Aqueous biphasic cancer cell migration assay enables robust,high‐throughput screening of anti‐cancer compounds

Migration of tumor cells is a fundamental event implicated in metastatic progression of cancer. Therapeutic compounds with the ability to inhibit the motility of cancer cells are critical for preventing cancer metastasis. Achieving this goal requires new technologies that enable high‐throughput drug screening against migration of cancer cells and expedite drug discovery. We report an easy‐to‐implement, robotically operated, cell migration microtechnology with the capability of simultaneous screening of multiple compounds. The technology utilizes a fully biocompatible polymeric aqueous two‐phase system to pattern a monolayer of cells containing a cell‐excluded gap that serves as the migration niche. We adapted this technology to a standard 96‐well plate format and parametrically optimized it to generate highly consistent migration niches. The analysis of migration is done automatically using computerized schemes. We use statistical metrics and show the robustness of this assay for drug screening and its sensitivity to identify effects of different drug compounds on migration of cancer cells. This technology can be employed in core centers, research laboratories, and pharmaceutical industries to evaluate the efficacy of compounds against migration of various types of metastatic cancer cells prior to expensive animal tests and thus, streamline anti‐migratory drug screening.     

四种小鼠便秘模型建立方法的比较

目的i初步探讨四种小鼠便秘模型建模方法的效果。方法：40只小鼠随机分为正常对照组（8只）和实验组（32只）,实验组分别采用经口灌胃止泻剂复方地芬诺酯、禁水不禁食、经口灌胃胃粘膜保护剂硫糖铝及经口灌胃冰水四种方法建立小鼠便秘模型,处理后观察四种不同方法建立的小鼠便秘模型首粒黑便排便时间、2小时内排便粒数及排便重量的差异。结果：与正常对照组比较,灌胃复方地芬诺酯、禁水不禁食、硫糖铝及冰水法四种造模方法均可使小鼠首粒黑便排便时间显著延长、2小时内排便粒数及排便重量显著减少（P〈0．05）。但采用复方地芬诺酯、禁水不禁食法处理的小鼠较经口灌胃硫糖铝及经口灌胃冰水诱导的小鼠首粒黑便排便时间显著延长、2小时内排便粒数及排便重量亦显著减少（P〈0．01）。结论：采用复方地芬诺酯、禁水不禁食方法建立小鼠便秘模型更加简单、有效。     

Multichannel surface electrodes increase the sensitivity of diagnosis of neuropathy in diabetic patients

This prospective study investigated the diagnostic sensitivity of a novel multichannel surface electrode for detecting electrophysiologic changes in symptomatic diabetic neuropathy. We recruited healthy subjects without neuropathic complaints and diabetic patients with distal symmetric sensory symptoms who had normal nerve conduction studies (NCS). Eight compound muscle action potentials (CMAPs) were recorded using a multichannel electrode from each subject’s abductor pollicis brevis muscle by stimulating the median nerve at the wrist. Latency- and amplitude-related variables were obtained and analyzed to compare the two groups. We used the Classification and Regression Tree (CART) algorithm to determine the cut-off values for selected predictors of diabetic neuropathy. All of the variables related to CMAP latency showed statistically significant differences between the median values for the diabetic group and the healthy control group. For example, the median value of the maximum latency and standard deviation of the eight CMAP onset latencies in diabetic patients (3.82 ms and 0.15 ms, respectively) were significantly larger than those in controls (3.26 ms and p < 0.001; 0.09 ms and p < 0.001, respectively). The CART analysis revealed that these variables were the most sensitive and specific variables for discriminating between patients with diabetic neuropathy and normal subjects. The multichannel surface electrode demonstrated both high sensitivity and specificity in detecting neurophysiologic abnormality of diabetic neuropathy, even when conventional NCS did not detect the abnormality.     

