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Hepatoblastoma (HB), a primary liver tumour, is notorious for its high metastatic potential and poor prognosis. Ganoderma lucidum, an edible mushroom species utilized in traditional Chinese medicine for addressing various tumour types, presents an intriguing avenue for HB treatment. However, the effectiveness of G. lucidum in managing HB and its underlying molecular mechanism necessitates further exploration. Standard in vitro assays were conducted to evaluate the impact of sporoderm-broken spores of G. lucidum (SBSGL) on the malignant characteristics of HB cells. The mechanism of SBSGL in treating HB and its tumour immunomodulatory effects were explored and validated by various experiments, including immunoprecipitation, Western blotting, mRFP-GFP-LC3 adenovirus transfection and co-localization analysis, as well as verified with in vivo experiments in this regard. The results showed that SBSGL effectively inhibited the malignant traits of HB cells and suppressed the O-GlcNAcylation of RACK1, thereby reducing its expression. In addition, SBSGL inhibited immune checkpoints and regulated cytokines. In conclusion, SBSGL had immunomodulatory effects and regulated the malignancy and autophagy of HB by regulating the O-GlcNAcylation of RACK1. These findings suggest that SBSGL holds promise as a potential anticancer drug for HB treatment.
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Hepatoblastoma , Neoplasias Hepáticas , Reishi , Hepatoblastoma/tratamiento farmacológico , Hepatoblastoma/genética , Esporas Fúngicas , Autofagia , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/genéticaRESUMEN
With the prevalence of coronavirus disease 2019, the administration of glucocorticoids (GCs) has become more widespread. Treatment with high-dose GCs leads to a variety of problems, of which steroid-induced osteonecrosis of the femoral head (SONFH) is the most concerning. Since hypoxia-inducible factor 1α (HIF-1α) is a key factor in cartilage development and homeostasis, it may play an important role in the development of SONFH. In this study, SONFH models were established using methylprednisolone (MPS) in mouse and its proliferating chondrocytes to investigate the role of HIF-1α in cartilage differentiation, extracellular matrix (ECM) homeostasis, apoptosis and glycolysis in SONFH mice. The results showed that MPS successfully induced SONFH in vivo and vitro, and MPS-treated cartilage and chondrocytes demonstrated disturbed ECM homeostasis, significantly increased chondrocyte apoptosis rate and glycolysis level. However, compared with normal mice, not only the expression of genes related to collagens and glycolysis, but also chondrocyte apoptosis did not demonstrate significant differences in mice co-treated with MPS and HIF-1α inhibitor. And the effects observed in HIF-1α activator-treated chondrocytes were similar to those induced by MPS. And HIF-1α degraded collagens in cartilage by upregulating its downstream target genes matrix metalloproteinases. The results of activator/inhibitor of endoplasmic reticulum stress (ERS) pathway revealed that the high apoptosis rate induced by MPS was related to the ERS pathway, which was also affected by HIF-1α. Furthermore, HIF-1α affected glucose metabolism in cartilage by increasing the expression of glycolysis-related genes. In conclusion, HIF-1α plays a vital role in the pathogenesis of SONFH by regulating ECM homeostasis, chondrocyte apoptosis, and glycolysis.
