eFood
·
2022
·Vol. 3
·期 4
·e26
·
IF 0
Yuan Liang,Tiehua Zhang
Epidermal growth factor receptor (EGFR) is among the first discovered transmembrane receptor tyrosine kinases. Ligand binding to the extracellular domain of EGFR results in the formation of homo-dimeric or hetero-dimeric complexes, activation, and auto-phosphorylation in the intracellular tyrosine kinase domain, then regulates the cell's proliferation, differentiation, migration, and invasion. It has been reported that EGFR is overexpressed, amplified, or mutated in several epithelial tumors. EGFR tyrosine kinase inhibitors (EGFR-TKIs) specifically bind to the tyrosine kinase domain of EGFR. Homogeneous time-resolved fluorescence (HTRF) assay is successfully used in kinase assays and is a crucial tool in the drug discovery of various kinases. Moreover, the HTRF assay has been determined to exhibit many advantages, such as being easy to miniaturize, simple to read, and excellent to repeat. In these guidelines, the theory, materials, and reagent preparation of HTRF kinase assay are described and emphasized. Furthermore, the protocol and optimization of the assay are determined to establish an HTRF screening platform, then an optimized HTRF kinase assay is performed to test the compounds for screening potential EGFR-TKIs. In addition, these guidelines can contribute to verifying the high-throughput screening results of HTRF and help to reveal the underlying mechanism of the EGFR kinase and tested compounds.
研究领域:
药理学
·
分子生物学
·
癌症研究
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DOI
ANALYTICAL AND BIOANALYTICAL CHEMISTRY
·
2022
·Vol. 414
·期 6
·2265-2273
·
IF 4.478
Zhao Yan,Hu Jin-Jie,Bai Xiao-Lin,Liu Hao-Peng,Qi Xu-Wei,Liao Xun
A simple and rapid method for screening of tyrosinase (TYR) inhibitors present in traditional Chinese medicines (TCMs) was developed by combining ligand fishing and the fluorescent enzymatic assay based on dopamine-functionalized carbon quantum dots (CQDs-Dopa). Ligands of the enzyme present in the TCM extractions were firstly adsorbed on the enzyme-modified magnetic beads, and then the beads were magnetically separated and subjected directly to the CQDs-Dopa-based fluorescent assay. Finally, compounds were desorbed from the “active” beads and identified with ultra-performance liquid chromatography-triple quadrupole mass spectrometry. A known natural TYR inhibitor quercetin was selected to assess the feasibility and quantification performance of this method, and good linearity in the range of 0.01–0.16 mM ( R 2 = 0.992) with a low detection limit of 0.004 mM was obtained. This method was then applied to screen TYR inhibitors present in Scutellaria baicalensis and Sophora flavescens . Six TYR inhibitors including baicalin ( 1 ), baicalein ( 2 ), wogonin ( 3 ), oroxylin A ( 4 ), kurarinone ( 5 ), and sophoraflavanone G ( 6 ) were found, among which 1 – 4 were firstly discovered in this work. This is the first report on the in situ assessment of the target compounds obtained by ligand fishing in the form of a mixture, which exhibited the combined advantages of specific extraction ability of ligand fishing and the high sensitivity of CQDs-based fluorescent assay, showing great potential for fast screening of enzyme inhibitors from TCMs. Graphical abstract
研究领域:
药理学
·
分析化学
·
中医药
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产品关联
DOI
PubMed
高影响因子
ONCOLOGY REPORTS
·
2022
·Vol. 48
·期 6
·1-11
·
IF 4.136
Haoran Miao,Hongliang Hui,Huaming Li,Yangui Lin,Dan Li,Min Luo,Bo Jiang,Yiqian Zhang
Current investigations suggest that pigment epithelial‑derived factor (PEDF) can mediate the progression of non‑small cell lung cancer (NSCLC) by regulating autophagy. However, the underlying mechanisms associated with autophagy remain poorly elucidated. The aim of the present study was to investigate the association between the PEDF/adenosine 5'‑monophosphate‑activated protein kinase (AMPK)/Unc‑51 like autophagy‑activated kinase 1 (ULK1) pathway and autophagy in NSCLC. Intracellular autophagy was evaluated using indicators such as the expression and activation of microtubule‑associated protein light chain 3‑I (LC3‑I), LC3‑II and p62, as well as the distribution and number of autophagosomes observed by confocal microscopy. In addition, the activity and proliferative capacity of NSCLC cells under PEDF overexpression was also examined using Cell Counting Kit‑8 and lactate dehydrogenase (LDH) assays, and western blotting (WB) of related proteins. The results revealed that PEDF significantly inhibited NSCLC cell proliferation and viability, and increased LDH release and intercellular adhesion. Furthermore, PEDF suppressed the expression and activation of LC‑3 and reduced the number and distribution of autophagosomes. The PEDF‑induced inhibition of autophagy exhibited a direct association with the suppressed proliferation capacity and cell viability of NSCLC cells. The results of WB showed that NSCLC cells regulated autophagy through the AMPK/ULK1 signaling pathway. PEDF downregulated the AMPK/ULK1 signaling pathway, and AMPK or ULK1 overexpression markedly reduced the inhibitory effect of PEDF on autophagy. In conclusion, PEDF overexpression significantly inhibited the proliferative capacity and cell viability of NSCLC cells, as PEDF exerted an inhibitory function by regulating autophagy in NSCLC cells. Finally, it was demonstrated that autophagy may be suppressed by inhibiting the AMPK/ULK1 signaling pathway, thereby revealing a mechanism of lung cancer progression.
