RESUMEN
Despite the well-studied effects of the full-length membrane-locating isoform Iso1 of Programmed Cell Death Protein-Ligand 1 (PD-L1) on immunosuppression, little is known about another membrane-locating isoform, Iso2. While expressional and survival analysis of liver cancer patients indicated that Iso2 plays a tumor-suppressive role, our results also indicated that the tumor-promoting and immune-suppressive effects of Iso1 depended on the positive expression of Iso2. Through mediation analysis, we discovered several downstream genes or pathways of Iso2 and investigated their effects on the Iso1-regulating survival. Among all potential downstream immune factors, Iso2 was inclined to activate the proliferation of T cells by regulating chemokine activity and increasing CD3 levels by promoting TNF expression. Similar results were confirmed in the Mongolian liver cancer cohort, and the Iso2/TNF/T-cell axis was verified in several other cancers in the TCGA cohort. Finally, we demonstrated the promoting effects of Iso2 in terms of producing TNF and increasing T cells both in vitro and in vivo. Our findings illustrate that PD-L1 Iso2 can increase the number of T cells in the tumor microenvironment by elevating TNF levels, which is a necessary part of the tumor-suppressive effects of Iso1 in liver cancer.
Asunto(s)
Antígeno B7-H1 , Neoplasias Hepáticas , Humanos , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Proliferación Celular/genética , Terapia de Inmunosupresión , Ligandos , Neoplasias Hepáticas/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Microambiente Tumoral/genética , Factores de Necrosis Tumoral/metabolismoRESUMEN
Chlorogenic acid (CGA) is a bioactive substance with anti-inflammatory activities. Clusters of CD36 have been suggested to be widely involved in inflammatory damage. However, the mechanism of CGA protecting against LPS-induced inflammation involving the CD36 regulation is unclear. Here, we demonstrated that CGA protected against LPS-induced cell death and decreased the production of ROS. Moreover, the SOD, CAT, and GSH-Px activities were also upregulated in CGA-treated cells during LPS stimulation. CGA reduced COX-2 and iNOS expression and IL-1ß, IL-6, and TNF-α secretion in LPS-stimulated RAW264.7 macrophages. In addition, CGA treatment widely involved in immune-related signaling pathways, including NF-κB signaling, NOD-like receptor signaling, and IL-17 signaling using transcriptomic analysis and CD36 also markedly reduced during CGA pretreatment in LPS-induced RAW264.7 cells. Furthermore, the CD36 inhibitor SSO attenuated inflammation and oxidative stress by enabling activation of the AMPK/PGC-1α cascade. These results indicate that CGA might provide benefits for the regulation of inflammatory diseases by modulating CD36/AMPK/PGC-1α to alleviate oxidative stress.
Asunto(s)
Proteínas Quinasas Activadas por AMP , Ácido Clorogénico , Humanos , Ácido Clorogénico/farmacología , Lipopolisacáridos/toxicidad , Macrófagos , Inflamación/inducido químicamente , Inflamación/tratamiento farmacológico , Estrés Oxidativo , Antígenos CD36RESUMEN
OBJECTIVE: Dysfunction of endoplasmic reticulum (ER) proteins is closely related to homeostasis disturbance and malignant transformation of hepatocellular carcinoma (HCC). Reticulons (RTN) are a family of ER-resident proteins critical for maintaining ER function. Nevertheless, the precise roles of RTN in HCC remain largely unclear. The aim of the study is to examine the effect of reticulon family member RTN3 on HCC development and explore the underlying mechanisms. DESIGN: Clinical HCC samples were collected to assess the relationship between RTN3 expression and patients' outcome. HCC cell lines were employed to examine the effects of RTN3 on cellular proliferation, apoptosis and signal transduction in vitro. Nude mice model was used to detect the role of RTN3 in modulating tumour growth in vivo. RESULTS: We found that RTN3 was highly expressed in normal hepatocytes but frequently downregulated in HCC. Low RTN3 expression predicted poor outcome in patients with HCC in TP53 gene mutation and HBV infection status-dependent manner. RTN3 restrained HCC growth and induced apoptosis by activating p53. Mechanism studies indicated that RTN3 facilitated p53 Ser392 phosphorylation via Chk2 and enhanced subsequent p53 nuclear localisation. RTN3 interacted with Chk2, recruited it to ER and promoted its activation in an ER calcium-dependent manner. Nevertheless, the tumour suppressive effects of RTN3 were abrogated in HBV-positive cells. HBV surface antigen competed with Chk2 for RTN3 binding and blocked RTN3-mediated Chk2/p53 activation. CONCLUSION: The findings suggest that RTN3 functions as a novel suppressor of HCC by activating Chk2/p53 pathway and provide more clues to better understand the oncogenic effects of HBV.