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Apoptosis , Cartílago , Condrocitos , Glucocorticoides , Glucólisis , Homeostasis , Subunidad alfa del Factor 1 Inducible por Hipoxia , Metilprednisolona , Animales , Masculino , Ratones , Apoptosis/efectos de los fármacos , Cartílago/metabolismo , Cartílago/patología , Cartílago/efectos de los fármacos , Condrocitos/metabolismo , Condrocitos/efectos de los fármacos , Condrocitos/patología , Modelos Animales de Enfermedad , Matriz Extracelular/metabolismo , Cabeza Femoral/patología , Cabeza Femoral/metabolismo , Necrosis de la Cabeza Femoral/inducido químicamente , Necrosis de la Cabeza Femoral/patología , Necrosis de la Cabeza Femoral/metabolismo , Necrosis de la Cabeza Femoral/genética , Glucocorticoides/efectos adversos , Glucocorticoides/farmacología , Glucólisis/efectos de los fármacos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Metilprednisolona/efectos adversos , Metilprednisolona/farmacología , Ratones Endogámicos C57BLRESUMEN
Achieving sensitive detection and accurate identification of cancer cells is vital for diagnosing and treating the disease. Here, we developed a logic signal amplification system using DNA tetrahedron-mediated three-dimensional (3D) DNA nanonetworks for sensitive electrochemiluminescence (ECL) detection and subtype identification of cancer cells. Specially designed hairpins were integrated into DNA tetrahedral nanostructures (DTNs) to perform a catalytic hairpin assembly (CHA) reaction in the presence of target microRNA, forming hyperbranched 3D nanonetworks. Benefiting from the "spatial confinement effect," the DNA tetrahedron-mediated catalytic hairpin assembly (DTCHA) reaction displayed significantly faster kinetics and greater cycle conversion efficiency than traditional CHA. The resulting 3D nanonetworks could load a large amount of Ru(phen)32+, significantly enhancing its ECL signal, and exhibit detection limits for both miR-21 and miR-141 at the femtomolar level. The biosensor based on modular logic gates facilitated the distinction and quantification of cancer cells and normal cells based on miR-21 levels, combined with miR-141 levels, to further identify different subtypes of breast cancer cells. Overall, this study provides potential applications in miRNA-related clinical diagnostics.
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Técnicas Biosensibles , Técnicas Electroquímicas , Mediciones Luminiscentes , MicroARNs , Humanos , MicroARNs/análisis , Técnicas Electroquímicas/métodos , Técnicas Biosensibles/métodos , ADN/química , Nanoestructuras/química , Límite de Detección , Línea Celular Tumoral , Neoplasias de la Mama/diagnóstico , Células MCF-7RESUMEN
Development of highly efficient, heavy-metal-free electrochemiluminescence (ECL) materials is attractive but still challenging. Herein, we report an aggregation-induced delayed ECL (AIDECL) active organic dot (OD) composed of a tert-butoxy-group-substituted benzophenone-dimethylacridine compound, which shows high ECL efficiency. The resultant ODs exhibit 2.1-fold higher ECL efficiency compared to control AIDECL-active ODs. Molecular stacking combined with theoretical calculations suggests that tert-butoxy groups effectively participate in the intermolecular interactions, further inhibiting the molecular motions in the aggregated states and thus accelerating radiative decay. On the basis of these ODs exhibiting excellent ECL performance, a proof-of-concept biosensor is constructed for the detection of miR-16 associated with Alzheimer's disease, which demonstrates excellent detection ability with the limit of detection of 1.7 fM. This work provides a new approach to improve the ECL efficiency and enriches the fundamental understanding of the structure-property relationship.
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Constructing versatile metal nanoclusters (NCs) assemblies through noncovalent weak interactions between inter-ligands is a long-standing challenge in interfacial chemistry, while compelling interfacial hydrogen-bond-driven metal NCs assemblies remain unexplored so far. Here, the study reports an amination-ligand o-phenylenediamine-coordinated copper NCs (CuNCs), demonstrating the impact of interfacial hydrogen-bonds (IHBs) motifs on the luminescent behaviors of metal NCs as the alteration of protic solvent. Experimental results supported by theoretical calculation unveil that the flexibility of interfacial ligand and the distance of cuprophilic CuI···CuI interaction between intra-/inter-NCs can be tailored by manipulating the cooperation between the diverse IHBs motifs reconstruction, therewith the IHBs-modulated fundamental structure-property relationships are established. Importantly, by utilizing the IHBs-mediated optical polychromatism of aminated CuNCs, portable visualization of humidity sensing test-strips with fast response is successfully manufactured. This work not only provides further insights into exploring the interfacial chemistry of NCs based on inter-ligands hydrogen-bond interactions, but also offers a new opportunity to expand the practical application for optical sensing of metal NCs.
