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1.
J Environ Pathol Toxicol Oncol ; 40(2): 55-63, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33822517

RESUMEN

BACKGROUND: SET domain-containing protein 5 (SETD5) could promote non-small cell lung cancer (NS-CLC) cell invasion, but the effect of SETD5 on NSCLC cell stemness characteristics is unknown. Thus we attempted to evaluate the effect of SETD5 on NSCLC stemness and its mechanism. METHODS: The expressions of SETD5 and stemness-related genes (SOX2, OCT4, ABCG2) were detected in NSCLC tissues by immunohistochemistry assay, qRT-PCR, and western blot. A SETD5 knockdown cell model was constructed by siRNA transfection in A549 and H1299 cells. A CCK8 assay was used to examine cell viability. A sphere-forming assay and side population cell assay were conducted to measure the cancer cell stem properties. The cells with SETD5 deletion were treated with an activator of AKT, SC79, and the protein expressions of Akt, p-Akt, mTOR, and p-mTOR were assessed. RESULTS: SETD5 and cancer stem-related genes SOX2, OCT4, and ABCG2 were co-expressed and co-localized in tumor tissues and cell lines of NSCLC. The deletion of SETD5 significantly reduced the cell viability, cancer stem properties, and activity of the PI3K/Akt/mTOR pathway, while the decreased SETD5-induced effects were partially restored with SC79 treatment. CONCLUSION: In this study, SETD5 promoted the cancer stem cell property of NSCLC through mitigating the activation of the PI3K/Akt/mTOR pathway, suggesting a candidate target role for SETD5 in NSCLC treatment.


Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/genética , Neoplasias Pulmonares/genética , Metiltransferasas/genética , Fosfatidilinositol 3-Quinasas , Proteínas Proto-Oncogénicas c-akt , Serina-Treonina Quinasas TOR , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/genética , Línea Celular , Humanos , Proteínas de Neoplasias/genética , Células Madre Neoplásicas , Factor 3 de Transcripción de Unión a Octámeros/genética , Factores de Transcripción SOXB1/genética , Transducción de Señal
2.
J Environ Pathol Toxicol Oncol ; 40(2): 81-87, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33822519

RESUMEN

Gastric cancer (GC) is the third leading cause of cancer-related deaths in the world. Tumor metastasis is considered one of the main factors for GC development. Nup62 is a member of the nuclear pore complex (NPC). It bridges the nuclear envelope, is important in nucleocytoplasmic exchange, and is associated with cancer. This study aimed to investigate the role of Nup62 in GC metastasis. The relationship between the expression level of Nup62 in GC and patient survival was evaluated using Kaplan-Meier analysis. Then Nup62 expression in GC tissues and matched normal gastric tissues was analyzed by immunohistochemistry and that in cell lines by Western blot analysis. Furthermore, clonogenic and Transwell migration assays were performed, and the expression of epithelial-mesenchymal transition (EMT) proteins was detected to determine the metastatic functional roles of Nup62 in GC. Compared with the adjacent normal tissues, Nup62 was found to be upregulated in GC tissues using software prediction and detecting clinical specimens and cell lines. Moreover, the downregulation of Nup62 suppressed colony formation and decreased the number of migrated cells. In contrast, Nup62 overexpression promoted colony formation and increased the number of migrated cells. Further functional studies showed that the abnormal expression of Nup62 influenced cell migration and EMT through wingless/ß-catenin (Wnt/ß-catenin) and transforming growth factor (TGF)-ß signaling pathways. In summary, the findings indicate that Nup62 regulates cell migration by interfering with Wnt/ß-catenin and TGF-ß signaling pathways in GC.


Asunto(s)
Glicoproteínas de Membrana/genética , Proteínas de Complejo Poro Nuclear/genética , Neoplasias Gástricas/patología , Factor de Crecimiento Transformador beta/metabolismo , Vía de Señalización Wnt , Línea Celular , Movimiento Celular , Transición Epitelial-Mesenquimal , Humanos , Estimación de Kaplan-Meier , Glicoproteínas de Membrana/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/mortalidad
3.
Sci Total Environ ; 764: 142902, 2021 Apr 10.
Artículo en Inglés | MEDLINE | ID: mdl-33757253

