RESUMEN
Nicaraven has been reported to inhibit the activity of poly (ADP-ribose) polymerase (PARP). In this study, we investigated the probable ability of nicaraven to attenuate cancer radioresistance during fractionated radiotherapy. Tumor models were established in C57BL/6 mice and BALB/c nude mice by subcutaneous injection of Lewis mouse lung carcinoma cancer cells and A549 human lung cancer cells, respectively. When the tumors had grown to approximately 100 mm3, we initiated fractionated radiotherapy. Nicaraven or saline was administered immediately after each irradiation exposure. Compared to saline treatment, nicaraven administration significantly induced gamma-H2AX foci formation and cell apoptosis in tumors at 1 or 3 days after an additional challenge exposure to 10 Gy and inhibited tumor growth during the short-term follow-up period, suggesting increased radiosensitivity of cancer cells. Moreover, the expression of PARP in tumor tissue was decreased by nicaraven administration. Our data suggest that nicaraven likely attenuates the acquired radioresistance of cancers through PARP inhibition.
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Histone acetyltransferases (HATs) and histone deacetylases (HDACs) conduct many critical functions through nonhistone substrates in metazoans, but only chromatin-associated nonhistone substrates are known in Saccharomyces cerevisiae. Using yeast proteome microarrays, we identified and validated many nonchromatin substrates of the essential nucleosome acetyltransferase of H4 (NuA4) complex. Among these, acetylation sites (Lys19 and 514) of phosphoenolpyruvate carboxykinase (Pck1p) were determined by tandem mass spectrometry. Acetylation at Lys514 was crucial for enzymatic activity and the ability of yeast cells to grow on nonfermentable carbon sources. Furthermore, Sir2p deacetylated Pck1p both in vitro and in vivo. Loss of Pck1p activity blocked the extension of yeast chronological life span caused by water starvation. In human hepatocellular carcinoma (HepG2) cells, human Pck1 acetylation and glucose production were dependent on TIP60, the human homolog of ESA1. Our findings demonstrate a regulatory function for the NuA4 complex in glucose metabolism and life span by acetylating a critical metabolic enzyme.
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Gluconeogénesis , Histona Acetiltransferasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetilación , Técnicas de Silenciamiento del Gen , Glucosa/metabolismo , Histona Acetiltransferasas/genética , Histona Desacetilasas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lisina Acetiltransferasa 5 , Complejos Multiproteicos/metabolismo , Fosfoenolpiruvato Carboxiquinasa (GTP)/metabolismo , Análisis por Matrices de Proteínas , Sirtuinas/metabolismo , Agua/metabolismoRESUMEN
BACKGROUND: Doxorubicin (DOX) is a first-line chemotherapeutic drug for various malignancies that causes cardiotoxicity. Plant-derived exosome-like nanovesicles (P-ELNs) are growing as novel therapeutic agents. Here, we investigated the protective effects in DOX cardiotoxicity of ELNs from Momordica charantia L. (MC-ELNs), a medicinal plant with antioxidant activity. RESULTS: We isolated MC-ELNs using ultracentrifugation and characterized them with canonical mammalian extracellular vesicles features. In vivo studies proved that MC-ELNs ameliorated DOX cardiotoxicity with enhanced cardiac function and myocardial structure. In vitro assays revealed that MC-ELNs promoted cell survival, diminished reactive oxygen species, and protected mitochondrial integrity in DOX-treated H9c2 cells. We found that DOX treatment decreased the protein level of p62 through ubiquitin-dependent degradation pathway in H9c2 and NRVM cells. However, MC-ELNs suppressed DOX-induced p62 ubiquitination degradation, and the recovered p62 bound with Keap1 promoting Nrf2 nuclear translocation and the expressions of downstream gene HO-1. Furthermore, both the knockdown of Nrf2 and the inhibition of p62-Keap1 interaction abrogated the cardioprotective effect of MC-ELNs. CONCLUSIONS: Our findings demonstrated the therapeutic beneficials of MC-ELNs via increasing p62 protein stability, shedding light on preventive approaches for DOX cardiotoxicity.
