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PURPOSE: Metastatic pulmonary large cell neuroendocrine carcinoma (LCNEC) is an aggressive cancer with generally poor outcomes. Effective methods for predicting survival in patients with metastatic LCNEC are needed. This study aimed to identify independent survival predictors and develop nomograms for predicting survival in patients with metastatic LCNEC. PATIENTS AND METHODS: We conducted a retrospective analysis using the Surveillance, Epidemiology, and End Results (SEER) database, identifying patients with metastatic LCNEC diagnosed between 2010 and 2017. To find independent predictors of cancer-specific survival (CSS), we performed Cox regression analysis. A nomogram was developed to predict the 6-, 12-, and 18-month CSS rates of patients with metastatic LCNEC. The concordance index (C-index), area under the receiver operating characteristic (ROC) curves (AUC), and calibration curves were adopted with the aim of assessing whether the model can be discriminative and reliable. Decision curve analyses (DCAs) were used to assess the model's utility and benefits from a clinical perspective. RESULTS: This study enrolled a total of 616 patients, of whom 432 were allocated to the training cohort and 184 to the validation cohort. Age, T staging, N staging, metastatic sites, radiotherapy, and chemotherapy were identified as independent prognostic factors for patients with metastatic LCNEC based on multivariable Cox regression analysis results. The nomogram showed strong performance with C-index values of 0.733 and 0.728 for the training and validation cohorts, respectively. ROC curves indicated good predictive performance of the model, with AUC values of 0.796, 0.735, and 0.736 for predicting the 6-, 12-, and 18-month CSS rates of patients with metastatic LCNEC in the training cohort, and 0.795, 0.801, and 0.780 in the validation cohort, respectively. Calibration curves and DCAs confirmed the nomogram's reliability and clinical utility. CONCLUSION: The new nomogram was developed for predicting CSS in patients with metastatic LCNEC, providing personalized risk evaluation and aiding clinical decision-making.
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Carcinoma Neuroendocrino , Neoplasias Pulmonares , Nomogramas , Programa de VERF , Humanos , Masculino , Femenino , Carcinoma Neuroendocrino/patología , Carcinoma Neuroendocrino/mortalidad , Persona de Mediana Edad , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/mortalidad , Estudios Retrospectivos , Pronóstico , Anciano , Carcinoma de Células Grandes/mortalidad , Carcinoma de Células Grandes/patología , Carcinoma de Células Grandes/secundario , Carcinoma de Células Grandes/terapia , Curva ROC , Estadificación de Neoplasias , Adulto , Tasa de SupervivenciaRESUMEN
Background: Chronic thromboembolic pulmonary hypertension (CTEPH) is a serious pulmonary vascular disease characterized by residual thrombi in the pulmonary arteries and distal pulmonary microvascular remodeling. The pathogenesis of CTEPH remains unclear, but many factors such as inflammation, immunity, coagulation and angiogenesis may be involved. Monocytes are important immune cells that can differentiate into macrophages and dendritic cells and play an important role in thrombus formation. However, the distribution, gene expression profile and differentiation trajectory of monocyte subsets in CTEPH patients have not been systematically studied. This study aims to reveal the characteristics and functions of monocytes in CTEPH patients using single-cell sequencing technology, and to provide new insights for the diagnosis and treatment of CTEPH. Methods: Single-cell RNA sequencing (scRNA-seq) were performed to analyze the transcriptomic features of peripheral blood mononuclear cells (PBMCs) from healthy controls, CTEPH patients and the tissues from CTEPH patients after the pulmonary endarterectomy (PEA). We established a CTEPH rat model with chronic pulmonary embolism caused by repeated injection of autologous thrombi through a central venous catheter, and used flow cytometry to detect the proportion changes of monocyte subsets in CTEPH patients and CTEPH rat model. We also observed the infiltration degree of macrophage subsets in thrombus tissue and their differentiation relationship with peripheral blood monocyte subsets by immunofluorescence staining. Results: The results showed that the monocyte subsets in peripheral blood of CTEPH patients changed significantly, especially the proportion of CD16+ monocyte subset increased. This monocyte subset had unique functional features at the transcriptomic level, involving processes such as cell adhesion, T cell activation, coagulation response and platelet activation, which may play an important role in pulmonary artery thrombus formation and pulmonary artery intimal remodeling. In addition, we also found that the macrophage subsets in pulmonary endarterectomy tissue of CTEPH patients showed pro-inflammatory and lipid metabolism reprogramming features, which may be related to the persistence and insolubility of pulmonary artery thrombi and the development of pulmonary hypertension. Finally, we also observed that CD16+ monocyte subset in peripheral blood of CTEPH patients may be recruited to pulmonary artery intimal tissue and differentiate into macrophage subset with high expression of IL-1ß, participating in disease progression. Conclusion: CD16+ monocytes subset had significant gene expression changes in CTEPH patients, related to platelet activation, coagulation response and inflammatory response. And we also found that these cells could migrate to the thrombus and differentiate into macrophages with high expression of IL-1ß involved in CTEPH disease progression. We believe that CD16+ monocytes are important participants in CTEPH and potential therapeutic targets.
