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PURPOSE: This investigation aims to explore the protective role of Naringenin (Nar) in bone cancer pain (BCP) via TNF-α-mediated NF-κB/uPA/PAR2 pathway. METHODS: BCP model was manipulated by the injection of LL2 cells into femur of mice. The levels of TNF-α and uPA in bone tissue and serum were studied by ELISA. The expressions of PAR2, PKC-γ, PKA and TRPV1 were determined by qPCR and western blot. Levels of p-IKKß, IKKß, p-p65, p65 were determined by western blot. Levels of p-p65 and uPA in bone tissue were studied by immunohistochemistry. Behavior tests in this investigation included paw withdrawal latency (PWL) and the paw withdrawal threshold (PWT). Radiological analysis and micro-CT were used to study bone structure. The lesions of bone tissue were determined by HE staining. The Dorsal root ganglia (DRG) isolated from mice were used to determine the level of PAR2 pathway. RESULTS: Naringenin improved the BCP-induced bone damage based on the increases of BV/TV, Conn. D, BMD and BMC and the decrease of bone destruction score. Naringenin repressed the reductions of PWT and PWL in BCP mice. Naringenin decreased the levels of PAR2, PKC-γ, PKA and TRPV1 of DRG and reduced the levels of p-IKKß, p-p65, and uPA in serum and bone tissue in BCP. Importantly, naringenin suppressed the enhancement of TNF-α in serum and bone tissue in BCP mice. CONCLUSION: Naringenin alleviated pain sensitization and bone damage of mice with BCP via TNF-α-mediated NF-κB/uPA/PAR2 pathway. We demonstrated a novel pathway for anti-BCP treatment with naringenin.
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Neoplasias Ósseas , Dor do Câncer , Flavanonas , NF-kappa B , Animais , Flavanonas/farmacologia , Camundongos , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/complicações , NF-kappa B/metabolismo , Dor do Câncer/tratamento farmacológico , Dor do Câncer/etiologia , Dor do Câncer/metabolismo , Transdução de Sinais/efeitos dos fármacos , Modelos Animais de Doenças , FemininoRESUMO
Inflammatory bowel diseases (IBD) are often associated with dysregulated gut microbiota and excessive inflammatory microenvironment. Probiotic therapy combined with inflammation management is a promising approach to alleviate IBD, but the efficacy is hindered by the inferior colonization of probiotics in mucus-depleted inflammatory bowel segments. Here, we present modified montmorillonite armed probiotic Escherichia coli Nissle 1917 (MMT-Fe@EcN) with enhanced intestinal colonization and hydrogen sulfide (H2S) scavenging for synergistic alleviation of IBD. The montmorillonite layer that can protect EcN against environmental assaults in oral delivery and improve on-site colonization of EcN in the mucus-depleted intestinal segment due to its strong adhesive capability and electronegativity, with a 22.6-fold increase in colonization efficiency compared to EcN. Meanwhile, MMT-Fe@EcN can manage inflammation by scavenging H2S, which allows for enhancing probiotic viability and colonization for restoring the gut microbiota. As a result, MMT-Fe@EcN exhibits extraordinary therapeutic effects in the dextran sulfate sodium-induced mouse colitis models, including alleviating intestinal inflammation and restoring disrupted intestinal barrier function, and gut microbiota. These findings provide a promising strategy for clinical IBD treatment and potentially other mucus-depletion-related diseases.
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As a typical biomarker of Alzheimer's disease, rapid and specific detection of tau protein can help improve the early diagnosis and prognosis of the disease. In this study, a simple sandwich electrochemical immunosensor was developed for rapid detection of tau protein. Primary monoclonal antibodies (mAb1) against the middle domain of tau protein (amino acids 189-195) were immobilized on the gold electrode surface through a self-assembled monolayer (SAM) of 3,3'-dithiobis (sulfosuccinimidyl propionate) (DTSSP). Then the tau protein was captured through the specific adsorption between the antigen and the antibody, resulting in a change in the impedance. Secondary monoclonal antibodies (mAb2) against the N-terminal region of tau protein were used for further amplification of the binding reaction between mAb1 and tau protein. A linear correlation between the total change in impedance and the logarithm of tau concentration was found from 2 × 10-6 mg mL-1 to 2 × 10-3 mg mL-1, with a detection limit as low as 1 × 10-6 mg mL-1. No significant interference was observed from human serum albumin. Furthermore, the fabricated sandwich immunosensor successfully detected target tau protein in artificial cerebrospinal fluid (aCSF) samples, indicating good potential for clinical applications in the future.