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Cardiotoxicidad , Doxorrubicina , Exosomas , Momordica charantia , Factor 2 Relacionado con NF-E2 , Animales , Cardiotoxicidad/prevención & control , Cardiotoxicidad/metabolismo , Momordica charantia/química , Exosomas/metabolismo , Ratas , Factor 2 Relacionado con NF-E2/metabolismo , Línea Celular , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Masculino , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Supervivencia Celular/efectos de los fármacos , Ratas Sprague-Dawley , Proteína Sequestosoma-1/metabolismoRESUMEN
Protein O-glycosylation, also known as mucin-type O-glycosylation, is one of the most abundant glycosylation in mammalian cells. It is initially catalyzed by a family of polypeptide GalNAc transferases (ppGalNAc-Ts). The trimeric spike protein (S) of SARS-CoV-2 is highly glycosylated and facilitates the virus's entry into host cells and membrane fusion of the virus. However, the functions and relationship between host ppGalNAc-Ts and O-glycosylation on the S protein remain unclear. Herein, we identify 15 O-glycosites and 10 distinct O-glycan structures on the S protein using an HCD-product-dependent triggered ETD mass spectrometric analysis. We observe that the isoenzyme T6 of ppGalNAc-Ts (ppGalNAc-T6) exhibits high O-glycosylation activity for the S protein, as demonstrated by an on-chip catalytic assay. Overexpression of ppGalNAc-T6 in HEK293 cells significantly enhances the O-glycosylation level of the S protein, not only by adding new O-glycosites but also by increasing O-glycan heterogeneity. Molecular dynamics simulations reveal that O-glycosylation on the protomer-interface regions, modified by ppGalNAc-T6, potentially stabilizes the trimeric S protein structure by establishing hydrogen bonds and non-polar interactions between adjacent protomers. Furthermore, mutation frequency analysis indicates that most O-glycosites of the S protein are conserved during the evolution of SARS-CoV-2 variants. Taken together, our finding demonstrate that host O-glycosyltransferases dynamically regulate the O-glycosylation of the S protein, which may influence the trimeric structural stability of the protein. This work provides structural insights into the functional role of specific host O-glycosyltransferases in regulating the O-glycosylation of viral envelope proteins.
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The majority of people in China have been immunized with the inactivated viral vaccine BBIBP-CorV. The emergence of the Omicron variant raised the concerns about protection efficacy of the inactivated viral vaccine in China. However, longitudinal neutralization data describing protection efficacy against Omicron variant is still lacking. Here we present one-year longitudinal neutralization data of BBIBP-CorV on authentic Omicron, Delta, and wild-type strains using 224 sera collected from 14 volunteers who have finished three doses BBIBP-CorV. The sera were also subjected for monitoring the SARS-CoV-2 specific IgG, IgA, and IgM responses on protein and peptide microarrays. The neutralization titers showed different protection efficacies against the three strains. By incorporating IgG and IgA signals of proteins and Spike protein derived peptide on microarray, panels as potential surrogate biomarkers for rapid estimation of neutralization titers were established. These data support the necessity of the 3rd dose of BBIBP-CorV vaccination. After further validation and assay development, the panels could be used for reliable, convenient and fast evaluation of the efficacy of vaccination.
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COVID-19 , Humanos , COVID-19/prevención & control , SARS-CoV-2 , Vacunas contra la COVID-19 , Inmunoglobulina G , Vacunación , Inmunoglobulina A , Anticuerpos AntiviralesRESUMEN
As a ubiquitous bacterial secondary messenger, c-di-GMP plays key regulatory roles in processes such as bacterial motility and transcription regulation. CobB is the Sir2 family protein deacetylase that controls energy metabolism, chemotaxis, and DNA supercoiling in many bacteria. Using an Escherichia coli proteome microarray, we found that c-di-GMP strongly binds to CobB. Further, protein deacetylation assays showed that c-di-GMP inhibits the activity of CobB and thereby modulates the biogenesis of acetyl-CoA. Interestingly, we also found that one of the key enzymes directly involved in c-di-GMP production, DgcZ, is a substrate of CobB. Deacetylation of DgcZ by CobB enhances its activity and thus the production of c-di-GMP. Our work establishes a novel negative feedback loop linking c-di-GMP biogenesis and CobB-mediated protein deacetylation.