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Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/virología , Virus de la Hepatitis B/patogenicidad , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/virología , Proteína p53 Supresora de Tumor/metabolismo , Animales , Apoptosis , Carcinogénesis/metabolismo , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Quinasa de Punto de Control 2/metabolismo , Retículo Endoplásmico/metabolismo , Hepatocitos/metabolismo , Humanos , Masculino , Proteínas de la Membrana/metabolismo , Ratones Endogámicos BALB C , Ratones Desnudos , Proteínas del Tejido Nervioso/metabolismo , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Red cabbage (Brassica oleracea L. var. capitata) continues to receive increasing attention on its health-promoting properties because of its high glucosinolate content. Glucosinolates are an unstable active substance; however, there are few studies on their changes in different cooking processes. In this study, we investigated the effects of processing methods (boiling, steaming, microwave heating, frying, stir-frying) and boiling time on glucosinolates in red cabbage. Ten glucosinolates, including 4-methoxyglucobrassicin, neoglucobrassicin, glucoalyssin, glucobrassicin, glucoraphanin, glucoiberin, progoitrin, gluconapin and sinigrin, in red cabbage were detected. Decreases of 32.36%, 24.83%, 25.27%, 81.11% and 84.29% for total glucosinolates were observed after boiling, microwaving, steaming, frying and stir-frying. Indole glucosinolates were more efficiently lost compared to aliphatic glucosinolates after boiling, while microwaving, steaming, frying and stir-frying also resulted in a greater reduction in indole glucosinolates than aliphatic glucosinolates. Glucoalyssin, glucoerucin and sinigrin were more thermal sensitive than other glucosinolates. It was confirmed that microwaving and steaming retained higher levels of glucosinolates than other methods and may be better for cooking red cabbage.
Asunto(s)
Brassica/química , Glucosinolatos/química , Cromatografía Liquida/métodos , Culinaria/métodos , Glucosa/análogos & derivados , Glucosa/química , Imidoésteres/química , Indoles/química , Microondas , Oximas/química , Sulfóxidos/química , Espectrometría de Masas en Tándem/métodosRESUMEN
Immune responses against antigens generally require an efficient activation of antigen-presenting cells (APCs). Currently, the targeting of vaccine antigens to APCs has emerged as a promising strategy for boosting vaccine immunogenicity. Here, we reported that the C-terminus of heat shock protein 60 (HSP60C) can activate mouse peritoneal macrophages to secret a series of cytokines, and phosphorylation of p38 mitogen-activated protein kinase (MAPK) and NF-κB p65 was involved in the pathway. We showed that the activation effect of HSP60C on macrophages was independent of toll-like receptor (TLR) 4 and the TLR-associated myeloide differentiation factor 88 (MyD88). Knockdown of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) reduced the activation of HSP60C-induced macrophage p38 MAPK, NF-κB p65 and cytokine secretion to some extent. Finally, we found that HSP60C up-regulated the expression of LOX-1 on macrophages and ovalbumin (OVA) model antigen fused with HSP60C markedly enhanced OVA-specific IgG responses. Thus, our results unravel a novel LOX-1-dependent pathway by which HSP60C can effectively activate macrophages and APCs targeting based on LOX-1 interaction is a promising approach to improve vaccines.
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Chaperonina 60/química , Chaperonina 60/metabolismo , Activación de Macrófagos , Macrófagos Peritoneales/metabolismo , Receptores Depuradores de Clase E/metabolismo , Animales , Formación de Anticuerpos , Células CHO , Cricetinae , Cricetulus , Citocinas/metabolismo , Endocitosis , Femenino , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/metabolismo , Ovalbúmina/metabolismo , Unión Proteica , Transducción de Señal , Relación Estructura-Actividad , Receptor Toll-Like 4/metabolismo , Regulación hacia Arriba , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
BACKGROUND: C-Type Lectin Domain Family 3 Member B (CLEC3B), is down-regulated in serum and tumor tissues in different cancers including hepatocellular carcinoma (HCC). However, the functions of CLEC3B in HCC remains elucidated. The aim of this study is to analyze the roles of CLEC3B in HCC. METHODS: The expression of genes was evaluated by immunohistochemistry, western blot, real-time PCR, enzyme-linked immunosorbent assays, and analysis on TCGA-LIHC database and gene expression omnibus. Transmission electron microscopy and immunofluorescence were applied to detect CLEC3B in exosomes. The function of exosomal CLEC3B in tumor progression were performed in vivo and in vitro. RESULTS: We determined that down-regulated CLEC3B in HCC indicated a poor prognosis. Exosomes derived from HCC with down-regulated CLEC3B promoted migration, invasion, epithelial-mesenchymal transition of both tumor cells and endothelial cells (ECs). Moreover, the downregulation CLEC3B in exosomes suppressed VEGF secretion in both HCC cells and ECs, and eventually inhibited angiogenesis. Mechanistically, CLEC3B-mediated VEGF expression in tumor cells and ECs depends on the activation of AMPK signal pathway. CONCLUSION: This study demonstrates that CLEC3B acts as a novel independent prognostic factor, and CLEC3B in exosomes might be a potential therapeutic target for hepatocellular carcinoma.