研究领域:
肿瘤学
·
分子生物学
·
细胞生物学
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DOI
PubMed
高影响因子
INDUSTRIAL CROPS AND PRODUCTS
·
2022
·
IF 6.449
Chuks Fidelis Nwanade,Min Wang,Hongxia Li,Abolfazl Masoudi,Zhijun Yu,Jingze Liu
Plant essential oils (EOs) are usually complex mixtures of volatile compounds, and these compounds can vary in composition as a result of biotic and abiotic factors. Thus, understanding the contribution of the individual compounds particularly their interactions with respect to acaricidal activity is important for the use of EOs as an alternative to synthetic acaricides . Herein, the acaricidal activity of constituents from EOs of cinnamon ( Cinnamomum zeylanicum and Cinnamomum cassia ) against Haemaphysalis longicornis was investigated using the nymphal and adult immersion tests. Among the test compounds, benzyl benzoate showed the highest toxicity against the nymphs (LC 50 = 24.41 µL/mL) and adults (LC 50 = 3.99 µL/mL). All combinations at their sublethal concentration (LC 30 ) exhibited synergy. The binary mixture of benzyl benzoate + cinnamaldehyde presented the highest synergy ratio (3.42) while tertiary mixtures of cinnamaldehyde + eugenol + linalool showed the least synergy ratio (1.20). Enzyme assays revealed that benzyl benzoate significantly inhibited AChE activity ( p < 0.05). The ecotoxicological assessments of benzyl benzoate and cinnamaldehyde indicate likely minimal effects on non-target terrestrial invertebrate, Tenebrio molitor . On the other hand, they were toxic to non-target aquatic species , Daphnia magna . Based on our results, the acaricidal activity of cinnamon EO against H . longicornis may be linked to the content of benzyl benzoate whose presence may be synergistic to the activity of other compounds.
研究领域:
昆虫学
·
药理学
·
毒理学
·
植物科学
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产品关联
DOI
高影响因子
ANALYTICAL CHEMISTRY
·
2022
·
IF 8.008
Rui Liu,Gaoyi Yi,Baian Ji,Xiuqiong Liu,Yuanqi Gui,Zhining Xia,Qifeng Fu
Enzyme assays are important for studying enzyme-mediated biochemical reactions and for clinical diagnosis and drug development. The technique of an immobilized enzyme microreactor (IMER) integrated with capillary electrophoresis (CE) has been frequently utilized in online enzyme assays. However, the traditional approaches for IMER-CE enzyme analysis have some defects such as low loading capacity and poor stability. Herein, metal–organic frameworks (MOFs), which have enormous potential in the fields of enzyme immobilization and capillary electrochromatographic (CEC) separation, were first explored as novel support materials with good enzyme immobilization performance and stationary phases with excellent separation abilities to construct an integrated MOFs-IMER-CEC microanalysis system for a high-efficiency online enzyme assay. As a proof-of-concept demonstration, acetylcholinesterase (AChE) was immobilized on a densely packed UiO-66-NH2 nanocrystal coating on a capillary inner surface with abundant intercrystalline mesoporosity and was employed as a highly effective and robust IMER for CEC-integrated online enzyme analysis. The excellent separation performance of the UiO-66-NH2-modified capillary was verified by high-efficiency separation of three types of neutral, acidic, and basic compounds. The Michaelis–Menten constant and enzyme inhibition kinetics of UiO-66-NH2–IMER were systematically assessed, exhibiting distinct advantages such as remarkably increased enzyme loadability, superior affinity for substrates, and greatly improved stability and repeatability compared to CE-integrated IMERs prepared by the traditional covalent bonding method. Furthermore, the developed method was successfully utilized for detecting organophosphorus pesticides in leguminous vegetable samples, demonstrating its strong practicality. The study not only proposed a novel support material and construction strategy for a high-performance microchannel-based IMER but also can be widely used in bioanalysis and biosensing research.