RESUMEN

To avoid potential risks of biofuels on the environment and human, ecotoxicity investigation should be integrated into the early design stage for promising biofuel candidates. In the present study, a green toxicology testing strategy combining experimental bioassays with in silico tools was established to investigate the potential ecotoxicity of biofuel candidates. Experimental results obtained from the acute immobilisation test, the fish embryo acute toxicity test and the in vitro micronucleus assay (Chinese hamster lung fibroblast cell line V79) were compared with model prediction results by ECOSAR and OECD QSAR Toolbox. Both our experimental and model prediction results showed that 1-Octanol (1-Oct) and Di-n-butyl ether (DNBE) were the most toxic to Daphnia magna and zebrafish among all the biofuel candidates we investigated, while Methyl ethyl ketone (MEK), Dimethoxymethane (DMM) and Diethoxymethane (DEM) were the least toxic. Moreover, both in vitro micronucleus assay and OECD QSAR Toolbox evaluation suggested that the metabolites present higher genotoxicity than biofuel candidates themselves. Overall, our results proved that this green toxicology testing strategy is a useful tool for assessing ecotoxicity of biofuel candidates.


Asunto(s)
Biocombustibles , Contaminantes Químicos del Agua , Animales , Biocombustibles/toxicidad , Línea Celular , Cricetinae , Daphnia , Humanos , Pruebas de Toxicidad Aguda , Pez Cebra
4.
Genome Med ; 13(1): 43, 2021 03 15.
Artículo en Inglés | MEDLINE | ID: mdl-33722288

RESUMEN

BACKGROUND: ChAdOx1 nCoV-19 is a recombinant adenovirus vaccine against SARS-CoV-2 that has passed phase III clinical trials and is now in use across the globe. Although replication-defective in normal cells, 28 kbp of adenovirus genes is delivered to the cell nucleus alongside the SARS-CoV-2 S glycoprotein gene. METHODS: We used direct RNA sequencing to analyse transcript expression from the ChAdOx1 nCoV-19 genome in human MRC-5 and A549 cell lines that are non-permissive for vector replication alongside the replication permissive cell line, HEK293. In addition, we used quantitative proteomics to study over time the proteome and phosphoproteome of A549 and MRC5 cells infected with the ChAdOx1 nCoV-19 vaccine. RESULTS: The expected SARS-CoV-2 S coding transcript dominated in all cell lines. We also detected rare S transcripts with aberrant splice patterns or polyadenylation site usage. Adenovirus vector transcripts were almost absent in MRC-5 cells, but in A549 cells, there was a broader repertoire of adenoviral gene expression at very low levels. Proteomically, in addition to S glycoprotein, we detected multiple adenovirus proteins in A549 cells compared to just one in MRC5 cells. CONCLUSIONS: Overall, the ChAdOx1 nCoV-19 vaccine's transcriptomic and proteomic repertoire in cell culture is as expected. The combined transcriptomic and proteomics approaches provide a detailed insight into the behaviour of this important class of vaccine using state-of-the-art techniques and illustrate the potential of this technique to inform future viral vaccine vector design.


Asunto(s)
/efectos adversos , /inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , /metabolismo , /virología , Línea Celular , Células Cultivadas , Expresión Génica , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Humanos , Poliadenilación , Proteómica/métodos , ARN Mensajero , ARN Viral , Glicoproteína de la Espiga del Coronavirus/genética , Transcripción Genética
5.
Sci Rep ; 11(1): 5890, 2021 03 15.
Artículo en Inglés | MEDLINE | ID: mdl-33723270

RESUMEN

To circumvent time-consuming clinical trials, testing whether existing drugs are effective inhibitors of SARS-CoV-2, has led to the discovery of Remdesivir. We decided to follow this path and screened approved medications "off-label" against SARS-CoV-2. Fluoxetine inhibited SARS-CoV-2 at a concentration of 0.8 µg/ml significantly in these screenings, and the EC50 was determined with 387 ng/ml. Furthermore, Fluoxetine reduced viral infectivity in precision-cut human lung slices showing its activity in relevant human tissue targeted in severe infections. Fluoxetine treatment resulted in a decrease in viral protein expression. Fluoxetine is a racemate consisting of both stereoisomers, while the S-form is the dominant serotonin reuptake inhibitor. We found that both isomers show similar activity on the virus, indicating that the R-form might specifically be used for SARS-CoV-2 treatment. Fluoxetine inhibited neither Rabies virus, human respiratory syncytial virus replication nor the Human Herpesvirus 8 or Herpes simplex virus type 1 gene expression, indicating that it acts virus-specific. Moreover, since it is known that Fluoxetine inhibits cytokine release, we see the role of Fluoxetine in the treatment of SARS-CoV-2 infected patients of risk groups.


