RESUMEN
Over the past few decades, the increased application of nanomaterials has raised questions regarding their safety and possible toxic effects. Organoids have been suggested as promising tools, offering efficient assays for nanomaterial-induced toxicity evaluation. However, organoid systems have some limitations, such as size heterogeneity and poor penetration of nanoparticles because of the extracellular matrix, which is necessary for organoid culture. Here, we developed a novel system for the improved safety assessment of nanomaterials by establishing a 3D floating organoid paradigm. In addition to overcoming the limitations of two-dimensional systems including the lack of in vitro-in vivo cross-talk, our method provides multiple benefits as compared with conventional organoid systems that rely on an extracellular matrix for culture. Organoids cultured using our method exhibited relatively uniform sizing and structural integrity and were more conducive to the internalization of nanoparticles. Our floating culture system will accelerate the research and development of safe nanomaterials.
Asunto(s)
Nanoestructuras , Organoides , Matriz ExtracelularRESUMEN
Autophagy is a lysosome-mediated degradative process that removes damaged proteins and organelles, during which autophagosome-lysosome fusion is a key step of the autophagic flux. Based on our observation that intermediate cytofilament keratin 8 (KRT8) enhances autophagic clearance in cells under oxidative stress condition, we investigated whether KRT8 supports the cytoplasmic architectural networks to facilitate the vesicular fusion entailing trafficking onto filamentous tracks. We found that KRT8 interacts with actin filaments via the cytolinker, plectin (PLEC) during trafficking of autophagosome. When PLEC was knocked down or KRT8 structure was collapsed by phosphorylation, autophagosome-lysosome fusion was attenuated. Inhibition of actin polymerization resulted in accumulation of autophagosomes owing to a decrease in autophagosome and lysosome fusion. Furthermore, myosin motor protein was found to be responsible for vesicular trafficking along the actin filaments to entail autolysosome formation. Thus, the autophagosome-lysosome fusion is aided by PLEC-stabilized actin filaments as well as intermediate cytofilament KRT8 that supports the structural integrity of actin filaments during macroautophagic process under oxidative stress condition.
Asunto(s)
Actinas/metabolismo , Autofagosomas/metabolismo , Queratina-8/metabolismo , Lisosomas/metabolismo , Plectina/metabolismo , Línea Celular , Humanos , Fusión de Membrana , Mapas de Interacción de ProteínasRESUMEN
RNase E is a component of the RNA degradosome complex and plays a key role in RNA degradation and maturation in Escherichia coli RNase E-mediated target RNA degradation typically involves the RNA chaperone Hfq and requires small guide RNAs (sRNAs) acting as a seed by binding to short (7-12-bp) complementary regions in target RNA sequences. Here, using recombinantly expressed and purified proteins, site-directed mutagenesis, and RNA cleavage and protein cross-linking assays, we investigated Hfq-independent RNA decay by RNase E. Exploring its RNA substrate preferences in the absence of Hfq, we observed that RNase E preferentially cleaves AU-rich sites of single-stranded regions of RNA substrates that are annealed to an sRNA that contains a monophosphate at its 5'-end. We further found that the quaternary structure of RNase E is also important for complete, Hfq-independent cleavage at sites both proximal and distal to the sRNA-binding site within target RNAs containing monophosphorylated 5'-ends. Of note, genetic RNase E variants with unstable quaternary structure exhibited decreased catalytic activity. In summary, our results show that RNase E can degrade its target RNAs in the absence of the RNA chaperone Hfq. We conclude that RNase E-mediated, Hfq-independent RNA decay in E. coli requires a cognate sRNA sequence for annealing to the target RNA, a 5'-monophosphate at the RNA 5'-end, and a stable RNase E quaternary structure.
