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The endoplasmic reticulum (ER) is selectively degraded by ER-phagy to maintain cell homeostasis. α-synuclein accumulates in the ER, causing ER stress that contributes to neurodegeneration in Parkinson's disease (PD), but the role of ER-phagy in α-synuclein modulation is largely unknown. Here, we investigated the mechanisms by which ER-phagy selectively recognizes α-synuclein for degradation in the ER. We found that ER-phagy played an important role in the degradation of α-synuclein and recovery of ER function through interaction with FAM134B, where calnexin is required for the selective FAM134B-mediated α-synuclein clearance via ER-phagy. Overexpression of α-synuclein in the ER of the substantia nigra (SN) resulted in marked loss of dopaminergic neurons and motor deficits, mimicking PD characteristics. However, enhancement of ER-phagy using FAM134B overexpression in the SN exerted neuroprotective effects on dopaminergic neurons and recovered motor performance. These data suggest that ER-phagy represents a specific ER clearance mechanism for the degradation of α-synuclein.
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Fármacos Neuroprotetores , Doença de Parkinson , Humanos , alfa-Sinucleína , Retículo Endoplasmático , AutofagiaRESUMO
Single-cell RNA-seq has led to novel designations for mesenchymal cells associated with bone as well as multiple designations for what appear to be the same cell type. The main goals of this study were to increase the amount of single-cell RNA sequence data for osteoblasts and osteocytes, to compare cells from the periosteum to those inside bone, and to clarify the major categories of cell types associated with murine bone. We created an atlas of murine bone-associated cells by harmonizing published datasets with in-house data from cells targeted by Osx1-Cre and Dmp1-Cre driver strains. Cells from periosteal bone were analyzed separately from those isolated from the endosteum and trabecular bone. Over 100,000 mesenchymal cells were mapped to reveal 11 major clusters designated fibro-1, fibro-2, chondrocytes, articular chondrocytes, tenocytes, adipo-Cxcl12 abundant reticular (CAR), osteo-CAR, preosteoblasts, osteoblasts, osteocytes, and osteo-X, the latter defined in part by periostin expression. Osteo-X, osteo-CAR, and preosteoblasts were closely associated with osteoblasts at the trabecular bone surface. Wnt16 was expressed in multiple cell types from the periosteum but not in cells from endocortical or cancellous bone. Fibro-2 cells, which express markers of stem cells, localized to the periosteum but not trabecular bone in adult mice. Suppressing bone remodeling eliminated osteoblasts and altered gene expression in preosteoblasts but did not change the abundance or location of osteo-X or osteo-CAR cells. These results provide a framework for identifying bone cell types in murine single-cell RNA-seq datasets and suggest that osteoblast progenitors reside near the surface of remodeling bone.
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Células-Tronco Mesenquimais , Osteoblastos , Osteócitos , Periósteo , Animais , Camundongos , Condrócitos/metabolismo , Condrócitos/citologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Osteoblastos/metabolismo , Osteoblastos/citologia , Osteócitos/metabolismo , Osteócitos/citologia , Periósteo/citologia , Periósteo/metabolismo , Análise de Célula Única , Camundongos Endogâmicos C57BLRESUMO
MOTIVATION: Prediction of T-cell receptor (TCR)-epitope interactions is important for many applications in biomedical research, such as cancer immunotherapy and vaccine design. The prediction of TCR-epitope interactions remains challenging especially for novel epitopes, due to the scarcity of available data. RESULTS: We propose TSpred, a new deep learning approach for the pan-specific prediction of TCR binding specificity based on paired chain TCR data. We develop a robust model that generalizes well to unseen epitopes by combining the predictive power of CNN and the attention mechanism. In particular, we design a reciprocal attention mechanism which focuses on extracting the patterns underlying TCR-epitope interactions. Upon a comprehensive evaluation of our model, we find that TSpred achieves state-of-the-art performances in both seen and unseen epitope specificity prediction tasks. Also, compared to other predictors, TSpred is more robust to bias related to peptide imbalance in the dataset. In addition, the reciprocal attention component of our model allows for model interpretability by capturing structurally important binding regions. Results indicate that TSpred is a robust and reliable method for the task of TCR-epitope binding prediction. AVAILABILITY AND IMPLEMENTATION: Source code is available at https://github.com/ha01994/TSpred.