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A central aim of community ecology is to understand how local species diversity is shaped. Agricultural activities are reshaping and filtering soil biodiversity and communities; however, ecological processes that structure agricultural communities have often overlooked the role of the regional species pool, mainly owing to the lack of large datasets across several regions. Here, we conducted a soil survey of 941 plots of agricultural and adjacent natural ecosystems (e.g., forest, wetland, grassland, and desert) in 38 regions across diverse climatic and soil gradients to evaluate whether the regional species pool of soil microbes from adjacent natural ecosystems is important in shaping agricultural soil microbial diversity and completeness. Using a framework of multiscales community assembly, we revealed that the regional species pool was an important predictor of agricultural bacterial diversity and explained a unique variation that cannot be predicted by historical legacy, large-scale environmental factors, and local community assembly processes. Moreover, the species pool effects were associated with microbial dormancy potential, where taxa with higher dormancy potential exhibited stronger species pool effects. Bacterial diversity in regions with higher agricultural intensity was more influenced by species pool effects than that in regions with low intensity, indicating that the maintenance of agricultural biodiversity in high-intensity regions strongly depends on species present in the surrounding landscape. Models for community completeness indicated the positive effect of regional species pool, further implying the community unsaturation and increased potential in bacterial diversity of agricultural ecosystems. Overall, our study reveals the indubitable role of regional species pool from adjacent natural ecosystems in predicting bacterial diversity, which has useful implication for biodiversity management and conservation in agricultural systems.
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Bacterias , Ecosistema , Biodiversidad , Suelo/química , Bosques , Microbiología del SueloRESUMEN
Directional surface plasmon polaritons (SPPs) are expected to promote the energy efficiency of plasmonic devices, via limiting the energy in a given spatial domain. The directional scattering of dielectric nanoparticles induced by the interference between electric and magnetic responses presents a potential candidate for directional SPPs. Magnetic nanoparticles can introduce permeability as an extra manipulation, whose directional scattered SPPs have not been investigated yet. In this work, we demonstrated the directional scattered SPPs by using single magnetic nanoparticles via simulation and experiment. By increasing the permeability and particle size, the high-order TEM modes are excited inside the particle and induce more forward directional SPPs. It indicated that the particle size manifests larger tuning range compared with the permeability. Experimentally, the maximum forward-to-backward (F-to-B) SPP scattering intensity ratio of 118.52:1 is visualized by using a single 1â µm Fe3O4 magnetic nanoparticle. The directional scattered SPPs of magnetic nanoparticles are hopeful to improve the efficiency of plasmonic devices and pave the way for plasmonic circuits on-chip.
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BACKGROUND: The aim of this study was to summarize the optimal strategy for early feeding in patients with acute pancreatitis. METHODS: The search was undertaken in electronic databases, which compared early with delayed feeding in acute pancreatitis. The primary outcome was the length of hospital stay (LOHS). The second outcomes were intolerance of refeeding, mortality, and total cost of each patient. This meta-analysis followed the "Preferred Reporting Items for Systematic Reviews and Meta-analyses" guideline. Research is registered by PROSPERO, CRD42020192133. RESULTS: A total of 20 trials involving 2168 patients were included, randomly assigned to the early feeding group (N = 1033) and delayed feeding group (N = 1135). The LOHS was significantly lower in the early feeding group than the delayed feeding group (mean difference: -2.35, 95% CI: -2.89 to -1.80; P < 0.0001), no matter the mild or severe subgroup ( Pint = 0.69). The secondary outcome of feeding intolerance and mortality were no significant difference (risk ratio: 0.96, 0.40 to 2.16, P = 0.87 and 0.91, 0.57 to 1.46, P = 0.69; respectively). Moreover, the hospitalization cost was significantly less in the early feeding group, resulting in an average savings of 50%. In patients with severe pancreatitis, early feeding after 24 hours may be beneficial ( Pint = 0.001). CONCLUSION: Early oral feeding can significantly reduce the LOHS and hospitalization costs in patients with acute pancreatitis without increasing feeding intolerance or mortality. In patients with severe pancreatitis, early feeding after 24 hours may be beneficial.