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Proteínas Quinasas Activadas por AMP/metabolismo , Carcinoma Hepatocelular/patología , Regulación hacia Abajo , Lectinas Tipo C/genética , Neoplasias Hepáticas/patología , Transducción de Señal/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Carcinoma Hepatocelular/irrigación sanguínea , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/genética , Línea Celular Tumoral , Exosomas/metabolismo , Humanos , Masculino , Ratones , Metástasis de la Neoplasia , Neovascularización Patológica/genética , PronósticoRESUMEN
Stable and low-cost carbon dots (C-dots) were prepared from polyethylenimine (PEI) by a hydrothermal method. It is found that the fluorescence of the C-dots (best measured at excitation/emission wavelengths of 365/473 nm) is quenched by selective oxidation of surface PEI by periodate but recovers in the presence of uric acid (UA). It is assumed that this is due to the selective reduction of the nitrone groups to hydroxylamine groups by UA. The findings were used to design a fluorometric method for determination of UA that has a 2.3 nM detection limit. This is lower than that of reported fluorometric and enzymatic assays. The performance of the method has been validated by determination of UA in samples of human saliva. It is found that the results agree well with those obtained by a commercial UA assay. Graphical abstract Schematic presentation of the polyethylenimine (PEI) carbon nanodots (C-dots) as a fluorescent probe for uric acid. Their fluorescence is quenched by periodate (IO4-) due to oxidative formation of nitrone groups, an subsequently restored due to reduction by uric acid (UA).
Asunto(s)
Fluorometría/métodos , Puntos Cuánticos/química , Ácido Úrico/análisis , Carbono , Fluorescencia , Humanos , Límite de Detección , Nanoestructuras , Nitrógeno , Saliva/químicaRESUMEN
BACKGROUND & AIMS: Aberrant oncogenic mRNA translation and protein O-linked ß-N-acetylglucosaminylation (O-GlcNAcylation) are general features during tumorigenesis. Nevertheless, whether and how these two pathways are interlinked remain unknown. Our previous study indicated that ribosomal receptor for activated C-kinase 1 (RACK1) promoted chemoresistance and growth in hepatocellular carcinoma (HCC). The aim of this study is to examine the role of RACK1 O-GlcNAcylation in oncogene translation and HCC carcinogenesis. METHODS: The site(s) of RACK1 for O-GlcNAcylation was mapped by mass spectrometry analysis. HCC cell lines were employed to examine the effects of RACK1 O-GlcNAcylation on the translation of oncogenic factors and behaviors of tumor cells in vitro. Transgenic knock-in mice were used to detect the role of RACK1 O-GlcNAcylation in modulating HCC tumorigenesis in vivo. The correlation of RACK1 O-GlcNAcylation with tumor progression and relapse were analyzed in clinical HCC samples. RESULTS: We found that ribosomal RACK1 was highly modified by O-GlcNAc at Ser122. O-GlcNAcylation of RACK1 enhanced its protein stability, ribosome binding and interaction with PKCßII (PRKCB), leading to increased eukaryotic translation initiation factor 4E phosphorylation and translation of potent oncogenes in HCC cells. Genetic ablation of RACK1 O-GlcNAcylation at Ser122 dramatically suppressed tumorigenesis, angiogenesis, and metastasis in vitro and in diethylnitrosamine (DEN)-induced HCC mouse model. Increased RACK1 O-GlcNAcylation was also observed in HCC patient samples and correlated with tumor development and recurrence after chemotherapy. CONCLUSIONS: These findings demonstrate that RACK1 acts as key mediator linking O-GlcNAc metabolism to cap-dependent translation during HCC tumorigenesis. Targeting RACK1 O-GlcNAcylation provides promising options for HCC treatment. LAY SUMMARY: O-GlcNAcylation of ribosomal receptor for activated C-kinase 1 at the amino acid serine122 promotes its stability, ribosome localization and interaction with the protein kinase, PKCßII, thus driving the translation of oncogenes and tumorigenesis of hepatocellular carcinoma. Increased O-GlcNAcylation of ribosomal receptor for activated C-kinase 1 is positively correlated with tumor growth, metastasis and recurrence in patients with hepatocellular carcinoma.