Asunto(s)
Antivirales/farmacología , Fluoxetina/farmacología , Pulmón/efectos de los fármacos , Pulmón/virología , Inhibidores de la Captación de Serotonina/farmacología , Animales , Antivirales/uso terapéutico , Línea Celular , Células Cultivadas , Fluoxetina/uso terapéutico , Humanos , Pulmón/patología , Inhibidores de la Captación de Serotonina/uso terapéutico , Replicación Viral/efectos de los fármacos
6.
FASEB J ; 35(4): e21360, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33749932

RESUMEN

The novel coronavirus disease, COVID-19, has grown into a global pandemic and a major public health threat since its breakout in December 2019. To date, no specific therapeutic drug or vaccine for treating COVID-19 and SARS has been FDA approved. Previous studies suggest that berberine, an isoquinoline alkaloid, has shown various biological activities that may help against COVID-19 and SARS, including antiviral, anti-allergy and inflammation, hepatoprotection against drug- and infection-induced liver injury, as well as reducing oxidative stress. In particular, berberine has a wide range of antiviral activities such as anti-influenza, anti-hepatitis C, anti-cytomegalovirus, and anti-alphavirus. As an ingredient recommended in guidelines issued by the China National Health Commission for COVID-19 to be combined with other therapy, berberine is a promising orally administered therapeutic candidate against SARS-CoV and SARS-CoV-2. The current study comprehensively evaluates the potential therapeutic mechanisms of berberine in preventing and treating COVID-19 and SARS using computational modeling, including target mining, gene ontology enrichment, pathway analyses, protein-protein interaction analysis, and in silico molecular docking. An orally available immunotherapeutic-berberine nanomedicine, named NIT-X, has been developed by our group and has shown significantly increased oral bioavailability of berberine, increased IFN-γ production by CD8+ T cells, and inhibition of mast cell histamine release in vivo, suggesting a protective immune response. We further validated the inhibition of replication of SARS-CoV-2 in lung epithelial cells line in vitro (Calu3 cells) by berberine. Moreover, the expression of targets including ACE2, TMPRSS2, IL-1α, IL-8, IL-6, and CCL-2 in SARS-CoV-2 infected Calu3 cells were significantly suppressed by NIT-X. By supporting protective immunity while inhibiting pro-inflammatory cytokines; inhibiting viral infection and replication; inducing apoptosis; and protecting against tissue damage, berberine is a promising candidate in preventing and treating COVID-19 and SARS. Given the high oral bioavailability and safety of berberine nanomedicine, the current study may lead to the development of berberine as an orally, active therapeutic against COVID-19 and SARS.


Asunto(s)
Antivirales/farmacología , Berberina/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Modelos Biológicos , Virus del SRAS/metabolismo , Síndrome Respiratorio Agudo Grave , Administración Oral , /metabolismo , Línea Celular , Simulación por Computador , Humanos , Pandemias , Síndrome Respiratorio Agudo Grave/tratamiento farmacológico , Síndrome Respiratorio Agudo Grave/metabolismo
7.
Nat Commun ; 12(1): 1659, 2021 03 12.
Artículo en Inglés | MEDLINE | ID: mdl-33712564

RESUMEN

Human cell conversion technology has become an important tool for devising new cell transplantation therapies, generating disease models and testing gene therapies. However, while transcription factor over-expression-based methods have shown great promise in generating cell types in vitro, they often endure low conversion efficiency. In this context, great effort has been devoted to increasing the efficiency of current protocols and the development of computational approaches can be of great help in this endeavor. Here we introduce a computer-guided design tool that combines a computational framework for prioritizing more efficient combinations of instructive factors (IFs) of cellular conversions, called IRENE, with a transposon-based genomic integration system for efficient delivery. Particularly, IRENE relies on a stochastic gene regulatory network model that systematically prioritizes more efficient IFs by maximizing the agreement of the transcriptional and epigenetic landscapes between the converted and target cells. Our predictions substantially increased the efficiency of two established iPSC-differentiation protocols (natural killer cells and melanocytes) and established the first protocol for iPSC-derived mammary epithelial cells with high efficiency.