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GMP Cíclico/análogos & derivados , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Liasas de Fósforo-Oxígeno/metabolismo , Sirtuinas/metabolismo , Acetilcoenzima A/metabolismo , Acetilación , GMP Cíclico/metabolismo , Retroalimentación Fisiológica , Regulación Bacteriana de la Expresión Génica , Análisis por Matrices de Proteínas/métodos , Proteómica/métodos , Sistemas de Mensajero SecundarioRESUMEN
Biomechanical forces are known to regulate the biological behaviors of cells. Although negative pressure has been used for wound healing, it is still unknown about its role in regulating cell plasticity. We investigated whether negative pressure could induce the dedifferentiation of hepatocytes. Using a commercial device, we found that the exposure of primary human hepatocytes to -50 mmHg quickly induced the formation of stress fibers and obviously changed cell morphology in 72 h. Moreover, the exposure of hepatocytes to -50 mmHg significantly upregulated RhoA, ROCK1, and ROCK2 in 1-6 h, and dramatically enhanced the expression of marker molecules on "stemness", such as OCT4, SOX2, KLF4, MYC, NANOG, and CD133 in 6-72 h. However, all these changes in hepatocytes induced by -50 mmHg stimulation were almost abrogated by ROCK inhibitor Y27623. Our data suggest that an appropriate force of negative pressure stimulation can effectively induce the dedifferentiation of hepatocytes via RhoA/ROCK pathway activation.
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Desdiferenciación Celular , Hepatocitos , Proteína de Unión al GTP rhoA , Humanos , Hepatocitos/metabolismo , Quinasas Asociadas a rho/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Transducción de Señal , Desdiferenciación Celular/genética , Desdiferenciación Celular/fisiologíaRESUMEN
Pleurodeles waltl is coming to light as a model animal, especially in regeneration studies, but deep studies on the molecular mechanisms have been limited due to the absence of primary tissue cells for wide usage. Therefore, we aimed to grow primary cells from limb tissue of P. waltl for in vitro experiments. Limb tissues were cut into small pieces and seeded as "explants" on culture dishes coated with fibronectin and gelatin. Compared to the control without coating, both fibronectin and gelatin supported quicker outgrowth of cells from explants and faster cell adhesion, and fibronectin showed significantly better performance than gelatin. Interestingly, the doubling time of cells on fibronectin- and gelatin-coated surfaces was almost the same (42.39 ± 2.79 h vs. 42.91 ± 3.69 h) and was not significantly different from that on non-coated plates (49.64 ± 3.63 h). The cryopreserved cells were successfully recovered and showed a multiplication capacity that was similar to that of fresh cells. Senescent cells were barely detected even after long-term sub-culture (>15 passages). Moreover, enhanced fluorescence of MitoSOX™ Red in cells under H2 O2 exposure confirmed the respondence to chemical stimuli. Collectively, our results show that we are able to grow enough good-quality cells from P. waltl limb tissue for in vitro experiments, and fibronectin coating provides the best biocompatible environment for cell outgrowth and attachment.