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Nutrición Enteral , Pancreatitis , Humanos , Recién Nacido , Nutrición Enteral/métodos , Enfermedad Aguda , Pancreatitis/terapia , Hospitalización , Tiempo de Internación , Ensayos Clínicos Controlados Aleatorios como AsuntoRESUMEN
In this paper, we present a novel three-dimensional (3D) coupled configuration of piezoelectric micromachined ultrasound transducers (pMUTs) by combing a curved and an annular diaphragm for transmit performance optimization in biomedical applications. An analytical equivalent circuit model (EQC) is developed with varied excitation methods to incorporate the acoustic-structure coupling of the curved and annular diaphragm-coupled pMUTs (CAC-pMUTs). The model-derived results align well with the reference simulated by the finite element method (FEM). Using this EQC model, we optimize the key design parameters of the CAC-pMUTs in order to improve the output sound pressure, including the width of the annular membrane, the thickness of the passive layer, and the phase difference of the driving voltage. In the anti-phase mode, the designed CAC-pMUTs demonstrate a transmit efficiency 285 times higher than that of single annular pMUTs. This substantial improvement underscores the potential of CAC-pMUTs for large array applications.
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BACKGROUND: Intestinal development and function are critical to maintaining sustained broiler growth. The present study aimed to evaluate the effects of coated sodium butyrate (CSB) and vitamin D3 (VD3) on the intestinal immunity, barrier, oxidative stress and microflora in early-stage broilers. In total, 192 one-day-old broilers were assigned to a 2 × 2 factorial design including two dietary supplements at two different levels, in which the main effects were VD3 (3000 or 5000 IU kg-1) and CSB (0 or 1 g kg-1). RESULTS: The results showed that CSB supplementation increased ileal goblet cells (GCs) numbers, villus height and decreased crypt depth in broilers. CSB increased ileal proliferating cell nuclear antigen expression and high-level VD3 decreased cluster of differentiation 3 expression. CSB reduced serum d-lactate, endotoxin (ET), adrenocorticotropic hormone, corticosterone and malondialdehyde (MDA) concentrations and increased total antioxidant capacity (T-AOC) level. Meanwhile, high-level VD3 decreased serum ET concentration. Furthermore, CSB increased ileal T-AOC, lysozyme (LYZ) and transforming growth factor (TGF)-ß and decreased MDA, whereas high-level VD3 decreased ileal MDA and increased secretory immunoglobulin A. CSB up-regulated ileal claudin1, superoxide dismutase 1, TGF-ß and LYZ mRNA expression and down-regulated interleukin-1ß mRNA expression. CSB combined with high-level VD3 increased ileal Faecalibaculum abundance. Spearman correlation analysis showed that Faecalibaculum was related to the immune and barrier function. CONCLUSION: Dietary supplementation with CSB and high-level VD3 improved early gut health in broilers by promoting intestinal development, enhancing antioxidant capacity, strengthening barrier function and enhancing the favorable composition of the gut bacterial flora. © 2024 Society of Chemical Industry.