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Hipertensión Pulmonar , Monocitos , Embolia Pulmonar , Receptores de IgG , Análisis de la Célula Individual , Humanos , Hipertensión Pulmonar/etiología , Hipertensión Pulmonar/inmunología , Hipertensión Pulmonar/metabolismo , Monocitos/inmunología , Monocitos/metabolismo , Receptores de IgG/metabolismo , Embolia Pulmonar/inmunología , Embolia Pulmonar/metabolismo , Animales , Masculino , Enfermedad Crónica , Ratas , Femenino , Persona de Mediana Edad , Proteínas Ligadas a GPI/metabolismo , Modelos Animales de Enfermedad , Transcriptoma , Anciano , Arteria Pulmonar/metabolismo , Arteria Pulmonar/inmunología , Arteria Pulmonar/patologíaRESUMEN
A precise regulation of gene expression depends on the accuracy of the three-dimensional (3D) structure of chromatin; however, the effects of 3D genome on gene expression in psoriasis remain unknown. In this study, we conducted Hi-C and RNA-seq on CD4+ T cells collected from five psoriasis patients and three healthy controls, and constructed a comprehensive 3D chromatin interaction map to delineate the genomic hierarchies including A/B compartments, topologically associated domains (TADs), and chromatin loops. Then, the specific super-enhancers (SEs) related to psoriasis were identified by Hi-C and H3K27ac ChIP-seq data. Subsequently, comprehensive analyses were carried out on the differentially expressed genes that are associated with altered TADs, loops and SEs in psoriasis. Lastly, we screened the candidate target genes and examined the potential functional single nucleotide polymorphism in psoriasis affected by disruptions of the spatial organization. This paper provides a comprehensive reference for examining the 3D genome interactions in psoriasis and elucidating the interplay between spatial organization disruption and gene regulation. We hope our findings can help clarify the mechanisms underlying the pathogenesis of psoriasis and shed light on the role of 3D genomic structure, therefore informing potential therapeutic approaches.
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INTRODUCTION: Genetically modified (GM) crops have been widely cultivated across the world and the development of rapid, ultrasensitive, visual multiplex detection platforms that are suitable for field deployment is critical for GM organism regulation. OBJECTIVE: In this study, we developed a novel one-pot system, termed MR-DCA (Multiplex RPA and Dual CRISPR assay), for the simultaneous detection of CaMV35S and NOS genetic targets in GM crops. This innovative approach combined Multiplex RPA (recombinase polymerase amplification) with the Dual CRISPR (clustered regularly interspaced short palindromic repeat) assay technique, to provide a streamlined and efficient method for GM crop detection. METHODS: The RPA reaction used for amplification CaMV35S and NOS targets was contained in the tube base, while the dual CRISPR enzymes were placed in the tube cap. Following centrifugation, the dual CRISPR (Cas13a/Cas12a) detection system was initiated. Fluorescence visualization was used to measure CaMV35S through the FAM channel and NOS through the HEX channel. When using lateral flow strips, CaMV35S was detected using rabbit anti-digoxin (blue line), whilst NOS was identified using anti-mouse FITC (red line). Line intensity was quantified using Image J and depicted graphically. RESULTS: Detection of the targets was completed in 35 min, with a limit of detection as low as 20 copies. In addition, two analysis systems were developed and they performed well in the MR-DCA assay. In an analysis of 24 blind samples from GM crops with a wide genomic range, MR-DCA gave consistent results with the quantitative PCR method, which indicated high accuracy, applicability and semi-quantitative ability. CONCLUSION: The development of MR-DCA represents a significant advancement in the field of GM detection, offering a rapid, sensitive and portable method for multiple target detection that can be used in resource-limited environments.
