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Osteogenic differentiation is important for fracture healing. Microfibrial-associated glycoprotein 2 (MAGP2) is found to function as a proangiogenic regulator in bone formation; however, its role in osteogenic differentiation during bone repair is not clear. Here, a mouse model of critical-sized femur fracture was constructed, and the adenovirus expressing MAGP2 was delivered into the fracture site. Mice with MAGP2 overexpression exhibited increased bone mineral density and bone volume fraction (BV/TV) at Day 14 postfracture. Within 7 days postfracture, overexpression of MAGP2 increased collagen I and II expression at the fracture callus, with increasing chondrogenesis. MAGP2 inhibited collagen II level but elevated collagen I by 14 days following fracture, accompanied by increased endochondral bone formation. In mouse osteoblast precursor MC3T3-E1 cells, MAGP2 treatment elevated the expression of osteoblastic factors (osterix, BGLAP and collagen I) and enhanced ALP activity and mineralization through activating ß-catenin signaling after osteogenic induction. Besides, MAGP2 could interact with lipoprotein receptor-related protein 5 (LRP5) and upregulated its expression. Promotion of osteogenic differentiation and ß-catenin activation mediated by MAGP2 was partially reversed by LRP5 knockdown. Interestingly, ß-catenin/transcription factor 4 (TCF4) increased MAGP2 expression probably by binding to MAGP2 promoter. These findings suggest that MAGP2 may interact with ß-catenin/TCF4 to enhance ß-catenin/TCF4's function and activate LRP5-activated ß-catenin signaling pathway, thus promoting osteogenic differentiation for fracture repair. mRNA sequencing identified the potential targets of MAGP2, providing novel insights into MAGP2 function and the directions for future research.
Assuntos
Fraturas Ósseas , Osteogênese , Animais , Camundongos , beta Catenina/genética , beta Catenina/metabolismo , Diferenciação Celular/genética , Colágeno/metabolismo , Consolidação da Fratura , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Osteoblastos/metabolismo , Via de Sinalização Wnt , Masculino , Camundongos Endogâmicos C57BL , Linhagem CelularRESUMO
Fracture healing is a complex physiological process in which angiogenesis plays an essential role. Microfibril-associated glycoprotein-2 (MAGP2) has been reported to possess a proangiogenic activity via integrin αvß3, yet its role in bone repair is unexplored. In this study, a critical-sized femoral defect (2 mm) was created in mice, followed by the delivery of an adenovirus-based MAGP2 overexpression vector or its negative control at the fracture site. At days 7, 14, 21, and 28 postfracture, bone fracture healing was evaluated by radiography, micro-computed tomography, and histopathologic analysis. Adenovirus-based MAGP2 overexpression vector-treated mice exhibited increased bone mineral density and bone volume fraction. MAGP2 overexpression contributed to an advanced stage of endochondral ossification and induced cartilage callus into the bony callus. Further analysis indicated that MAGP2 was associated with enhanced angiogenesis, as evidenced by marked MAGP2 and integrin αvß3 costaining and increased endothelial cell markers such as endomucin and CD31 levls, as well as elevated phosphorylation of protein tyrosine kinase 2 (PTK2) and AKT serine/threonine kinase 1 (AKT) in the callus. In vitro, recombinant human MAGP2 treatment enhanced the viability, migration, and tube formation ability of human microvascular endothelial cells, which was partially reversed by integrin αvß3 inhibition or MK-2206, a specific AKT inhibitor. Inhibition of integrin αvß3 abolished MAGP2-induced PTK2 and AKT activation. Taken together, our data provide the first evidence that MAGP2 promotes angiogenesis and bone formation by activating the integrin αvß3/PTK2/AKT signaling pathway.