Asunto(s)
Carcinoma Hepatocelular/etiología , Neoplasias Hepáticas/etiología , Proteínas de Neoplasias/metabolismo , Receptores de Cinasa C Activada/metabolismo , Sustitución de Aminoácidos , Animales , Carcinógenos/química , Carcinógenos/metabolismo , Carcinoma Hepatocelular/metabolismo , Línea Celular Tumoral , Progresión de la Enfermedad , Glicosilación , Humanos , Neoplasias Hepáticas/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Mutantes , Ratones Desnudos , Ratones Transgénicos , Mutagénesis Sitio-Dirigida , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Trasplante de Neoplasias , Proteína Quinasa C beta/metabolismo , Estabilidad Proteica , Receptores de Cinasa C Activada/química , Receptores de Cinasa C Activada/genética , Serina/químicaRESUMEN
BACKGROUND: Rabies is a fatal disease that is preventable when post exposure prophylaxis (PEP) is administered in a timely fashion. CpG oligodeoxynucleotides (ODNs) can trigger cells that express Toll-like receptor 9, and their immunopotentiation activity in an inactivated aluminum-adjuvanted rabies vaccine for dogs has been identified using mouse and dog models. METHODS: A human diploid cell rabies vaccine (HDCV) of humans and a CpG ODNs with cross-immunostimulatory activity in humans and mice were used to evaluate the immunogenicity and protective efficacy of CpG ODN in a mouse model that simulates human PEP. RESULTS: HDCV combined with CpG ODN (HDCV-CpG) stimulated mice to produce rabies virus-specific neutralizing antibody (RVNA) earlier and increased the seroconversion rate. Compared with HDCV alone, either HDCV-1.25 µg CpG or HDCV-5 µg CpG increased the levels of RVNA. In particular, 5 µg CpG ODN per mouse significantly boosted the levels of RVNA compared with HDCV alone. IFN-γ producing splenocytes generated in the HDCV-5 µg CpG group were significantly increased compared to the group treated with HDCV alone. When the immunization regimen was reduced to three injections or the dose was reduced to half of the recommended HDCV combined with CpG ODN, the RVNA titers were still higher than those induced by HDCV alone. After viral challenge, 50% of mice immunized with a half-dose HDCV-CpG survived, while the survival rate of mice immunized with HDCV alone was 30%. CONCLUSIONS: The immunopotentiation activity of CpG ODNs for a commercially available human rabies vaccine was first evaluated in a mouse model on the basis of the Essen regimen. Our results suggest that the CpG ODN used in this study is a potential adjuvant to rabies vaccines for human use.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Oligodesoxirribonucleótidos/inmunología , Profilaxis Posexposición , Vacunas Antirrábicas/inmunología , Virus de la Rabia/inmunología , Rabia/inmunología , Animales , Anticuerpos Antivirales/sangre , Modelos Animales de Enfermedad , Femenino , Humanos , Inmunogenicidad Vacunal , Ratones , Ratones Endogámicos BALB C , Oligodesoxirribonucleótidos/administración & dosificación , Rabia/prevención & control , Vacunas Antirrábicas/administración & dosificación , Vacunas de Productos Inactivados/administración & dosificación , Vacunas de Productos Inactivados/inmunologíaRESUMEN
BACKGROUND & AIMS: C-type lectin-like receptor 2 (CLEC2) is a transmembrane receptor expressed on platelets and several hematopoietic cells. CLEC2 regulates platelet aggregation and the immune response. We investigated its expression and function in normal and transformed gastric epithelial cells from human tissues. METHODS: We performed tissue microarray analyses of gastric carcinoma samples collected from 96 patients who underwent surgery at Zhongshan Hospital of Fudan University in Shanghai, China and performed real-time polymerase chain reaction assays from an independent group of 60 patients; matched nontumor gastric mucosa tissues were used as the control. Full-length and mutant forms of CLEC2 were expressed in gastric cancer cell line (MGC80-3), or CLEC2 protein was knocked down using small-hairpin RNAs in gastric cancer cell lines (NCI-N87 and AGS). CLEC2 signaling was stimulated by incubation of cells with recombinant human podoplanin or an antibody agonist of CLEC2; cell migration and invasion were assessed by transwell and wound-healing assays. Immunoblot, immunofluorescence microscopy, and real-time polymerase chain reaction assays were used to measure expression of markers of the epithelial to mesenchymal transition and activation of signaling pathways. Immunoprecipitation experiments were performed with an antibody against spleen tyrosine kinase (SYK). Cells were injected into lateral tail vein of BALB/C nude mice; some mice were also given injections of the phosphoinositide 3-kinase (PI3K) inhibitor LY294002. Lung and liver tissues were collected and analyzed for metastases. RESULTS: Levels of CLEC2 were higher in nontumor gastric mucosa (control) than in gastric tumor samples. Levels of CLEC2 protein in gastric tumor tissues correlated with depth of tumor invasion, metastasis to lymph node, tumor TNM stage, and 5-year survival of patients. Activation of CLEC2 in gastric cancer cells reduced their invasive activities in vitro and expression of epithelial to mesenchymal transition markers; these tumor-suppressive effects of CLEC2 required SYK. CLEC2 and SYK interacted physically, and SYK maintained the stability of CLEC2 in cells. AGS cells with CLEC2 knockdown had increased levels of phosphorylated AKT and glycogen synthase kinase-3 beta, increased expression of Snail, reduced levels of E-cadherin, and formed more metastases in mice than AGS cells that expressed CLEC2; these knockdown changes were prevented by the PI3K inhibitor LY294002. Activation of CLEC2 in AGS cells reduced protein and messenger RNA levels of PI3K subunits p85 and p110; this effect was blocked by SYK inhibitor R406. Levels of CLEC2 and SYK proteins and messenger RNAs correlated in gastric tumor samples. CONCLUSIONS: CLEC2 suppresses metastasis of gastric cancer cells injected into mice, and prevents activation of AKT and glycogen synthase kinase-3 beta signaling, as well as invasiveness and expression of epithelial to mesenchymal transition markers in gastric cancer cell lines. CLEC2 prevents expression of PI3K subunits, in a SYK-dependent manner.