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Antioxidantes , Dieta , Animales , Dieta/veterinaria , Antioxidantes/metabolismo , Pollos/metabolismo , Ácido Butírico/metabolismo , Colecalciferol/farmacología , Suplementos Dietéticos/análisis , ARN Mensajero/metabolismo , Alimentación Animal/análisisRESUMEN
BACKGROUND: Rice vinegar is a popular cereal vinegar worldwide and is typically produced in an open environment, and the ecosystem of solid-state fermentation is complicated and robust. The present study aimed to reveal the shaping force of the establishment of the ecosystem of Beijing rice vinegar, the core function microbiota and their correlation with critical environmental factors. [Correction added after first online publication on 29 May 2024; the word "worldwide" has been removed from the first sentence under the section Background.] RESULTS: The experimental findings revealed the changes in environmental factors, major metabolites and microbial patterns during Beijing rice vinegar fermentation were obtained. The major metabolites accumulated at the middle and late acetic acid fermentation (AAF) periods. Principal coordinates and t-test analyses revealed the specific bacterial and fungal species at corresponding stages. Kosakonia, Methlobacterium, Sphingomonas, unidentified Rhizobiaceae, Pseudozyma and Saccharomycopsis dorminated during saccharification and alcohol fermentation and early AAF, whereas Lactococcus, Acetobacter, Rhodotorula and Kazachstania dominated the later AAF stages. Canonical correspondence analysis of environmental factors with core microbiota. Temperature and total acid were the most significant factors correlated with the SAF bacterial profile (Pediococcus, Weissella, Enterococcus and Kosakonia). Ethanol was the most significant factor between AAF1 and AAF3, and mainly affected Acetobacter and Lactobacillus. Conversely, ethanol was the most significant factor in the SAF, AAF1 and AAF3 fungi communities; typical microorganisms were Saccharomyces and Malassezia. Furthermore, the predicted phenotypes of bacteria and their response to environmental factors were evaluated. CONCLUSION: In conclusion, the present study has provided insights into the process regulation of spontaneous fermentation and distinguished the key driving forces in the microbiota of Beijing rice vinegar fermentation. © 2024 Society of Chemical Industry.
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Ácido Acético , Bacterias , Fermentación , Hongos , Microbiota , Oryza , Ácido Acético/metabolismo , Ácido Acético/análisis , Oryza/microbiología , Oryza/metabolismo , Oryza/química , Bacterias/metabolismo , Bacterias/clasificación , Bacterias/aislamiento & purificación , Bacterias/genética , Hongos/metabolismo , Hongos/clasificación , Hongos/aislamiento & purificación , Beijing , Microbiología de Alimentos , Etanol/metabolismo , Etanol/análisisRESUMEN
Aggregation-induced emission (AIE) enables "Turn-On" imaging generally through single aggregation of the AIE luminogen (AIEgen). Dual aggregrations of the AIEgen might further enhance the imaging intensity and the consequent sensitivity. Herein, we rationally designed a near-infrared (NIR) AIEgen Ac-Trp-Glu-His-Asp-Cys(StBu)-Pra(QMT)-CBT (QMT-CBT) which, upon caspase1 (Cas1) activation, underwent a CBT-Cys click reaction to form cyclic dimers QMT-Dimer (the first aggregation) and assembled into nanoparticles (the second aggregation), turning the AIE signal "on" for enhanced imaging of Alzheimer's disease (AD). Molecular dynamics simulations validated that the fluorogen QMT in QMT-NPs stacked much tighter with each other than in the single aggregates of the control compound Ac-Trp-Glu-His-Asp-Cys(tBu)-Pra(QMT)-CBT (QMT-CBT-Ctrl). Dual aggregations of QMT rendered 1.9-, 1.7-, and 1.4-fold enhanced fluorescence intensities of its single aggregation in vitro, in cells, and in a living AD mouse model, respectively. We anticipate this smart fluorogen to be used for sensitive diagnosis of AD in the clinic in the near future.