Assuntos
Consolidação da Fratura , Proteínas Proto-Oncogênicas c-akt , Animais , Humanos , Camundongos , Calo Ósseo/metabolismo , Calo Ósseo/patologia , Células Endoteliais/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Consolidação da Fratura/fisiologia , Integrina alfaVbeta3/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Microtomografia por Raio-XRESUMO
Dictyophora rubrovolvata volva, an agricultural by-product, is often directly discarded resulting in environmental pollution and waste of the proteins' resources. In this study, D. rubrovolvata volva proteins (DRVPs) were recovered using the ultrasound-assisted extraction (UAE) method. Based on one-way tests, orthogonal tests were conducted to identify the effects of the material-liquid ratio, pH, extraction time, and ultrasonic power on the extraction rate of DRVPs. Moreover, the impact of UAE on the physicochemical properties, structure characteristics, intermolecular forces, and functional attributes of DRVPs were also examined. The maximum protein extraction rate was achieved at 43.34% under the best extraction conditions of UAE (1:20 g/mL, pH 11, 25 min, and 550 W). UAE significantly altered proteins' morphology and molecular size compared to the conventional alkaline method. Furthermore, while UAE did not affect the primary structure, it dramatically changed the secondary and tertiary structure of DRVPs. Approximately 13.42% of the compact secondary structures (α-helices and ß-sheets) underwent a transition to looser structures (ß-turns and random coils), resulting in the exposure of hydrophobic groups previously concealed within the molecule's core. In addition, the driving forces maintaining and stabilizing the sonicated protein aggregates mainly involved hydrophobic forces, disulfide bonding, and hydrogen bonding interactions. Under specific pH and temperature conditions, the water holding capacity, oil holding capacity, foaming capacity and stability, emulsion activity, and stability of UAE increased significantly from 2.01 g/g to 2.52 g/g, 3.90 g/g to 5.53 g/g, 92.56% to 111.90%, 58.97% to 89.36%, 13.85% to 15.37%, and 100.22% to 136.53%, respectively, compared to conventional alkali extraction. The findings contributed to a new approach for the high-value utilization of agricultural waste from D. rubrovolvata.
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Molybdenum disulfide (MoS2), a typical layered material, has important applications in various fields, such as optoelectronics, catalysis, electronic devices, sensors, and supercapacitors. Extensive research has been carried out on few-layered MoS2 in the field of electrochemistry due to its large specific surface area, abundant active sites and short electron transport path. However, the preparation of few-layered MoS2 is a significant challenge. This work presents a simple one-pot hydrothermal method for synthesizing few-layered MoS2. Furthermore, it investigates the exfoliation effect of different amounts of sodium borohydride (NaBH4) as a stripping agent on the layer number of MoS2. Na+ ions, as alkali metal ions, can intercalate between layers to achieve the purpose of exfoliating MoS2. Additionally, NaBH4 exhibits reducibility, which can effectively promote the formation of the metallic phase of MoS2. Few-layered MoS2, as an electrode for supercapacitor, possesses a wide potential window of 0.9 V, and a high specific capacitance of 150 F g-1 at 1 A g-1. This work provides a facile method to prepare few-layered two-dimensional materials for high electrochemical performance.
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Implantation timing is critical for a successful pregnancy. A short delay in embryo implantation caused by targeted gene ablation produced a cascading problem in the later stages of the pregnancy. Although several delayed implantation models have been established in wild mice, almost none of them is suitable for investigating the early delay's effects on the late events of pregnancy. Here, we report a new delayed implantation model established by the intraperitoneal administration of letrozole at 5 mg/kg body weight on day 3 of pregnancy. In these mice, initiation of implantation was induced at will by the injection of estradiol (E2). When the estradiol (3 ng) was injected on day 4 of pregnancy (i.e., without delay), the embryo implantation restarted, and the pregnancy continued normally. However, 25 ng estrogen caused compromised implantation. We also found that 67% of the female mice could be pregnant normally and finally gave birth when the estradiol injection (3 ng) was on day 5 of pregnancy (i.e., 1-day delay). Most failed pregnancies had impaired decidualization, decreased serum progesterone levels, and compromised angiogenesis. Progesterone supplementation could rescue decidualization failure in the mice. Collectively, we established a new model of delayed implantation by letrozole, which can be easily applied to study the effect and mechanisms of delay of embryo implantation on the progression of late pregnancy events.