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Movimiento Celular , Fosfatidilinositol 3-Quinasa Clase Ia/metabolismo , Lectinas Tipo C/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Neoplasias Gástricas/enzimología , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Fosfatidilinositol 3-Quinasa Clase Ia/genética , Transición Epitelial-Mesenquimal , Femenino , Regulación Neoplásica de la Expresión Génica , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Humanos , Lectinas Tipo C/genética , Neoplasias Hepáticas/enzimología , Neoplasias Hepáticas/prevención & control , Neoplasias Hepáticas/secundario , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/prevención & control , Neoplasias Pulmonares/secundario , Masculino , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , Invasividad Neoplásica , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Interferencia de ARN , Transducción de Señal/efectos de los fármacos , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Quinasa Syk/antagonistas & inhibidores , Quinasa Syk/genética , Quinasa Syk/metabolismo , Factores de Tiempo , Transfección , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Heat shock proteins (HSPs) were originally identified as stress-responsive proteins and serve as molecular chaperones in different intracellular compartments. Translocation of HSPs to the cell surface and release of HSPs into the extracellular space have been observed during the apoptotic process and in response to a variety of cellular stress. Here, we report that UV irradiation and cisplatin treatment rapidly induce the expression of membrane-bound Hsp60, Hsp70, and Hsp90 upstream the phosphatidylserine exposure. Membrane-bound Hsp60, Hsp70 and Hsp90 could promote the release of IL-6 and IL-1ß as well as DC maturation by the evaluation of CD80 and CD86 expression. On the other hand, Hsp60, Hsp70 and Hsp90 on cells could facilitate the uptake of dying cells by bone marrow-derived dendritic cells. Lectin-like oxidized LDL receptor-1 (LOX-1), as a common receptor for Hsp60, Hsp70, and Hsp90, is response for their recognition and mediates the uptake of dying cells. Furthermore, membrane-bound Hsp60, Hsp70 and Hsp90 could promote the cross-presentation of OVA antigen from E.G7 cells and inhibition of the uptake of dying cells by LOX-1 decreases the cross-presentation of cellular antigen. Therefore, the rapid exposure of HSPs on dying cells at the early stage allows for the recognition by and confers an activation signal to the immune system.