方右旋糖酐40注射液联合甘露醇治疗下肢软组织开放性损伤负压封闭引流术后患者的疗效及对血液流变学的影响

目的:探讨复方右旋糖酐40注射液联合甘露醇治疗下肢软组织开放性损伤负压封闭引流(VSD)术后的治疗效果及对血液流变学的影响,为下肢软组织开放性损伤的治疗提供临床依据。方法:选取下肢软组织开放性损伤VSD术后患者80例,随机分为对照组和观察组各40例,对照组单纯应用甘露醇治疗,观察组应用复方右旋糖酐40注射液及甘露醇治疗,观察两组的临床疗效,比较两组治疗前后国际骨关节炎评分标准(Lequesne指数)、视觉模拟评分法(VAS)、美国食品药品管理局(FDA)皮肤评分、Lysholm膝关节功能评分(Lysholm)、血液流变学的变化以及不良反应发生情况。结果:治疗后,对照组和观察组的总有效率分别为85.00%、97.50%,比较差异具有统计学意义(P<0.05)。治疗后,两组VAS评分和Lequesne指数评分均降低,Lysholm评分及FDA皮肤评分较治疗前显著升高(P<0.05);治疗后,观察组VAS评分和Lequesne指数评分低于对照组,Lysholm评分及FDA皮肤评分高于对照组(P<0.05)。治疗后,两组血细胞比容较治疗前升高,全血比高切黏度、全血比低切黏度、血浆比黏度较治疗前降低(P<0.05);治疗后,观察组血细胞比容高于对照组,全血比高切黏度、全血比低切黏度、血浆比黏度低于对照组(P<0.05)。两组不良反应发生率比较无差异(P>0.05)。结论:复方右旋糖酐40注射液联合甘露醇治疗下肢软组织开放性损伤VSD术后患者疗效确切,可减轻膝关节疼痛,促进皮肤软组织恢复,改善膝关节功能和血液流变学,安全性较好。     

复方多糖口服液的制备及质量研究

目的制备复方多糖口服液,并进行定量分析,对各项指标进行检测。方法采用水提醇沉法制备复方多糖口服液,薄层色谱法进行定性鉴别,苯酚-硫酸法测定含量。结果检测波长为490 nm,在4.86～24.7mg范围内多糖呈线性关系,加样回收率为99.84%。结论本法简便易行,结果稳定,可做复方多糖口服液中多糖的含量测定方法。     

复方861对大鼠肝脏卵圆细胞分化的影响

目的探讨复方861对大鼠肝脏卵圆细胞分化的影响,了解其在肝纤维化治疗过程中促进肝细胞再生的可能机制。方法不同浓度(1.95,3.90,7.81,15.62,31.25,62.50,125,250,500,1000μg/mL)的复方861在无血清培养条件下作用于WB-F344细胞24 h,MTT法分析法检测细胞生长情况。500μg/mL复方861在无血清条件下作用WB-F344细胞72 h后,通过RT-PCR观察CK-19、AFP、ALB、αmRNA表达的变化。以同期未作处理的WB-F344作为空白对照组。结果 WB-F344细胞经过不同复方861作用后,除1000μg/mL外,各组细胞生长均未受到抑制,500μg/mL时细胞生存活性最佳。无血清条件下作用72 h后,半定量RT-PCR发现861组AFP mRNA的表达显著增加,CK-19 mRNA的表达显著减少,同时发现861组有ALB mRNA的表达。结论复方861可能诱导WB-F344细胞主要向肝细胞方向分化。     

Human Indoleamine 2,3-Dioxygenase Is a Catalyst of Physiological Heme Peroxidase Reactions: IMPLICATIONS FOR THE INHIBITION OF DIOXYGENASE ACTIVITY BY HYDROGEN PEROXIDE*

The heme enzyme indoleamine 2,3-dioxygenase (IDO) is a key regulator of immune responses through catalyzing l-tryptophan (l-Trp) oxidation. Here, we show that hydrogen peroxide (H₂O₂) activates the peroxidase function of IDO to induce protein oxidation and inhibit dioxygenase activity. Exposure of IDO-expressing cells or recombinant human IDO (rIDO) to H₂O₂ inhibited dioxygenase activity in a manner abrogated by l-Trp. Dioxygenase inhibition correlated with IDO-catalyzed H₂O₂ consumption, compound I-mediated formation of protein-centered radicals, altered protein secondary structure, and opening of the distal heme pocket to promote nonproductive substrate binding; these changes were inhibited by l-Trp, the heme ligand cyanide, or free radical scavengers. Protection by l-Trp coincided with its oxidation into oxindolylalanine and kynurenine and the formation of a compound II-type ferryl-oxo heme. Physiological peroxidase substrates, ascorbate or tyrosine, enhanced rIDO-mediated H₂O₂ consumption and attenuated H₂O₂-induced protein oxidation and dioxygenase inhibition. In the presence of H₂O₂, rIDO catalytically consumed nitric oxide (NO) and utilized nitrite to promote 3-nitrotyrosine formation on IDO. The promotion of H₂O₂ consumption by peroxidase substrates, NO consumption, and IDO nitration was inhibited by l-Trp. This study identifies IDO as a heme peroxidase that, in the absence of substrates, self-inactivates dioxygenase activity via compound I-initiated protein oxidation. l-Trp protects against dioxygenase inactivation by reacting with compound I and retarding compound II reduction to suppress peroxidase turnover. Peroxidase-mediated dioxygenase inactivation, NO consumption, or protein nitration may modulate the biological actions of IDO expressed in inflammatory tissues where the levels of H₂O₂ and NO are elevated and l-Trp is low.     

