The neural substrates of anxiety are poorly understood, which hinders treatment of anxiety disorders. Here we found, αCaMKII+ neurons in the ventral medial hypothalamic nucleus (VMH) responded to stressors with increased activity in male mice, both under physiological conditions and after repeated restraint stress. Activation of VMH αCaMKII+ neurons were necessary and sufficient to ameliorate stress-induced anxiety. The peripheral metabolic hormone ghrelin and receptor GHS-R1a play a complex role in emotion regulation; however, the mechanism is uncertain. A delayed increase in GHS-R1a expression in VMH αCaMKII+ neurons coincided with the development of stress-induced enhancement of anxiety-related behavior. GHS-R1a expression in VMH αCaMKII+ neurons promoted anxiety-related behavior, whereas GHS-R1a knockdown had the opposite effect. GHS-R1a upregulation inhibited the excitability of VMH αCaMKII+ neurons. We conclude that GHSR1a signaling drives stress-induced anxiety by shaping the activity of VMH αCaMKII+ neurons. GHS-R1a may be a therapeutic target for treating anxiety disorders such as post-traumatic stress disorder.

Despite significant advancements in the development of hydrogel dressings for wound healing, there remains a highly urgent demand to create multifunctional hydrogels with satisfying biocompatibility, self-healing ability, adhesiveness, appropriate mechanical properties, antibacterial activity, and therapeutic effects. Herein, a versatile hydrogel dressing was prepared through the cross-linking of terminally aldehyde-modified Pluronic-F127 (AF127) micelles with dihydrocaffeic acid-functionalized chitosan (CA) via the formation of a dynamic Schiff base. The cooperation of chitosan grafted with multiple catechol groups and the dynamic nature of the Schiff base imparted the hydrogel with superior antioxidant properties, adhesiveness, rapid self-healing, intrinsic antibacterial activity, and pH-dependent responsiveness. The good adhesiveness and self-healing abilities, derived from the hydrogen bonding interactions of catechol groups and dynamic covalent bonds, enhance hydrogels' resistance to deformation. Moreover, after loading hydrophobic rhein into AF127 micelles, the hydrogel demonstrated potent anti-inflammatory effects and pH-responsive drug release, making it suitable for treating infected and inflammatory skin wounds. In vivo experiments showed that rhein-loaded CA/AF hydrogels significantly accelerated tissue regeneration, with an intact epidermis, enhanced collagen disposition, and increased angiogenesis in a full-thickness skin defect model infected by Staphylococcus aureus. Overall, these versatile chitosan/pluronic hydrogels, possessing intrinsic antibacterial, antioxidant, and anti-inflammatory properties, hold significant potential for treating infected and chronic inflammatory wounds.

Objective:Coronavirus disease-2019 (COVID-19) can cause not only respiratory symptoms but also facial paralysis. Lianhua Qingwen (LHQW) has been reported to have therapeutic effects on COVID-19 and facial neuritis (FN). We explored the potential mechanism of LHQW in the treatment of COVID-19 and FN through a network-pharmacology approach. Methods:Active compounds and relevant targets of LHQW were obtained from the databases of Traditional Chinese Medicine Systems Pharmacology Database, HERB, UniProt Knowledge Base, SwissADME, and Swiss Target Prediction. Disease targets of COVID-19 and FN were acquired from Gene Cards. Database For Annotation, Visualization And Integrated Discovery and Metascape were used to search the biological functions of intersecting targets. After identifying the core targets and their corresponding ingredients, KEGG Mapper analyzes the localization of core targets in key pathways. AutoDock were employed to conduct molecular docking of the core targets and their corresponding ingredients. Results:We obtained four core genes: interleukin (IL)-8, IL-1B, IL-6, and tumor necrosis factor (TNF)-α. Database searching revealed the anti-inflammatory and antiviral effects of LHQW may be related to the action of aleo-emodin, hyperforin, kaempferol, luteolin, and quercetin on these four genes by regulating the pathways of IL-17 and NOD-like receptor. The molecular-docking results of the four core targets and their corresponding active ingredients showed good binding activity between receptors and ligands. Conclusions:We uncovered the active ingredients, potential targets, and biological pathways of LHQW for COVID-19 and FN coinfection. Our data provide a theoretical basis for further exploration of the mechanism of action of LHQW in treatment of COVID-19 and FN.