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The new generation of gene editing technologies, primarily based on CRISPR/Cas9 and its derivatives, allows for more precise editing of organisms. However, when the editing efficiency is low, only a small fraction of gene fragments is edited, leaving behind minimal traces and making it difficult to detect and evaluate the editing effects. Although a series of technologies and methods have been developed, they lack the ability for precise quantification and quantitative analysis of these products. Digital polymerase chain reaction (dPCR) offers advantages such as high precision and sensitivity, making it suitable for absolute quantification of nucleic acid samples. In the present study, we developed a novel platform for precise quantification of gene editing products based on microfluidic chip-based dPCR. The results indicated that our assay accurately identified different types of edited samples within a variety of different types, including more complex genomic crops such as tetraploid rapeseed and soybean (highly repetitive sequence). The sensitivity of this detection platform was as low as 8.14 copies per µL, with a detection limit of 0.1%. These results demonstrated the superior performance of the platform, including high sensitivity, low detection limit, and wide applicability, enabling precise quantification and assessment of gene editing efficiency. In conclusion, microfluidic chip-based dPCR was used as a powerful tool for precise quantification and assessment of gene editing products.
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Sistemas CRISPR-Cas , Edición Génica , Reacción en Cadena de la Polimerasa , Edición Génica/métodos , Reacción en Cadena de la Polimerasa/métodos , Sistemas CRISPR-Cas/genética , Dispositivos Laboratorio en un Chip , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodos , Glycine max/genética , Brassica napus/genéticaRESUMEN
OBJECTIVES: To investigate the correlation between innate lymphoid cell (ILC) subsets with T-helper (Th) cells and to explore the effect of ILCs on T cells in rheumatoid arthritis (RA). METHODS: We analysed the frequencies of ILC subsets in RA patients with varying disease activity and their correlation with Th cell subsets. We further investigated this correlation in various organs of collagen-induced arthritis (CIA) mice. The effects of ILCs on CD4+ T cells were determined by in vitro cell co-culture experiments. RESULTS: ILCs were less frequent in RA patients than in healthy controls, with higher levels of group 3 ILCs (ILC3s) in RA (p<0.05). ILC3s correlated positively with Th1 and Th17 cells in RA peripheral blood (p<0.05). In the peripheral blood, spleen, and lymph nodes of CIA, ILC3s decreased and then increased during arthritis progression. ILC3s correlated positively with Th1 and Th17 cells in the spleen and lymph nodes of CIA (p<0.05). NKp46+ ILC3s in the spleen positively correlated with Th1 and Th17 cells (p<0.05). Under Th17 cell differentiation conditions, co-culturing CIA-derived ILC3s directly with naive CD4+ T cells promoted Th17 differentiation and increased IL-17 secretion. However, co-culturing through a transwell insert impeded Th17 differentiation without affecting IL-17 secretion. CONCLUSIONS: ILC3s positively correlated with Th1 and Th17 cells in RA. In CIA, the frequencies of ILC3s changed with disease development and showed a positive correlation with Th1 and Th17 cells. ILC3s may facilitate the differentiation of Th17 cells through direct cell-cell contact.