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Fibronectinas , Pleurodeles , Animales , Fibronectinas/farmacología , Fibronectinas/metabolismo , Pleurodeles/metabolismo , Gelatina/farmacología , Gelatina/metabolismoRESUMEN
Angiotensin receptor blockers (ARBs) have been reported to be beneficial of renal fibrosis, but the molecular and cellular mechanisms are still unclear. In this study, we investigated the effectiveness and relevant mechanism of ARBs in alleviating renal fibrosis, especially by focusing on biomechanical stress-induced epithelial to mesenchymal transition (EMT) of renal epithelial cells. Unilateral ureteral obstruction (UUO) renal fibrosis model was established in mice by ligating the left ureter, and then randomly received losartan at a low dose (1 mg/kg) or a regular dose (3 mg/kg) for 2 weeks. Compared to the control, histological analysis showed that losartan treatment at either a low dose or a regular dose effectively attenuated renal fibrosis in the UUO model. To further understand the mechanism, we ex vivo loaded primary human renal epithelial cells to 50 mmHg hydrostatic pressure. Western blot and immunostaining analyses indicated that the loading to 50 mmHg hydrostatic pressure for 24 h significantly upregulated vimentin, ß-catenin and α-SMA, but downregulated E-cadherin in renal epithelial cells, suggesting the EMT. The addition of 10 or 100 nM losartan in medium effectively attenuated the EMT of renal epithelial cells induced by 50 mmHg hydrostatic pressure loading. Our in vivo and ex vivo experimental data suggest that losartan treatment, even at a low dose can effectively alleviate renal fibrosis in mouse UUO model, at least partly by inhibiting the biomechanical stress-induced EMT of renal epithelial cells. A low dose of ARBs may repurpose for renal fibrosis treatment.
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Enfermedades Renales , Obstrucción Ureteral , Humanos , Ratones , Animales , Transición Epitelial-Mesenquimal , Losartán/farmacología , Losartán/uso terapéutico , Antagonistas de Receptores de Angiotensina/farmacología , Antagonistas de Receptores de Angiotensina/uso terapéutico , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Inhibidores de la Enzima Convertidora de Angiotensina/uso terapéutico , Enfermedades Renales/patología , Obstrucción Ureteral/complicaciones , Obstrucción Ureteral/tratamiento farmacológico , Células Epiteliales/patología , Fibrosis , Factor de Crecimiento Transformador beta1/farmacologíaRESUMEN
Rhizoctonia solani and Xanthomonas oryzae pv. oryzae (Xoo) are the two major diseases affecting the quality and quantity of rice production. In the current study, volatile organic compounds (VOCs) of Bacillus spp. were used as green biocontrol agents for plant diseases. In in vitro experiments, Bacillus spp. FZB42, NMTD17, and LLTC93-VOCs displayed strong antimicrobial volatile activity with inhibition rates of 76, 66, and 78% for R. solani and 78, 81, and 76% for Xoo, respectively, compared to control. The synthetic VOCs, namely Pentadecane (PDC), Benzaldehyde (BDH), 1,2-Benz isothiazol-3(2H)-one (1,2-BIT), and mixture (MIX) of VOCs showed high volatile activity with inhibition rates of 86, 86, 89, and 92% against R. solani and 81, 81, 82, and 86%, respectively, against Xoo as compared to control. In addition, the scanning and transmission electron microscopes (SEM and TEM) analyses were performed to examine the effect of Bacillus and synthetic VOC treatments on R. solani and Xoo morphology. The analysis revealed the deformed and irregularized morphology of R. solani mycelia and Xoo cells after VOC treatments. The microscopic analysis showed that the rapid inhibition was due to severe oxidative productions inside the R. solani mycelia and Xoo cells. By using molecular docking, it was determined that the synthetic VOCs entered the active binding site of trehalase and NADH dehydrogenase proteins, causing R. solani and Xoo cells to die prematurely and an accumulation of ROS. In the greenhouse experiment, FZB42, NMTD17, and LLTC93-VOCs significantly reduced the lesions of R. solani 8, 7, and 6 cm, and Xoo 7, 6, and 6 cm, respectively, then control. The synthetic VOCs demonstrated that the PDC, BDH, 1,2-BIT, and MIX-VOCs significantly reduced R. solani lesions on leaves 6, 6, 6, and 5 cm and Xoo 6, 5, 5, and 4 cm, respectively, as compared to control. Furthermore, plant defence-related genes and antioxidant enzymes were upregulated in rice plants. These findings provide novel mechanisms by which Bacillus antimicrobial VOCs control plant diseases.
