BACKGROUND & AIMS: :Novel treatment strategies for tuberculosis are urgently needed. Many different preclinical models assessing anti-tuberculosis drug activity are available, but it is yet unclear which combination of models is most predictive of clinical treatment efficacy. The aim of this study was to determine the role of our in vitro time kill-kinetics assay as an asset to a predictive preclinical modeling framework assessing anti-tuberculosis drug activity. The concentration- and time-dependent mycobacterial killing capacities of six anti-tuberculosis drugs were determined during exposure as single drugs or in dual, triple and quadruple combinations towards a Mycobacterium tuberculosis Beijing genotype strain and drug resistance was assessed. Streptomycin, rifampicin and isoniazid were most active against fast-growing M. tuberculosis. Isoniazid with rifampicin or high dose ethambutol were the only synergistic drug combinations. The addition of rifampicin or streptomycin to isoniazid prevented isoniazid resistance. In vitro ranking showed agreement with early bactericidal activity in tuberculosis patients for some but not all anti-tuberculosis drugs. The time-kill kinetics assay provides important information on the mycobacterial killing dynamics of anti-tuberculosis drugs during the early phase of drug exposure. As such, this assay is a valuable component of the preclinical modeling framework.

背景与目标： 迫切需要新的结核病治疗策略。有许多不同的评估抗结核药物活性的临床前模型，但尚不清楚哪种模型组合最能预测临床治疗效果。这项研究的目的是确定我们的体外时间杀伤动力学测定法作为评估抗结核药物活性的预测性临床前建模框架的资产的作用。在暴露于结核分枝杆菌北京基因型菌株的过程中，确定了六种抗结核药物的浓度和时间依赖性的分枝杆菌杀伤能力，并评估了耐药性。链霉素，利福平和异烟肼对快速生长的结核分枝杆菌最有效。异烟肼与利福平或高剂量乙胺丁醇是唯一的协同药物组合。在异烟肼中添加利福平或链霉素可防止异烟肼耐药性。体外排名显示，对于某些 (但不是所有) 抗结核药物，结核病患者的早期杀菌活性一致。时间杀伤动力学测定法提供了有关药物暴露早期抗结核药物的分枝杆菌杀伤动力学的重要信息。因此，该测定是临床前建模框架的有价值的组成部分。

BACKGROUND & AIMS: OBJECTIVE:The aim of this study was the evaluation of the first commercially available in-vitro diagnostic (IVD)- mass spectrometric immunosuppressant assay from Chromsystems (MassTox Immunosuppressants ONEMINUTE Test) and the comparison to a routinely used online solid phase extraction liquid chromatography-tandem mass spectrometric assay method for the measurement of cyclosporine A, everolimus, sirolimus, and tacrolimus in patient whole blood samples. METHODS:An API 4000 [tandem mass spectrometer (AB SCIEX)] combined with a CTC Pal autosampler (CTC Analytics AG) and a Shimadzu ultra-fast liquid chromatography (UFLC) system were applied for the direct liquid chromatography-tandem mass spectrometric assay analysis using electrospray ionization in positive ion mode. Isotope-labeled internal standards were used for the commercial assay. Within- and between-day variation, accuracy, and limits of quantification were determined. Samples from external international proficiency testing schemes were measured to assess the accuracy of the commercial assay. About 100 patient samples were reanalyzed with the commercial test kit, and the results were compared with our in-house results. RESULTS:The limit of quantification for the commercial assay was 0.5 ng/mL for everolimus, sirolimus, and tacrolimus and 5 ng/mL for cyclosporine A. The coefficient of variation for all immunosuppressants was lower than 7% (within day) and 12% (between days) for all 5 concentration levels. Accuracy ranged between 82% and 111% for quality control samples and between 89% and 112% for samples from the external quality assurance program. Both methods showed a very good agreement (r > 0.91) in patient samples over the whole concentration range for all immunosuppressants. CONCLUSION:The commercial immunosuppressant assay from Chromsystems represents a standardized IVD-certified alternative to our in-house developed assay.

背景与目标：

BACKGROUND & AIMS: :An automated microparticle enzyme immunoassay (IMx Rubella IgG Antibody Assay; Abbott Laboratories, North Chicago, Ill.) was compared with a conventional enzyme-linked immunosorbent assay (ELISA) for detection of rubella-specific immunoglobulin G (IgG) in 400 consecutive antenatal patients. There was complete agreement between the two tests in this population, which had a positivity rate of 99% for rubella-specific IgG antibodies. The performance of the IMx was also evaluated at the cutoff zone by assaying 64 selected antenatal serum samples with low or negative rubella antibody titers as determined by ELISA. Overall, the IMx was found to be a specific, sensitive assay for the detection of rubella-specific IgG and is virtually fully automated for easy performance.