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The Papaya ringspot virus (PRSV)-resistant genetically modified (GM) papaya 'Huanong No.1' has been certified as safe for consumption and widely planted in China for about 18 years. To protect consumers' rights and facilitate government supervision and monitoring, it is necessary to establish a simple, rapid, and specific detection method for 'Huanong No.1'. Herein, we developed a platform based on recombinase polymerase amplification (RPA) coupled with CRISPR-Cas12a for the detection of 'Huanong No.1'. The RPA-CRISPR-Cas12a platform was found to have high specificity, with amplification signals only present in 'Huanong No.1'. Additionally, the platform was highly sensitive, with a limit of detection (LOD) of approximately 20 copies. The detection process was fast and could be completed in less than 1 h. This novel platform enables the rapid on-site visualization detection of 'Huanong No.1', eliminating dependence on laboratory conditions and specialized instruments, and can serve as a technical reference for the rapid detection of other GM plants.
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Sistemas CRISPR-Cas , Carica , Técnicas de Amplificación de Ácido Nucleico , Plantas Modificadas Genéticamente , Carica/genética , Carica/virología , Sistemas CRISPR-Cas/genética , Plantas Modificadas Genéticamente/genética , Técnicas de Amplificación de Ácido Nucleico/métodos , Potyvirus/genética , Potyvirus/aislamiento & purificación , Recombinasas/metabolismo , Límite de Detección , Proteínas Bacterianas , Endodesoxirribonucleasas , Proteínas Asociadas a CRISPRRESUMEN
Maleic anhydride (MAH)-modified polymers are used as tie layers for binding dissimilar polymers in multilayer polymer films. The MAH chemistry which promotes adhesion is well characterized in the bulk; however, only recently has the interfacial chemistry been studied. Sum frequency generation vibrational spectroscopy (SFG) is an interfacial spectroscopy technique which provides detailed information on interfacial chemical reactions, species, and molecular orientations and has been essential for characterizing the MAH chemistry in both nylon and ethyl vinyl alcohol copolymer (EVOH) model systems and coextruded multilayer films. Here, we further characterize the interfacial chemistry between MAH-modified polyethylene tie layers and both EVOH and nylon by investigating the model systems over a range of MAH concentrations. We can detect the interfacial chemical reaction products between MAH and the barrier layer at MAH concentrations of ≥0.022 wt % for nylon and ≥0.077 wt % for EVOH. Additionally, from the concentration-dependent reaction reactant/product SFG peak positions and the product imide or ester/acid CâO group tilt angles extracted from the polarization-dependent SFG spectra, we quantitatively observe concentration-dependent changes to both the interfacial chemistry and interfacial structure. The interfacial chemistry and molecular orientation as a function of MAH concentration are well correlated with the adhesion strength, providing important quantitative information for the future design of MAH-modified tie layers for a variety of important applications.
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Cellulose plays a significant role in designing efficient and stable cellulose-based metallic catalysts, owing to its surface functionalities. Its hydroxyl groups are used as anchor sites for the nucleation and growth of metallic nanoparticles and, as a result, improve the stability and catalytic activity. Meanwhile, cellulose is also amenable to surface modifications to be more suitable for incorporating and stabilizing metallic nanoparticles. Herein, the Ag-/Bi-doped Mo(S,O)3 trimetallic sulfo-oxide anchored on B and N codoped cellulose (B-N-C) synthesized by a facile approach showed excellent stability and catalytic activity for PHER at 573.28 µmol/h H2 with 25 mg of catalyst under visible light, and 92.3% of the 4-nitrophenol (4-NP) reduction was achieved within 135 min by in situ-generated protons. In addition to B and N codoping, our use of the calcination method for B-N-C preparation further increases the structural disorders and defects, which act as anchoring sites for Ag-/Bi-doped Mo(S,O)3 nanoparticles. The Ag-/Bi-doped Mo(S,O)3@B-N-C surface active site also stimulates H2O molecule adsorption and activation kinetics and reduces the photogenerated charge carrier's recombination rate. The Mo4+ â Mo6+ electron hopping transport and the O 2p and Bi 6s orbital overlap facilitate the fast electron transfer by enhancing the electron's lifetime and photoinduced charge carrier mobility, respectively. In addition to acting as a support, B-N-C provides a highly conductive network that enhances charge transport, and the relocated electron in B-N-C activates the H2O molecule, which enables Ag-/Bi-doped Mo(S,O)3@B-N-C to have appreciable PHER performance.