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Antiinfecciosos , Bacillus , Oryza , Compuestos Orgánicos Volátiles , Xanthomonas , Compuestos Orgánicos Volátiles/farmacología , Compuestos Orgánicos Volátiles/metabolismo , Simulación del Acoplamiento Molecular , Enfermedades de las Plantas/genética , Oryza/metabolismo , Antiinfecciosos/metabolismo , Antiinfecciosos/farmacologíaRESUMEN
Antibodies play essential roles in both diagnostics and therapeutics. Epitope mapping is essential to understand how an antibody works and to protect intellectual property. Given the millions of antibodies for which epitope information is lacking, there is a need for high-throughput epitope mapping. To address this, we developed a strategy, Antibody binding epitope Mapping (AbMap), by combining a phage displayed peptide library with next-generation sequencing. Using AbMap, profiles of the peptides bound by 202 antibodies were determined in a single test, and linear epitopes were identified for >50% of the antibodies. Using spike protein (S1 and S2)-enriched antibodies from the convalescent serum of one COVID-19 patient as the input, both linear and potentially conformational epitopes of spike protein specific antibodies were identified. We defined peptide-binding profile of an antibody as the binding capacity (BiC). Conceptually, the BiC could serve as a systematic and functional descriptor of any antibody. Requiring at least one order of magnitude less time and money to map linear epitopes than traditional technologies, AbMap allows for high-throughput epitope mapping and creates many possibilities.
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COVID-19/inmunología , Mapeo Epitopo/métodos , Glicoproteína de la Espiga del Coronavirus/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Anticuerpos Antivirales/metabolismo , Ensayo de Inmunoadsorción Enzimática , Epítopos/metabolismo , Proteínas de Escherichia coli/inmunología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Sueros Inmunes/sangre , Sueros Inmunes/inmunología , Biblioteca de PéptidosRESUMEN
BACKGROUND: Mechanical ventilation is a supportive therapy used to maintain respiratory function in several clinical and surgical cases but is always accompanied by lung injury risk due to improper treatment. We investigated how tidal volume and oxygen delivery would contribute independently or synergistically to ventilator-induced lung injury (VILI). METHODS: Under general anesthesia and tracheal intubation, healthy female C57BL/6 N mice (9 weeks old) were randomly ventilated for 2 h by standard (7 ml/kg) or high (14 ml/kg) tidal volume at positive end-expiratory pressure (PEEP) of 2 cmH2O, with room air, 50% O2 (moderate hyperoxia), or 100% O2 (severe hyperoxia); respectively. Mice were sacrificed 4 h after mechanical ventilation, and lung tissues were collected for experimental assessments on lung injury. RESULTS: Compared with the healthy control, severe hyperoxia ventilation by either standard or high tidal volume resulted in significantly higher wet-to-dry lung weight ratio and higher levels of IL-1ß and 8-OHdG in the lungs. However, moderate hyperoxia ventilation, even by high tidal volume did not significantly increase the levels of IL-1ß and 8-OHdG in the lungs. Western blot analysis showed that the expression of RhoA, ROCK1, MLC2, and p-MLC2 was not significantly induced in the ventilated lungs, even by high tidal volume at 2 cmH2O PEEP. CONCLUSION: Severe hyperoxia ventilation causes inflammatory response and oxidative damage in mechanically ventilated lungs, while high tidal volume ventilation at a reasonable PEEP possibly does not cause VILI.
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Hiperoxia , Lesión Pulmonar Inducida por Ventilación Mecánica , Femenino , Animales , Ratones , Ratones Endogámicos C57BL , Volumen de Ventilación Pulmonar , Hiperoxia/complicaciones , Respiración , 8-Hidroxi-2'-DesoxicoguanosinaRESUMEN
CRISPR-based detection technologies have been widely explored for molecular diagnostics. However, the challenge lies in converting the signal of different biomolecules, such as nucleic acids, proteins, small molecules, exosomes, and ions, into a CRISPR-based nucleic acid detection signal. Understanding the detection of different biomolecules using CRISPR technology can aid in the development of practical and promising detection approaches. Unfortunately, existing reviews rarely provide an overview of CRISPR-based molecular diagnostics from the perspective of different biomolecules. Herein, we first introduce the principles and characteristics of various CRISPR nucleases for molecular diagnostics. Then, we focus on summarizing and evaluating the latest advancements in CRISPR-based detection of different biomolecules. Through a comparison of different methods of amplification and signal readout, we discuss how general detection methods can be integrated with CRISPR. Finally, we conclude by identifying opportunities for the improvement of CRISPR in quantitative, amplification-free, multiplex, all-in-one, and point-of-care testing (POCT) purposes.