Assuntos
Receptores de Antígenos de Linfócitos T , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/imunologia , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/química , Epitopos de Linfócito T/metabolismo , Humanos , Aprendizado Profundo , Biologia Computacional/métodos , Software , Ligação ProteicaRESUMO
An ideal cancer therapeutic strategy involves the selective killing of cancer cells without affecting the surrounding normal cells. However, researchers have failed to develop such methods for achieving selective cancer cell death because of shared features between cancerous and normal cells. In this study, we have developed a therapeutic strategy called the cancer-specific insertions-deletions (InDels) attacker (CINDELA) to selectively induce cancer cell death using the CRISPR-Cas system. CINDELA utilizes a previously unexplored idea of introducing CRISPR-mediated DNA double-strand breaks (DSBs) in a cancer-specific fashion to facilitate specific cell death. In particular, CINDELA targets multiple InDels with CRISPR-Cas9 to produce many DNA DSBs that result in cancer-specific cell death. As a proof of concept, we demonstrate here that CINDELA selectively kills human cancer cell lines, xenograft human tumors in mice, patient-derived glioblastoma, and lung patient-driven xenograft tumors without affecting healthy human cells or altering mouse growth.
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Sistemas CRISPR-Cas , Mutação INDEL , Neoplasias/genética , Animais , Morte Celular/genética , Quebras de DNA de Cadeia Dupla , Xenoenxertos , Humanos , CamundongosRESUMO
We demonstrate excitatory and inhibitory properties in a single heterostructure consisting of two quantum dots/graphene synaptic elements using linearly polarized monochromatic light. Perovskite quantum dots and PbS quantum dots were used to increase and decrease photocurrent weights, respectively. The polarization-dependent photocurrent was realized by adding a polarizer in the middle of the PbS quantum dots/graphene and perovskite quantum dots/graphene elements. When linearly polarized light passed through the polarizer, both the lower excitatory and upper inhibitory devices were activated, with the lower device with the stronger response dominating to increase the current weight. In contrast, the polarized light was blocked by the polarizer, and the above device was only operated, reducing the current weight. Furthermore, two orthogonal polarizations of light were used to perform the sequential processes of potentiation and habituation. By adjustment of the polarization angle of light, not only the direction of the current weight but also its level was altered.
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Advanced microelectronics in the future may require semiconducting channel materials beyond silicon. Two-dimensional (2D) semiconductors, with their atomically thin thickness, hold great promise for future electronic devices. One challenge to achieving high-performance 2D semiconductor field effect transistors (FET) is the high contact resistance at the metal-semiconductor interface. In this study, we develop a charge-transfer doping strategy with WSe2/α-RuCl3 heterostructures to achieve low-resistance ohmic contact for p-type monolayer WSe2 transistors. We show that hole doping as high as 3 × 1013 cm-2 can be achieved in the WSe2/α-RuCl3 heterostructure due to its type-III band alignment, resulting in an ohmic contact with resistance of 4 kΩ µm. Based on that, we demonstrate p-type WSe2 transistors with an on-current of 35 µA·µm-1 and an ION/IOFF ratio exceeding 109 at room temperature.
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Hem1 (hematopoietic protein 1), a hematopoietic cell-specific member of the Hem family of cytoplasmic adaptor proteins, is essential for lymphopoiesis and innate immunity as well as for the transition of hematopoiesis from the fetal liver to the bone marrow. However, the role of Hem1 in bone cell differentiation and bone remodeling is unknown. Here, we show that deletion of Hem1 resulted in a markedly increase in bone mass because of defective bone resorption in mice of both sexes. Hem1-deficient osteoclast progenitors were able to differentiate into osteoclasts, but the osteoclasts exhibited impaired osteoclast fusion and decreased bone-resorption activity, potentially because of decreased mitogen-activated protein kinase and tyrosine kinase c-Abl activity. Transplantation of bone marrow hematopoietic stem and progenitor cells from wildtype into Hem1 knockout mice increased bone resorption and normalized bone mass. These findings indicate that Hem1 plays a pivotal role in the maintenance of normal bone mass.