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Fibroblasts are crucial components of the skin structure. They were traditionally believed to maintain the skin's structure by producing extracellular matrix and other elements. Recent research illuminated that fibroblasts can respond to external stimuli and exhibit diverse functions, such as the secretion of pro-inflammatory factors, adipogenesis, and antigen presentation, exhibiting remarkable heterogeneity and plasticity. This revelation positions fibroblasts as active contributors to the pathogenesis of skin diseases, challenging the traditional perspective that views fibroblasts solely as structural entities. Based on their diverse functions, fibroblasts can be categorized into six subtypes: pro-inflammatory fibroblasts, myofibroblasts, adipogenic fibroblasts, angiogenic fibroblasts, mesenchymal fibroblasts, and antigen-presenting fibroblasts. Cytokines, metabolism, and epigenetics regulate functional abnormalities in fibroblasts. The dynamic changes fibroblasts exhibit in different diseases and disease states warrant a comprehensive discussion. We focus on dermal fibroblasts' aberrant manifestations and pivotal roles in inflammatory and autoimmune skin diseases, including psoriasis, vitiligo, lupus erythematosus, scleroderma, and atopic dermatitis, and propose targeting aberrantly activated fibroblasts as a potential therapeutic strategy for inflammatory and autoimmune skin diseases.
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Silicones have excellent material properties and are used extensively in many applications, ranging from adhesives and lubricants to electrical insulation. To ensure strong adhesion of silicone adhesives to a wide variety of substrates, silane-based adhesion promotors are typically blended into the silicone adhesive formulation. However, little is known at the molecular level about the true silane adhesion promotion mechanism, which limits the ability to develop even more effective adhesion promoters. To understand the adhesion promotion mechanism of silane molecules at the molecular level, this study has used sum frequency generation vibrational spectroscopy (SFG) to determine the behavior of (3-glycidoxypropyl)trimethoxy silane (γ-GPS) at the buried interface between poly(ethylene terephthalate) (PET) and a bulk silicone adhesive. To complement and extend the SFG results, atomistic molecular dynamics (MD) simulations were applied to investigate molecular behavior and interfacial interaction of γ-GPS at the silicone/PET interface. Free energy computations were used to study the γ-GPS interaction in the sample system and determine the γ-GPS interfacial segregation mechanism. Both experiments and simulations consistently show that γ-GPS molecules prefer to segregate at the interface between PET and PDMS. The methoxy groups on γ-GPS molecules orient toward the PDMS polymer phase. The consistent picture of interfacial structure emerging from both simulation and experiment provides enhanced insight on how γ-GPS behaves in the silicone - PET system and illustrates why γ-GPS could improve the adhesion of silicone adhesive, leading to further understanding of silicone adhesion mechanisms useful in the design of silicone adhesives with improved performance.
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Nutrient enrichment is a major global change component that often disrupts the relationship between aboveground biodiversity and ecosystem functions by promoting species dominance, altering trophic interactions, and reducing ecosystem stability. Emerging evidence indicates that nutrient enrichment also reduces soil biodiversity and weakens the relationship between belowground biodiversity and ecosystem functions, but the underlying mechanisms remain largely unclear. Here, we explore the effects of nutrient enrichment on soil properties, soil biodiversity, and multiple ecosystem functions through a 13-year field experiment. We show that soil acidification induced by nutrient enrichment, rather than changes in mineral nutrient and carbon (C) availability, is the primary factor negatively affecting the relationship between soil diversity and ecosystem multifunctionality. Nitrogen and phosphorus additions significantly reduce soil pH, diversity of bacteria, fungi and nematodes, as well as an array of ecosystem functions related to C and nutrient cycling. Effects of nutrient enrichment on microbial diversity also have negative consequences at higher trophic levels on the diversity of microbivorous nematodes. These results indicate that nutrient-induced acidification can cascade up its impacts along the soil food webs and influence ecosystem functioning, providing novel insight into the mechanisms through which nutrient enrichment influences soil community and ecosystem properties.