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Enfermedad de Alzheimer , Nanopartículas , Animales , Ratones , Enfermedad de Alzheimer/diagnóstico por imagen , Imagen Óptica/métodos , Simulación de Dinámica Molecular , Colorantes FluorescentesRESUMEN
Circular RNAs (circRNAs) are a type of non-coding RNA that plays a vital role in biology. circRNAs appear to have a role in the development and progression of several malignancies, according to research. However, circRNAs that regulate prostate cancer (PCa) progression are still largely unknown and deserve further exploration. The aim of this study was to investigate the effect of hsa_circ_0070512 on the function and mechanism of PCa. hsa_circ_0070512 was increased in PCa tissues and cells and was mostly found in the cytoplasm of PCa cells. Overexpression of hsa_circ_0070512 considerably increased PCa cell proliferation and migration, whereas silencing of hsa_circ_0070512 greatly decreased PCa cell proliferation and migration. Mechanistically, we show that hsa_circ_0070512 acts as a "molecular sponge" for miR-338-3p and that the miR-338-3p mimics partially block the pro-tumor effects of hsa_circ_0070512. RNA sequencing analysis of PC3 cells stably overexpressing hsa_circ_0070512 revealed that hedgehog was downstream of the signaling pathways of hsa_circ_0070512 and miR-338-3p. Our results implied that hsa_circ_0070512 regulated the hedgehog signaling pathways through miR-338-3p to enhance PCa growth and migration, providing a new diagnostic and therapeutic target for PCa.
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MicroARNs , Neoplasias de la Próstata , Masculino , Humanos , MicroARNs/genética , MicroARNs/metabolismo , ARN Circular/genética , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Neoplasias de la Próstata/genética , Transducción de Señal/genética , Proliferación Celular/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión GénicaRESUMEN
Anthrax lethal toxin (LT) is known to induce NLRP1B inflammasome activation and pyroptotic cell death in macrophages from certain mouse strains in its metalloprotease activity-dependent manner, but the underlying mechanism is unknown. Here, we establish a simple but robust cell system bearing dual-fluorescence reporters for LT-induced ASC specks formation and pyroptotic lysis. A genome-wide siRNA screen and a CRISPR-Cas9 knockout screen were applied to this system for identifying genes involved in LT-induced inflammasome activation. UBR2, an E3 ubiquitin ligase of the N-end rule degradation pathway, was found to be required for LT-induced NLRP1B inflammasome activation. LT is known to cleave NLRP1B after Lys44. The cleaved NLRP1B, bearing an N-terminal leucine, was targeted by UBR2-mediated ubiquitination and degradation. UBR2 partnered with an E2 ubiquitin-conjugating enzyme UBE2O in this process. NLRP1B underwent constitutive autocleavage before the C-terminal CARD domain. UBR2-mediated degradation of LT-cleaved NLRP1B thus triggered release of the noncovalent-bound CARD domain for subsequent caspase-1 activation. Our study illustrates a unique mode of inflammasome activation in cytosolic defense against bacterial insults.
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Antígenos Bacterianos/efectos adversos , Proteínas Reguladoras de la Apoptosis/química , Proteínas Reguladoras de la Apoptosis/metabolismo , Toxinas Bacterianas/efectos adversos , Macrófagos/efectos de los fármacos , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Sistemas CRISPR-Cas , Caspasa 1/metabolismo , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Inflamasomas/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Dominios Proteicos , Proteolisis/efectos de los fármacos , Células RAW 264.7 , ARN Interferente Pequeño/farmacología , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitinación/efectos de los fármacosRESUMEN
A microfluidic gradient array is a widely used screening and analysis device, which has characteristics of high efficiency, high automation, and low consumption. Bipolar electrode electrochemiluminescence (BPE-ECL) has special value in microfluidic array chips. The combination of the microfluidic gradient and BPE arrays has potential for high-throughput screening. In this article, a microfluidic BPE array chip for gradient culture and conditional screening of cancer cells was designed. The generation of concentration gradients, continuous culture of cancer cells with high throughput, and drug screening through BPE-ECL of the Ru(bpy)32+/TPrA system can be performed in one chip. We tested gradient pro-oxidation of MCF-7 by ascorbic acid and the synergistic effect of pro-oxidation on doxorubicin. The method achieves high analysis efficiency through a BPE array while simplifying the tedious procedures required by cell culture methods.