Assuntos
Progesterona , Útero , Animais , Implantação do Embrião , Estradiol/farmacologia , Estrogênios/farmacologia , Feminino , Letrozol , Camundongos , Gravidez , Progesterona/farmacologiaRESUMO
Rational design of separators is especially critical to solve the "shuttle effect" of lithium polysulfides (LiPSs) and the sluggish redox kinetics in lithium-sulfur batteries (LSBs). Here, the multi-functional nanocomposite involving Co-doped molybdenum phosphide (Co-MoP) nanofibers and porous carbon nanofibers (PCNFs) is designed and prepared through electro-blow spinning and phosphating process, which possesses multiple adsorption and catalytic sites and is acted as the functional material for LSBs separators. In this multifunctional nanocomposite, the prepared Co-MoP nanofibers can provide internal adsorption and catalytic sites for LiPSs conversion. And the interconnected nitrogen-doped PCNFs can be elaborated an efficient LiPSs mediator and accommodate the huge volume changes in the reaction process for LSBs. Benefiting from the multiple adsorptive and catalytic sites of the developed functional materials, the assembled LSBs with a Co-MoP/PCNFs modified separator display outstanding electrochemical performances, including an admirable capacity retention of 770.4 mAh g-1 after 400 cycles at 1.0 C, only 0.08 % capacity decay per cycle at 2.0 C, rate performance up to 5 C, and also decent areal capacity even under a high sulfur loading of 4.9 mg cm-2. The work provides a facile pathway towards multifunctional separators in LSBs, and it may also help deepen preparation method of MoP through the electrostatic blowing/electrospinning technology in other related energy storage fields.
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Electro-mechanical brakes (EMBs) are the future of braking systems, particularly in commercial vehicles. Therefore, it is important to design a simple EMB scheme and establish its clamping force control strategy to satisfy the demands of commercial vehicle braking systems. This study proposes a pneumatic disc-brake-based EMB for an electric bus. Its working principle was established, and the system model was analyzed. Subsequently, the hidden Markov models (HMMs) of driver decelerate and brake intentions were built and recognized based on the analytic hierarchy process (AHP). Given the time-consuming behavior of the proposed EMB to eliminate brake clearance due to the leverage effect of the arm and motor performance limitation, a clamping force control strategy factoring in the driver intentions was developed to improve the response performance without changing the structure or size of the EMB. Furthermore, simulation analyses were performed using MATLAB/Simulink. The results confirmed that under the action of a step and 5 Hz triangular sawtooth signals, the clamping force output from the EMB corresponds well with the target signal. The clamping force gradually increases when approaching the target without overshoot and jitter during the process. The overall clamping force response time is decreased by approximately 0.25 s under the driver emergency brake than the conventional control method. Hence, the response performance of the EMB is improved.
Assuntos
Automóveis/normas , Simulação por Computador , Desaceleração , Intenção , Condução de Veículo/psicologia , Desenho de Equipamento/métodos , HumanosRESUMO
Milk fat globule membrane (MFGM) proteins have recently gained increasing attention, due to their significant biological function. However, the glycosylation of proteins in human MFGM during lactation has not been studied in detail. In this study, through mass spectroscopy-based N-glycoproteomics, we analyzed protein glycosylation of human MFGM. A total of 912 N-glycosylation sites on 506 N-glycoproteins were identified in human colostrum and mature milk MFGM. Among them, 220 N-glycoproteins with 304 N-glycosylation sites were differentially expressed in colostrum and mature milk MFGM. Gene Ontology (GO) analysis revealed various biological processes, cellular components, and molecular functions of the differentially expressed N-glycoproteins. Specifically, these glycoproteins were involved in biological processes such as single-organism processes, biological regulation, regulation of biological processes, response to stimulus and localization; were cellular components in organelles, membranes, and the extracellular region; and had different molecular functions such as protein binding, receptor activity, and hydrolase activity. KEGG pathway analysis suggested that the majority of the differentially expressed N-glycoproteins were associated with phagosome, cell adhesion molecule and some disease-related pathways. Our results provide an in-depth understanding of the quantitative changes in N-glycosylation of proteins in human colostrum and mature MFGM, and extend our knowledge of the N-glycoproteome and of the distribution of N-glycosylation sites in human MFGM during lactation, providing insight into the biological functions of the highlighted glycoproteins.
