lower in the RPE layer of animals fed with ATAC. In mice where caspase-1 cleavage was impaired by the genetic deficiency in caspase-4, the XIAP protein level increased in eye tissues. In RPE cell culture, Leu-Leu-OMe stimulation led to caspase-1 cleavage, cytokine secretion, and XIAP reduction, which can be abolished by Z-YVAD-FMK. When XIAP siRNA was given as a pre-treatment to RPE in vitro, Leu-Leu-OMe induced IL-1ß/IL-18 secretion was enhanced, whereas overexpressing XIAP reduced IL-1ß secretion under inflammasome activation, both compared to controls cells. CONCLUSIONS: Together, these data suggest XIAP-mediated inhibition of inflammasome activity in RPE may provide insights into the biological consequences of inflammasome activation in RPE and reveals the caspase-1/XIAP/IL-1ß/IL-18 axis as a target for broader applications in AMD biology and treatment design.
Assuntos
Inflamassomos/metabolismo , Proteínas Inibidoras de Apoptose/deficiência , Degeneração Macular/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Animais , Células Cultivadas , Humanos , Inflamassomos/genética , Mediadores da Inflamação/metabolismo , Proteínas Inibidoras de Apoptose/genética , Degeneração Macular/genética , Degeneração Macular/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Ratos , Epitélio Pigmentado da Retina/patologiaRESUMO
BACKGROUND The TMEFF2 gene encodes the transmembrane protein with EGF like and two follistatin-like domains 2 and has been reported to be a tumor suppressor gene, but its role remains unknown in pancreatic cancer. This study aimed to investigate the expression of TMEFF2 in human pancreatic cancer tissue and the effects of knockdown of TMEFF2 on cell, proliferation, and apoptosis in human pancreatic cell lines. MATERIAL AND METHODS Thirty-five samples of human pancreatic tissue and adjacent normal pancreatic tissue, and five human pancreatic cancer cell lines, CAPAN1, ASPC1, BXPC3, SW1990, and CFPAC were studied. RNA expression, protein expression, cell proliferation, and apoptosis were studied using real-time polymerase chain reaction (RT-PCR), Western blot, the cell counting kit-8 (CCK-8) assay, and ï¬ow cytometry, respectively. A co-immunoprecipitation assay evaluated protein interactions. RESULTS TMEFF2 expression was down-regulated in pancreatic cancer tissue compared with normal pancreas. In human pancreatic cancer cell lines, overexpression of TMEFF2 suppressed cell proliferation and enhanced apoptosis, suppressed the expression of p-STAT3, MCL1, VEGF and increased the expression of the tyrosine-specific protein phosphatase, SHP-1. The co-immunoprecipitation assay showed that TMEFF2 interacted with SHP-1. Knockdown of expression of TMEFF2 resulted in the increased expression of p-STAT3, MCL1, and VEGF, increased cell proliferation and decreased cell apoptosis, which were reversed by overexpression of SHP-1. CONCLUSIONS In pancreatic cancer, TMEFF2 exerted as a tumor suppressor effect by regulating p-STAT3, MCL1, and VEGF via SHP-1.