Asunto(s)
Diferenciación Celular/genética , Reprogramación Celular , Biología Computacional/métodos , Redes Reguladoras de Genes , Línea Celular , Células Epiteliales/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo
8.
Nat Commun ; 12(1): 1611, 2021 03 12.
Artículo en Inglés | MEDLINE | ID: mdl-33712590

RESUMEN

Genome-wide association studies of Systemic Lupus Erythematosus (SLE) nominate 3073 genetic variants at 91 risk loci. To systematically screen these variants for allelic transcriptional enhancer activity, we construct a massively parallel reporter assay (MPRA) library comprising 12,396 DNA oligonucleotides containing the genomic context around every allele of each SLE variant. Transfection into the Epstein-Barr virus-transformed B cell line GM12878 reveals 482 variants with enhancer activity, with 51 variants showing genotype-dependent (allelic) enhancer activity at 27 risk loci. Comparison of MPRA results in GM12878 and Jurkat T cell lines highlights shared and unique allelic transcriptional regulatory mechanisms at SLE risk loci. In-depth analysis of allelic transcription factor (TF) binding at and around allelic variants identifies one class of TFs whose DNA-binding motif tends to be directly altered by the risk variant and a second class of TFs that bind allelically without direct alteration of their motif by the variant. Collectively, our approach provides a blueprint for the discovery of allelic gene regulation at risk loci for any disease and offers insight into the transcriptional regulatory mechanisms underlying SLE.


Asunto(s)
Alelos , Predisposición Genética a la Enfermedad/genética , Lupus Eritematoso Sistémico/genética , Linfocitos B , Línea Celular , Cromatina , Regulación de la Expresión Génica , Estudio de Asociación del Genoma Completo , Genotipo , Herpesvirus Humano 4 , Humanos , Sitios de Carácter Cuantitativo , Sinaptogirinas/genética , Linfocitos T
9.
Nat Commun ; 12(1): 1624, 2021 03 12.
Artículo en Inglés | MEDLINE | ID: mdl-33712610

RESUMEN

Adult Schwann cells (SCs) possess an inherent plastic potential. This plasticity allows SCs to acquire repair-specific functions essential for peripheral nerve regeneration. Here, we investigate whether stromal SCs in benign-behaving peripheral neuroblastic tumors adopt a similar cellular state. We profile ganglioneuromas and neuroblastomas, rich and poor in SC stroma, respectively, and peripheral nerves after injury, rich in repair SCs. Indeed, stromal SCs in ganglioneuromas and repair SCs share the expression of nerve repair-associated genes. Neuroblastoma cells, derived from aggressive tumors, respond to primary repair-related SCs and their secretome with increased neuronal differentiation and reduced proliferation. Within the pool of secreted stromal and repair SC factors, we identify EGFL8, a matricellular protein with so far undescribed function, to act as neuritogen and to rewire cellular signaling by activating kinases involved in neurogenesis. In summary, we report that human SCs undergo a similar adaptive response in two patho-physiologically distinct situations, peripheral nerve injury and tumor development.


Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Diferenciación Celular/fisiología , Familia de Proteínas EGF/genética , Familia de Proteínas EGF/metabolismo , Neurogénesis/fisiología , Células de Schwann/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Proteínas de Unión al Calcio/genética , Línea Celular , Plasticidad de la Célula/fisiología , Proliferación Celular , Técnicas de Cocultivo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Regeneración Nerviosa , Neuroblastoma/patología , Neurogénesis/genética , Traumatismos de los Nervios Periféricos , Transcriptoma , Adulto Joven
10.
Nat Commun ; 12(1): 1626, 2021 03 12.
Artículo en Inglés | MEDLINE | ID: mdl-33712616

RESUMEN

Minichromosome maintenance protein 10 (MCM10) is essential for eukaryotic DNA replication. Here, we describe compound heterozygous MCM10 variants in patients with distinctive, but overlapping, clinical phenotypes: natural killer (NK) cell deficiency (NKD) and restrictive cardiomyopathy (RCM) with hypoplasia of the spleen and thymus. To understand the mechanism of MCM10-associated disease, we modeled these variants in human cell lines. MCM10 deficiency causes chronic replication stress that reduces cell viability due to increased genomic instability and telomere erosion. Our data suggest that loss of MCM10 function constrains telomerase activity by accumulating abnormal replication fork structures enriched with single-stranded DNA. Terminally-arrested replication forks in MCM10-deficient cells require endonucleolytic processing by MUS81, as MCM10:MUS81 double mutants display decreased viability and accelerated telomere shortening. We propose that these bi-allelic variants in MCM10 predispose specific cardiac and immune cell lineages to prematurely arrest during differentiation, causing the clinical phenotypes observed in both NKD and RCM patients.