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Doença de Alzheimer , Biomarcadores , Técnicas Biossensoriais , Técnicas Eletroquímicas , Proteínas tau , Doença de Alzheimer/diagnóstico , Humanos , Anticorpos Monoclonais , Ouro/química , Imunoensaio/métodos , Limite de Detecção , EletrodosRESUMO
BACKGROUND: Pluripotent states of embryonic stem cells (ESCs) with distinct transcriptional profiles affect ESC differentiative capacity and therapeutic potential. Although single-cell RNA sequencing has revealed additional subpopulations and specific features of naive and primed human pluripotent stem cells (hPSCs), the underlying mechanisms that regulate their specific transcription and that control their pluripotent states remain elusive. RESULTS: By single-cell analysis of high-resolution, three-dimensional (3D) genomic structure, we herein demonstrate that remodeling of genomic structure is highly associated with the pluripotent states of human ESCs (hESCs). The naive pluripotent state is featured with specialized 3D genomic structures and clear chromatin compartmentalization that is distinct from the primed state. The naive pluripotent state is achieved by remodeling the active euchromatin compartment and reducing chromatin interactions at the nuclear center. This unique genomic organization is linked to enhanced chromatin accessibility on enhancers and elevated expression levels of naive pluripotent genes localized to this region. In contradistinction, the primed state exhibits intermingled genomic organization. Moreover, active euchromatin and primed pluripotent genes are distributed at the nuclear periphery, while repressive heterochromatin is densely concentrated at the nuclear center, reducing chromatin accessibility and the transcription of naive genes. CONCLUSIONS: Our data provide insights into the chromatin structure of ESCs in their naive and primed states, and we identify specific patterns of modifications in transcription and chromatin structure that might explain the genes that are differentially expressed between naive and primed hESCs. Thus, the inversion or relocation of heterochromatin to euchromatin via compartmentalization is related to the regulation of chromatin accessibility, thereby defining pluripotent states and cellular identity.
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Células-Tronco Pluripotentes , Análise de Célula Única , Humanos , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/citologia , Genoma Humano , Eucromatina/genética , Eucromatina/metabolismo , Cromatina/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Heterocromatina/metabolismo , Células-Tronco Embrionárias/metabolismo , Montagem e Desmontagem da CromatinaRESUMO
Background: Pediatric cerebral palsy (CP) is a non-progressive brain injury syndrome characterized by central motor dysfunction and insufficient brain coordination ability. The etiology of CP is complex and often accompanied by diverse complications such as intellectual disability and language disorders, making clinical treatment difficult. Despite the availability of pharmacological interventions, rehabilitation programs, and spasticity relief surgery as treatment options for CP, their effectiveness is still constrained. Electroacupuncture (EA) stimulation has demonstrated great improvements in motor function, but its comprehensive, objective therapeutic effects on pediatric CP remain to be clarified. Methods: We present a case of a 5-year-old Chinese female child who was diagnosed with CP at the age of 4. The patient exhibited severe impairments in motor, language, social, and cognitive functions. We performed a 3-month period of EA rehabilitation, obtaining resting state functional magnetic resonance imaging (rs-fMRI) of the patient at 0 month, 3 months and 5 months since treatment started, then characterized brain functional connectivity patterns in each phase for comparison. Results: After a 12-month follow-up, notable advancements were observed in the patient's language and social symptoms. Changes of functional connectivity patterns confirmed this therapeutic effect and showed specific benefits for different recovery phase: starting from language functions then modulating social participation and other developmental behaviors. Conclusion: This is a pioneering report demonstrating the longitudinal effect of EA stimulation on functional brain connectivity in CP patients, suggesting EA an effective intervention for developmental disabilities (especially language and social dysfunctions) associated with pediatric CP.