A Chrysin Derivative Suppresses Skin Cancer Growth by Inhibiting Cyclin-dependent Kinases

Chrysin (5,7-dihydroxyflavone), a natural flavonoid widely distributed in plants, reportedly has chemopreventive properties against various cancers. However, the anticancer activity of chrysin observed in in vivo studies has been disappointing. Here, we report that a chrysin derivative, referred to as compound 69407, more strongly inhibited EGF-induced neoplastic transformation of JB6 P⁺ cells compared with chrysin. It attenuated cell cycle progression of EGF-stimulated cells at the G₁ phase and inhibited the G₁/S transition. It caused loss of retinoblastoma phosphorylation at both Ser-795 and Ser-807/811, the preferred sites phosphorylated by Cdk4/6 and Cdk2, respectively. It also suppressed anchorage-dependent and -independent growth of A431 human epidermoid carcinoma cells. Compound 69407 reduced tumor growth in the A431 mouse xenograft model and retinoblastoma phosphorylation at Ser-795 and Ser-807/811. Immunoprecipitation kinase assay results showed that compound 69407 attenuated endogenous Cdk4 and Cdk2 kinase activities in EGF-stimulated JB6 P⁺ cells. Pulldown and in vitro kinase assay results indicated that compound 69407 directly binds with Cdk2 and Cdk4 in an ATP-independent manner and inhibited their kinase activities. A binding model between compound 69407 and a crystal structure of Cdk2 predicted that compound 69407 was located inside the Cdk2 allosteric binding site. The binding was further verified by a point mutation binding assay. Overall results indicated that compound 69407 is an ATP-noncompetitive cyclin-dependent kinase inhibitor with anti-tumor effects, which acts by binding inside the Cdk2 allosteric pocket. This study provides new insights for creating a general pharmacophore model to design and develop novel ATP-noncompetitive agents with chemopreventive or chemotherapeutic potency.     

乳源性复合益生菌对糖尿病大鼠肾组织 TLR2、TLR4/NFκB信号通路的影响

目的研究乳源性复合益生菌对糖尿病大鼠肾组织Toll样受体2(TLR2)、Toll样受体4(TLR4)、核转录因子κB(NFκB)表达的影响。方法将高脂饮食和链脲佐菌素(STZ)诱导的糖尿病SD大鼠模型随机分为模型组、二甲双胍组、利拉鲁肽组、复合益生菌低剂量组和复合益生菌高剂量组,正常SD大鼠为对照组,每组8只。血糖仪检测不同时段血糖值,ELISA法检测糖化血红蛋白(HbA1c)含量,生化仪检测大鼠尿素氮(BUN)、肌酐(Scr)、24 h尿蛋白定量(24h Alb)的变化,HE染色观察肾脏组织形态,qPCR法检测肾组织TLR2、TLR4的mRNA的表达以及Western blot检测TLR2、NFκBpp65蛋白表达量。结果与模型组相比,复合益生菌组大鼠HbA1c、BUN、Scr及24h Alb水平明显下降,并且复合益生菌能够明显改善肾脏的组织形态,显著降低TLR2、TLR4的mRNA的表达和TLR2、NFκBpp65蛋白表达量。结论复合益生菌可显著改善糖尿病大鼠早期肾脏损害,其机制与部分抑制糖尿病大鼠肾组织中TLR2、TLR4/NFκB信号通路有关。