Asunto(s)
Alelos , Cardiomiopatías/genética , Proteínas de Mantenimiento de Minicromosoma/genética , Proteínas de Mantenimiento de Minicromosoma/inmunología , Acortamiento del Telómero , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Replicación del ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endonucleasas/genética , Endonucleasas/metabolismo , Humanos , Células Asesinas Naturales
11.
mBio ; 12(2)2021 03 16.
Artículo en Inglés | MEDLINE | ID: mdl-33727353

RESUMEN

The angiotensin-converting enzyme 2 (ACE2) receptor is a major severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) host range determinant, and understanding SARS-CoV-2-ACE2 interactions will provide important insights into COVID-19 pathogenesis and animal model development. SARS-CoV-2 cannot infect mice due to incompatibility between its receptor binding domain and the murine ACE2 receptor. Through molecular modeling and empirical in vitro validation, we identified 5 key amino acid differences between murine and human ACE2 that mediate SARS-CoV-2 infection, generating a chimeric humanized murine ACE2. Additionally, we examined the ability of the humanized murine ACE2 receptor to permit infection by an additional preemergent group 2B coronavirus, WIV-1, providing evidence for the potential pan-virus capabilities of this chimeric receptor. Finally, we predicted the ability of these determinants to inform host range identification of preemergent coronaviruses by evaluating hot spot contacts between SARS-CoV-2 and additional potential host receptors. Our results identify residue determinants that mediate coronavirus receptor usage and host range for application in SARS-CoV-2 and emerging coronavirus animal model development.IMPORTANCE SARS-CoV-2 (the causative agent of COVID-19) is a major public health threat and one of two related coronaviruses that have caused epidemics in modern history. A method of screening potential infectible hosts for preemergent and future emergent coronaviruses would aid in mounting rapid response and intervention strategies during future emergence events. Here, we evaluated determinants of SARS-CoV-2 receptor interactions, identifying key changes that enable or prevent infection. The analysis detailed in this study will aid in the development of model systems to screen emergent coronaviruses as well as treatments to counteract infections.


Asunto(s)
/química , Betacoronavirus/fisiología , Replicación Viral , Secuencia de Aminoácidos , Animales , Betacoronavirus/metabolismo , Sitios de Unión , Línea Celular , Infecciones por Coronavirus/virología , Especificidad del Huésped , Humanos , Ratones , Modelos Moleculares , Mutación , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , /fisiología , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismo
12.
Nat Commun ; 12(1): 1689, 2021 03 16.
Artículo en Inglés | MEDLINE | ID: mdl-33727548

RESUMEN

Administration of drugs via the buccal route has attracted much attention in recent years. However, developing systems with satisfactory adhesion under wet conditions and adequate drug bioavailability still remains a challenge. Here, we propose a mussel-inspired mucoadhesive film. Ex vivo models show that this film can achieve strong adhesion to wet buccal tissues (up to 38.72 ± 10.94 kPa). We also demonstrate that the adhesion mechanism of this film relies on both physical association and covalent bonding between the film and mucus. Additionally, the film with incorporated polydopamine nanoparticles shows superior advantages for transport across the mucosal barrier, with improved drug bioavailability (~3.5-fold greater than observed with oral delivery) and therapeutic efficacy in oral mucositis models (~6.0-fold improvement in wound closure at day 5 compared with that observed with no treatment). We anticipate that this platform might aid the development of tissue adhesives and inspire the design of nanoparticle-based buccal delivery systems.


Asunto(s)
Biomimética , Bivalvos/química , Sistemas de Liberación de Medicamentos , Mucosa Bucal/fisiología , Adhesividad , Administración Bucal , Animales , Línea Celular , Dexametasona/farmacología , Dihidroxifenilalanina/química , Liberación de Fármacos , Humanos , Indoles/toxicidad , Masculino , Mucinas/química , Moco/química , Nanopartículas/química , Nanopartículas/ultraestructura , Tamaño de la Partícula , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/toxicidad , Polímeros/toxicidad , Alcohol Polivinílico/química , Alcohol Polivinílico/toxicidad , Ratas Sprague-Dawley , Espectrofotometría Ultravioleta , Porcinos , Distribución Tisular
13.
Cell Rep ; 34(11): 108872, 2021 03 16.
Artículo en Inglés | MEDLINE | ID: mdl-33730572

RESUMEN

Viruses need to hijack the translational machinery of the host cell for a productive infection to happen. However, given the dynamic landscape of tRNA pools among tissues, it is unclear whether different viruses infecting different tissues have adapted their codon usage toward their tropism. Here, we collect the coding sequences of 502 human-infecting viruses and determine that tropism explains changes in codon usage. Using the tRNA abundances across 23 human tissues from The Cancer Genome Atlas (TCGA), we build an in silico model of translational efficiency that validates the correspondence of the viral codon usage with the translational machinery of their tropism. For instance, we detect that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is specifically adapted to the upper respiratory tract and alveoli. Furthermore, this correspondence is specifically defined in early viral proteins. The observed tissue-specific translational efficiency could be useful for the development of antiviral therapies and vaccines.