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Accurate prediction of the efficacy of immunotherapy for cancer patients through the characterization of both genetic and phenotypic heterogeneity in individual patient cells holds great promise in informing targeted treatments, and ultimately in improving care pathways and clinical outcomes. Here, we describe the nanoplatform for interrogating living cell host-gene and (micro-)environment (NICHE) relationships, that integrates micro- and nanofluidics to enable highly efficient capture of circulating tumor cells (CTCs) from blood samples. The platform uses a unique nanopore-enhanced electrodelivery system that efficiently and rapidly integrates stable multichannel fluorescence probes into living CTCs for in situ quantification of target gene expression, while on-chip coculturing of CTCs with immune cells allows for the real-time correlative quantification of their phenotypic heterogeneities in response to immune checkpoint inhibitors (ICI). The NICHE microfluidic device provides a unique ability to perform both gene expression and phenotypic analysis on the same single cells in situ, allowing us to generate a predictive index for screening patients who could benefit from ICI. This index, which simultaneously integrates the heterogeneity of single cellular responses for both gene expression and phenotype, was validated by clinically tracing 80 non-small cell lung cancer patients, demonstrating significantly higher AUC (area under the curve) (0.906) than current clinical reference for immunotherapy prediction.
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Células Neoplásicas Circulantes , Humanos , Células Neoplásicas Circulantes/patologia , Células Neoplásicas Circulantes/metabolismo , Microfluídica/métodos , Análise de Célula Única/métodos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/sangue , Fenótipo , Linhagem Celular Tumoral , Imunoterapia/métodos , Perfilação da Expressão Gênica/métodos , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/sangue , Técnicas Analíticas Microfluídicas/métodos , Técnicas Analíticas Microfluídicas/instrumentaçãoRESUMO
PURPOSE: To examine the knowledge, attitudes, and practices (KAP) of caregivers of children with Kawasaki disease toward Kawasaki disease. METHODS: This cross-sectional study was conducted at four hospitals in China from March 2023 to June 2023. The KAP scores were evaluated using a self-designed questionnaire (Cronbach's α = 0.840; KMO = 0.7381). Correlations between dimension scores were evaluated by Pearson correlation analysis. A structural equation model (SEM) was used to examine the relationships among factors. RESULTS: Of 643 surveyed, 49.50% were male caregivers. The mean knowledge, attitude, and practice scores were 7.12 ± 2.34 (possible range, 0-11), 29.23 ± 5.67 (possible range, 12-60), and 21.57 ± 5.34 (possible range, 6-30). Knowledge correlated with attitude (r = 0.172, P < 0.001) and practice (r = 0.280, P < 0.001). Attitude was significantly related to practice (r = 0.598, P < 0.001). SEM showed knowledge had a positive effect on attitudes (ß = 0.581, P < 0.001) and practices (ß = 0.786, P < 0.001). In addition, attitudes also positively affected practices (ß = 0.554, P < 0.001). Occupation type (ß = 0.598, P = 0.025) and monthly per capita income (ß=-0.750, P = 0.020) had different effects on attitudes, while monthly per capita income also had negative effects on practices (ß=-0.410, P = 0.021). CONCLUSION: Caregivers of children with Kawasaki disease have moderate knowledge and unfavorable attitudes but proactive practices toward this disease. The results could help design an educational intervention to improve KAP, which could translate into better patient management and outcomes. TRIAL REGISTRATION: Not applicable.
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Cuidadores , Síndrome de Linfonodos Mucocutâneos , Criança , Humanos , Masculino , Feminino , Estudos Transversais , Conhecimentos, Atitudes e Prática em Saúde , Inquéritos e QuestionáriosRESUMO
ARL3 is essential for cilia development, and mutations in ARL3 are closely associated with ciliopathies. In a previous study, we observed distinct phenotypes of retinal dystrophy in patients with heterozygous ARL3T31A and compound heterozygous ARL3T31A/C118F mutations, indicating that different mutation types may exert diverse effects on their functions. Here, we generated transformed immortal fibroblast cells from patients carrying heterozygous ARL3T31A and compound heterozygous ARL3T31A/C118F mutations, and systematically evaluated their cilia morphology and function, which were further validated in ARPE-19 cells. Results showed that both ARL3T31A and ARL3T31A/C118F mutations led to a decrease in cilium formation. The ARL3T31A/C118F mutations caused significantly elongated cilia and impaired retrograde transport, whereas the ARL3T31A mutation did not induce significant changes in fibroblasts. RNA-sequencing results indicated that compared to ARL3T31A , ARL3T31A/C118F fibroblasts exhibited a higher enrichment of biological processes related to neuron projection development, tissue morphogenesis, and extracellular matrix (ECM) organization, with noticeable alterations in pathways such as ECM-receptor interaction, focal adhesion, and TGF-ß signaling. Similar changes were observed in the proteomic results in ARPE-19 cells. Core regulated genes including IQUB, UNC13D, RAB3IP, and GRIP1 were specifically downregulated in the ARL3T31A/C118F group, and expressions of IQUB, NPM2, and SLC38A4 were further validated. Additionally, IQUB showed a rescuing effect on the overlong cilia observed in ARL3T31A/C118F fibroblasts. Our results not only enhance our understanding of ARL3-related diseases but also provide new insights into the analysis of heterozygous and compound heterozygous mutations in genetics.