Assuntos
Colostro/química , Glicolipídeos/química , Glicoproteínas/química , Leite Humano/química , Adulto , Colostro/metabolismo , Feminino , Glicolipídeos/metabolismo , Glicoproteínas/metabolismo , Glicosilação , Humanos , Lactação , Gotículas Lipídicas , Proteínas do Leite/química , Proteínas do Leite/metabolismo , Leite Humano/metabolismo , Gravidez , Espectrometria de Massas em Tandem , Adulto JovemRESUMO
We investigated the cytoprotective effects of anthocyanins in Aronia melanocarpa against apoptosis induced by Aß1-42, a key mediator of AD pathophysiology. We measured intracellular calcium with a colorimetric kit, cellular apoptosis with DAPI, intracellular ROS with the fluorescent marker 2,3-dimethoxy-1,4-naphthoquinone, mitochondrial membrane potential with JC-1, and ATP with a colorimetric kit. Gene transcription and protein expression levels of calmodulin, cytochrome c, caspase-9, cleaved caspase-3, Bcl-2, and Bax were analyzed by RT-PCR and Western blotting. The results showed that pretreatment with anthocyanins significantly inhibited Aß1-42-induced apoptosis, decreased intracellular calcium and ROS, and increased ATP and mitochondrial membrane potential. RT-PCR and Western blotting revealed that anthocyanins upregulated the gene transcription and protein expression of calmodulin and Bcl-2 and downregulated those of cytochrome c, caspase-9, cleaved caspase-3, and Bax. A. melanocarpa anthocyanins protected SH-SY5Y cells against Aß1-42-induced apoptosis by regulating Ca2+ homeostasis and apoptosis-related genes and inhibiting mitochondrial dysfunction.
Assuntos
Peptídeos beta-Amiloides/farmacologia , Antocianinas/farmacologia , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Mitocôndrias/efeitos dos fármacos , Photinia/química , Trifosfato de Adenosina/metabolismo , Doença de Alzheimer , Antocianinas/isolamento & purificação , Apoptose/genética , Cálcio/análise , Linhagem Celular Tumoral , Expressão Gênica/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/fisiologia , Neuroblastoma , Fármacos NeuroprotetoresRESUMO
We measured changes in the main physical and chemical properties, flavour compounds and microbial diversity in suan-cai during natural fermentation. The results showed that the pH and concentration of soluble protein initially decreased but were then maintained at a stable level; the concentration of nitrite increased in the initial fermentation stage and after reaching a peak it decreased significantly to a low level by the end of fermentation. Suan-cai was rich in 17 free amino acids. All of the free amino acids increased in concentration to different degrees, except histidine. Total free amino acids reached their highest levels in the mid-fermentation stage. The 17 volatile flavour components identified at the start of fermentation increased to 57 by the mid-fermentation stage; esters and aldehydes were in the greatest diversity and abundance, contributing most to the aroma of suan-cai. Bacteria were more abundant and diverse than fungi in suan-cai; 14 bacterial species were identified from the genera Leuconostoc, Bacillus, Pseudomonas and Lactobacillus. The predominant fungal species identified were Debaryomyces hansenii, Candida tropicalis and Penicillium expansum.