研究领域:
分析化学
·
生物化学
·
生物技术
·
环境科学
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产品关联
DOI
PubMed
高影响因子
SENSORS AND ACTUATORS A-PHYSICAL
·
2022
·
IF 4.291
Cheng Bai,Wen Zhou,Shifeng Yu,Tengfei Zheng,Chaohui Wang
Microfluidic mixing is a critical issue in the lab-on-a-chip system, which has been applied in various biomedical research. Recently, acoustic wave-based active micromixers have been applied to increase the mixing efficiency via acoustic streaming. However, along with the acoustic streaming, the acoustic thermal effect will generate additional heat to increase the temperature of the microfluid. In this work, we present an integrated thermal control unit applied to a surface acoustic wave (SAW)-assisted micromixer to address this problem. A metal microwire as the thermal resistance sensor is fabricated under the microchannel to measure the temperature in different conditions, together with the Peltier cooler beneath allowing the active temperature control of the microfluid. For characterization, neutralization reaction and alkaline phosphatase assay were successfully conducted on this device. It shows the integrated thermal control unit applied to a SAW-assisted micromixer is highly effective in temperature-sensitive biomedical research.
研究领域:
微流控技术
·
生物医学工程
·
声学
·
热能工程
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产品关联
DOI
高影响因子
IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY-ANIMAL
·
2022
·Vol. 59
·期 1
·1-9
·
IF 2.723
Chen Sunyu,Xiao Zhanhao,Jiang Wenjin
Osteogenic differentiation is a crucial process of new bone formation. This study aimed to explore the roles and mechanism of SRY-Box Transcription Factor 2 (SOX2) on proliferation and osteogenic differentiation of MC3T3-E1 cells. Bone morphogenetic protein 2 (BMP2) was used to induce the osteogenic differentiation of MC3T3-E1 cells. The expression of SOX2 was determined by quantitative real-time PCR (RT-PCR) at different time points after induction. The SOX2 overexpression plasmids were constructed and transfected into MC3T3-E1 cells. Osteogenic differentiation was evaluated by Alizarin Red S staining and alkaline phosphatase (ALP) assay. The expressions of osteogenic differentiation markers including runt-related transcription factor 2 (Runx2), osteopontin (OPN), and osteocalcin (OCN) were detected by western blot assay. Luciferase reporter and CHIP assays were used to confirm that SOX2 regulated the transcriptional activation of leucine-rich repeat-containing G protein–coupled receptor 4 (LGR4). We found that SOX2 was down-regulated upon BMP2-induced osteogenic differentiation in MC3T3-E1 cells. Overexpression of SOX2 effectively inhibited osteogenic differentiation with decreased ALP activity, calcification, and osteogenic differentiation markers’ expression including Runx2, OPN, and OCN. LGR4 was identified as a target of SOX2, and the inhibitory effect of SOX2 on osteogenic differentiation was reversed by knockdown of LGR4. The present study confirmed that SOX2 suppressed osteogenic differentiation of MC3T3-E1 cells through targeting LGR4, which possesses a therapeutic strategy for bone formation and generation.