Asunto(s)
Chaperonina 60/genética , Reactividad Cruzada , Células Dendríticas/inmunología , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/genética , Proteínas Mitocondriales/genética , Animales , Apoptosis/efectos de los fármacos , Antígeno B7-1/genética , Antígeno B7-1/inmunología , Antígeno B7-2/genética , Antígeno B7-2/inmunología , Células CHO , Membrana Celular/efectos de los fármacos , Membrana Celular/inmunología , Membrana Celular/metabolismo , Chaperonina 60/inmunología , Cricetulus , Células Dendríticas/citología , Células Dendríticas/efectos de los fármacos , Regulación de la Expresión Génica , Células HL-60 , Proteínas HSP70 de Choque Térmico/inmunología , Proteínas HSP90 de Choque Térmico/inmunología , Humanos , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Interleucina-6/genética , Interleucina-6/inmunología , Ratones , Ratones Endogámicos C57BL , Proteínas Mitocondriales/inmunología , Ovalbúmina/farmacología , Fosfatidilserinas/inmunología , Fosfatidilserinas/metabolismo , Unión Proteica , Receptores Depuradores de Clase E/genética , Receptores Depuradores de Clase E/inmunología , Transducción de SeñalRESUMEN
We have developed a simple, sensitive, and rapid fluorescence assay for the detection of cancer cells, based on "turn-on" retro-self-quenched fluorescence inside the cells. 1,3-Phenylenediamine resin (DAR) nanoparticles (NPs) containing rhodamine 6G (R6G) are conjugated with aptamer (apt) sgc8c to prepare sgc8c-R6GDAR NPs, while that containing rhodamine 101 (R101) are conjugated with TD05 for the preparation of TD05-R101DAR NPs. The sgc8c-R6GDAR and TD05-R101DAR NPs separately recognize CCRF-CEM and Ramos cells. The fluorescence intensities of the two apt-DAR NPs are both weak due to self-quenching, but they increase inside the cells as a result of release of the fluorophores from the apt-DAR NPs. The apt-DAR NPs' structure becomes less compact at low pH value, leading to the release of the fluorophores. The sgc8c-R6GDAR and TD05-R101DAR NPs allow detection of as low as 44 CCRF-CEM cells and 79 Ramos cells mL(-1), respectively, using a commercial reader within 10 min. Practicality of the two probes have been validated by the quantitation and identification of CCRF-CEM and Ramos cells spiked in blood samples through conventional fluorescence and flow cytometry analysis, with advantages of sensitivity, selectivity, and rapidity.
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Aptámeros de Nucleótidos/química , Separación Celular/métodos , Fluorescencia , Nanopartículas/química , Neoplasias/patología , Polímeros/química , Animales , Humanos , Ratones , Células 3T3 NIH , Neoplasias/diagnóstico , Células Tumorales CultivadasRESUMEN
Hepatocellular carcinoma (HCC) remains the second leading cause of cancer-related death worldwide, and elevated rates of reactive oxygen species (ROS) have long been considered as a hallmark of almost all types of cancer including HCC. Protein kinase C alpha (PKCα), a serine/threonine kinase among conventional PKC family, is recognized as a major player in signal transduction and tumor progression. Overexpression of PKCα is commonly observed in human HCC and associated with its poor prognosis. However, how PKCα is involved in hepatocellular carcinogenesis remains not fully understood. In this study, we found that among the members of conventional PKC family, PKCα, but not PKCßI or ßII, promoted ROS production in HCC cells. PKCα stimulated generation of ROS by up-regulating DUOX2 at post-transcriptional level. Depletion of DUOX2 abrogated PKCα-induced activation of AKT/MAPK pathways as well as cell proliferation, migration and invasion in HCC cells. Moreover, the expression of DUOX2 and PKCα was well positively correlated in both HCC cell lines and patient samples. Collectively, our findings demonstrate that PKCα plays a critical role in HCC development by inducing DUOX2 expression and ROS generation, and propose a strategy to target PKCα/DUOX2 as a potential adjuvant therapy for HCC treatment.
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Carcinoma Hepatocelular/enzimología , Neoplasias Hepáticas/enzimología , NADPH Oxidasas/metabolismo , Proteína Quinasa C-alfa/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Secuencia de Bases , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Oxidasas Duales , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Proteína Quinasa C-alfa/genética , ARN Interferente Pequeño , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
The complicated delivery mechanism of group II membrane proteins makes it difficult to decide the fusion pattern of their extracellular domains (ECDs) with Fc moiety. In this study, we compared the expression of ECDs of three group II membrane proteins including CLEC-2, Dectin-1, and LOX-1 by fusion of Fc moiety. We found that the pattern of ECD-Fc fusion order produced the functionally active recombinant proteins while the pattern of Fc-ECD fusion order led to the altered glycosylation which abolished the binding of these proteins with their ligands. Meanwhile, our results indicated that the secretion of mouse Fc (mFc)-fused ECD of CLEC-2 was more efficient than that of rabbit Fc (rFc)-fused protein, while rFc moiety was more sensitive for detection compared with mFc moiety. Altogether, we provide a favorable fusion pattern of Fc moiety with the ECDs of group II transmembrane proteins.