背景与目标： : 将自动微粒酶免疫测定法 (IMx风疹IgG抗体测定法; Abbott Laboratories，North Chicago，ill。) 与常规酶联免疫吸附测定法 (ELISA) 进行比较，以检测400连续产前患者的风疹特异性免疫球蛋白G (IgG)。在该人群中，两种测试之间完全一致，其风疹特异性IgG抗体的阳性率为99%。还通过测定64个选定的产前血清样品，通过ELISA测定的风疹抗体滴度低或阴性，在临界区评估了IMx的性能。总体而言，IMx被发现是用于检测风疹特异性IgG的特异性，灵敏的检测方法，并且实际上是全自动的，易于执行。

BACKGROUND & AIMS: :In a small number of patients treated with botulinum toxin (BT) antibody (Ab) formation occurs. BT Ab can be detected by the mouse protection assay (MPA) or by the mouse diaphragm assay (MDA). Both methods, however, have major drawbacks. We tested a method for detecting BT Ab which measures the BT-induced reduction in the electromyographic amplitude of the mean maximal voluntary activation (M-EMG) of the sternocleidomastoid muscle. The M-EMG reduction was compared in 17 patients with cervical dystonia and secondary BT therapy failure to the M-EMG reduction previously measured in controls. Values more than 2 SD below the mean of controls were considered abnormal. Six patients showed BT Ab on the MPA and MDA; all of these had abnormal M-EMG reductions. Eleven patients showed no BT Ab on MPA and MDA testing; in ten of these the M-EMG reduction was normal, and in one it was pathological, but MDA testing later changed to positive under continued BT therapy. The sternocleidomastoid test is easy to perform and produces quantitative results. Since its sensitivity and specificity are at least as good as those of the MDA and the MPA, it can replace them.

背景与目标： : 在少数接受肉毒杆菌毒素 (BT) 治疗的患者中，抗体 (Ab) 形成。BT Ab可以通过小鼠保护试验 (MPA) 或小鼠隔膜试验 (MDA) 检测。然而，这两种方法都有主要缺点。我们测试了一种检测BT Ab的方法，该方法测量了BT引起的胸锁乳突肌平均最大自愿激活 (m-emg) 的肌电图幅度的降低。将17例宫颈肌张力障碍和继发性BT治疗失败的患者的m-emg降低与先前在对照组中测量的m-emg降低进行了比较。低于对照组平均值超过2 SD的值被认为是异常的。六名患者在MPA和MDA上显示BT Ab; 所有这些都有异常的M-EMG降低。11例患者在MPA和MDA测试中没有显示BT Ab; 其中10例m-emg降低是正常的，其中1例是病理性的，但随后在持续的BT治疗下MDA测试变为阳性。胸锁乳突肌试验易于进行并产生定量结果。由于其敏感性和特异性至少与MDA和MPA一样好，因此可以替代它们。

BACKGROUND & AIMS: :We studied the use of the INNO-LIA syphilis score assay in the resolution of discordant positive screening results of the Murex ICE Syphilis enzyme immunoassay (EIA) with the confirmatory results of both the Serodia Treponema pallidum particle agglutination (TPPA) and the fluorescent treponemal antibody-absorption (FTA-Abs) assays, for the serological diagnosis of syphilis. This was an observational study on the serum samples received by the Syphilis Laboratory, Hong Kong, during the period from January 2006 to December 2012. A total of 801 serum samples with discordant positive screening EIA results were used. Consensus results of such serum samples were derived from results of the EIA, TPPA and FTA-abs assays. The age range of the individuals was 14 to 104 years (median of 52). There were 369 males and 432 females. Of 378 serum samples, 139 showed agreement among positive results, 23 of 310 showed agreement among indeterminate results and 277 of 465 showed agreement among negative results. The proportions of agreement among positive, indeterminate and negative results were 0.37 (95% CI 0.32-0.42), 0.07 (95% CI 0.05-0.11) and 0.60 (95% CI 0.55-0.64), respectively; kappa 0.55 (95% CI 0.49-0.60). There were 60 serum samples with positive consensus results but negative INNO-LIA syphilis score results and 10 with negative consensus results but positive INNO-LIA syphilis score results. Although the INNO-LIA syphilis score assay can be considered a valid alternative confirmatory test for the serological diagnosis of syphilis, the present study showed that its use in the resolution of discordant positive screening EIA results was moderate. A more extensive characterization of serum samples with discordant reactive screening treponemal test results is necessary.