Asunto(s)
Endorribonucleasas/metabolismo , Estabilidad del ARN/fisiología , Sitios de Unión , Endorribonucleasas/fisiología , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/fisiología , Proteína de Factor 1 del Huésped/química , Proteína de Factor 1 del Huésped/metabolismo , Proteína de Factor 1 del Huésped/fisiología , Chaperonas Moleculares/metabolismo , Conformación de Ácido Nucleico , ARN Bacteriano/metabolismo , ARN Mensajero/genética , ARN Pequeño no Traducido/metabolismo , Ribonucleasa Pancreática , Ribonucleasas/metabolismoRESUMEN
Graphene oxide (GO) is known to strongly bind single-stranded nucleic acids with fluorescence quenching near the GO surface. However, GO exhibits weak biocompatibility characteristics, such as low dispersibility in cell culture media and significant cytotoxicity. To improve dispersibility in cell culture media and cell viability of GO, we prepared nanosized GO (nGO) constructs and modified the nGO surface using polyethylene glycol (PEG-nGO). Single-stranded peptide nucleic acid (PNA) was adsorbed onto the PEG-nGO and was readily desorbed by adding complementary RNA or under low pH conditions. PNA adsorbed on the PEG-nGO was efficiently delivered into lung cancer cells via endocytosis without affecting cell viability. Furthermore, antisense PNA delivered using PEG-nGO effectively downregulated the expression of the target gene in cancer cells. Our results suggest that PEG-nGO is a biocompatible carrier useful for PNA delivery into cells and serves as a promising gene delivery tool.
Asunto(s)
Materiales Biocompatibles/química , Portadores de Fármacos/química , Grafito/química , Nanoestructuras/química , Ácidos Nucleicos de Péptidos/administración & dosificación , Polietilenglicoles/química , Línea Celular Tumoral , Endocitosis , Humanos , Nanoestructuras/ultraestructura , Óxidos/química , Ácidos Nucleicos de Péptidos/farmacocinética , Propiedades de SuperficieRESUMEN
We have developed a facile fluorometric system for the detection of microRNA (miRNA), using rolling circle amplification (RCA), graphene oxide (GO), and fluorescently labeled peptide nucleic acid (F-PNA). The padlock probe DNA complementary to a target miRNA was selectively ligated to form circular DNA that was then used as the template for RCA. F-PNAs complementary to the target miRNA were annealed to multiple sites of the isothermally amplified single-stranded RCA product (RCAP) containing multiple target miRNA sequences. This F-PNA/RCAP duplex is less adsorbed onto the GO monolayer, thus attenuating the quenching of F-PNA fluorescence by GO. In the absence of target miRNA (and hence the absence of RCA and duplex formation), the free F-PNA is completely adsorbed onto the GO monolayer and fluorescence quenching ensues. Thus, GO-based fluorescence detection coupled with isothermal gene amplification would be a simple and convenient method for the quantitative detection of miRNA.
Asunto(s)
Fluorometría/métodos , Amplificación de Genes , Grafito/química , MicroARNs/química , Óxidos/químicaRESUMEN
Intravitreal injection of biodegradable implant drug carriers shows promise in reducing the injection frequency for neovascular retinal diseases. However, current intravitreal ocular devices have limitations in adjusting drug release rates for individual patients, thereby affecting treatment effectiveness. Accordingly, we developed mesoporous silica nanoparticles (MSNs) featuring a surface that reverse its charge in response to reactive oxygen species (ROS) for efficient delivery of humanin peptide (HN) to retinal epithelial cells (ARPE-19). The MSN core, designed with a pore size of 2.8 nm, ensures a high HN loading capacity 64.4% (w/w). We fine-tuned the external surface of the MSNs by incorporating 20% Acetyl-L-arginine (Ar) to create a partial positive charge, while 80% conjugated thioketal (TK) methoxy polyethylene glycol (mPEG) act as ROS gatekeeper. Ex vivo experiments using bovine eyes revealed the immobilization of Ar-MSNs-TK-PEG (mean zeta potential: 2 mV) in the negatively charged vitreous. However, oxidative stress reversed the surface charge to -25 mV by mPEG loss, facilitating the diffusion of the nanoparticles impeded with HN. In vitro studies showed that ARPE-19 cells effectively internalize HN-loaded Ar-MSNs-TK, subsequently releasing the peptide, which offered protection against oxidative stress-induced apoptosis, as evidenced by reduced TUNEL and caspase3 activation. The inhibition of retinal neovascularization was further validated in an in vivo oxygen-induced retinopathy (OIR) mouse model.
RESUMEN
Dysregulation of inflammation induced by noninfectious stress conditions, such as nutrient deprivation, causes tissue damage and intestinal permeability, resulting in the development of inflammatory bowel diseases. We studied the effect of autophagy on cytokine secretion related to intestinal permeability under nutrient deprivation. Autophagy removes NLRP3 inflammasomes via ubiquitin-mediated degradation under starvation. When autophagy was inhibited, starvation-induced NLRP3 inflammasomes and their product, IL-1ß, were significantly enhanced. A prolonged nutrient deprivation resulted in an increased epithelial mesenchymal transition (EMT), leading to intestinal permeability. Under nutrient deprivation, IL-17E/25, which is secreted by IL-1ß, demolished the intestinal epithelial barrier. Our results suggest that an upregulation of autophagy maintains the intestinal barrier by suppressing the activation of NLRP3 inflammasomes and the release of their products, including proinflammatory cytokines IL-1ß and IL-17E/25, under nutrient deprivation. [BMB Reports 2021; 54(5): 260-265].