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Proteínas Adaptadoras de Transdução de Sinal , Reabsorção Óssea , Osteoclastos , Animais , Feminino , Masculino , Camundongos , Reabsorção Óssea/genética , Reabsorção Óssea/metabolismo , Diferenciação Celular , Hematopoese , Transplante de Células-Tronco Hematopoéticas , Camundongos Knockout , Osteoclastos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismoRESUMO
BACKGROUND: Activation of vascular smooth muscle cell (VSMC) inflammation is vital to initiate vascular disease. The role of human-specific long noncoding RNAs in VSMC inflammation is poorly understood. METHODS: Bulk RNA sequencing in differentiated human VSMCs revealed a novel human-specific long noncoding RNA called inflammatory MKL1 (megakaryoblastic leukemia 1) interacting long noncoding RNA (INKILN). INKILN expression was assessed in multiple in vitro and ex vivo models of VSMC phenotypic modulation as well as human atherosclerosis and abdominal aortic aneurysm. The transcriptional regulation of INKILN was verified through luciferase reporter and chromatin immunoprecipitation assays. Loss-of-function and gain-of-function studies and multiple RNA-protein and protein-protein interaction assays were used to uncover a mechanistic role of INKILN in the VSMC proinflammatory gene program. Bacterial artificial chromosome transgenic mice were used to study INKILN expression and function in ligation injury-induced neointimal formation. RESULTS: INKILN expression is downregulated in contractile VSMCs and induced in human atherosclerosis and abdominal aortic aneurysm. INKILN is transcriptionally activated by the p65 pathway, partially through a predicted NF-κB (nuclear factor kappa B) site within its proximal promoter. INKILN activates proinflammatory gene expression in cultured human VSMCs and ex vivo cultured vessels. INKILN physically interacts with and stabilizes MKL1, a key activator of VSMC inflammation through the p65/NF-κB pathway. INKILN depletion blocks interleukin-1ß-induced nuclear localization of both p65 and MKL1. Knockdown of INKILN abolishes the physical interaction between p65 and MKL1 and the luciferase activity of an NF-κB reporter. Furthermore, INKILN knockdown enhances MKL1 ubiquitination through reduced physical interaction with the deubiquitinating enzyme USP10 (ubiquitin-specific peptidase 10). INKILN is induced in injured carotid arteries and exacerbates ligation injury-induced neointimal formation in bacterial artificial chromosome transgenic mice. CONCLUSIONS: These findings elucidate an important pathway of VSMC inflammation involving an INKILN/MKL1/USP10 regulatory axis. Human bacterial artificial chromosome transgenic mice offer a novel and physiologically relevant approach for investigating human-specific long noncoding RNAs under vascular disease conditions.
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Aneurisma da Aorta Abdominal , RNA Longo não Codificante , Animais , Humanos , Camundongos , Aneurisma da Aorta Abdominal/metabolismo , Proliferação de Células , Células Cultivadas , Inflamação/genética , Inflamação/metabolismo , Luciferases/metabolismo , Camundongos Transgênicos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , NF-kappa B/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Ubiquitina Tiolesterase/metabolismoRESUMO
Acylglycerophosphate acyltransferases (AGPATs) catalyze the de novo formation of phosphatidic acid to synthesize glycerophospholipids and triglycerides. AGPATs demonstrate unique physiological roles despite a similar biochemical function. AGPAT3 is highly expressed in the testis, kidney, and liver, with intermediate expression in adipose tissue. Loss of AGPAT3 is associated with reproductive abnormalities and visual dysfunction. However, the role of AGPAT3 in adipose tissue and whole body metabolism has not been investigated. We found that male Agpat3 knockout (KO) mice exhibited reduced body weights with decreased white and brown adipose tissue mass. Such changes were less pronounced in the female Agpat3-KO mice. Agpat3-KO mice have reduced plasma insulin growth factor 1 (IGF1) and insulin levels and diminished circulating lipid metabolites. They manifested intact glucose homeostasis and insulin sensitivity despite a lean phenotype. Agpat3-KO mice maintained an energy balance with normal food intake, energy expenditure, and physical activity, except for increased water intake. Their adaptive thermogenesis was also normal despite reduced brown adipose mass and triglyceride content. Mechanistically, Agpat3 was elevated during mouse and human adipogenesis and enriched in adipocytes. Agpat3-knockdown 3T3-L1 cells and Agpat3-deficient mouse embryonic fibroblasts (MEFs) have impaired adipogenesis in vitro. Interestingly, pioglitazone treatment rescued the adipogenic deficiency in Agpat3-deficient cells. We conclude that AGPAT3 regulates adipogenesis and adipose development. It is possible that adipogenic impairment in Agpat3-deficient cells potentially leads to reduced adipose mass. Findings from this work support the unique role of AGPAT3 in adipose tissue.NEW & NOTEWORTHY AGPAT3 deficiency results in male-specific growth retardation. It reduces adipose tissue mass but does not significantly impact glucose homeostasis or energy balance, except for influencing water intake in mice. Like AGPAT2, AGPAT3 is upregulated during adipogenesis, potentially by peroxisome proliferator-activated receptor gamma (PPARγ). Loss of AGPAT3 impairs adipocyte differentiation, which could be rescued by pioglitazone. Overall, AGPAT3 plays a significant role in regulating adipose tissue mass, partially involving its influence on adipocyte differentiation.