Assuntos
Proteínas de Membrana/biossíntese , Proteínas de Neoplasias/biossíntese , Neoplasias Pancreáticas/metabolismo , Antineoplásicos Fitogênicos/farmacologia , Apoptose/fisiologia , Pontos de Checagem do Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Regulação para Baixo , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Metástase Neoplásica , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Fator de Transcrição STAT3/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND: The membrane attack complex (MAC) is a key player in the pathogenesis of age-related macular degeneration (AMD) and is a putative activator of the NLRP3 inflammasome. Amyloid beta (Aß), a component of drusen deposits, has also been implicated in inflammasome activation by our work and those of others. However, the interactions of MAC and Aß are still poorly understood, especially their roles in aging and retinal degenerative pathologies. Since inflammasome activation may represent a key cellular pathway underlying age-related chronic inflammation in the eye, the purpose of this study is to identify the effects associated with MAC and inflammasome activation in the retinal pigment epithelium (RPE)/choroid and to evaluate the therapeutic merits of MAC suppression. METHODS: Adult Long-Evans rats were divided into treatment and control groups. Treatment groups received oral aurin tricarboxylic acid complex (ATAC), a MAC inhibitor, in drinking-water, and control groups received drinking-water alone (No ATAC). Groups were sacrificed at 7.5 or 11.5 months, after approximately 40 days of ATAC treatment. To study age-related changes of Aß and MAC in RPE/choroid, naive animals were sacrificed at 2.5, 7.5, and 11.5 months. Eye tissues underwent immunohistochemistry and western blot analysis for MAC, Aß, NF-κB activation, as well as cleaved caspase-1 and IL-18. Vitreal samples were collected and assessed by multiplex assays for secreted levels of IL-18 and IL-1ß. Statistical analyses were performed, and significance level was set at p ≤ 0.05. RESULTS: In vivo studies demonstrated an age-dependent increase in MAC, Aß, and NF-κB activation in the RPE/choroid. Systemic ATAC resulted in a prominent reduction in MAC formation and a concomitant reduction in inflammasome activation measured by cleaved caspase-1 and secreted levels of IL-18 and IL-1ß, but not in NF-κB activation. In vitro studies demonstrated Aß-induced MAC formation on RPE cells. CONCLUSIONS: Age-dependent increases in Aß and MAC are present in the rodent outer retina. Our results suggest that suppressing MAC formation and subsequent inflammasome activation in the RPE/choroid may reduce chronic low-grade inflammation associated with IL-18 and IL-1ß in the outer retina.
Assuntos
Envelhecimento/metabolismo , Peptídeos beta-Amiloides/metabolismo , Proteínas de Transporte/metabolismo , Corioide/metabolismo , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Inflamassomos/metabolismo , Retina/metabolismo , Animais , Ácido Aurintricarboxílico/farmacologia , Corioide/efeitos dos fármacos , Modelos Animais de Doenças , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Degeneração Macular/metabolismo , NF-kappa B/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR , Ratos , Ratos Long-Evans , Retina/efeitos dos fármacosRESUMO
PURPOSE: Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in people 50 years of age or older in developed countries. The homozygous CC genotype in the complement factor H (CFH) Y402H single nucleotide polymorphism (SNP; rs1061170) is widely recognized as a risk factor for the development of AMD. In this study, we examined vitreal levels of granulocyte macrophage colony-stimulating factor (GM-CSF), a hematopoietic cytokine, and macrophages in the choroid of postmortem human eyes genotyped for the CFH Y402H SNP. METHODS: Twenty-two pairs of postmortem, non-diseased, human donor eyes were obtained. The vitreous and retinal tissues of the left eyes were collected for GM-CSF level measurement and CFH Y402H genotyping, respectively. The right eyes were paraffin-embedded and sectioned for immunohistochemistry using a macrophage and microglia marker, CD68. Cell cultures of RPE cells were stimulated with complement C3a, C5a, 4-hydroxynonenal (HNE), or tumor necrosis factor alpha (TNF-α), and GM-CSF expression was measured with a suspension assay or quantitative PCR. RESULTS: Eyes genotyped with the CC or the CT risk variant of the CFH Y402H SNP showed significantly increased levels of GM-CSF in the vitreous compared to eyes with the protective TT variant (mean ± standard error of mean, 607.54±85.83 pg/ml or 656.32±15.20 pg/ml versus 286.69±81.96 pg/ml, p<0.05). The choroid of eye tissues genotyped with the CC variant showed higher levels of CD68 immunoreactivity than the tissues genotyped with the TT variant (p<0.05). The GM-CSF levels detected in the supernatant of RPE cells in culture treated with HNE or TNF-α were significantly higher compared to the non-treated control (145.88±5.06 pg/ml and 149.32±3.76 pg/ml versus 123.27±4.05 pg/ml, p<0.05). Furthermore, the gene expression of GM-CSF detected in the lysate of RPE cells stimulated with complement C3a or C5a showed significantly increased fold changes compared to the non-treated control (C3a: 2.38±0.31 fold, p<0.05; C5a: 2.84±0.54 fold, p<0.01). CONCLUSIONS: Our data showed a relationship between the CFH Y402H polymorphism and GM-CSF levels in the vitreous and accumulation of choroidal macrophages in the postmortem eye. These data suggest that the at-risk variant of the CFH gene may contribute to the dysregulation of proinflammatory cytokines locally in the eye.