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Cílios , Proteômica , Humanos , Cílios/genética , Cílios/metabolismo , Transporte Proteico , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Mutação , Fibroblastos/metabolismo , Proteínas de Membrana/metabolismoRESUMO
Recently, lanthanide (Ln) luminescent nanocrystals have attracted increasing attention in various fields such as biomedical imaging, lasers, and anticounterfeiting. However, due to the forbidden 4f-4f transition of lanthanide ions, the absorption cross-section and luminescence brightness of lanthanide nanocrystals are limited. To address the challenge, we constructed an optical oscillator-like system to repeatedly simulate lanthanide nanocrystals to enhance the absorption efficiency of lanthanide ions on excitation photons. In this optical system, the upconversion luminescence (UCL) of Tm3+ emission of ~450 nm excited by a 980 nm laser can be amplified by a factor beyond 104 . The corresponding downshifting luminescence of Tm3+ at 1460 nm was enhanced by three orders of magnitude. We also demonstrated that the significant luminescence enhancement in the designed optical oscillator-like system was general for various lanthanide nanocrystals including NaYF4 :Yb3+ /Ln3+ , NaErF4 @NaYF4 and NaYF4 :Yb3+ /Ln3+ @NaYF4 :Yb3+ @NaYF4 (Ln = Er, Tm, Ho) regardless of the wavelengths of excitation sources (808 and 980 nm). The mechanism study revealed that both elevated laser power in the optical system and multiple excitations on lanthanide nanocrystals were the main reason for the luminescence amplification. Our findings may benefit the future development of low-threshold upconversion and downshifting luminescence of lanthanide nanocrystals and expand their applications.
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Elementos da Série dos Lantanídeos , Nanopartículas , Elementos da Série dos Lantanídeos/química , Luminescência , Nanopartículas/química , Luz , ÍonsRESUMO
Aflatoxins (AFs) exhibit hepatotoxicity, immunotoxicity, and carcinogenicity, and their detection in food has attracted widespread concern. An ordered macroporous metal-organic framework (OM-ZIF-8) based on solid-phase extraction (SPE) was used to extract six AFs from milk products. The SPE conditions, including eluting solvent, eluting volume, amounts of OM-ZIF-8, pH of loading solution, loading solvent, ionic strength, loading flow rate, and elution flow rate, were exhaustively optimized. Under optimal parameters, the six AFs were detected by ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The OM-ZIF-8 exhibited satisfactory AFs extraction performance through ordered macropore structure, π-π interaction, coordination interaction, and electrostatic interaction. Furthermore, linearity in the range of 0.01-100 ng mL-1 with low detection limits of 0.002-0.0150 ng mL-1 was obtained, and the relative recoveries of AFs were 80.3-110 % with relative standard deviation ≤8.7 %. Thus, this research provides a promising platform for the analysis of trace AFs in complex foods.