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Técnicas Biosensibles , Microfluídica , Mediciones Luminiscentes/métodos , Técnicas Biosensibles/métodos , Fotometría , ElectrodosRESUMEN
Plasmon resonance energy transfer (PRET), which occurs between plasmonic nanoparticles (NPs) and organic dyes, shows significant potential in sensing chemistry due to its high sensitivity at the single-particle level. In this work, a PRET-based sensing strategy was presented for the ultrasensitive sensing of nitric oxide (NO) in living cells. Supramolecular cyclodextrin (CD) molecules that exhibited different binding abilities to various molecules due to its unique rigid structure and annular cavity was applied and modified on gold NPs (GNPs) to construct the PRET nanosensors. NO-reactive molecules, rhodamine B-derived molecules (RdMs), were further inserted into the cavity of CD molecules through hydrophobic interactions to form host-guest structures. In the presence of NO, RdMs reacted with the target and generated rhodamine (RdB). Due to the spectral overlap between GNPs@CD and RdB molecules, PRET occurred and further led to a decrease in the scattering intensity of GNPs@CD, which was sensitive to the concentration of NO. The proposed sensing platform not only provides quantitative detection of NO in solution but also realized the single-particle imaging analysis of exogenous and endogenous NO in living cells. The single-particle plasmonic probes exhibit great potential in the in vivo sensing of biomolecules and metabolic processes.
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Nanopartículas , Óxido Nítrico , Transferencia de Energía , Supervivencia CelularRESUMEN
Photosynthesis, as the core of solar energy biotransformation, is driven by photosynthetic membrane protein complexes in plants and algae. Current methods for intracellular photosynthetic membrane protein complex analysis mostly require the separation of specific chloroplasts or the change of the intracellular environment, which causes the missing of real-time and on-site information. Thus, we explored a method for in vivo crosslinking and mapping of photosynthetic membrane protein complexes in the chloroplasts of living Chlamydomonas reinhardtii (C. reinhardtii) cells under cultural conditions. Poly(lactic-co-glycolic acid) (PLGA) and poly(lactic-co-glycolic acid)-poly(ethylene glycol) (PLGA-PEG) nanoparticles were fabricated to deliver bis(succinimidyl)propargyl with a nitro compound (BSPNO) into the chloroplasts to crosslink photosynthetic membrane protein complexes. After the in vivo crosslinked protein complexes were extracted and digested, mass spectrometry was employed to detect lysine-specific crosslinked peptides for further elucidating the protein conformations and interactions. With this method, the weak interactions between extrinsic proteins in the luminal side (PsbL and PsbH) and the core subunits (CP47 and CP43) in photosynthetic protein complexes were directly captured in living cells. Additionally, the previously uncharacterized protein (Cre07.g335700) was bound to the light-harvesting proteins, which was related to the biosynthesis of light-harvesting antennae. These results indicated that in vivo analysis of photosynthetic protein complexes based on crosslinker nanocarriers was expected to not only figure out the difficulty in the study of photosynthetic protein complexes in living cells but also provide an approach to explore transient and weak interactions and the function of uncharacterized proteins.