Asunto(s)
Fragmentos Fc de Inmunoglobulinas/metabolismo , Lectinas Tipo C/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Células CHO , Membrana Celular/metabolismo , Cricetinae , Cricetulus , Glicosilación , Células HEK293 , Humanos , Fragmentos Fc de Inmunoglobulinas/genética , Lectinas Tipo C/química , Lectinas Tipo C/genética , Ratones , Estructura Terciaria de Proteína , Transporte de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Receptores Depuradores de Clase E/química , Receptores Depuradores de Clase E/genética , Receptores Depuradores de Clase E/metabolismoRESUMEN
In this study, highly hydrophilic and photoluminescent sheets of reduced graphene oxide decorated with carbon dots (C-dots@RGO), methylene blue (MB), and a probe DNA have been used for the detection of DNA. The photoluminescence of C-dots@RGO is quenched by MB, which is restored in the presence of a target DNA. The combination of the C-dots@RGO, MB, and a DNA probe is selective for perfectly matched DNA over mismatched DNA, mainly because relative to single-stranded DNA, double-stranded DNA intercalates more strongly with MB, but interacts more weakly with RGO. In the presence of a target DNA, MB intercalates with the as-formed double-stranded DNA and is released from the surface of C-dots@RGO, leading to "turn-on" photoluminescence. The practicality of this assay has been validated by the determination of tumor suppressor gene BRCA1, with linearity over the concentration range from 25 to 250 nM and a limit of detection (LOD, at a signal-to-noise ratio of 3) of 14.6 nM. The C-dots@RGO probe provides higher specificity towards target DNA than towards common salts, carbohydrates, amino acids, and proteins found in real samples. Having the advantages of simplicity, cost-effectiveness, selectivity, and sensitivity, the DNA-P/C-dots@RGO-MB probe on microwells has been successfully employed for the detection of DNA, suggesting its potential for multiple analyses of DNA targets when various DNA probes are employed.
Asunto(s)
Sondas de ADN , Secuencia de Bases , Luminiscencia , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Datos de Secuencia MolecularRESUMEN
Early recognition of malnutrition is essential to improve the prognosis of older patients with hip fracture. The Nutritional Risk Screening 2002 (NRS-2002), the Short-Form Mini Nutritional Assessment (MNA-SF) and the Global Leadership Initiative on Malnutrition (GLIM) are widely used in malnutrition diagnosis. However, criteria for predicting postoperative hip joint motor function in older patients with hip fractures are still necessary. The objective of this study was to select the most appropriate criteria from the NRS-2002, the MNA-SF and the GLIM in predicting the postoperative hip joint motor function recovery 1 year after surgery. This retrospective observational study included 161 patients agedâ ≥â 65 years with hip fractures. The nutritional status of patients was determined by the NRS-2002, MNA-SF and GLIM. The Harris hip joint score (HHS), the primary outcome of this study, was used to evaluate hip joint motor function. HHS was classified as excellent (HHSâ >â 75) or non-excellent outcomes (HHSâ ≤â 75). Logistic regression models for hip joint motor function recovery were constructed. Both the receiver operating characteristic curve and the decision curve analysis were used to select the most predictive criteria. The overall mean age of the 161 patients was 77.90â ±â 8.17. As a result, NRS-2002 (OR:0.06, 95%CI [0.01, 0.17]), MNA-SF (OR:0.05, 95%CI [0.00, 0.23]) and GLIM (OR of moderate: 0.03, 95%CI [0.01, 0.11]; OR of severe: 0.02 [0.00, 0.07]) were predictive for recovery of hip joint motor function. Additionally, both the area under curve of the receiver operating characteristic curve (NRS-2002: 81.2 [73.8, 88.6], MNA-SF: 76.3 [68.5, 84.2], GLIM: 86.2 [79.6,92.8]) and the decision curve analysis showed the GLIM was better than others. Compared with NRS-2002 and MNA-SF, GLIM was a more suitable nutritional assessment criteria to predict the postoperative recovery of hip joint motor function for older patients with hip fracture 1 year after surgery.
Asunto(s)
Fracturas de Cadera , Desnutrición , Humanos , Anciano , Estado Nutricional , Estudios Retrospectivos , Recuperación de la Función , Liderazgo , Desnutrición/diagnóstico , Evaluación Nutricional , Fracturas de Cadera/cirugíaRESUMEN
Obtaining a microorganism strain with a broad-spectrum resistance property and highly efficient antifungal activity is important to the biocontrol strategy. Herein, a marine Streptomyces sp. HNBCa1 demonstrated a broad-spectrum resistance to 17 tested crop pathogenic fungi and exhibited a high biocontrol efficiency against mango anthracnose and banana fusarium wilt. To uncover the critical bioactive secondary metabolites basis, genome assembly and annotation, metabolomic analysis, and a semipreparative HPLC-based activity-guide method were employed. Finally, geldanamycin and ectoine involved in codifferential secondary metabolites were also found to be related to biosynthetic gene clusters in the genome of HNBCa1. Reblastatin and geldanamycin were uncovered in response to broad-spectrum resistance to the 17 crop pathogenic fungi. Our results suggested that reblastatin and geldanamycin were critical to maintaining the broad-spectrum resistance property and highly efficient antifungal activity of HNBCa1, which could be further developed as a biological control agent to control crop fungal diseases.