研究领域:
分子生物学
·
细胞生物学
·
骨骼生物学
·
转录调控
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产品关联
DOI
PubMed
高影响因子
BIOSENSORS & BIOELECTRONICS
·
2023
·
IF 12.6
Weiqiang Yang,Jiajing Xu,Qingda Yao,Xiaoguang Xu,Xiaoping Chen,Jiancong Ni,Qingxiang Wang,Zhenyu Lin
Prolidase (PLD) plays a crucial role as a dipeptidase in various physiological processes , specifically involved in the cleavage of proline-containing dipeptides for efficient recycling of proline . The accurate determination of PLD activity holds significant importance in clinical diagnosis. Herein, a solid-state electrochemiluminescence (ECL) biosensor was developed to address the urgent need for PLD assay. The Ru(bpy) 3 2+ was electrophoretically deposited within the nanochannels of vertically-ordered mesoporous silica film (VMSF) on indium tin oxide (ITO) electrodes. The Ru(bpy) 3 2+ -deposited VMSF/ITO (Ru-VMSF/ITO) exhibited a remarkable ECL response towards proline, attributed to the enhanced concentration of the reactants and improved electron transfer resulting from the nanoconfinement effect. As PLD specifically enzymolyzed the Gly-Pro dipeptide to release proline, a proline-mediated biosensor was developed for PLD assay. Increased PLD activity led to enhanced release of proline into the porous solid-state ECL sensors, resulting in a more robust ECL signal. There was a linear relationship between Δ ECL intensity and logarithmic concentration of PLD in the range of 10–10000 U/L, with a detection limit of 1.98 U/L. Practical tests demonstrated the reliability and convenience of the proposed bioassay, making it suitable for widespread application in PLD assays.
研究领域:
分析化学
·
生物传感器
·
电化学
·
纳米技术
·
生物医学工程
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产品关联
DOI
PubMed
高影响因子
INTERNATIONAL IMMUNOPHARMACOLOGY
·
2018
·
IF 3.118
Jing Xing,Zhenlong Yu,Xiangyu Zhang,Wenyang Li,Dongna Gao,Jian Wang,Xiaochi Ma,Xinshi Nie,Wei Wang
The p38 MAPK signaling pathway plays a key role in lung inflammation and the development of acute lung injury (ALI). We previously reported that the phenolic compound procyanidin B1 inhibits inflammation by suppressing the p38 MAPK signaling pathway. Here, we asked whether the monomer of procyanidin B1, epicatechin (EC), can alleviate LPS-induced ALI in mice, and if so, whether EC acts by inhibiting p38 MAPK. C57BL/6 mice were randomly divided into four groups ( n = 8) and received EC alone, vehicle (sham group), LPS alone, or LPS and EC. LPS was administered via intraperitoneal injection and EC via nasogastric feeding. Lung histopathology , alveolocapillary membrane permeability , inflammation, and p38 MAPK pathway activation were assessed by immunohistochemistry , tissue wet/dry weight analysis, quantitative PCR, protein assays, ELISA , and western blot analysis using lung tissue and/or bronchoalveolar fluid. We also performed molecular modeling and in vitro enzymatic assays to examine the potential interaction between EC and p38 MAPK at the molecular level. We found that LPS caused an increase in ALI-associated lung pathology accompanied by activation of p-p38 pathway components and the transcription factor AP1. All of these effects were substantially reduced by treatment with EC. Furthermore, molecular modeling suggested that EC suppressed p38 MAPK signaling by hydrogen bonding with Glu71, Ala 111, Asp112, and Leu171 in the active site of p38α. In vitro kinase assays confirmed the ability of EC to directly inhibit purified p38 MAPK. Collectively, our data suggest that the naturally occurring compound EC could be a new therapeutic option for ALI.
研究领域:
药理学
·
免疫学
·
分子生物学
·
病理学
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产品关联
DOI
PubMed
高影响因子
ACS Applied Materials & Interfaces
·
2016
·
IF 7.145
Yeping Wu,Anzhi Wang,Xiaokang Ding,Fu-Jian Xu
The development of new materials for fast and sensitive protease assay is in demand for timely diagnosis of diseases, such as cardiovascular disease, cancers, and Alzheimer disease. Herein, poly(methacrylic acid) (PMAA) brushes were synthesized from the surfaces of silica nanoparticles via surface-initiated atom transfer radical polymerization (ATRP), and functionalized with series of proteolytically cleavable peptides for highly sensitive protease assay. Upon the proteolytic cleavage of the peptides, a short peptide fragment with fluorescent tag (GGK-FITC) is released to the solution, which can be easily detected with a benchtop fluorescence microscope. The grafting densities of PMAA brushes and peptides can be readily tuned by controlling the monomer concentrations of sodium methacrylate in the ATRP reaction. Because of the three-dimensional architecture of PMAA brushes, the loading amount of peptides can reach 21.4% of the total weight of functionalized silica particles (22.4 peptides/nm2), which is much higher than direct immobilization on silica nanoparticles without polymer brushes. Because of the high loading density of peptides, the limit of detection (LOD) of trypsin can reach 1.4 pM in buffer solution or 2.6 nM in nondiluted serum. By rational design of peptide substrates, the peptide-functionalized PMAA brushes can be readily expanded to detect other proteases, such as matrix metalloproteinase-2 (MMP-2), a virtual biomarker for many cancers, with an LOD of 1.1 pM. The proteolytically cleavable peptide-functionalized PMAA brushes offer a starting point for fast and sensitive protease assay.