Asunto(s)
Biosíntesis de Proteínas/genética , Virosis/genética , Virus/genética , Línea Celular , Línea Celular Tumoral , Uso de Codones/genética , Genes Relacionados con las Neoplasias/genética , Células HCT116 , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Alveolos Pulmonares/virología , ARN de Transferencia/genética , Infecciones del Sistema Respiratorio/virología , Tropismo/genética , Proteínas Virales/genética , Virosis/virología
14.
Nat Commun ; 12(1): 1706, 2021 03 17.
Artículo en Inglés | MEDLINE | ID: mdl-33731712

RESUMEN

Our incomplete understanding of osteoarthritis (OA) pathogenesis has significantly hindered the development of disease-modifying therapy. The functional relationship between subchondral bone (SB) and articular cartilage (AC) is unclear. Here, we found that the changes of SB architecture altered the distribution of mechanical stress on AC. Importantly, the latter is well aligned with the pattern of transforming growth factor beta (TGFß) activity in AC, which is essential in the regulation of AC homeostasis. Specifically, TGFß activity is concentrated in the areas of AC with high mechanical stress. A high level of TGFß disrupts the cartilage homeostasis and impairs the metabolic activity of chondrocytes. Mechanical stress stimulates talin-centered cytoskeletal reorganization and the consequent increase of cell contractile forces and cell stiffness of chondrocytes, which triggers αV integrin-mediated TGFß activation. Knockout of αV integrin in chondrocytes reversed the alteration of TGFß activation and subsequent metabolic abnormalities in AC and attenuated cartilage degeneration in an OA mouse model. Thus, SB structure determines the patterns of mechanical stress and the configuration of TGFß activation in AC, which subsequently regulates chondrocyte metabolism and AC homeostasis.


Asunto(s)
Cartílago Articular/metabolismo , Cartílago Articular/patología , Estrés Mecánico , Factor de Crecimiento Transformador beta/metabolismo , Animales , Huesos/patología , Línea Celular , Condrocitos/metabolismo , Citoesqueleto/metabolismo , Homeostasis , Humanos , Integrina alfaV/genética , Integrina alfaV/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Osteoartritis/metabolismo , Osteoartritis/patología , Transducción de Señal , Talina/metabolismo
15.
Nat Commun ; 12(1): 1378, 2021 03 02.
Artículo en Inglés | MEDLINE | ID: mdl-33654081

RESUMEN

Glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) and GITR ligand (GITRL) are members of the tumor necrosis superfamily that play a role in immune cell signaling, activation, and survival. GITR is a therapeutic target for directly activating effector CD4 and CD8 T cells, or depleting GITR-expressing regulatory T cells (Tregs), thereby promoting anti-tumor immune responses. GITR activation through its native ligand is important for understanding immune signaling, but GITR structure has not been reported. Here we present structures of human and mouse GITR receptors bound to their cognate ligands. Both species share a receptor-ligand interface and receptor-receptor interface; the unique C-terminal receptor-receptor enables higher order structures on the membrane. Human GITR-GITRL has potential to form a hexameric network of membrane complexes, while murine GITR-GITRL complex forms a linear chain due to dimeric interactions. Mutations at the receptor-receptor interface in human GITR reduce cell signaling with in vitro ligand binding assays and minimize higher order membrane structures when bound by fluorescently labeled ligand in cell imaging experiments.