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Aflatoxinas , Estruturas Metalorgânicas , Animais , Leite/química , Aflatoxinas/análise , Espectrometria de Massas em Tandem/métodos , Extração em Fase Sólida/métodos , Cromatografia Líquida de Alta Pressão/métodos , Solventes/análiseRESUMO
Nanotechnology brings hope for targeted drug delivery. However, most current drug delivery systems use passive delivery strategies with limited therapeutic efficiency. Over the past two decades, research on micro/nanomotors (MNMs) has flourished in the biomedical field. Compared with other driven methods, light-driven MNMs have the advantages of being reversible, simple to control, clean, and efficient. Under light irradiation, the MNMs can overcome several barriers in the body and show great potential in the treatment of various diseases, such as tumors, and gastrointestinal, cardiovascular and cerebrovascular diseases. Herein, the classification and mechanism of light-driven MNMs are introduced briefly. Subsequently, the applications of light-driven MNMs in overcoming physiological and pathological barriers in the past five years are highlighted. Finally, the future prospects and challenges of light-driven MNMs are discussed as well. This review will provide inspiration and direction for light-driven MNMs to overcome biological barriers in vivo and promote the clinical application of light-driven MNMs in the biomedical field.
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Nanoestruturas , Neoplasias , Humanos , Nanoestruturas/efeitos da radiação , Nanotecnologia/métodos , Sistemas de Liberação de Medicamentos , Neoplasias/diagnóstico , Neoplasias/tratamento farmacológicoRESUMO
High-resolution in vivo optical multiplexing in second near-infrared window (NIR-II, 1000-1700â nm) is vital to biomedical research. Presently, limited by bio-tissue scattering, only luminescent probes located at NIR-IIb (1500-1700â nm) window can provide high-resolution in vivo multiplexed imaging. However, the number of available luminescent probes in this narrow NIR-IIb region is limited, which hampers the available multiplexed channels of in vivo imaging. To overcome the above challenges, through theoretical simulation we expanded the conventional NIR-IIb window to NIR-II long-wavelength (NIR-II-L, 1500-1900â nm) window on the basis of photon-scattering and water-absorption. We developed a series of novel lanthanide luminescent nanoprobes with emission wavelengths from 1852â nm to 2842â nm. NIR-II-L nanoprobes enabled high-resolution in vivo dynamic multiplexed imaging on blood vessels and intestines, and provided multi-channels imaging on lymph tubes, tumors and intestines. The proposed NIR-II-L probes without mutual interference are powerful tools for high-contrast in vivo multiplexed detection, which holds promise for revealing physiological process in living body.
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Elementos da Série dos Lantanídeos , Nanopartículas , Neoplasias , Humanos , Elementos da Série dos Lantanídeos/química , Imagem Óptica/métodos , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Nanopartículas/químicaRESUMO
To minimize and control the transmission of infectious diseases, a sensitive, accurate, rapid, and robust assay strategy for application on-site screening is critical. Here, we report single-molecule RNA capture-assisted digital RT-LAMP (SCADL) for point-of-care testing of infectious diseases. Target RNA was captured and enriched by specific capture probes and oligonucleotide probes conjugated to magnetic beads, replacing laborious RNA extraction. Droplet generation, amplification, and the recording of results are all integrated on a microfluidic chip. In assaying commercial standard samples, quantitative results precisely corresponded to the actual concentration of samples. This method provides a limit of detection of 10 copies mL-1 for the N gene within 1 h, greatly reducing the need for skilled personnel and precision instruments. The ultrasensitivity, specificity, portability, rapidity and user-friendliness make SCADL a competitive candidate for the on-site screening of infectious diseases.
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Oral administration is a convenient administration route for gastrointestinal disease therapy with good patient compliance. But the nonspecific distribution of the oral drugs may cause serious side effects. In recent years, oral drug delivery systems (ODDS) have been applied to deliver the drugs to the gastrointestinal disease sites with decreased side effects. However, the delivery efficiency of ODDS is tremendously limited by physiological barriers in the gastrointestinal sites, such as the long and complex gastrointestinal tract, mucus layer, and epithelial barrier. Micro/nanomotors (MNMs) are micro/nanoscale devices that transfer various energy sources into autonomous motion. The outstanding motion characteristics of MNMs inspired the development of targeted drug delivery, especially the oral drug delivery. However, a comprehensive review of oral MNMs for the gastrointestinal diseases therapy is still lacking. Herein, the physiological barriers of ODDS were comprehensively reviewed. Afterward, the applications of MNMs in ODDS for overcoming the physiological barriers in the past 5 years were highlighted. Finally, future perspectives and challenges of MNMs in ODDS are discussed as well. This review will provide inspiration and direction of MNMs for the therapy of gastrointestinal diseases, pushing forward the clinical application of MNMs in oral drug delivery.