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The organization of modifiable and functional building components into various superstructures is of great interest due to their broad applications. Supramolecular self-assembly, based on rationally designed building blocks and appropriately utilized driving forces, is a promising and widely used strategy for constructing superstructures with well-defined nanostructures and diverse morphologies across multiple length scales. In this study, two homogeneous organohydrogels with distinct appearances were constructed by simply mixing polyoxometalate (phosphomolybdic acid, HPMo) and a double-tailed zwitterionic quaternary ammonium amphiphile in a binary solvent of water and dimethyl sulfoxide (DMSO). The delicate balance between electrostatic attraction and repulsion of anionic HPMo clusters and zwitterionic structures drove them to co-assemble into homogeneous organohydrogels with diverse microstructures. Notably, the morphologies of the organohydrogels, including unilamellar vesicles, onion-like vesicles, and spherical aggregates, can be controlled by adjusting the ionic interactions between the zwitterionic amphiphiles and phosphomolybdic acid clusters. Furthermore, we observed an organohydrogel fabricated with densely stacked onion-like structures (multilamellar vesicles) consisting of more than a dozen layers at certain proportions. Additionally, the relationships between the self-assembled architectures and the intermolecular interactions among the polyoxometalate, zwitterionic amphiphile, and solvent molecules were elucidated. This study offers valuable insights into the mechanisms of polyoxometalate-zwitterionic amphiphile co-assembly, which are essential for the development of materials with specific structures and emerging functionalities.

IntroductionIsorhynchophylline is one of the main active ingredients from Uncaria rhynchophylla, the effects and mechanisms of isorhynchophylline on stress-induced emotional disorders and cognitive impairment remain unclear.MethodsLong-term potentiation (LTP) in vivo was used for synaptic plasticity evaluation; chronic unpredictable mild stress (CUMS) model was used to evaluate the effect of isorhynchophylline on stress induced emotional disorders and cognitive impairment; sucrose preference test (SPT), open field test (OFT), and elevated plus maze (EPM) were used to evaluate emotional disorders; morris water maze (MWM) test was used to evaluate cognitive impairment; Western blotting (WB) was used to the expression of proteins; high performance liquid chromatography (HPLC) was used to quantify neurotransmitters; Nissl staining was used to identify pathological changes induced by stress.ResultsIn this study, we found that isorhynchophylline improved corticosterone-induced in vivo LTP impairment significantly, indicating positive effects on stress. Therefore, 28-day CUMS model was adopted to evaluate the anti-stress effects of isorhynchophylline. The results showed that isorhynchophylline improved CUMS-induced weight loss, anxiety- and depression-like behaviors, and spatial memory impairment. Isorhynchophylline reduced CUMS-induced corticosterone elevation. N-methyl-D-aspartic acid (NMDA) receptors play an important role in the process of emotion and memory. Glutamate and the expression of GluN2B increased in the CUMS mice, while D-serine and the expression of serine racemase (SR) decreased significantly, and isorhynchophylline restored these changes to normal level.ConclusionThese results indicated that isorhynchophylline ameliorated stress-induced emotional disorders and cognitive impairment, modulating NMDA receptors might be one of the underlying mechanisms.

Stress can induce learning and memory impairment; corticosterone is often used to study the effects and mechanisms of stress in animal models. Long-term potentiation (LTP) has been widely used for tackling the mechanisms of memory. Liuwei Dihuang decoction-active fraction combination (LW-AFC) can improve stress-induced LTP and cognition impairment; stachyose is an oligosaccharide in LW-AFC. The effects and mechanisms of stachyose on stress are unknown. In this study, stachyose showed protective effects against LTP impairment by corticosterone in vivo only via intragastric administration for 7 consecutive days, but there was little effect even after direct intracerebroventricular injection; the protective effect of stachyose could be canceled by non-absorbable antibiotics (ATB) which disturbed gut flora. 16S rRNA sequencing, alpha diversity, and principal coordinate analysis (PCoA) revealed that the gut flora in corticosterone-treated mice was disturbed and stachyose could improve corticosterone-induced gut flora disturbance. Bacteroidetes were decreased and Deferribacteres were increased significantly in corticosterone-treated mice, and stachyose restored Bacteroidetes and Deferribacteres to the normal level. D-serine, a coactivator of NMDA receptors, plays an important role in synaptic plasticity and cognition. Here, corticosterone had little effect on the content of D-serine and L-serine (the precursor of D-serine), but it reduced the D-serine release-related proteins, Na+-independent alanine-serine-cysteine transporter-1 (ASC-1), and vesicle-associated membrane protein 2 (VAMP2) significantly in hippocampus; stachyose significantly increased ASC-1 and VAMP2 in corticosterone-treated mice, and ATB blocked stachyose's effects on ASC-1 and VAMP2. NMDA receptors co-agonists L-serine, D-serine, and glycine significantly improved LTP impairment by corticosterone. These results indicated that stachyose might indirectly increase D-serine release through the gut-brain axis to improve LTP impairment by corticosterone in the hippocampus in vivo.