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1-Acilglicerol-3-Fosfato O-Aciltransferase , Adipócitos , Camundongos Knockout , Animais , Feminino , Masculino , Camundongos , 1-Acilglicerol-3-Fosfato O-Aciltransferase/genética , 1-Acilglicerol-3-Fosfato O-Aciltransferase/metabolismo , Adipócitos/metabolismo , Adipogenia/genética , Adipogenia/fisiologia , Tecido Adiposo Marrom/metabolismo , Diferenciação Celular , Metabolismo Energético/genética , Resistência à Insulina/genética , Camundongos Endogâmicos C57BL , Fenótipo , Termogênese/genética , Magreza/metabolismo , Magreza/genéticaRESUMO
Interleukin-2 (IL-2), a cytokine with pleiotropic immune effects, was the first approved cancer immunotherapy agent. However, IL-2 is associated with systemic toxicity due to binding with its ligand IL-2Rα, such as vascular leakage syndrome, limiting its clinical applications. Despite efforts to extend the half-life of IL-2 and abolish IL-2Rα interactions, the risk of toxicity remains unresolved. In this study, we developed the bispecific fusion protein MB2033, comprising a novel IL-2 variant (IL-2v) connected to anti-programmed death ligand 1 (PD-L1) via a silenced Fc domain. The IL-2v of MB2033 exhibits attenuated affinity for IL-2Rßγ without binding to IL-2Rα. The binding affinity of MB2033 for PD-L1 is greater than that for IL-2Rßγ, indicating its preferential targeting of PD-L1+ tumor cells to induce tumor-specific immune activation. Accordingly, MB2033 exhibited significantly reduced regulatory T cell activation, while inducing comparable CD8+ T cell activation to recombinant human IL-2 (rhIL-2). MB2033 induced lower immune cell expansion and reduced cytokine levels compared with rhIL-2 in human peripheral blood mononuclear cells, indicating a decreased risk of peripheral toxicity. MB2033 exhibited superior anti-tumor efficacy, including tumor growth inhibition and complete responses, compared with avelumab monotherapy in an MC38 syngeneic mouse model. In normal mice, MB2033 was safer than non-α IL-2v and tolerable up to 30 mg/kg. These preclinical results provide evidence of the dual advantages of MB2033 with an enhanced safety and potent clinical efficacy for cancer treatment.
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Antígeno B7-H1 , Interleucina-2 , Proteínas Recombinantes de Fusão , Animais , Camundongos , Humanos , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Recombinantes de Fusão/genética , Antígeno B7-H1/metabolismo , Antígeno B7-H1/antagonistas & inibidores , Feminino , Camundongos Endogâmicos C57BL , Imunoterapia/métodos , Linhagem Celular Tumoral , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/imunologiaRESUMO
Early neutralizing antibodies against hepatitis C virus (HCV) and CD8 + T cell effector responses can lead to viral clearance. However, these functions alone are not sufficient to protect patients against HCV infection, thus undefined additional antiviral immune mechanisms are required. In recent years, Fc-receptor-dependent antibody effector functions, particularly, antibody-dependent cellular phagocytosis (ADCP) were shown to offer immune protection against several RNA viruses. However, its development and clinical role in patients with HCV infection remain unknown. In this study, we found that patients with chronic GT1a or GT3a HCV infection had significantly higher concentrations of anti-envelope 2 (E2) antibodies, predominantly IgG1 subclass, than patients that cleared the viruses while the latter had antibodies with higher affinities. 97% of the patients with HCV had measurable ADCP of whom patients with chronic disease showed significantly higher ADCP than those who naturally cleared the virus. Epitope mapping studies showed that patients with antibodies that target antigenic domains on the HCV E2 protein that are known to associate with neutralization function are also strongly associated with ADCP, suggesting antibodies with overlapping/dual functions. Correlation studies showed that ADCP significantly correlated with plasma anti-E2 antibody levels and neutralization function regardless of clinical outcome and genotype of infecting virus, while a significant correlation between ADCP and affinity was only evident in patients that cleared the virus. These results suggest ADCP was mostly driven by antibody titer in patients with chronic disease while maintained in clearers due to the quality (affinity) of their anti-E2 antibodies despite having lower antibody titers.