Asunto(s)
Autofagia , Regulación hacia Abajo , Células Epiteliales/metabolismo , Inflamación/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Nutrientes/metabolismo , Células CACO-2 , HumanosRESUMEN
Dysregulation of mitochondrial homeostasis and accumulation of damaged mitochondria cause degenerative diseases such as age-related macular degeneration (AMD). We studied the effects of the intermediate cytofilament KRT8 (keratin 8) on mitochondrial homeostasis in relation to the morphology and function of mitochondria in retinal pigment epithelial cells under oxidative stress. When the mitochondria were damaged owing to oxidative stress, the damaged mitochondria were readily disposed of via mitophagy following mitochondrial fission. During this process, KRT8 was found to physically interact with the mitochondria through PLEC (plectin) and facilitate the mitochondrial fission-mediated mitophagy. However, the association between PLEC-anchoring mitochondria and KRT8 was dwindled by KRT8 phosphorylation under oxidative stress. The efficient KRT8-facilitated mitophagy flux suppressed the accumulation of damaged mitochondria and consequently diminished necrotic cell death under oxidative stress. Thus, by facilitating mitophagy, KRT8 protects RPE cells against necrotic cell death due to oxidative stress.Abbreviations: 3-MA: 3-methyladenine; AMD: age-related macular degeneration; DDIT3: DNA damage inducible transcript 3; DNM1L: dynamin 1 like; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GCN1: GCN1 activator of EIF2AK4; IF: intermediate filament; KRT8: keratin 8; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3; MFF: mitochondrial fission factor; MMP: mitochondrial membrane potential; OCR: oxygen consumption rate; PLEC: plectin; ROS: reactive oxygen species; RPE: retinal pigment epithelium; SQSTM1/p62: sequestosome 1; TOMM20: translocase of outer mitochondrial membrane 20.
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Dinámicas Mitocondriales , Mitofagia , Autofagia , Muerte Celular , Queratina-8/metabolismo , Mitofagia/genética , Plectina , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The surface-enhanced Raman scattering (SERS) technique, that uses magnetic plasmonic particles (MPPs), is an advanced SERS detection platform owing to the synergetic effects of the particles' magnetic and plasmonic properties. As well as being an ultrasensitive and reliable SERS material, MPPs perform various functions, such as aiding in separation, drug delivery, and acting as a therapeutic material. This literature discusses the structure and multifunctionality of MPPs, which has enabled the novel application of MPPs to various biological fields.
RESUMEN
The immunoproteasome (iP), an inducible proteasome variant harboring three immunosubunits, low molecular mass polypeptide-2 (LMP2), multicatalytic endopeptidase complex subunit-1, and low molecular mass polypeptide-7 (LMP7), is involved in multiple facets of inflammatory responses. We recently reported that YU102, a dual inhibitor of the iP subunit LMP2 and the constitutive proteasome catalytic subunit ß1, ameliorates cognitive impairments in mouse models of Alzheimer's disease (AD) independently of amyloid deposits. To investigate whether inhibition of LMP2 is sufficient to improve the cognitive functions of AD mice, here we prepared 37 YU102 analogues and identified a potent LMP2 inhibitor DB-310 (28) (IC50: 80.6 nM) with improved selectivity and permeability in cells overexpressing ABCB1 transporters. We show that DB-310 induces suppression of IL-1α production in microglia cells and improves cognitive functions in the Tg2576 transgenic mouse model of AD. This study supports that inhibition of LMP2 is a promising therapeutic strategy for treatment of AD.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/uso terapéutico , Nootrópicos/uso terapéutico , Oligopéptidos/uso terapéutico , Animales , Línea Celular Transformada , Inhibidores de Cisteína Proteinasa/síntesis química , Inhibidores de Cisteína Proteinasa/toxicidad , Transición Epitelial-Mesenquimal/efectos de los fármacos , Humanos , Interleucina-1alfa/metabolismo , Ratones Transgénicos , Microglía/efectos de los fármacos , Estructura Molecular , Nootrópicos/síntesis química , Nootrópicos/toxicidad , Oligopéptidos/síntesis química , Oligopéptidos/toxicidad , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Bibliotecas de Moléculas Pequeñas/toxicidad , Relación Estructura-ActividadRESUMEN
The Alu element, the most abundant transposable element, is transcribed to Alu RNA. We hypothesized that the PIWI protein regulates the expression of Alu RNA in retinal pigment epithelial (RPE) cells, where accumulated Alu RNA leads to macular degeneration. Alu transcription was induced in RPE cells treated with H2O2. At an early stage of oxidative stress, PIWIL4 was translocated into the nucleus; however, subsequently it was sequestered into cytoplasmic stress granules, resulting in the accumulation of Alu RNA. An elevated amount of Alu RNA was positively correlated with the disruption of the epithelial features of RPE via induction of mesenchymal transition. Therefore, we suggest that oxidative stress causes Alu RNA accumulation via PIWIL4 sequestration into the cytoplasmic stress granules. [BMB Reports 2019; 52(3): 196-201].