Assuntos
Corioide/metabolismo , Fator H do Complemento/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Macrófagos/citologia , Polimorfismo de Nucleotídeo Único , Corpo Vítreo/metabolismo , Aldeídos/farmacologia , Substituição de Aminoácidos , Autopsia , Células Cultivadas , Corioide/química , Corioide/citologia , Complemento C3a/farmacologia , Complemento C5a/farmacologia , Fator H do Complemento/metabolismo , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Expressão Gênica , Genótipo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Macrófagos/metabolismo , Masculino , Pessoa de Meia-Idade , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Corpo Vítreo/química , Corpo Vítreo/citologiaRESUMO
Age-related macular degeneration (AMD) is the leading cause of legal blindness in the elderly in industrialized countries. AMD is a multifactorial disease influenced by both genetic and environmental risk factors. Progression of AMD is characterized by an increase in the number and size of drusen, extracellular deposits, which accumulate between the retinal pigment epithelium (RPE) and Bruch's membrane (BM) in outer retina. The major pathways associated with its pathogenesis include oxidative stress and inflammation in the early stages of AMD. Little is known about the interactions among these mechanisms that drive the transition from early to late stages of AMD, such as geographic atrophy (GA) or choroidal neovascularization (CNV). As part of the innate immune system, inflammasome activation has been identified in RPE cells and proposed to be a causal factor for RPE dysfunction and degeneration. Here, we will first review the classic model of inflammasome activation, then discuss the potentials of AMD-related factors to activate the inflammasome in both nonocular immune cells and RPE cells, and finally introduce several novel mechanisms for regulating the inflammasome activity.
Assuntos
Proteínas de Transporte/metabolismo , Inflamassomos/metabolismo , Degeneração Macular/metabolismo , Animais , Lâmina Basilar da Corioide/metabolismo , Lâmina Basilar da Corioide/patologia , Humanos , Degeneração Macular/patologia , Proteína 3 que Contém Domínio de Pirina da Família NLR , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologiaRESUMO
Altered expression of TROP2 is observed in various types of human cancers. However, the clinical significance and pathological role of TROP2 in gallbladder cancer (GBC) remains unclear. The main objective of this investigation was to clarify the relationships between TROP2 expression and the clinicopathological features of patients with GBC. Immunohistochemistry was performed to investigate the expression of TROP2 and epithelial-mesenchymal transition (EMT) indicator proteins in 93 patients with GBC. Immunohistochemistry showed that the protein expression level of TROP2 was significantly higher in GBC tissues than in adjacent noncancerous tissues. In addition, immunohistochemistry analysis showed that TROP2 expression was significantly correlated with histologic grade (P=0.038), tumor stage (P=0.015), and lymph node metastasis (P=0.007). Furthermore, high TROP2 expression was significantly associated with a loss of the epithelial marker E-cadherin (P=0.013) and acquisition of expression of the mesenchymal marker vimentin (P=0.031). Kaplan-Meier analysis and Cox proportional hazards regression models were used to investigate the correlation between TROP2 expression and prognosis of GBC patients. Kaplan-Meier analysis indicated that patients with high TROP2 expression had poor overall survival (P<0.001). Multivariate analysis showed that high TROP2 expression was an independent predictor of overall survival. In conclusion, our data suggest for the first time that the increased expression of TROP2 in GBC is associated significantly with aggressive progression and poor prognosis. In conclusion, this study confirmed that TROP2 might be involved in regulating the EMT and malignant progression in GBC. It also provided the first evidence that TROP2 expression in GBC was an independent prognostic factor of patients, which might be a potential diagnostic and therapeutic target of GBC.