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Hepacivirus , Hepatite C , Humanos , Anticorpos Anti-Hepatite C , Anticorpos Neutralizantes , Proteínas do Envelope Viral , Fagocitose , Doença CrônicaRESUMO
BACKGROUND: Neoadjuvant chemoradiation therapy (nCRT) is recommended when lymph node metastasis is evident or strongly suspected on preoperative imaging studies, even for a completely resectable (cT1-2) tumor with minimal lymph node involvement (cN1). We evaluated the validity of upfront surgical approach in this patient group. METHODS: We retrospectively reviewed data from 247 patients with cT1-2 esophageal squamous cell carcinoma (ESCC) who underwent upfront radical esophagectomy followed by the pathology-based adjuvant treatment. Oncologic outcomes of cN1 patients were compared with those of cN0 patients. RESULTS: There were 203 cN0 and 44 cN1 patients. The lymph node yield was 62.0 (interquartile range [IQR], 51.0-76.0) in cN0 and 65.5 (IQR, 57.5-85.0) in cN1 patients (p = 0.033). The size of metastatic node was 0.6 cm (IQR, 0.4-0.9 cm) in cN0 and 0.8 cm (IQR, 0.5-1.3 cm) in cN1 patients (p = 0.001). Nodal upstaging was identified in 29.1% of cN0 and 40.9% of cN1 patients, whereas 18.2% of the cN1 had no actual lymph node metastasis (pN0). The 5-year disease-free survival rate was not significantly different between the groups (cN0, 74.4%; cN1, 71.8%; p = 0.529). Survival rates were closely correlated with pN stage, and a multivariate analysis revealed that pN2-3 stage was a risk factor for poor disease-free survival. CONCLUSIONS: Upfront radical surgery provided accurate nodal staging information, potentially sparing some cN1 patients from unnecessary nCRT while demonstrating comparable survival rates. It might be a valid option for the treatment of cT1-2N1 ESCC.
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Neoplasias Esofágicas , Carcinoma de Células Escamosas do Esôfago , Humanos , Carcinoma de Células Escamosas do Esôfago/patologia , Terapia Neoadjuvante/métodos , Metástase Linfática/patologia , Neoplasias Esofágicas/patologia , Estudos Retrospectivos , Linfonodos/patologia , Estadiamento de Neoplasias , Excisão de Linfonodo/métodosRESUMO
Long-term glucocorticoids (GCs) treatment is associated with osteoporosis and fractures. We investigated whether low-dose GC treatment also increased the risk of osteoporotic fractures, and the results showed that even low-dose GC treatment increased the risk of osteoporotic fractures, especially spine fractures. PURPOSE: The effect of low-dose glucocorticoid (GC) therapy on the fracture risk in postmenopausal women with low bone mass was investigated. METHODS: 119,790 66-year-old postmenopausal women with low bone mass based on bone mineral density (BMD) results were included. GC group consisted of patients who had been prescribed oral GCs within 6 months of BMD testing. In GC group, GCs dosage was calculated by a defined daily dose (DDD), and divided into five groups according to GC usage (Group 1[G1]; < 11.25 DDDs, G2; ≥ 11.25, < 22.5 DDDs, G3; ≥ 22.5, < 45 DDDs, G4; ≥ 45, < 90 DDDs, G5; ≥ 90 DDDs). The risk of major osteoporotic fractures (MOF) and non-MOF was analyzed and compared with that of the control group during the 1-year follow-up. RESULTS: The risk of total fracture was higher in G3-G5 than in the control group (G3, hazard ratio (HR) 1.25, 95% confidence interval [CI] 1.07-1.46; G4, 1.37 [1.13-1.66]; G5 1.45 [1.08-1.94]). The risk of MOF was higher in all groups except G2 than in the control group (G1, 1.23 [1.05-1.45]; G3, 1.37 [1.11-1.68]; G4, 1.41 [1.09-1.83]; G5, 1.66 [1.14-2.42]). The risk of spine fracture was significantly higher in all GC groups except G2 than in the control group. The risk of non-MOF was higher only in G4 than in the control group (G4, 1.48 [1.13-1.94]). CONCLUSION: Low-dose GC therapy can increase the risk of osteoporotic fractures, particularly spine fractures, in postmenopausal women with low bone mass.