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Sistemas de Liberação de Medicamentos , Nanotecnologia , Humanos , Trato Gastrointestinal , Administração OralRESUMO
Loop-mediated isothermal amplification (LAMP) is a rapid and high-yield amplification technology for specific DNA or RNA molecules. In this study, we designed a digital loop-mediated isothermal amplification (digital-LAMP)-functioning microfluidic chip to achieve higher sensitivity for detection of nucleic acids. The chip could generate droplets and collect them, based on which we could perform Digital-LAMP. The reaction only took 40 min at a constant temperature of 63 °C. The chip enabled highly accurate quantitative detection, with the limit of detection (LOD) down to 102 copies µL-1. For better performance while reducing the investment of money and time in chip structure iterations, we used COMSOL Multiphysics to simulate different droplet generation ways by including flow-focusing structure and T-junction structure. Moreover, the linear structure, serpentine structure, and spiral structure in the microfluidic chip were compared to study the fluid velocity and pressure distribution. The simulations provided a basis for chip structure design while facilitating chip structure optimization. The digital-LAMP-functioning chip proposed in the work provides a universal platform for analysis of viruses.
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To efficiently eliminate highly polar organic pollutants from water has always been a difficult issue, especially in the case of ultralow concentrations. Herein, we present the facile synthesis of quinolinecarboxylic acid-linked COF (QCA-COF) via the Doebner multicomponent reaction, possessing multifunction, high specific surface area, robust physicochemical stability, and excellent crystallinity. The marked feature lies in the quinolinyl and carboxyl functions incorporated simultaneously to QCA-COF in one step. The major cis-orientation of carboxyl arms in QCA-COF was speculated by powder X-ray diffraction and total energy analysis. QCA-COF demonstrates excellent adsorption capacity for water-soluble organic pollutants such as rhodamine B (255.7 mg/g), methylene blue (306.1 mg/g), gentamycin (338.1 mg/g), and 2,4-dichlorophenoxyacetic acid (294.1 mg/g) in water. The kinetic adsorptions fit the pseudo-second order model and their adsorption isotherms are Langmuir model. Remarkably, QCA-COF can capture the above four water-soluble organic pollutants from real water samples at ppb level with higher than 95% removal efficiencies and excellent recycling performance.
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LHON is a common blinding inherited optic neuropathy caused by mutations in mitochondrial genes. In this study, by using skin fibroblasts derived from LHON patients with the most common m.G11778A mutation and healthy objects, we performed proteomic analysis to document changes in molecular proteins, signaling pathways and cellular activities. Furthermore, the results were confirmed by functional studies. A total of 860 differential expression proteins were identified, containing 624 upregulated and 236 downregulated proteins. Bioinformatics analysis revealed increased glycolysis in LHON fibroblasts. A glycolysis stress test showed that ECAR (extra-cellular acidification rate) values increased, indicating an enhanced level of glycolysis in LHON fibroblasts. Downregulated proteins were mainly enriched in oxidoreductase activity. Cellular experiments verified high levels of ROS in LHON fibroblasts, indicating the presence of oxidative damage. KEGG analysis also showed the metabolic disturbance of fatty acid in LHON cells. This study provided a proteomic profile of skin fibroblasts derived from LHON patients bearing m.G11778A. Increased levels of glycolysis, decreased oxidoreductase activity and fatty acid metabolism could represent the in-depth mechanisms of mitochondrial dysfunction mediated by the mutation. The results provided further evidence that LHON fibroblast could be an alternative model for investigating the devastating disease.