Asunto(s)
Proteína Relacionada con TNFR Inducida por Glucocorticoide/química , Factores de Necrosis Tumoral/metabolismo , Animales , Fenómenos Biofísicos , Línea Celular , Membrana Celular/metabolismo , Proteína Relacionada con TNFR Inducida por Glucocorticoide/metabolismo , Humanos , Ratones , Modelos Moleculares , Unión Proteica , Reproducibilidad de los Resultados , Factores de Necrosis Tumoral/química
16.
Nat Commun ; 12(1): 1413, 2021 03 03.
Artículo en Inglés | MEDLINE | ID: mdl-33658493

RESUMEN

pH-sensitive fluorescent proteins (FPs) are highly advantageous for the non-invasive monitoring of exocytosis events. Superecliptic pHluorin (SEP), a green pH-sensitive FP, has been widely used for imaging single-vesicle exocytosis. However, the docking step cannot be visualized using this FP, since the fluorescence signal inside vesicles is too low to be observed during docking process. Among the available red pH-sensitive FPs, none is comparable to SEP for practical applications due to unoptimized pH-sensitivity and fluorescence brightness or severe photochromic behavior. In this study, we engineer a bright and photostable red pH-sensitive FP, named pHmScarlet, which compared to other red FPs has higher pH sensitivity and enables the simultaneous detection of vesicle docking and fusion. pHmScarlet can also be combined with SEP for dual-color imaging of two individual secretory events. Furthermore, although the emission wavelength of pHmScarlet is red-shifted compared to that of SEP, its spatial resolution is high enough to show the ring structure of vesicle fusion pores using Hessian structured illumination microscopy (Hessian-SIM).


Asunto(s)
Exocitosis/fisiología , Proteínas Luminiscentes/metabolismo , Animales , Línea Celular , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hipocampo/citología , Humanos , Concentración de Iones de Hidrógeno , Proteínas Luminiscentes/genética , Mutación , Neuronas/citología , Ratas Sprague-Dawley , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Vesículas Sinápticas/fisiología , Imagen de Lapso de Tiempo , Proteína 2 de Membrana Asociada a Vesículas/genética , Proteína 2 de Membrana Asociada a Vesículas/metabolismo
17.
Nat Commun ; 12(1): 1443, 2021 03 04.
Artículo en Inglés | MEDLINE | ID: mdl-33664260

RESUMEN

Heterogeneous ribonucleoproteins (hnRNPs) are RNA binding molecules that are involved in key processes such as RNA splicing and transcription. One such hnRNP protein, hnRNP L, regulates alternative splicing (AS) by binding to pre-mRNA transcripts. However, it is unclear what factors contribute to hnRNP L-regulated AS events. Using proteomic approaches, we identified several key factors that co-purify with hnRNP L. We demonstrate that one such factor, the histone methyltransferase SETD2, specifically interacts with hnRNP L in vitro and in vivo. This interaction occurs through a previously uncharacterized domain in SETD2, the SETD2-hnRNP Interaction (SHI) domain, the deletion of which, leads to a reduced H3K36me3 deposition. Functionally, SETD2 regulates a subset of hnRNP L-targeted AS events. Our findings demonstrate that SETD2, by interacting with Pol II as well as hnRNP L, can mediate the crosstalk between the transcription and the splicing machinery.


Asunto(s)
Empalme Alternativo/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo L/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Precursores del ARN/genética , ARN Mensajero/genética , Línea Celular , Células HEK293 , N-Metiltransferasa de Histona-Lisina/genética , Histonas/metabolismo , Humanos , Dominios Proteicos/fisiología , ARN Polimerasa II/metabolismo
18.
Int J Nanomedicine ; 16: 1663-1680, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33688184

RESUMEN

Background: Intracellular tension plays a crucial role in the destruction of the blood-brain barrier (BBB) in response to lesion stimuli. Tight junction structure could be primarily affected by tension activity. In this study, we aimed to determine the effects of extracellular BBB damage on intracellular tension activity, and elucidate the mechanism underlying the effects of intracellular protein nanoparticle-related osmotic pressure on BBB permeability. Methods: The intracellular tension for tight junction proteins occludin and ZO1 was evaluated using the fluorescence resonance energy transfer (FRET)-based tension probes and cpstFRET analysis. The changes in mobility ratios of occludin were evaluated via the fluorescence recovery after photobleaching (FRAP) test. The cytoplasmic osmotic pressure (OP) was measured using Osmometer. The count rate of cytoplasmic nanoparticles was detected by Nanosight NS300. The activation of cofilin and stathmin was examined by Western blot analysis. The BBB permeability in vivo was determined via the changes of Evans Blue (EB) injected into SD rats. The tight junction formation was assessed by the measurement of transendothelial electrical resistance (TEER). Intracellular calcium or chloride ions were measured using Fluo-4 AM or MQAE dyes. Results: BBB lesions were accompanied by changes in occludin/ZO1 tension. Increases in intracellular osmotic pressure were involved in alteration of BBB permeability, possibly through the depolymerization of microfilaments or microtubules and mass production of protein nanoparticles according to the Donnan effect. Recovery of protein nanoparticle-related osmotic pressure could effectively reverse the effects of changes in occludin/ZO1 tension under BBB lesions. Outward tension of intracellular osmotic potential also caused upregulation of membrane fluidity, which promoted nonselective drug influx. Conclusion: Our results suggest a crucial mechanical mechanism underlying BBB lesions, and protein nanoparticle-related osmotic pressure could be a novel therapeutic target for BBB lesion-related brain diseases.