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Ecosistema , Suelo , Suelo/química , Biodiversidad , Microbiología del Suelo , Nutrientes , Concentración de Iones de HidrógenoRESUMEN
Oxygen is essential for aerobic organisms, but little is known about its role in antiviral immunity. Here, we report that during responses to viral infection, hypoxic conditions repress antiviral-responsive genes independently of HIF signaling. EGLN1 is identified as a key mediator of the oxygen enhancement of antiviral innate immune responses. Under sufficient oxygen conditions, EGLN1 retains its prolyl hydroxylase activity to catalyze the hydroxylation of IRF3 at proline 10. This modification enhances IRF3 phosphorylation, dimerization and nuclear translocation, leading to subsequent IRF3 activation. Furthermore, mice and zebrafish with Egln1 deletion, treatment with the EGLN inhibitor FG4592, or mice carrying an Irf3 P10A mutation are more susceptible to viral infections. These findings not only reveal a direct link between oxygen and antiviral responses, but also provide insight into the mechanisms by which oxygen regulates innate immunity.
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Prolina Dioxigenasas del Factor Inducible por Hipoxia , Inmunidad Innata , Factor 3 Regulador del Interferón , Oxígeno , Prolina , Pez Cebra , Animales , Prolina Dioxigenasas del Factor Inducible por Hipoxia/metabolismo , Prolina Dioxigenasas del Factor Inducible por Hipoxia/genética , Factor 3 Regulador del Interferón/metabolismo , Hidroxilación , Humanos , Prolina/metabolismo , Ratones , Oxígeno/metabolismo , Células HEK293 , Fosforilación , Ratones Noqueados , Transducción de Señal , Ratones Endogámicos C57BLRESUMEN
To effectively protect the host from viral infection while avoiding excessive immunopathology, the innate immune response must be tightly controlled. However, the precise regulation of antiviral innate immunity and the underlying mechanisms remain unclear. Here, we find that sirtuin3 (SIRT3) interacts with mitochondrial antiviral signaling protein (MAVS) to catalyze MAVS deacetylation at lysine residue 7 (K7), which promotes MAVS aggregation, as well as TANK-binding kinase I and IRF3 phosphorylation, resulting in increased MAVS activation and enhanced type I interferon signaling. Consistent with these findings, loss of Sirt3 in mice and zebrafish renders them more susceptible to viral infection compared to their wild-type (WT) siblings. However, Sirt3 and Sirt5 double-deficient mice exhibit the same viral susceptibility as their WT littermates, suggesting that loss of Sirt5 in Sirt3-deficient mice may counteract the increased viral susceptibility displayed in Sirt3-deficient mice. Thus, we not only demonstrate that SIRT3 positively regulates antiviral immunity in vitro and in vivo, likely via MAVS, but also uncover a previously unrecognized mechanism by which SIRT3 acts as an accelerator and SIRT5 as a brake to orchestrate antiviral innate immunity.
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Sirtuina 3 , Sirtuinas , Virosis , Animales , Ratones , Proteínas Adaptadoras Transductoras de Señales/genética , Inmunidad Innata , Lisina , Sirtuina 3/genética , Sirtuinas/genética , Pez Cebra , Proteínas de Pez CebraRESUMEN
The gene editing technology represented by clustered rule-interspersed short palindromic repeats (CRISPR)/Cas9 has developed as a common tool in the field of biotechnology. Many gene-edited products in plant varieties have recently been commercialized. However, the rapid on-site visual detection of gene-edited products without instrumentation remains challenging. This study aimed to develop a novel and efficient method, termed the CRISPR/SpRY detection platform, for the rapid screening of CRISPR/Cas9-induced mutants based on CRISPR/SpRY-mediated in vitro cleavage using rice (Oryza sativa L.) samples genetically edited at the TGW locus as an example. We designed the workflow of the CRISPR/SpRY detection platform and conducted a feasibility assessment. Subsequently, we optimized the reaction system of CRISPR/SpRY, and developed a one-pot CRISPR/SpRY assay by integrating recombinase polymerase amplification (RPA). The sensitivity of the method was further verified using recombinant plasmids. The proposed method successfully identified various types of mutations, including insertions, deletions (indels), and nucleotide substitutions, with excellent sensitivity. Finally, the applicability of this method was validated using different rice samples. The entire process was completed in less than an hour, with a limit of detection as low as 1%. Compared with previous methods, our approach is simple to operate, instrumentation-free, cost-effective, and time-efficient. The primary significance lies in the liberation of our developed system from the limitations imposed using protospacer adjacent motif sequences. This expands the scope and versatility of the CRISPR-based detection platform, making it a promising and groundbreaking platform for detecting mutations induced by gene editing.