研究领域:
材料科学
·
生物化学
·
纳米技术
·
生物医学工程
·
分析化学
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产品关联
DOI
PubMed
高影响因子
TOXICOLOGY IN VITRO
·
2021
·
IF 3.5
Meiting Wang,Jiaxian Wang,Alex Y.P. Tsui,Zhaomin Li,Yizhe Zhang,Qi Zhao,Hongyan Xing,Xijie Wang
The awareness of the long-term toxicities of cancer survivors after chemotherapy treatment has been gradually strengthened as the population of cancer survivors grows. Generally, chemotherapy-induced peripheral neurotoxicity (CIPN) is studied by animal models which are not only expensive and time-consuming, but also species-specific differences. The generation of human induced pluripotent stem cells (hiPSCs) and differentiation of peripheral neurons have provided an in vitro model to elucidate the risk of CIPN. Here, we developed a drug-induced peripheral neurotoxicity model using hiPSC-derived peripheral neurons (hiPSC-PNs) to study the mechanisms of different chemotherapeutic agents on neuronal viability using LDH assay, a cell apoptosis assay determined by caspase 3/7 activation, neurite outgrowth, ion channel expression and neurotransmitter release following treatment of cisplatin, bortezomib, ixabepilone, or pomalidomide. Our data showed that the multiple endpoints of the hiPSC-PNs model had different sensitivity to various chemotherapeutic agents. Furthermore, the chemotherapeutics separated cell viability from the decrease in neurite lengthand changed levels of ion channels and neurotransmitters to a certain extent. Thus, we study the mechanisms of peripheral neurotoxicity induced by chemotherapeutic agents through changes in these indicators.
研究领域:
肿瘤学
·
神经科学
·
干细胞生物学
·
毒理学
✓ 已验证
DOI
PubMed
高影响因子
International Journal of Polymeric Materials and Polymeric Biomaterials
·
2022
·
IF 3.221
Zhao Wang,Wenli Song,Ruilong Sheng,Xinyu Guo,Lingyun Hao,Xiaojuan Zhang
A new series of cholesterol-bearing poly(methacrylate)s with pendant cationic (l-)-lysine (Chol-PHML) was synthesized via reversible addition–fragmentation chain transfer (RAFT) polymerization, followed by Boc-deprotection. Their structures were characterized by nuclear magnetic resonance (NMR) spectroscopy and gel permeation chromatography (GPC). Then, the average particle sizes and zeta potentials of the Chol-PHML/pDNA polyplexes were examined using dynamic light scattering (DLS), and their plasmid DNA (pDNA)-binding ability was analyzed using agarose gel retardation assay. In vitro cytotoxicity of the polycations was determined using a cell counting kit-8 (CCK-8) assay and lactate dehydrogenase (LDH) kits in HeLa cells. The polyplex’s gene transfection effectiveness was investigated using a luciferase assay. The results revealed that the Chol-PHML series could efficiently bind/load pDNA with a high binding affinity (N/P < 3.0) and subsequently form nanoparticles with the sizes of 90–200 nm and zeta potentials of ∼+40 mV in an aqueous solution. The in vitro cytotoxicity and gene transfection efficiency considerably depended on the Chol-PHML vector’s molecular weight. The optimal Chol-PHML27 exhibited a substantially higher gene transfection efficiency and lower cytotoxicity than those of the linear structural PLL (15 K–30K) and PHML26 control. This study presents a simple method for synthesizing biocompatible and controllable cationic gene vectors by introducing hydrophobic cholesterol moieties, which will function as a promising foundation for gene delivery.
研究领域:
高分子化学
·
基因治疗
·
生物医学工程
·
分子生物学
✓ 已验证
产品关联
DOI
高影响因子
危险品化学品经营许可证（带存储）