Asunto(s)
Barrera Hematoencefálica/metabolismo , Fluidez de la Membrana , Nanopartículas/química , Presión Osmótica , Proteínas/química , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/metabolismo , Animales , Barrera Hematoencefálica/efectos de los fármacos , Línea Celular , Azul de Evans/metabolismo , Humanos , Masculino , Fluidez de la Membrana/efectos de los fármacos , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Ocludina/metabolismo , Presión Osmótica/efectos de los fármacos , Permeabilidad , Fitoquímicos/farmacología , Polimerizacion , Ratas Sprague-Dawley , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/metabolismo , Proteína de la Zonula Occludens-1/metabolismo
19.
Science ; 371(6535)2021 03 19.
Artículo en Inglés | MEDLINE | ID: mdl-33737461

RESUMEN

Interleukin-10 (IL-10) is an immunoregulatory cytokine with both anti-inflammatory and immunostimulatory properties and is frequently dysregulated in disease. We used a structure-based approach to deconvolute IL-10 pleiotropy by determining the structure of the IL-10 receptor (IL-10R) complex by cryo-electron microscopy at a resolution of 3.5 angstroms. The hexameric structure shows how IL-10 and IL-10Rα form a composite surface to engage the shared signaling receptor IL-10Rß, enabling the design of partial agonists. IL-10 variants with a range of IL-10Rß binding strengths uncovered substantial differences in response thresholds across immune cell populations, providing a means of manipulating IL-10 cell type selectivity. Some variants displayed myeloid-biased activity by suppressing macrophage activation without stimulating inflammatory CD8+ T cells, thereby uncoupling the major opposing functions of IL-10. These results provide a mechanistic blueprint for tuning the pleiotropic actions of IL-10.


Asunto(s)
Interleucina-10/química , Interleucina-10/metabolismo , Animales , Sitios de Unión , Linfocitos T CD8-positivos/inmunología , Línea Celular , Microscopía por Crioelectrón , Citocinas/metabolismo , Evolución Molecular Dirigida , Humanos , Inflamación , Interleucina-10/agonistas , Subunidad alfa del Receptor de Interleucina-10/química , Subunidad alfa del Receptor de Interleucina-10/metabolismo , Subunidad beta del Receptor de Interleucina-10/química , Subunidad beta del Receptor de Interleucina-10/metabolismo , Activación de Macrófagos , Ratones , Modelos Moleculares , Monocitos/inmunología , Monocitos/metabolismo , Células Mieloides/inmunología , Células Mieloides/metabolismo , Unión Proteica , Ingeniería de Proteínas , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Factor de Transcripción STAT3/metabolismo , Sepsis/inmunología , Transducción de Señal
20.
Science ; 371(6535): 1245-1248, 2021 03 19.
Artículo en Inglés | MEDLINE | ID: mdl-33737484

RESUMEN

Mosaic mutations can be used to track cell lineages in humans. We used cell cloning to analyze embryonic cell lineages in two living individuals and a postmortem human specimen. Of 10 reconstructed postzygotic divisions, none resulted in balanced contributions of daughter lineages to tissues. In both living individuals, one of two lineages from the first cleavage was dominant across tissues, with 90% frequency in blood. We propose that the efficiency of DNA repair contributes to lineage imbalance. Allocation of lineages in postmortem brain correlated with anterior-posterior axis, associating lineage history with cell fate choices in embryos. We establish a minimally invasive framework for defining cell lineages in any living individual, which paves the way for studying their relevance in health and disease.


Asunto(s)
Blastómeros/citología , División Celular , Linaje de la Célula , Desarrollo Embrionario , Adulto , Anciano , Blastocisto/citología , Células Sanguíneas , Diferenciación Celular , Línea Celular , Reparación del ADN , Femenino , Feto/citología , Variación Genética , Genoma Humano , Humanos , Mutación INDEL , Células Madre Pluripotentes Inducidas/citología , Masculino , Células-Madre Neurales/citología , Polimorfismo de Nucleótido Simple
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