Background: Cranial radiotherapy is clinically used in the treatment of brain tumours; however, the consequent cognitive and emotional dysfunctions seriously impair the life quality of patients. LW-AFC, an active fraction combination extracted from classical traditional Chinese medicine prescription Liuwei Dihuang decoction, can improve cognitive and emotional dysfunctions in many animal models; however, the protective effect of LW-AFC on cranial irradiation–induced cognitive and emotional dysfunctions has not been reported. Recent studies indicate that impairment of adult hippocampal neurogenesis (AHN) and alterations of the neurogenic microenvironment in the hippocampus constitute critical factors in cognitive and emotional dysfunctions following cranial irradiation. Here, our research further investigated the potential protective effects and mechanisms of LW-AFC on cranial irradiation–induced cognitive and emotional dysfunctions in mice. Methods: LW-AFC (1.6 g/kg) was intragastrically administered to mice for 14 days before cranial irradiation (7 Gy γ-ray). AHN was examined by quantifying the number of proliferative neural stem cells and immature neurons in the dorsal and ventral hippocampus. The contextual fear conditioning test, open field test, and tail suspension test were used to assess cognitive and emotional functions in mice. To detect the change of the neurogenic microenvironment, colorimetry and multiplex bead analysis were performed to measure the level of oxidative stress, neurotrophic and growth factors, and inflammation in the hippocampus. Results: LW-AFC exerted beneficial effects on the contextual fear memory, anxiety behaviour, and depression behaviour in irradiated mice. Moreover, LW-AFC increased the number of proliferative neural stem cells and immature neurons in the dorsal hippocampus, displaying a regional specificity of neurogenic response. For the neurogenic microenvironment, LW-AFC significantly increased the contents of superoxide dismutase, glutathione peroxidase, glutathione, and catalase and decreased the content of malondialdehyde in the hippocampus of irradiated mice, accompanied by the increase in brain-derived neurotrophic factor, insulin-like growth factor-1, and interleukin-4 content. Together, LW-AFC improved cognitive and emotional dysfunctions, promoted AHN preferentially in the dorsal hippocampus, and ameliorated disturbance in the neurogenic microenvironment in irradiated mice. Conclusion: LW-AFC ameliorates cranial irradiation–induced cognitive and emotional dysfunctions, and the underlying mechanisms are mediated by promoting AHN in the dorsal hippocampus and improving the neurogenic microenvironment. LW-AFC might be a promising therapeutic agent to treat cognitive and emotional dysfunctions in patients receiving cranial radiotherapy.

OBJECTIVE Previous studies showed that over activation of NMDA receptors may be a crucial cause of long-term potentiation (LTP) and cognitive impairment induced by stress or corticosterone. However, other studies showed that the function of NMDA receptors is insufficient since the NMDA receptors co-agonist D-serine could improve stress-induced cognitive impairment. The purpose of this study is to clarify whether over activation of NMDA receptors or hypofunction of NMDA receptors is involved in hippocampal impairment of LTP by corticosterone and the underlying mechanisms. METHODS Cort was injected subcutaneously 1 h before the high-frequency stimulation (HFS) to induce LTP impairment. NMDA receptor antagonists and agonists were administrated by icv. RESULTS Hippocampal LTP and object location recognition memory were impaired in corticosterone-treated mice. Corticosterone increased the gluta?mate level in hippocampal tissues, neither NMDA receptors antagonist nor its subtype antagonists alleviated impairment of LTP, while enhancing the function of NMDA receptors by D-serine did alleviate impairment of LTP by corticosterone, suggesting that hypofunction of NMDA receptors might be one of the main reasons for impairment of LTP by corticoste?rone. Further results showed that the level of D-serine and its precursor L-serine did not change. D-serine release-related protein Na+-independent alanine-serine-cysteine transporter-1 (ASC-1) in the cell membrane was decreased and increas?ing D-serine release by the selective activator of ASC-1 antiporter activity alleviated impairment of LTP by corticoste?rone. CONCLUSION Taken together, this study demonstrates that hypofunction of NMDA receptors may be involved in impairment of LTP by corticosterone and reduced D-serine release may be an important reason for its hypofunction, which is an important complement to existing mechanisms of corticosterone-induced LTP and cognitive impairment.