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Exosomas , Ácidos Nucleicos , Exosomas/genética , Ácidos Nucleicos/genética , Endonucleasas , Sistemas CRISPR-Cas/genéticaRESUMEN
Retinoic acid-inducible gene I (RIG-I) is up-regulated during granulocytic differentiation of acute promyelocytic leukemia (APL) cells induced by all-trans retinoic acid (ATRA). It has been reported that RIG-I recognizes virus-specific 5'-ppp-double-stranded RNA (dsRNA) and activates the type I interferons signaling pathways in innate immunity. However, the functions of RIG-I in hematopoiesis remain unclear, especially regarding its possible interaction with endogenous RNAs and the associated pathways that could contribute to the cellular differentiation and maturation. Herein, we identified a number of RIG-I-binding endogenous RNAs in APL cells following ATRA treatment, including the tripartite motif-containing protein 25 (TRIM25) messenger RNA (mRNA). TRIM25 encodes the protein known as an E3 ligase for ubiquitin/interferon (IFN)-induced 15-kDa protein (ISG15) that is involved in RIG-I-mediated antiviral signaling. We show that RIG-I could bind TRIM25 mRNA via its helicase domain and C-terminal regulatory domain, enhancing the stability of TRIM25 transcripts. RIG-I could increase the transcriptional expression of TRIM25 by caspase recruitment domain (CARD) domain through an IFN-stimulated response element. In addition, RIG-I activated other key genes in the ISGylation pathway by activating signal transducer and activator of transcription 1 (STAT1), including the modifier ISG15 and several enzymes responsible for the conjugation of ISG15 to protein substrates. RIG-I cooperated with STAT1/2 and interferon regulatory factor 1 (IRF1) to promote the activation of the ISGylation pathway. The integrity of ISGylation in ATRA or RIG-I-induced cell differentiation was essential given that knockdown of TRIM25 or ISG15 resulted in significant inhibition of this process. Our results provide insight into the role of the RIG-I-TRIM25-ISGylation axis in myeloid differentiation.
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Diferenciación Celular , Citocinas/metabolismo , Proteína 58 DEAD Box/metabolismo , Granulocitos/fisiología , Factores de Transcripción/metabolismo , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinas/metabolismo , Línea Celular Tumoral , Citocinas/genética , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Estabilidad del ARN , ARN Mensajero/metabolismo , Receptores Inmunológicos , Factores de Transcripción/genética , Proteínas de Motivos Tripartitos/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitinas/genética , Regulación hacia ArribaRESUMEN
The synthesis of bench-stable conjugated π-radicals is challenging owing to the lack of modular approaches, which greatly hampers their practical material screens and applications. Here, we demonstrate a spin-distribution-directed regioselective substitution strategy to introduce substituents into the specific positions of an olympicenyl radical in a stepwise manner, resulting in a series of highly stable radical species. The substituents can also adjust the crystal packing by means of steric and electronic factors, enabling the changing from a π-dimer to a pseudo-one-dimensional chain. The first single crystal organic field-effect transistor device based on a graphenic radical is fabricated in air, showing a hole mobility of up to 0.021â cm2 V-1 s-1 and excellent device stability. This approach may be generalized to diverse spin-delocalized open-shell organic radicals.
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Mechanical forces can modulate the immune response, mostly described as promoting the activation of immune cells, but the role and mechanism of pathological levels of mechanical stress in lymphocyte activation have not been focused on before. By an ex vivo experimental approach, we observed that mechanical stressing of murine spleen lymphocytes with 50 mmHg for 3 h induced the nuclear localization of NFAT1, increased C-Jun, and increased the expression of early activation marker CD69 in resting CD8+ cells. Interestingly, 50 mmHg mechanical stressing induced the nuclear localization of NFAT1; but conversely decreased C-Jun and inhibited the expression of CD69 in lymphocytes under lipopolysaccharide or phorbol 12-myristate 13-acetate/ionomycin stimulation. Additionally, we observed similar changes trends when comparing RNA-seq data of hypertensive and normotensive COVID-19 patients. Our results indicate a biphasic effect of mechanical stress on lymphocyte activation, which provides insight into the variety of immune responses in pathologies involving elevated mechanical stress.