Asunto(s)
Elementos Alu/genética , Proteínas Argonautas/fisiología , Proteínas Argonautas/metabolismo , Línea Celular , Regulación de la Expresión Génica/genética , Humanos , Peróxido de Hidrógeno/metabolismo , Degeneración Macular , Estrés Oxidativo/genética , Estrés Oxidativo/fisiología , ARN/metabolismo , Proteínas de Unión al ARN , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/fisiologíaRESUMEN
The immunoproteasome (iP) is a variant of the constitutive proteasome (cP) that is abundantly expressed in immune cells which can also be induced in somatic cells by cytokines such as TNF-α or IFN-γ. Accumulating evidence support that the iP is closely linked to multiple facets of inflammatory response, eventually leading to the development of several iP inhibitors as potential therapeutic agents for autoimmune diseases. Recent studies also found that the iP is upregulated in reactive glial cells surrounding amyloid ß (Aß) deposits in brains of Alzheimer's disease (AD) patients, but the role it plays in the pathogenesis of AD remains unclear. In this study, we investigated the effects of several proteasome inhibitors on cognitive function in AD mouse models and found that YU102, a dual inhibitor of the iP catalytic subunit LMP2 and the cP catalytic subunit Y, ameliorates cognitive impairments in AD mouse models without affecting Aß deposition. The data obtained from our investigation revealed that YU102 suppresses the secretion of inflammatory cytokines from microglial cells. Overall, this study indicates that there may exist a potential link between LMP2/Y and microglia-mediated neuroinflammation and that inhibition of these subunits may offer a new therapeutic strategy for AD.
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Enfermedad de Alzheimer/tratamiento farmacológico , Encéfalo/efectos de los fármacos , Disfunción Cognitiva/tratamiento farmacológico , Cisteína Endopeptidasas/genética , Neuroglía/efectos de los fármacos , Inhibidores de Proteasoma/farmacología , Enfermedad de Alzheimer/enzimología , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/metabolismo , Animales , Encéfalo/enzimología , Encéfalo/patología , Línea Celular , Disfunción Cognitiva/enzimología , Disfunción Cognitiva/genética , Disfunción Cognitiva/patología , Cisteína Endopeptidasas/metabolismo , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Humanos , Interleucina-1alfa/genética , Interleucina-1alfa/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Hígado/efectos de los fármacos , Hígado/enzimología , Hígado/patología , Aprendizaje por Laberinto/efectos de los fármacos , Ratones , Ratones Endogámicos ICR , Ratones Transgénicos , Proteínas Quimioatrayentes de Monocitos/genética , Proteínas Quimioatrayentes de Monocitos/metabolismo , Neuroglía/enzimología , Neuroglía/patología , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Bazo/efectos de los fármacos , Bazo/enzimología , Bazo/patologíaRESUMEN
Contribution of autophagy and regulation of related proteins to the degeneration of retinal pigment epithelium (RPE) in age-related macular degeneration (AMD) remain unknown. We report that upregulation of KRT8 (keratin 8) as well as its phosphorylation are accompanied with autophagy and attenuated with the inhibition of autophagy in RPE cells under oxidative stress. KRT8 appears to have a dual role in RPE pathophysiology. While increased expression of KRT8 following autophagy provides a cytoprotective role in RPE, phosphorylation of KRT8 induces pathologic epithelial-mesenchymal transition (EMT) of RPE cells under oxidative stress, which is mediated by MAPK1/ERK2 (mitogen-activated protein kinase 1) and MAPK3/ERK1. Inhibition of autophagy further promotes EMT, which can be reversed by inhibition of MAPK. Thus, regulated enhancement of autophagy with concurrent increased expression of KRT8 and the inhibition of KRT8 phosphorylation serve to inhibit oxidative stress-induced EMT of RPE cells as well as to prevent cell death, suggesting that pharmacological manipulation of KRT8 upregulation through autophagy with combined inhibition of the MAPK1/3 pathway may be attractive therapeutic strategies for the treatment of AMD.