目的 通过单次皮下注射不同剂量皮质酮模拟不同程度急性应激刺激,并评价小鼠情绪症状与认知功能的变化,拟建立一种稳定可靠的急性应激障碍(ASD)模型.方法 SPF级BALB/c小鼠经自主活动性筛选后,按自主活动及体质量随机分为对照组、皮质酮给药组(50、100和200 mg/kg).行为学实验均在皮质酮注射1h后进行.采用旷场实验、高架十字迷宫实验和悬尾实验评价单次皮质酮注射对小鼠情绪症状的影响;采用新物体识别实验和Morris水迷宫实验评价单次皮质酮注射对小鼠认知功能的影响.结果 与对照组相比,皮质酮50 mg/kg组小鼠情绪及认知行为无明显改变.皮质酮100 mg/kg可使小鼠在旷场实验中中心区域进入时间从(17.99±2.80)s降低为(6.36±1.98) s(P＜0.05),中心区域进入次数从(13.80±1.76)次降低为(7.22±1.72)次(P＜0.01);对高架十字迷宫的开放臂进入时间和次数及悬尾实验的不动时间无明显影响;小鼠对新物体的偏好指数从0.56±0.06降低为0.37±0.04(P＜0.05);水迷宫实验中小鼠的目标象限停留时间从(31.53±3.51)s降低为(18.55±2.98) s(P＜0.05),目标象限运动距离从(4.82±0.43)m降低为(2.69±0.38) m(P＜0.01).当皮质酮剂量达到200 mg/kg时小鼠在旷场中活动的总距离从(16.98±1.15)m降低为(11.72±1.48)m.结论 单次皮下注射100 mg/kg皮质酮可诱导类ASD症状,出现焦虑样精神障碍,物体识别记忆和空间记忆等认知功能损伤,可作为一种稳定可靠的ASD动物模型用于抗ASD药物的筛选和相关机制研究.

Previous studies reported that ginsenoside Rg1 (Rg1) exerts antidepressant-like effect in animal models of depression. However, its effect on post-traumatic stress disorder (PTSD) remains elusive; PTSD is a common and costly psychiatric condition with negative cognitive and affective dysfunctions, such as anxiety and depression. In this study, we evaluated the role of Rg1 in a validated mice model of PTSD induced by single-prolonged stress (SPS). Sertraline, one of the FDA-approved medications for PTSD was used as a positive control. Our results showed that SPS exposure led to increased anxiety-like and despair-like behaviors. SPS exposure also caused enhanced contextual fear memory and overgeneralization of learned fear. Sertraline significantly ameliorated those abnormal behaviors induced by SPS, while Rg1 did not. Meanwhile, we found that sertraline but not Rg1 blocked the suppressive effect of SPS on adult neurogenesis in the hippocampus. Consistently, we found that SPS elevated adrenocorticotropic hormone (ACTH) level in the serum, which was inhibited by sertraline but not Rg1. Our results thus demonstrate that Rg1 at a dose used to treat depression may not be effective to rescue behavioral deficits associated with PTSD.