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Activación de Linfocitos/inmunología , Estrés Mecánico , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/metabolismo , Biomarcadores/metabolismo , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , COVID-19/complicaciones , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Comorbilidad , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Hipertensión/complicaciones , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Canales Iónicos/metabolismo , Lectinas Tipo C/metabolismo , Lipopolisacáridos/farmacología , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/genética , Masculino , Ratones Endogámicos C57BL , Factores de Transcripción NFATC/metabolismo , Transporte de Proteínas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-jun/metabolismo , Transducción de Señal/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Angiotensin receptor blockers have been reported to be beneficial to liver fibrosis, but the relevant molecular and cellular mechanisms remain unclear. We herein investigated whether low-dose angiotensin receptor blocker alleviated liver fibrosis through mechanotransduction regulation. Hydrostatic pressure-induced liver fibrosis model was established in mice by ligating partially the inferior vena cava, and then randomly received a very low dose of losartan (0.5 mg/kg) or placebo treatment for 8 weeks. We found that losartan administration interfered the expression of several mechanotransductive molecules, and effectively alleviated liver fibrosis. Using a commercial device, we further confirmed that ex vivo loading of hepatic stellate cells to 50 mmHg hydrostatic pressure for 24 h significantly upregulated RhoA, ROCK, AT1R, and p-MLC2, which was effectively attenuated by adding 10 nM losartan in medium. Our in vivo and ex vivo experimental data suggest that low-dose angiotensin receptor blockers may alleviate hydrostatic pressure-induced liver fibrosis by altering the mechanotransduction properties of hepatic stellate cells.NEW & NOTEWORTHY Our ex vivo and in vivo experiments clearly indicated that low-dose losartan alleviated liver fibrosis, likely by modulating the mechanotransduction properties of HSCs. Uncovering the biomechanical signaling pathway of ARB treatment on liver fibrosis will be helpful to develop novel molecular targeting therapy for liver diseases.
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Antagonistas de Receptores de Angiotensina , Células Estrelladas Hepáticas , Antagonistas de Receptores de Angiotensina/metabolismo , Antagonistas de Receptores de Angiotensina/farmacología , Antagonistas de Receptores de Angiotensina/uso terapéutico , Inhibidores de la Enzima Convertidora de Angiotensina/metabolismo , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Inhibidores de la Enzima Convertidora de Angiotensina/uso terapéutico , Animales , Células Estrelladas Hepáticas/metabolismo , Hígado/metabolismo , Cirrosis Hepática/metabolismo , Losartán/farmacología , Losartán/uso terapéutico , Mecanotransducción Celular , RatonesRESUMEN
BACKGROUND: Mounting evidence has revealed the dynamic variations in the cellular status and phenotype of the smooth muscle cell (SMC) are vital for shaping the atherosclerotic plaque microenvironment and ultimately mapping onto heterogeneous clinical outcomes in coronary artery disease. Currently, the underlying clinical significance of SMC evolutions remains unexplored in atherosclerosis. METHODS: The dissociated cells from diseased segments within the right coronary artery of four cardiac transplant recipients and 1070 bulk samples with atherosclerosis from six bulk cohorts were retrieved. Following the SMC fate trajectory reconstruction, the MOVICS algorithm integrating the nearest template prediction was used to develop a stable and robust molecular classification. Subsequently, multi-dimensional potential biological implications, molecular features, and cell landscape heterogeneity among distinct clusters were decoded. RESULTS: We proposed an SMC cell fate decision signature (SCFDS)-based atherosclerosis stratification system and identified three SCFDS subtypes (C1-C3) with distinguishing features: (i) C1 (DNA-damage repair type), elevated base excision repair (BER), DNA replication, as well as oxidative phosphorylation status. (ii) C2 (immune-activated type), stronger immune activation, hyper-inflammatory state, the complex as well as varied lesion microenvironment, advanced stage, the most severe degree of coronary stenosis severity. (iii) C3 (stromal-rich type), abundant fibrous content, stronger ECM metabolism, immune-suppressed microenvironment. CONCLUSIONS: This study uncovered atherosclerosis complex cellular heterogeneity and a differentiated hierarchy of cell populations underlying SMC. The novel high-resolution stratification system could improve clinical outcomes and facilitate individualized management.