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BACKGROUND: Oestrogen deficiency increases bone resorption, contributing to osteoporosis development. Yet, the mechanisms mediating the effects of oestrogen on osteoclasts remain unclear. This study aimed to elucidate the early metabolic alteration induced by RANKL, the essential cytokine in osteoclastogenesis and 17-beta-oestradiol (E2) on osteoclast progenitor cells, using RAW 264.7 macrophage cell line and primary bone marrow-derived macrophages as biological models. RESULTS: This research demonstrated that, in osteoclast precursors, RANKL stimulates complex I activity, oxidative phosphorylation (OXPHOS) and mitochondria-derived ATP production as early as 3 h of exposure. This effect on mitochondrial bioenergetics is associated with an increased capacity to oxidize TCA cycle substrates, fatty acids and amino acids. E2 inhibited all effects of RANKL on mitochondria metabolism. In the presence of RANKL, E2 also decreased cell number and stimulated the mitochondrial-mediated apoptotic pathway, detected as early as 3 h. Further, the pro-apoptotic effects of E2 during osteoclast differentiation were associated with an accumulation of p392S-p53 in mitochondria. CONCLUSIONS: These findings elucidate the early effects of RANKL on osteoclast progenitor metabolism and suggest novel p53-mediated mechanisms that contribute to postmenopausal osteoporosis.
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Diferenciação Celular , Estradiol , Mitocôndrias , Osteoclastos , Proteína Supressora de Tumor p53 , Animais , Camundongos , Trifosfato de Adenosina/metabolismo , Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Estradiol/farmacologia , Macrófagos/metabolismo , Mitocôndrias/metabolismo , Osteoclastos/metabolismo , Osteoclastos/efeitos dos fármacos , Fosforilação Oxidativa/efeitos dos fármacos , Ligante RANK/metabolismo , Células RAW 264.7 , Proteína Supressora de Tumor p53/metabolismoRESUMO
Bovine intestinal alkaline phosphatase (biALP), a membrane-bound plasma metalloenzyme, maintains intestinal homeostasis, regulates duodenal surface pH, and protects against infections caused by pathogenic bacteria. The N-glycans of biALP regulate its enzymatic activity, protein folding, and thermostability, but their structures are not fully reported. In this study, the structures and quantities of the N-glycans of biALP were analyzed by liquid chromatography-electrospray ionization-high energy collision dissociation-tandem mass spectrometry. In total, 48 N-glycans were identified and quantified, comprising high-mannose [6 N-glycans, 33.1 % (sum of relative quantities of each N-glycan)], hybrid (6, 11.9 %), and complex (36, 55.0 %) structures [bi- (13, 26.1 %), tri- (16, 21.5 %), and tetra-antennary (7, 7.4 %)]. These included bisecting N-acetylglucosamine (33, 56.6 %), mono-to tri-fucosylation (32, 53.3 %), mono-to tri-α-galactosylation (16, 20.7 %), and mono-to tetra-ß-galactosylation (36, 58.5 %). No sialylation was identified. N-glycans with non-bisecting GlcNAc (9, 10.3 %), non-fucosylation (10, 13.6 %), non-α-galactosylation (26, 46.2 %), and non-ß-galactosylation (6, 8.4 %) were also identified. The activity (100 %) of biALP was reduced to 37.3 ± 0.2 % (by de-fucosylation), 32.7 ± 2.9 % (by de-α-galactosylation), and 0.2 ± 0.2 % (by de-ß-galactosylation), comparable to inhibition by 10-4 to 101 mM EDTA, a biALP inhibitor. These results indicate that fucosylated and galactosylated N-glycans, especially ß-galactosylation, affected the activity of biALP. This study is the first to identify 48 diverse N-glycan structures and quantities of bovine as well as human intestinal ALP and to demonstrate the importance of the role of fucosylation and galactosylation for maintaining the activity of biALP.