Assuntos
Antígenos de Neoplasias/metabolismo , Biomarcadores Tumorais/metabolismo , Moléculas de Adesão Celular/metabolismo , Neoplasias da Vesícula Biliar/metabolismo , Caderinas/metabolismo , Feminino , Seguimentos , Neoplasias da Vesícula Biliar/mortalidade , Neoplasias da Vesícula Biliar/patologia , Humanos , Técnicas Imunoenzimáticas , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Invasividade Neoplásica , Estadiamento de Neoplasias , Prognóstico , Taxa de Sobrevida , Vimentina/metabolismoRESUMO
Chronic inflammation is a key pathogenic process in age-related macular degeneration (AMD). Amyloid-beta (Aß) is a constituent of AMD drusen and promotes the activation of NLRP3 inflammasome which facilitates the production of cytokines. We investigated the role of transcription factor NF-κB in the activation of inflammasome in the RPE and the effect of vinpocetine, a dietary supplement with inhibitory effect on NF-κΒ. ARPE19/NF-κB-luciferase reporter cells treated with Aß demonstrated enhanced NF-κB activation that was significantly suppressed by vinpocetine. Intraperitoneal injection of vinpocetine (15 mg/kg) inhibited NF-κB nuclear translocation and reduced the expression and activation of NLRP3, caspase-1, IL-1ß, IL-18, and TNF-α in the RPE of adult rats that received intraocular Αß, as measured by retinal immunohistochemistry and Western blot. Cytokine level in the vitreous was assayed using multiplex suspension arrays and revealed significantly lower concentration of MIP-3α, IL-6, IL-1α, IL-1ß, IL-18, and TNF-α in vinpocetine treated animals. These results suggest that the NF-κB pathway is activated by Aß in the RPE and signals the priming of NLRP3 inflammasome and the expression of pro-inflammatory cytokines including the inflammasome substrates IL-1ß and IL-18. NF-κB inhibition may be an effective approach to stem the chronic inflammatory milieu that underlies the development of AMD. Vinpocetine is a potentially useful anti-inflammatory agent that is well-tolerated in long term use.
Assuntos
Peptídeos beta-Amiloides/antagonistas & inibidores , Citocinas/metabolismo , Inflamassomos/metabolismo , NF-kappa B/metabolismo , Epitélio Pigmentado da Retina/efeitos dos fármacos , Vasodilatadores/farmacologia , Alcaloides de Vinca/farmacologia , Peptídeos beta-Amiloides/farmacologia , Animais , Western Blotting , Proteínas de Transporte , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Injeções Intraperitoneais , Proteína 3 que Contém Domínio de Pirina da Família NLR , Ratos , Ratos Long-Evans , Receptores Citoplasmáticos e Nucleares/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Corpo Vítreo/metabolismoRESUMO
Introduction: Sleep apnoea syndrome (SAS) is a serious sleep disorder and early detection of sleep apnoea not only reduces treatment costs but also saves lives. Conventional polysomnography (PSG) is widely regarded as the gold standard diagnostic tool for sleep apnoea. However, this method is expensive, time-consuming and inherently disruptive to sleep. Recent studies have pointed out that ECG analysis is a simple and effective diagnostic method for sleep apnea, which can effectively provide physicians with an aid to diagnosis and reduce patients' suffering. Methods: To this end, in this paper proposes a LightGBM hybrid model based on ECG signals for efficient detection of sleep apnea. Firstly, the improved Isolated Forest algorithm is introduced to remove abnormal data and solve the data sample imbalance problem. Secondly, the parameters of LightGBM algorithm are optimised by the improved TPE (Tree-structured Parzen Estimator) algorithm to determine the best parameter configuration of the model. Finally, the fusion model TPE_OptGBM is used to detect sleep apnoea. In the experimental phase, we validated the model based on the sleep apnoea ECG database provided by Phillips-University of Marburg, Germany. Results: The experimental results show that the model proposed in this paper achieves an accuracy of 95.08%, a precision of 94.80%, a recall of 97.51%, and an F1 value of 96.14%. Discussion: All of these evaluation indicators are better than the current mainstream models, which is expected to assist the doctor's diagnostic process and provide a better medical experience for patients.