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Miocitos del Músculo LisoRESUMEN
Type 2 diabetes mellitus (T2DM) is recognized as a serious public health concern with increasing incidence. The dipeptidyl peptidase-4 (DPP-4) inhibitor sitagliptin has been used for the treatment of T2DM worldwide. Although sitagliptin has excellent therapeutic outcome, adverse effects are observed. In addition, previous studies have suggested that sitagliptin may have pleiotropic effects other than treating T2DM. These pieces of evidence point to the importance of further investigation of the molecular mechanisms of sitagliptin, starting from the identification of sitagliptin-binding proteins. In this study, by combining affinity purification mass spectrometry (AP-MS) and stable isotope labeling by amino acids in cell culture (SILAC), we discover seven high-confidence targets that can interact with sitagliptin. Surface plasmon resonance (SPR) assay confirms the binding of sitagliptin to three proteins, i. e., LYPLAL1, TCP1, and CCAR2, with binding affinities (K D) ranging from 50.1â µM to 1490â µM. Molecular docking followed by molecular dynamic (MD) simulation reveals hydrogen binding between sitagliptin and the catalytic triad of LYPLAL1, and also between sitagliptin and the P-loop of ATP-binding pocket of TCP1. Molecular mechanics Poisson-Boltzmann Surface Area (MMPBSA) analysis indicates that sitagliptin can stably bind to LYPLAL1 and TCP1 in active sites, which may have an impact on the functions of these proteins. SPR analysis validates the binding affinity of sitagliptin to TCP1 mutant D88A is ~10 times lower than that to the wild-type TCP1. Our findings provide insights into the sitagliptin-targets interplay and demonstrate the potential of sitagliptin in regulating gluconeogenesis and in anti-tumor drug development.
Asunto(s)
Diabetes Mellitus Tipo 2 , Inhibidores de la Dipeptidil-Peptidasa IV , Fosfato de Sitagliptina , Humanos , Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras , Diabetes Mellitus Tipo 2/inducido químicamente , Inhibidores de la Dipeptidil-Peptidasa IV/farmacología , Hipoglucemiantes/farmacología , Simulación del Acoplamiento Molecular , Fosfato de Sitagliptina/farmacologíaRESUMEN
Excessive mechanical stress causes fibrosis-related atrial arrhythmia. Herein, we tried to investigate the mechanism of atrial fibrogenesis in response to mechanical stress by ex vivo approach. We collected atrial tissues from mice and then cultured them as "explants" under atmospheric pressure (AP group) or 50 mmHg hydrostatic pressure loading (HP group) conditions. Pathway-specific PCR array analysis on the expression of fibrosis-related genes indicated that the loading of atrial tissues to 50 mmHg for 24 hours extensively upregulated a series of profibrotic genes. qRT-PCR data also showed that loading atrial tissues to 50 mmHg enhanced Rhoa, Rock2, and Thbs1 expression at different time points. Interestingly, the enhanced expression of Thbs1 at 1 hour declined at 6-24 hours and then increased again at 72 hours. In contrast, an enhanced expression of Tgfb1 was observed at 72 hours. In contrast, daily loading to 50 mmHg for 3 hours significantly accelerated the outgrowth of mesenchymal stem-like stromal cells from atrial tissues; however, we did not observe significant phenotypic changes in these outgrowing cells. Our ex vivo experimental data clearly show the induction of profibrotic transcription of atrial tissues by HP loading, which confirms the common pathological feature of atrial fibrosis following pressure overload.