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Oryza , Oryza/genética , Sistemas CRISPR-Cas/genética , Edición Génica , Bioensayo , Biotecnología , ARNRESUMEN
RNA is an important information and functional molecule. It can respond to the regulation of life processes and is also a key molecule in gene expression and regulation. Therefore, RNA detection technology has been widely used in many fields, especially in disease diagnosis, medical research, genetic engineering and other fields. However, the current RT-qPCR for RNA detection is complex, costly and requires the support of professional technicians, resulting in it not having great potential for rapid application in the field. PCR-free techniques are the most attractive alternative. They are a low-cost, simple operation method and do not require the support of large instruments, providing a new concept for the development of new RNA detection methods. This article reviews current PCR-free methods, overviews reported RNA biosensors based on electrochemistry, SPR, microfluidics, nanomaterials and CRISPR, and discusses their challenges and future research prospects in RNA detection.
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Técnicas Biosensibles , ARN , ARN/análisis , Humanos , Técnicas Electroquímicas , Reacción en Cadena de la Polimerasa/métodos , Nanoestructuras , Resonancia por Plasmón de Superficie , MicrofluídicaRESUMEN
Herein, Cellulose-templated Zn1-XCuXO/Ag2O nanocomposites were prepared using biological renewable cellulose extracted from water hyacinth (Eichhornia crassipes). Cellulose-templated Cu-doped ZnO catalysts with different amounts of Cu as the dopants (1, 2, 3, and 4%) were prepared and denoted CZ-1, CZ-2, CZ-3, and CZ-4, respectively, for simplicity. The prepared catalysts were tested for the degradation of methylene blue (MB), and 2% Cu-doped ZnO (CZ-2) showed the best catalytic performance (82%), while the pure ZnO, CZ-1, CZ-3, and CZ-4 catalysts exhibited MB dye degradation efficiencies of 54, 63, 65, and 60%, respectively. The best catalyst (CZ-2) was chosen to further improve the degradation efficiency. Different amounts of AgNO3 (10, 15, 30, and 45 mg) were used for the deposition of Ag2O on the surface of CZ-2 and denoted CZA-10, CZA-15, CZA-30, and CZA-45, respectively. Among the composite catalysts, CZA-15 showed remarkable degradation efficiency and degraded 94% of MB, while the CZA-10, CZA-30, and CZA-45 catalysts showed 90, 81, and 79% degradation efficiencies, respectively, under visible light within 100 min of irradiation. The enhanced catalytic performance could be due to the smaller particle size, the higher electron and hole separation and charge transfer efficiencies, and the lower agglomeration in the composite catalyst system. The results also demonstrated that the Cu-doped ZnO prepared with cellulose as a template, followed by the optimum amount of Ag2O deposition, could have promising applications in the degradation of organic pollutants.
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Current research endeavors have focused on the combination of various isothermal nucleic acid amplification methods with CRISPR/Cas systems, aiming to establish a more sensitive and reliable molecular diagnostic approach. Nevertheless, most assays adopt a two-step procedure, complicating manual operations and heightening the risk of contamination. Efforts to amalgamate both assays into a single-step procedure have faced challenges due to their inherent incompatibility. Furthermore, the presence of the protospacer adjacent motif (PAM) motif (e.g., TTN or TTTN) in the target double-strand DNA (dsDNA) is an essential prerequisite for the activation of the Cas12-based method. This requirement imposes constraints on crRNA selection. To overcome such limitations, we have developed a novel PAM-free one-step asymmetric recombinase polymerase amplification (RPA) coupled with a CRISPR/Cas12b assay (OAR-CRISPR). This method innovatively merges asymmetric RPA, generating single-stranded DNA (ssDNA) amenable to CRISPR RNA binding without the limitations of the PAM site. Importantly, the single-strand cleavage by PAM-free crRNA does not interfere with the RPA amplification process, significantly reducing the overall detection times. The OAR-CRISPR assay demonstrates sensitivity comparable to that of qPCR but achieves results in a quarter of the time required by the latter method. Additionally, our OAR-CRISPR assay allows the naked-eye detection of as few as 60 copies/µL DNA within 8 min. This innovation marks the first integration of an asymmetric RPA into one-step CRISPR-based assays. These advancements not only support the progression of one-step CRISPR/Cas12-based detection but also open new avenues for the development of detection methods capable of targeting a wide range of DNA targets.