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Autofagia , Queratina-8/metabolismo , Estrés Oxidativo , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patología , Apoptosis , Autofagosomas/metabolismo , Autofagosomas/ultraestructura , Línea Celular , Movimiento Celular , Núcleo Celular/metabolismo , Citoprotección , Transición Epitelial-Mesenquimal , Humanos , Lisosomas/metabolismo , Lisosomas/ultraestructura , Fusión de Membrana , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Modelos Biológicos , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Epitelio Pigmentado de la Retina/ultraestructura , Transducción de Señal , Superóxidos/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Regulación hacia ArribaRESUMEN
A magnetic material that consists of silica-coated magnetic beads conjugated with graphene oxide (GO) was successfully prepared for facile ribonucleic acid (RNA) extraction. When the GO-modified magnetic beads were applied to separate the RNA from the lysed cell, the cellular RNAs were readily adsorbed to and readily desorbed from the surface of the GO-modified magnetic beads by urea. The amount of RNA extracted by the GO-modified magnetic beads was ≈170 % as much as those of the control extracted by a conventional phenol-based chaotropic solution. These results demonstrate that the facile method of RNA separation by using GO-modified magnetic beads as an adsorbent is an efficient and simple way to purify intact cellular RNAs and/or microRNA from cell lysates.
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Técnicas de Química Analítica/métodos , Grafito/química , Magnetismo , ARN/aislamiento & purificación , Electroforesis en Gel Bidimensional , Microscopía Electrónica de Rastreo , Nanopartículas/química , ARN/química , Factores de TiempoRESUMEN
Polymerase chain reaction (PCR), a versatile DNA amplification method, is a fundamental technology in modern life sciences and molecular diagnostics. After multiple rounds of PCR, however, nonspecific DNA fragments are often produced and the amplification efficiency and fidelity decrease. Here, we demonstrated that poly(ethylene glycol)-engrafted nanosized graphene oxide (PEG-nGO) can significantly improve the PCR specificity and efficiency. PEG-nGO allows the specificity to be maintained even after multiple rounds of PCR, allowing reliable amplification at low annealing temperatures. PEG-nGO decreases the nonspecific annealing of single-stranded DNA (ssDNA), such as primer dimerization and false priming, by adsorbing excess primers. Moreover, PEG-nGO interrupts the reannealing of denatured template DNA by preferentially binding to ssDNA. Thus, PEG-nGO enhances the PCR specificity by preferentially binding to ssDNA without inhibiting DNA polymerase, which is analogous to the role of ssDNA binding proteins.
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Grafito/química , Cartilla de ADN , Proteínas de Unión al ADN , Óxidos , Polietilenglicoles , Reacción en Cadena de la PolimerasaRESUMEN
We report a simple fluorometric method for detection of single-nucleotide changes in RNA using graphene oxide (GO) and RNA-cleaving DNAzyme. The fluorescent DNA probe (F-DNA) was annealed to RNA fragments generated by RNA cleavage with DNAzyme specific to mutant RNA. The F-DNA-RNA duplex attenuated the quenching of F-DNA fluorescence by GO.
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ADN Catalítico/química , ADN Catalítico/metabolismo , Fluorescencia , Grafito/química , Óxidos/química , ARN/química , Colorantes Fluorescentes/análisis , Colorantes Fluorescentes/química , Fluorometría , Mutación , ARN/genética , ARN/metabolismoRESUMEN
We report a simple, direct fluorometric assay based on graphene oxide (GO) for RNA polymerase-mediated RNA synthesis. In principle, fluorescent peptide nucleic acid (PNA) probes were designed, and annealed with RNA products and the resultant RNA-PNA hybrids induced the recovery of fluorescence intensity of the PNA probes adsorbed onto the GO surface.