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BACKGROUND: Recent genomic technologies have propelled our understanding of the mechanisms underlying complex eye diseases such as age-related macular degeneration (AMD). Genotyping postmortem eye tissues for known single nucleotide polymorphisms (SNPs) associated with AMD may prove valuable, especially when combined with information obtained through other methods such as immunohistochemistry, western blot, enzyme-linked immunosorbent assay (ELISA), and proteomics. Initially intending to genotype postmortem eye tissues for AMD-related SNPs, our group became interested in isolating and comparing the quality of DNA from the iris and retina of postmortem donor eyes. Since there is no previously published protocol in the literature on this topic, we present a protocol suitable for isolating high-quality DNA from postmortem eye tissues for genomic studies. METHODS: DNA from 33 retinal samples and 35 iris samples was extracted using the phenol-chloroform-isoamyl method from postmortem donor eye tissues. The quantity of DNA was measured with a spectrophotometer while the quality was checked using gel electrophoresis. The DNA samples were then amplified with PCR for the complement factor H (CFH) gene. The purified amplified products were then genotyped for the SNPs in the CFH gene. RESULTS: Regarding concentration, the retina yielded 936 ng/µl of DNA, while the iris yielded 78 ng/µl of DNA. Retinal DNA was also purer than iris DNA (260/280=1.78 vs. 1.46, respectively), and produced superior PCR results. Retinal tissue yielded significantly more DNA than the iris tissue per mg of sample (21.7 ng/µl/mg vs. 7.42 ng/µl/mg). Retinal DNA can be readily amplified with PCR, while iris DNA can also be amplified by adding bovine serum albumin. Overall, retinal tissues yielded DNA of superior quality, quantity, and suitability for genotyping and genomic studies. CONCLUSIONS: The protocol presented here provides a clear and reliable method for isolating total DNA from postmortem eye tissues. Retinal tissue provides DNA of excellent quantity and quality for genotyping and downstream genomic studies. However, DNA isolated from iris tissues, and treated with bovine serum albumin, may also be a valuable source of DNA for genotyping and genomic studies.
Assuntos
DNA/isolamento & purificação , Genótipo , Iris/metabolismo , Polimorfismo de Nucleotídeo Único , Retina/metabolismo , Adulto , Idoso , Animais , Autopsia/estatística & dados numéricos , Sequência de Bases , Bovinos , Fator H do Complemento/genética , DNA/genética , Eletroforese em Gel de Ágar , Técnicas de Genotipagem , Humanos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Soroalbumina Bovina/químicaRESUMO
We show that an innate defense-regulator peptide (IDR-1) was protective in mouse models of infection with important Gram-positive and Gram-negative pathogens, including methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus and Salmonella enterica serovar Typhimurium. When given from 48 h before to 6 h after infection, the peptide was effective by both local and systemic administration. Because protection by IDR-1 was prevented by in vivo depletion of monocytes and macrophages, but not neutrophils or B- and T-lymphocytes, we conclude that monocytes and macrophages are key effector cells. IDR-1 was not directly antimicrobial: gene and protein expression analysis in human and mouse monocytes and macrophages indicated that IDR-1, acting through mitogen-activated protein kinase and other signaling pathways, enhanced the levels of monocyte chemokines while reducing pro-inflammatory cytokine responses. To our knowledge, an innate defense regulator that counters infection by selective modulation of innate immunity without obvious toxicities has not been reported previously.