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Sistemas CRISPR-Cas , Recombinasas , Sistemas CRISPR-Cas/genética , ARN Guía de Sistemas CRISPR-Cas , Nucleotidiltransferasas , ADN/genética , ADN de Cadena Simple , ADN Complementario , Técnicas de Amplificación de Ácido NucleicoRESUMEN
Silicone elastomers are widely used in many industrial applications, including coatings, adhesives, and sealants. Room-temperature vulcanized (RTV) silicone, a major subcategory of silicone elastomers, undergoes molecular structural transformations during condensation curing, which affect their mechanical, thermal, and chemical properties. The role of reactive hydroxyl (-OH) groups in the curing reaction of RTV silicone is crucial but not well understood, particularly when multiple sources of hydroxyl groups are present in a formulated product. This work aims to elucidate the interfacial molecular structural changes and origins of interfacial reactive hydroxyl groups in RTV silicone during curing, focusing on the methoxy groups at interfaces and their relationship to adhesion. Sum frequency generation (SFG) vibrational spectroscopy is an in situ nondestructive technique used in this study to investigate the interfacial molecular structure of select RTV formulations at the buried interface at different levels of cure. The primary sources of hydroxyl groups required for interfacial reactions in the initial curing stage are found to be those on the substrate surface rather than those from the ingress of ambient moisture. The silylation treatment of silica substrates eliminates interfacial hydroxyl groups, which greatly impact the silicone interfacial behavior and properties (e.g., adhesion). This study establishes the correlation between interfacial molecular structural changes in RTV silicones and their effect on adhesion strength. It also highlights the power of SFG spectroscopy as a unique tool for studying chemical and structural changes at RTV silicone/substrate interface in situ and in real time during curing. This work provides valuable insights into the interfacial chemistry of RTV silicone and its implications for material performance and application development, aiding in the development of improved silicone adhesives.
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The application of network formulaology and network pharmacology has significantly advanced the scientific understanding of traditional Chinese medicine (TCM) treatment mechanisms in disease. The field of herbal biology is experiencing a surge in data generation. However, researchers are encountering challenges due to the fragmented nature of the data and the reliance on programming tools for data analysis. We have developed TCMNPAS, a comprehensive analysis platform that integrates network formularology and network pharmacology. This platform is designed to investigate in-depth the compatibility characteristics of TCM formulas and their potential molecular mechanisms. TCMNPAS incorporates multiple resources and offers a range of functions designed for automated analysis implementation, including prescription mining, molecular docking, network pharmacology analysis, and visualization. These functions enable researchers to analyze and obtain core herbs and core formulas from herbal prescription data through prescription mining. Additionally, TCMNPAS facilitates virtual screening of active compounds in TCM and its formulas through batch molecular docking, allowing for the rapid construction and analysis of networks associated with "herb-compound-target-pathway" and disease targets. Built upon the integrated analysis concept of network formulaology and network pharmacology, TCMNPAS enables quick point-and-click completion of network-based association analysis, spanning from core formula mining from clinical data to the exploration of therapeutic targets for disease treatment. TCMNPAS serves as a powerful platform for uncovering the combinatorial rules and mechanism of TCM formulas holistically. We distribute TCMNPAS within an open-source R package at GitHub ( https://github.com/yangpluszhu/tcmnpas ), and the project is freely available at http://54.223.75.62:3838/ .