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Fosfatase Alcalina , Galactose , Polissacarídeos , Animais , Bovinos , Polissacarídeos/metabolismo , Polissacarídeos/química , Fosfatase Alcalina/metabolismo , Fosfatase Alcalina/química , Galactose/metabolismo , Fucose/metabolismo , Fucose/química , Intestinos/enzimologia , GlicosilaçãoRESUMO
BACKGROUND: Mesenchymal stem cells (MSCs) are widely used in the development of therapeutic tools in regenerative medicine. However, their quality decreases during in vitro expansion because of heterogeneity and acquired cellular senescence. We investigated the potential role of podoplanin (PDPN) in minimizing cellular senescence and maintaining the stemness of tonsil-derived MSCs (TMSCs). METHODS: TMSCs were isolated from human tonsil tissues using an enzymatic method, expanded, and divided into two groups: early-passaged TMSCs, which were cultured for 3-7 passages, and late-passaged TMSCs, which were passaged more than 15 times. The TMSCs were evaluated for cellular senescence and MSC characteristics, and PDPN-positive and -negative cells were identified by fluorescence-activated cell sorting. In addition, MSC features were assessed in siRNA-mediated PDPN-depleted TMSCs. RESULTS: TMSCs, when passaged more than 15 times and becoming senescent, exhibited reduced proliferative rates, telomere length, pluripotency marker (NANOG, OCT4, and SOX2) expression, and tri-lineage differentiation potential (adipogenesis, chondrogenesis, or osteogenesis) compared to cells passaged less than five times. Furthermore, PDPN protein levels significantly decreased in a passage-dependent manner. PDPN-positive cells maintained their stemness characteristics, such as MSC-specific surface antigen (CD14, CD34, CD45, CD73, CD90, and CD105) and pluripotency marker expression, and exhibited higher tri-lineage differentiation potential than PDPN-negative cells. SiRNA-mediated silencing of PDPN led to decreased cell-cycle progression, proliferation, and migration, indicating the significance of PDPN as a preliminary senescence-related factor. These reductions directly contributed to the induction of cellular senescence via p16Ink4a/Rb pathway activation. CONCLUSION: PDPN may serve as a novel biomarker to mitigate cellular senescence in the clinical application of MSCs.
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Senescência Celular , Inibidor p16 de Quinase Dependente de Ciclina , Glicoproteínas de Membrana , Células-Tronco Mesenquimais , Tonsila Palatina , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Humanos , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/genética , Tonsila Palatina/citologia , Tonsila Palatina/metabolismo , Diferenciação Celular , Proliferação de Células , Transdução de Sinais , Células CultivadasRESUMO
Cationic polysaccharides have been extensively studied for drug delivery via the bloodstream, yet few have progressed to clinical use. Endothelial cells lining the blood vessel wall are coated in an anionic extracellular matrix called the glycocalyx. However, we do not fully comprehend the charged polysaccharide interactions with the glycocalyx. We reveal that the cationic polysaccharide poly(acetyl, arginyl) glucosamine (PAAG) exhibits the highest association with the endothelial glycocalyx, followed by dextran (neutral) and hyaluronan (anionic). Furthermore, we demonstrate that PAAG binds heparan sulfate (HS) within the glycocalyx, leading to intracellular accumulation. Using an in vitro glycocalyx model, we demonstrate a charge-based extent of association of polysaccharides with HS. Mechanistically, we observe that PAAG binding to HS occurs via a condensation reaction and functionally protects HS from degradation. Together, this study reveals the interplay between polysaccharide charge properties and interactions with the endothelial cell glycocalyx toward improved delivery system design and application.
Assuntos
Cátions , Matriz Extracelular , Glicocálix , Heparitina Sulfato , Heparitina Sulfato/química , Heparitina Sulfato/metabolismo , Humanos , Glicocálix/metabolismo , Glicocálix/química , Matriz Extracelular/metabolismo , Cátions/química , Células Endoteliais/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Ácido Hialurônico/química , Ácido Hialurônico/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismoRESUMO
Cardiovascular diseases (CVDs) are the leading cause of death worldwide, and morbidity and mortality rates continue to rise. Atherosclerosis constitutes the principal etiology of CVDs. Endothelial injury, inflammation, and dysfunction are the initiating factors of atherosclerosis. Recently, we reported that endothelial adenosine receptor 2â¯A (ADORA2A), a G protein-coupled receptor (GPCR), plays critical roles in neovascularization disease and cerebrovascular disease. However, the precise role of endothelial ADORA2A in atherosclerosis is still not fully understood. Here, we showed that ADORA2A expression was markedly increased in the aortic endothelium of humans with atherosclerosis or Apoe-/- mice fed a high-cholesterol diet. In vivo studies unraveled that endothelial-specific Adora2a deficiency alleviated endothelial-to-mesenchymal transition (EndMT) and prevented the formation and instability of atherosclerotic plaque in Apoe-/- mice. Moreover, pharmacologic inhibition of ADORA2A with KW6002 recapitulated the anti-atherogenic phenotypes observed in genetically Adora2a-deficient mice. In cultured human aortic endothelial cells (HAECs), siRNA knockdown of ADORA2A or KW6002 inhibition of ADORA2A decreased EndMT, whereas adenoviral overexpression of ADORA2A induced EndMT. Mechanistically, ADORA2A upregulated ALK5 expression via a cAMP/PKA/CREB axis, leading to TGFß-Smad2/3 signaling activation, thereby promoting EndMT. In conclusion, these findings, for the first time, demonstrate that blockade of ADORA2A attenuated atherosclerosis via inhibition of EndMT induced by the CREB1-ALK5 axis. This study discloses a new link between endothelial ADORA2A and EndMT and indicates that inhibiting endothelial ADORA2A could be an effective novel strategy for the prevention and treatment of atherosclerotic CVDs.