Assuntos
Anti-Infecciosos/farmacologia , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/imunologia , Peptídeos/farmacologia , Animais , Anti-Infecciosos/uso terapêutico , Anti-Infecciosos/toxicidade , Infecções Bacterianas/tratamento farmacológico , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação/imunologia , Lipopolissacarídeos/farmacologia , Camundongos , Modelos Imunológicos , Peptídeos/toxicidade , Resultado do TratamentoRESUMO
Transmembrane protein with epidermal growth factor (EGF)-like and two follistatin motifs 2 (TMEFF2) is downregulated in human gastric cancer, and its levels are associated with tumor aggressiveness. Herein, a positive correlation was identified between serum vitamin C levels (µg/ml) and mRNA levels of TMEFF2 in gastric cancer tissue. TMEFF2 silencing promotes cell proliferation in GES-1 normal human gastric epithelial cells and AGS human gastric adenocarcinoma cells. Notably, vitamin C and AG490 exerted antiproliferative effects on the two cell lines. The present study demonstrated that small interfering (si)-RNA-TMEFF2 exerts pro-proliferative effects on GES-1 and AGS cells. The results revealed that vitamin C significantly inhibited the growth of GES-1 and AGS cells by reducing cell viability, decreasing the expression of proliferating cell nuclear antigen (PCNA), and blocking the STAT3 pathway. Moreover, siRNA-TMEFF2-induced enhanced cell viability and PCNA expression were significantly reversed by additional vitamin C treatment; notably, markedly enhanced TMEFF2 expression was observed. Upregulated TMEFF2 expression was observed in association with the antiproliferative effect of AG490. In conclusion, serum vitamin C content (µg/ml) was positively correlated with the mRNA levels of TMEFF2 in gastric cancer tissue. Exploring novel drugs that target TMEFF2 is a potential therapeutic strategy for blocking human GC.
RESUMO
Aquaporin 1 (AQP1) plays an important role in endothelial functions and is regulated by MEF2C. However, how AQP1 level is stabilized to maintain endothelial water homeostasis is still not clear. Here, we show that AQP1 expression is significantly upregulated by MEF2C transcriptionally and inhibited by miR-133a-3p.1 post-transcriptionally. Meanwhile, MEF2C activates the expression of miR-133a1. Simultaneous overexpression of MEF2C and miR-133a-3p.1 suppresses the aptitude of changes in AQP1 expression caused by either MEF2C or miR-133a-3p.1. Accordingly, the changes in migration and tube formation of human umbilical vein endothelial cells (HUVECs) caused by MEF2C or miR133a-3p.1 are blunted by coexpression of both of them. These data demonstrate that the homeostasis and physiological function of AQP1 in endothelial health are maintained by the MEF2C and miR-133a-3p.1 regulatory circuit.
Assuntos
Aquaporina 1/genética , Regulação da Expressão Gênica/genética , Homeostase/genética , Células Endoteliais da Veia Umbilical Humana/metabolismo , MicroRNAs/genética , Água/metabolismo , Sequência de Bases , Movimento Celular/genética , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Fatores de Transcrição MEF2/metabolismo , Transcrição GênicaRESUMO
MicroRNAs (miRNA/miRs) are small, non-coding RNA molecules (19-25 nucleotides in length), which function to regulate gene expression. It has been reported that miR-128 serves an important role in regulating cancer cell growth; increasing evidence has indicated that the expression of miR-128 is decreased in pancreatic cancer (PC) cells. However, the specific mechanisms of miR-128 in regulating PC cell growth are unclear. In the present study, it was confirmed that the expression of miR-128 was significantly decreased within PC tissues compared with adjacent normal tissues via reverse transcription-quantitative polymerase chain reaction analysis. In addition, miR-128 mimics inhibited PC MIA-PaCa2 cell growth by enhancing cell apoptosis in a caspase-dependent manner. Furthermore, the results of the present study demonstrated that double minute 4 (MDM4) may be a direct target for miR-128 via a dual luciferase report assay; miR-128 may inhibit MDM4 expression, and increase p53 and cleaved caspase-3 protein expression levels. In summary, the present study indicated that miR-128 is downregulated in PC, and it may be a promising target for future PC diagnosis and treatment.