Asunto(s)
Fusarium , Lactamas Macrocíclicas , Enfermedades de las Plantas , Metabolismo Secundario , Streptomyces , Streptomyces/genética , Streptomyces/metabolismo , Streptomyces/química , Enfermedades de las Plantas/microbiología , Lactamas Macrocíclicas/farmacología , Lactamas Macrocíclicas/metabolismo , Lactamas Macrocíclicas/química , Fusarium/efectos de los fármacos , Benzoquinonas/farmacología , Benzoquinonas/metabolismo , Benzoquinonas/química , Hongos/genética , Antifúngicos/farmacología , Antifúngicos/metabolismo , Antifúngicos/químicaRESUMEN
To investigate the role of gastrointestinal (GI) polysaccharide fermentation in alleviating constipation, two polysaccharide fractions were isolated from a soluble fiber extract with determined anti-constipation activity: a 2.04 kDa neutral fraction (SSP-1) contained 99.29% glucose, and a 41.66 kDa acidic fraction (SSP-2) contained 63.85% uronic acid. After mice were given loperamide for 14 d to induce constipation, the GI transit rate increased significantly in the SSP-1 group (p < 0.05) but not in the SSP-2 group. The stool weight in the SSP-2 group was significantly higher than that in SSP-1 (383.60 mg vs. 226.23 mg) (p < 0.05). Both SSP-1 and SSP-2 groups had significantly increased serum gastrin and motilin levels (p < 0.05) and changes in their fecal short-chain fatty acid (SCFA) profiles, while SSP-1 showed better fermentation properties than SSP-2 in terms of statistically higher fecal contents of acetic acid and total SCFAs (p < 0.05). Bioinformatic analysis indicated that SSP-1 upregulated bacteria such as Oscillibacter to improve SCFA metabolism and stimulate GI hormone secretion, while SSP-2 had less influence on the gut microbiota. These results suggest that the neutral polysaccharide with superior GI fermentation properties exerted beneficial effects on constipation, while the less fermentable pectic fraction might act as a stool-bulking agent.
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Estreñimiento , Loperamida , Ratones , Animales , Loperamida/efectos adversos , Estreñimiento/inducido químicamente , Estreñimiento/tratamiento farmacológico , Polisacáridos/efectos adversos , Ácidos Grasos Volátiles/análisis , Heces/microbiologíaRESUMEN
FBN1 mutation promotes the degeneration of microfibril structures and extracellular matrix (ECM) integrity in the tunica media of the aorta in Marfan syndrome. However, whether FBN1 modulates cervical artery dissection (CAD) development and the potential molecular mechanisms of abnormal FBN1 in CAD remains elusive. In this study, FBN1 deficiency participated in the development of CAD and influenced the proliferation, apoptosis, and migration of vascular smooth muscle cells. FBN1 knockout induced alternations in mRNA levels of the transcriptome, protein expression of the proteome, and abundance of N-glycosylation of the N-glycoproteome. Comprehensive analysis of multiple omics showed up-regulation in mRNA levels of ECM proteins; yet, both the ECM protein levels and relative abundance of N-glycosylation were decreased. Moreover, we performed in vivo experiments to confirm the altered glycosylation of proteins in vascular smooth muscle cells. In conclusion, FBN1 deletion in vascular smooth muscle cells can result in altered N-glycosylation of ECM protein, which were critical for the stability of ECM and the process of CAD. This may open the way for a novel therapeutic strategy to treat people with CAD.
Asunto(s)
Proteínas de la Matriz Extracelular , Fibrilina-1 , Músculo Liso Vascular , Animales , Ratas , Aorta/metabolismo , Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Fibrilina-1/genética , Fibrilina-1/metabolismo , Glicosilación , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , ARN Mensajero/metabolismoRESUMEN
The influence of steaming treatment on the soluble dietary fiber (SDF) of sweet potato was investigated. The SDF content increased from 2.21 to 4.04 g/100 g (in dry basis) during 20 min of steaming. The microcosmic morphology of the fractured cell wall indicated the release of SDF components during steaming. The SDF from fresh (SDF-F) and 20 min steamed (SDF-S) sweet potato was characterized. The neutral carbohydrates and uronic acid levels in SDF-S were significantly higher than SDF-F (59.31% versus 46.83%, and 25.36% versus 9.60%, respectively) (p < 0.05). The molecular weight of SDF-S was smaller than SDF-F (5.32 kDa versus 28.79 kDa). The probiotic property was evaluated by four Lactobacillus spp. fermentation in vitro with these SDF as carbon source, using inulin as the references. SDF-F showed the best proliferation effects on the four Lactobacillus spp. in terms of the OD600 and pH in cultures, and the highest production of propanoic acid and butyric acid after 24 h fermentation. SDF-S presented higher Lactobacillus proliferation effects, but slight lower propanoic acid and butyric acid production than inulin. It was concluded that 20 min of steaming released SDF with inferior probiotic properties, which might derive from the degraded pectin, cell wall components, and resistant dextrin.