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Chlamydomonas reinhardtii , Proteínas del Complejo del Centro de Reacción Fotosintética , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Chlamydomonas reinhardtii/metabolismo , Proteínas de la Membrana/metabolismo , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Complejos de Proteína Captadores de Luz/metabolismo , CloroplastosRESUMEN
Chemical cross-linking coupled with mass spectrometry (XL-MS) is an important technique for the structural analysis of protein complexes where the coverage of amino acids and the identification of cross-linked sites are crucial. Photo-cross-linking has multisite reactivity and is valuable for the structural analysis of chemical cross-linking. However, a high degree of heterogeneity results from this multisite reactivity, which results in samples with higher complexity and lower abundance. Additionally, the applicability of photo-cross-linking is limited to purified protein complexes. In this work, we demonstrate a photo-cross-linker, alkynyl-succinimidyl-diazirine (ASD) with the reactive groups of N-hydroxysuccinimide ester and diazirine, as well as the click-enrichable alkyne group. Photo-cross-linkers can provide higher site reactivity for proteins that contain only a small number of lysine residues, thereby complementing the more commonly used lysine-targeting cross-linkers. By systematically analyzing proteins with differing lysine contents and differing flexibilities, we demonstrated clear enhancement in structure elucidation for proteins containing less lysine and with high flexibility. In addition, enrichment approaches of alkynyl-azide click chemistry conjugated with biotin-streptavidin purification (coinciding with parallel orthogonal digestion) improved the identification coverage of cross-links. We show that this photo-cross-linking approach can be used for membrane proteome-wide complex analysis. This method led to the identification of a total of 14066 lysine-X cross-linked site pairs from a total of 2784 proteins. Thus, this cross-linker is a valuable addition to a photo-cross-linking toolkit and improves the identification coverage of XL-MS in functional structure analysis.
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Diazometano , Lisina , Lisina/química , Aminoácidos/química , Espectrometría de Masas/métodos , Proteoma , Reactivos de Enlaces Cruzados/químicaRESUMEN
Meloxicam (Mel), as a powerful and effective anti-inflammatory drug, is commonly employed for the treatment of various inflammatory diseases; however, the use of Mel at high doses or for extended periods could cause severe side effects in human visceral organs. Therefore, a simple, rapid, and reliable method is urgently needed to monitor Mel in biological samples. Herein, novel water-soluble luminescent nano-carbon dots (nano-Cdots) with outstanding physicochemical properties were prepared by a one-pot high-temperature hydrothermal process of ellagic acid and guanidine. The nano-Cdots were further used as an optical probe for the sensitive detection of Mel in serum samples through the cooperative mechanisms of the inner filter effect and photoelectron transfer. By employing this sensor, an excellent linear correlation was achieved between the relative luminescent intensity [(PL0 - PL)/PL0] and the concentration of Mel in the range of 0.1 to 200 µM, with a limit of detection of 34.68 nM (3σ/k). This sensor was effectively employed for the analysis of Mel in real serum samples, implying its potential development prospects for the advancement of drug analysis with carbon-based probes.
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Puntos Cuánticos , Agua , Humanos , Meloxicam/uso terapéutico , Fluorometría , Agua/química , Espectrometría de Fluorescencia/métodos , Carbono/química , Puntos Cuánticos/química , Colorantes FluorescentesRESUMEN
Aqueous zinc-ion batteries (AZIBs) have been getting lots of attention in the field of large scale energy storage owing to their low cost, large capacity and excellent safety. However, Zn anodes have serious dendritic growth and corrosion hydrogen evolution issues, which hinder their further application. Herein, a simple drop-coating technique was used to build a thin sulfate poly(ether ketone) (SPEEK) solid-electrolyte interphase (SEI) on the surface of the Zn anode to address these issues. The sulfonated group (-SO3-ï¼ in SPEEK can provide rich coordination sites for Zn2+, controlling the uniform deposition of Zn2+. Therefore, the polymer SEI can block electrolytes and homogenize the Zn2+flux, resulting that the modified Zn (SPEEK@Zn) anode could effectively limit the formation of dendrites and side reactions. At a current density of 0.5 mA cm-2, SPEEK@Zn electrodes can maintain an ultra-long plating/stripping cycle life of 1000 h. Full batteries based on SPEEK@Zn have more superior cycle stability than the bare ones. This approach offers a straightforward and scalable remedy for high-performance Zn anode batteries.