RESUMO
Our previous studies, using differential mRNA display, suggested that the mouse Nip21 gene may be involved in myocarditis development in the coxsackievirus B3 (CVB3)-infected mouse heart. Sequence comparison indicated that the mouse Nip21 gene shares high sequence homology to human Nip2. This human protein is known to interact with both the apoptosis inhibitor Bcl-2 and a homologous protein, the adenovirus E1B 19-kDa protein. Such interactions implicate Nip21 gene in cell death pathways. To study the function of this gene, we have cloned Nip21 from mouse hearts and established a Tet-On doxycycline-inducible HeLa cell line and a cardiomyocyte H9c2 cell line expressing Nip21 to characterize gene function in relation to apoptosis. We demonstrated that Nip21 expression could induce apoptosis via caspase-depended mitochondria activation. To further determine the function of Nip21 in CVB3-induced apoptosis, the Tet-On/Nip21 HeLa cell line was induced by doxycycline followed by CVB3 infection. We found that activation of caspase-3 and cleavage of poly-(ADP-ribose) polymerase occurred 2 hours earlier than in vector-transfected control cells, suggesting that Nip21 expression enhances CVB3-induced apoptosis. We also demonstrated a significant decrease in HeLa cell and H9c2 cell viability. Particularly, as illustrated by viral plaque assay, CVB3 replication was dramatically reduced in Tet-On HeLa cells, due at least in part to the earlier killing of the host cells by Nip21 overexpression.
Assuntos
Apoptose , Enterovirus Humano B/crescimento & desenvolvimento , Mitocôndrias/fisiologia , Miocárdio/metabolismo , Proteínas/fisiologia , Sequência de Aminoácidos , Animais , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3 , Proteínas de Ligação ao Cálcio/genética , Proteínas de Transporte/metabolismo , Caspase 3 , Caspases/metabolismo , Linhagem Celular , Sobrevivência Celular , Clonagem Molecular , Grupo dos Citocromos c/metabolismo , Expressão Gênica , Células HeLa , Humanos , Camundongos , Dados de Sequência Molecular , Miocárdio/citologia , Proteínas/genética , Proteínas/metabolismo , Alinhamento de Sequência , Transdução de Sinais , Replicação ViralRESUMO
PURPOSE: To investigate the relationship between systemic cytokines, the complement factor H (CFH) Y402H polymorphism, drusen load, and subfoveal choroidal thickness in patients with dry age-related macular degeneration (AMD). DESIGN: Cross-sectional study. METHODS: Forty-four dry AMD patients under care of the Retina Service at the University of British Columbia were enrolled. Drusen load was measured with an automated software algorithm in spectral-domain optical coherence tomography; subfoveal choroidal thickness was measured manually using enhanced depth imaging. Bio-Plex suspension assays (Bio-Rad Laboratories) were used to analyze cytokines in plasma and CFH Y402H was genotyped. Statistical analyses included analysis of covariance and Pearson correlation, corrected for multiple comparisons. RESULTS: The levels of 3 of 4 studied cytokines were significantly different among patients with CC, CT, or TT variants of the CFH Y402H polymorphism (P < .01). Patients with the at-risk CC variant had higher systemic levels of interleukin-6, interleukin-18, and tumor necrosis factor α than those with the CT variants, the TT variant, or both (P < .01). Interleukin-1ß did not reach significance (P = .02), but did demonstrate a consistent trend. No correlation was found between plasma cytokines and drusen load or choroidal thickness (all P > .15). CONCLUSIONS: The elevated systemic levels of selected proinflammatory cytokines, including those representing products of inflammasome activation, were associated with the CC at-risk variant of the Y402H polymorphism and suggest that genetic factors regulate the inflammatory status in dry AMD patients. Our data support the central role of inflammation in the pathogenesis of AMD and provide further evidence of a systemic involvement in AMD etiology.