目的 研究单次腹腔注射肿瘤坏死因子α(TNF-α)对小鼠认知功能及焦虑情绪的影响及可能的作用途径.方法 根据自主活动性和体质量将BALB/c小鼠随机分为对照组和TNF-α处理组.采用Morris水迷宫空间探索实验、高架十字迷宫实验、在体电生理实验,分别观察TNF-α0.1、0.2和0.4 mg/kg单次腹腔注射1 h对小鼠空间记忆提取过程、焦虑样行为、突触可塑性的影响;采用Morris水迷宫定向航行实验,观察在学习训练的第1、3、5天训练前1 h腹腔注射TNF-α0.2 mg/kg对小鼠空间记忆获取与巩固功能的影响;采用尼氏染色实验,观察单次腹腔注射TNF-α0.2 mg/kg 1 h对小鼠不同脑区神经元尼氏体数目的影响.结果 Morris水迷宫实验中,定向航行实验阶段给予TNF-α后小鼠逃避潜伏期和穿环次数无明显变化,提示定向航行阶段给予TNF-α对小鼠空间学习记忆能力无明显损害.空间探索实验前1 h腹腔注射TNF-α0.1和0.4 mg/kg后小鼠逃避潜伏期明显延长(均P<0.05),给予TNF-α0.2和0.4 mg/kg 1 h后小鼠穿环次数明显减少(P<0.05,P<0.01),提示空间探索阶段单次腹腔注射TNF-α可致小鼠空间记忆提取功能明显受损;高架十字迷宫实验中,与对照组相比,单次腹腔注射TNF-α0.2 mg/kg 1 h后测试发现小鼠进入开放臂时间以及在开放臂时间占在开闭臂总时间百分比显著降低(P<0.01),给予TNF-α0.4 mg/kg后小鼠在开放臂时间占在开闭臂总时间百分比显著降低(P<0.01),提示小鼠出现焦虑样行为;在体电生理实验和组织病理学实验结果表明,与对照组相比,单次腹腔注射TNF-α0.2和0.4 mg/kg 1 h后小鼠平均群峰电位明显降低(P<0.05,P<0.01),给予TNF-α0.2 mg/kg 1 h后小鼠海马神经元尼氏体数量明显减少(P<0.01),提示小鼠长时程增强(LTP)及海马神经元明显受损.结论 TNF-α的急性升高可致小鼠空间学习记忆功能障碍并表现出焦虑样行为,可能与TNF-α对海马LTP及神经元的损伤密切相关.

Neuroinflammation is known as a typical feature associated with many neurodegenerative diseases including Alzheimer's disease (AD) and impairs the synaptic plasticity of the hippocampus. LW-AFC is an active fraction combination being extracted from Liuwei Dihuang decoction, a classic traditional Chinese medicine prescription. This study aimed to investigate the effects of LW-AFC on synaptic plasticity in mice with lipopolysaccharide (LPS) treatment. The results showed that the administration of LPS caused fever and long-term potentiation (LTP) impairment in mice. The pretreatment with LW-AFC had an antipyretic effect on fever and improved the impaired LTP induced by LPS, alleviated the microglia and astrocytes activation in the hippocampus, regulated the abnormal T-lymphocyte subpopulation in the spleen and blood caused by LPS, and reduced the aberrant secretion of cytokines in the brain and plasma. The compounds paeoniflorin, morroniside, and loganic acid in LW-AFC regulated the TNF-α secretion in non-LPS- and LPS-stimulated BV-2 cells. These data suggest that LW-AFC improves the LPS-induced impairment of LTP and alleviates the activation of glial cells in the hippocampus, which might be associated with modulating immune responses.

Phlegmadine A (1), a Lycopodium alkaloid with a unique cyclobutane ring and featuring a complex tetracyclo[4.2.2.03,8.03,10]decane-bridged system, together with three biogenetically related known compounds, was isolated from the Phlegmariurus phlegmaria. The structures and absolute configurations of 1 and 2 were determined by NMR and single-crystal X-ray analysis. Among them, compound 2 exhibited noticeable protective effects for long-term potentiation impairment by corticosterone induced in mice. Moreover, we succeeded in the efficient synthesis of 1 from 3 by a biomimetic synthesis method.

Procaspase-3-activating compound 1 (PAC-1) induces procaspase-3 activation via zinc chelation. However, whether PAC-1 employs other mechanisms remains unknown. Here we systematically screened for potent PAC-1 targets using 29 enhanced green fluorescent protein-labeled reporter cell lines and identified hypoxia-inducible factor 1α (HIF1α) and RAD51 pathways as PAC-1 targets. These results were verified in HepG2 cells and two other cancer cell lines. Mechanistically, PAC-1 specifically blocked HIF1α hydroxylation and upregulated HIF1α target genes. In addition, DNA damage, G1/S cell cycle arrest, and the inhibition of DNA synthesis were induced following PAC-1 administration. Interestingly, by using ferrozine-iron sequestration and iron titration assays, we uncovered the iron sequestering capacity of PAC-1. Additionally, the expression levels of iron shortage-related genes were also increased in PAC-1-treated cells, and iron (II) supplementation reversed all of the observed cellular responses. Thus, our results indicate that PAC-1 induces HIF1α stabilization and DNA damage by sequestering ferrous iron.