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Atrofia Óptica Hereditária de Leber , Humanos , Atrofia Óptica Hereditária de Leber/genética , Atrofia Óptica Hereditária de Leber/metabolismo , DNA Mitocondrial/genética , Proteômica , Oxirredutases/metabolismo , Mutação , Fibroblastos/metabolismo , Glicólise , Ácidos Graxos/metabolismoRESUMO
Enhancers involved in the upregulation of multiple oncogenes play a fundamental role in tumorigenesis and immortalization. Exploring the activity of enhancers in living cells has emerged as a critical path to a deep understanding of cancer properties, further providing important clues to targeted therapy. However, identifying enhancer activity in living cells is challenging due to the double biological barriers of a cell cytoplasmic membrane and a nuclear membrane, limiting the sensitivity and responsiveness of conventional probing methods. In this work, we developed a nanoelectroporation-probing (NP) platform, which enables intranuclear probe delivery for sensitive interrogation of enhancer activity in living cells. The nanoelectroporation biochip achieved highly focused perforation of the cell cytoplasmic membrane and brought about additional driving force to expedite the delivery of probes into the nucleus. The probes targeting enhancer activity (named "PH probe") are programmed with a cyclic amplification strategy and enable an increase in the fluorescence signals over 100-fold within 1 h. The platform was leveraged to detect the activity of CCAT1 enhancers (CCAT1, colon cancer-associated transcript-1, a long noncoding RNA that functions in tumor invasion and metastasis) in cell samples from clinical lung cancer patients, as well as reveal the heterogeneity of enhancers among different patients. The observations may extend the linkages between enhancers and cancer cells while validating the robustness and reliability of the platform for the assay of enhancer activity. This platform will be a promising toolbox with wide applicable potential for the intranuclear study of living cells.
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Neoplasias Pulmonares , Humanos , Reprodutibilidade dos TestesRESUMO
Background: In Leber's hereditary optic neuropathy (LHON), mtDNA mutations mediate mitochondrial dysfunction and apoptosis of retinal ganglion cells. Mitochondrial superoxide dismutase 2 (SOD2) is a crucial antioxidase against reactive oxygen species (ROS). This study aims to investigate whether SOD2 could ameliorate mtDNA mutation mediated mitochondrial dysfunction in skin fibroblasts of LHON patients and explore the underlying mechanisms. Methods: The skin of normal healthy subjects and severe LHON patients harboring m.11778G > A mutation was taken to prepare immortalized skin fibroblast cell lines (control-iFB and LHON-iFB). LHON-iFB cells were transfected with SOD2 plasmid or negative control plasmid, respectively. In addition, human neuroblastoma SH-SY5Y cells and human primary retinal pigmental epithelium (hRPE) cells were stimulated by H2O2 after gene transfection. The oxygen consumption rate (OCR) was measured with a Seahorse extracellular flux analyzer. The level of ATP production, mitochondrial membrane potential, ROS and malondialdehyde (MDA) were measured separately with the corresponding assay kits. The expression level of SOD2, inflammatory cytokines and p-IκBα/IκBα was evaluated by western-blot. Assessment of apoptosis was performed by TUNEL assay. Results: LHON-iFB exhibited lower OCR, ATP production, mitochondrial membrane potential but higher level of ROS and MDA than control-iFB. Western-blot revealed a significantly increased expression of IL-6 and p-IκBα/IκBα in LHON-iFB. Compared with the negative control, SOD2 overexpression increased OCR, ATP production and elevated mitochondrial membrane potential, but impaired ROS and MDA production. Besides, western-blot demonstrated exogenous SOD2 reduced the protein level of IL-6 and p-IκBα/IκBα. TUNEL assays suggested SOD2 inhibited cells apoptosis. Analogously, in SH-SY5Y and hRPE cells, SOD2 overexpression increased ATP production and mitochondrial membrane potential, but decreased ROS, MDA levels and suppressed apoptosis. Conclusion: SOD2 upregulation inhibited cells apoptosis through ameliorating mitochondrial dysfunction and reducing NF-κB associated inflammatory response. This study further support exogenous SOD2 may be a promising therapy for the treatment of LHON.
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Dominant optic atrophy (DOA) is the most common hereditary optic neuropathy. Although DOA is caused by mutations in several genes, there are still many cases that have not been diagnosed or misdiagnosed. Herein, we present a large family of 11 patients with DOA. To identify potential pathogenic mutations, whole exome sequencing (WES) was performed on the proband, a 35-year-old woman. WES revealed a novel pathogenic mutation (c.524T>C, p.F175S) in the AFG3L2 intermembrane space domain, rather than in the ATPase domain, which is the hot mutation region associated with most of the previously reported DOA cases. Functional studies on skin fibroblasts generated from patients and HEK293T cells showed that the mutation may impair mitochondrial function and decrease the ability of AFG3L2 protein to enter the mitochondrial inner membrane. In addition, this novel mutation led to protein degradation and reduced the stability of the AFG3L2 protein, which appeared to be associated with the proteasome-ubiquitin pathway.