Assuntos
Aterosclerose , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , Transição Epitelial-Mesenquimal , Camundongos Endogâmicos C57BL , Receptor A2A de Adenosina , Receptor do Fator de Crescimento Transformador beta Tipo I , Animais , Humanos , Masculino , Camundongos , Antagonistas do Receptor A2 de Adenosina/farmacologia , Aterosclerose/metabolismo , Aterosclerose/patologia , Aterosclerose/genética , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Camundongos Knockout , Receptor A2A de Adenosina/metabolismo , Receptor A2A de Adenosina/genética , Receptor do Fator de Crescimento Transformador beta Tipo I/genética , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Transdução de SinaisRESUMO
Deubiquitinating enzyme A (DUBA), a member of the ovarian tumor (OTU) subfamily of deubiquitinases (DUBs), is recognized for its negative regulatory role in type I interferon (IFN) expression downstream of Toll-like receptor 3 (TLR3). However, its involvement in the TLR3 signaling pathway in fish remains largely unexplored. In this study, we investigated the regulatory role of DUBA (OmDUBA) in the TLR3 response in rainbow trout (Oncorhynchus mykiss). OmDUBA features a conserved OTU domain, and its expression increased in RTH-149 cells following stimulation with the TLR3 agonist poly(I:C). Gain- and loss-of-function experiments demonstrated that OmDUBA attenuated the activation of TANK-binding kinase 1 (TBK1), resulting in a subsequent reduction in type I IFN expression and IFN-stimulated response element (ISRE) activation in poly(I:C)-stimulated cells. OmDUBA interacted with TRAF3, a crucial mediator in TLR3-mediated type I IFN production. Under poly(I:C) stimulation, there was an augmentation in the K63-linked polyubiquitination of TRAF3, a process significantly inhibited upon OmDUBA overexpression. These findings suggest that OmDUBA may function similarly to its mammalian counterparts in downregulating the poly(I:C)-induced type I IFN response in rainbow trout by removing the K63-linked ubiquitin chain on TRAF3. Our study provides novel insights into the role of fish DUBA in antiviral immunity.
Assuntos
Proteínas de Peixes , Interferon Tipo I , Oncorhynchus mykiss , Poli I-C , Transdução de Sinais , Fator 3 Associado a Receptor de TNF , Animais , Oncorhynchus mykiss/imunologia , Fator 3 Associado a Receptor de TNF/genética , Fator 3 Associado a Receptor de TNF/metabolismo , Fator 3 Associado a Receptor de TNF/imunologia , Interferon Tipo I/imunologia , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Proteínas de Peixes/metabolismo , Transdução de Sinais/imunologia , Poli I-C/farmacologia , Imunidade Inata , Regulação da Expressão Gênica/imunologia , Ubiquitinação , Receptor 3 Toll-Like/genética , Receptor 3 Toll-Like/metabolismo , Receptor 3 Toll-Like/imunologiaRESUMO
Transition metal dichalcogenides (TMDs) are actively studied in various fields of optics and optoelectronics, including nonlinear optics of second-harmonic generation (SHG). By stacking two different TMD materials to form a heterobilyaer, unique optical properties emerge, with stronger SHG at a twist angle of 0° between TMDs and weaker SHG at a twist angle of 60°. In this work, we demonstrate the enhancement of SHG in a heterobilayer consisting of WSe2and WS2monolayers stacked at a twist angle of 64.1°, using a nanoparticle to induce local strain. The interatomic spacing of the heterobilayer is deformed by the nanoparticle, breaking the inversion symmetry, resulting in a substantial increase in the SHG of the heterobilayer at room temperature. The SHG increases depending on the polarization of the pump laser: 15-fold for linear polarization, 9-fold for right-circular polarization, and up to 100-fold for left-circular polarization. In addition, the SHG enhanced in the heterobilayer with local strain satisfies the same chiral selection rule as in the unstrained TMD region, demonstrating that the chiral selection rule of SHG is insensitive to local strain. Our findings will increase the applicability of TMD heterobilayers in nonlinear optoelectronics and valleytronics.