Procaspase-3-activating compound 1 (PAC-1) induces procaspase-3 activation via zinc chelation. However, whether PAC-1 employs other mechanisms remains unknown. Here we systematically screened for potent PAC-1 targets using 29 enhanced green fluorescent protein-labeled reporter cell lines and identified hypoxia-inducible factor 1 alpha (HIF1 alpha) and RAD51 pathways as PAC-1 targets. These results were verified in HepG2 cells and two other cancer cell lines. Mechanistically, PAC-1 specifically blocked HIF1 alpha hydroxylation and upregulated HIF1 alpha target genes. In addition, DNA damage, G(1)/S cell cycle arrest, and the inhibition of DNA synthesis were induced following PAC-1 administration. Interestingly, by using ferrozine-iron sequestration and iron titration assays, we uncovered the iron sequestering capacity of PAC-1. Additionally, the expression levels of iron shortage-related genes were also increased in PAC-1 treated cells, and iron (II) supplementation reversed all of the observed cellular responses. Thus, our results indicate that PAC-1 induces HIF1 alpha stabilization and DNA damage by sequestering ferrous iron.

Cancer cells have an increased requirement for iron than normal cells, and iron chelators are under active consideration for cancer treatment. The metal-sequestering potential and antiproliferative mechanisms of a novel hydroxyphenyl hydrazone derivate YCL0426 were investigated here. Antiproliferative activity of YCL0426 was detected by MTT assay. The iron-sequestering potential was evaluated by ferrozine-Fe(II) sequestering assay and Fe(II) titration assay. Cell-cycle-arresting profile was checked by flow cytometry and the DNA synthesis status was evaluated by BrdU incorporation assay. SW480 cells stably expressing Rad51-EGFP fusion protein were used to evaluate the DNA damaging potential of the compound. The impact of extra Fe (II) supplement on compound activities was also examined. YCL0426 shows significant antiproliferative activity on 15 cancer cell lines with mean IC50 values of 5.25 mu mol/l. YCL0426 displayed concentration-dependent Fe(II) sequestering ability in ferrozine-Fe(II) sequestering assay, and induced upregulation of transferrin receptor 1 and divalent metal transporter 1 expression in HepG2 cells, which are genes responsible for Fe(II) uptake. YCL0426 blocked DNA synthesis in BrdU incorporation assay, and arrested cell cycle at S or G1 phase. Besides, YCL0426 induced Rad51 foci formation and histone H2AX phosphorylation with EC50 values of 1.35 and 2.29 mu mol/l, respectively, indicating the emergence of DNA damage. All these cellular responses, and even the growth-inhibiting activity of YCL0426, can be readily reversed by Fe(II) repletion, indicating that iron sequestering is responsible, at least in part, for the antiproliferative activity of YCL0426. YCL0426 is a potent iron chelator that exerts significant antiproliferative activities by inducing G1/S arrest and DNA damage. Copyright (C) 2017 Wolters Kluwer Health, Inc. All rights reserved.

Research involving non-adherent cell lines, primary cells and blood cells is definitely important, but its application in image-based assays, especially in high-content systems, is highly limited. Accordingly, efficient high-content methods to study non-adherent cells are needed not only to improve diagnostics but also for early screening of targeted drugs. A plate-based assay using adhesion reagents for multiparametric measurement with single non-adherent and non-anchored cells in a large cell population in high-content cytometry was developed and optimized. The cells preserved their identity even during extensive biomanipulations. The proposed method is highly robust for better imaging and can be used in various assays in different cellular backgrounds. Furthermore, as exemplary experiments, novel optimized assay protocols were used to study extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) activity after cell inhibition with imatinib in chronic myelocytic leukemia K562 cells, revealing the phosphorylation kinetics of ERK MAPK. The results showed that the proposed assay detects kinase phosphorylation with good sensitivity and may be used in rapid drug screening.