背景与目标： : 我们研究了INNO-LIA梅毒评分测定法在解决Murex冰梅毒酶免疫测定法 (EIA) 的不一致阳性筛查结果中的使用，并确定了梅毒螺旋体颗粒凝集 (TPPA) 和荧光梅毒螺旋体抗体吸收 (FTA-Abs) 测定法，用于梅毒的血清学诊断。这是一项对2006年1月至2012年12月期间香港梅毒实验室收到的血清样本的观察性研究。总共使用了801份具有不一致阳性筛选EIA结果的血清样品。此类血清样品的共识结果来自EIA，TPPA和FTA-abs测定的结果。个体的年龄范围为14至104岁 (中位数为52)。有369名男性和432名女性。在378份血清样品中，139份阳性结果一致，310份23份不确定结果一致，277份465份阴性结果一致。阳性，不确定和阴性结果之间的一致性比例分别为0.37 (95% CI 0.32-0.42)，0.07 (95% CI 0.05-0.11) 和0.60 (95% CI 0.55-0.64); kappa 0.55 (95% CI 0.49-0.60)。有60份血清样本的一致性结果为阳性，但INNO-LIA梅毒评分结果为阴性，而10份的一致性结果为阴性，但INNO-LIA梅毒评分结果为阳性。尽管INNO-LIA梅毒评分测定法可被认为是梅毒血清学诊断的有效替代验证性试验，但本研究表明，其在解决不一致的阳性筛查EIA结果中的使用是中等的。需要使用不一致的反应性筛选密螺旋体测试结果对血清样品进行更广泛的表征。

BACKGROUND & AIMS: :Concanavalin A inhibits serum 5'-nucleotidase activity, without causing significant inhibition of alkaline phosphatase activity. This observation serves as the basis for a new method for assaying the 5'-nucleotidase activity in serum, which depends upon the difference between the enzymic hydrolysis of adenosine-5'-monophosphate in the presence and absence of concanavalin A. A denosine released by the 5'-nucleotidase reaction is deaminated by a coupled reaction with adenosine deaminase to liberate inosine and ammonia, and ammonia is measured colorimetrically by the Berthelot reaction. In sera from 40 healthy adult persons, 5'-nucleotidase activity averaged 6.4 U/liter (SD, +/-2.0; range, 3-12). In sera from 100 patients, measurements of 5'-nucleotidase activity by the new assay averaged 8% lower than by a generally accepted method in which phenyl phosphate is used to suppress hydrolysis of adenosine-5'-monophosphate by alkaline phosphatase activity. The clinical validy of the new assay was tested by measuring serum 5'-nucleotidase activities in rats with bile duct ligation and in rats treated with thioacetamide to induce hepatocellular injury.

背景与目标： : 伴刀豆球蛋白A抑制血清5 '-核苷酸酶活性，而不会显着抑制碱性磷酸酶活性。该观察结果是测定血清中5 '-核苷酸酶活性的新方法的基础，该方法取决于在存在和不存在伴刀豆球蛋白a的情况下腺苷-5'-单磷酸的酶水解之间的差异。通过与腺苷脱氨酶的偶联反应，将5 '-核苷酸反应释放的脱氨脱氨，释放出肌苷和氨，并通过Berthelot反应比色法测量氨。在来自40个健康成年人的血清中，5 '-核苷酸酶活性平均为6.4 U/l (SD，+/-2.0; 范围，3-12)。在来自100患者的血清中，通过新测定法对5 '-核苷酸酶活性的测量平均8% 低于通过通常接受的方法 (其中使用磷酸苯酯来抑制碱性磷酸酶活性的腺苷-5'-单磷酸水解)。通过测量胆管结扎大鼠和硫代乙酰胺治疗诱发肝细胞损伤的大鼠的血清5 '-核苷酸酶活性，测试了新方法的临床有效性。

BACKGROUND & AIMS: :Menadione (2-methyl-1,4-naphthoquinone, MQ), a component of multivitamin drugs with antihemorrhagic, antineoplastic, and antimalarial activity, is frequently used to investigate quinone-induced cytotoxicity. The formation of MQ conjugates with glutathione (GSH) by Michael addition and subsequent biotransformation to yield N-acetyl-l-cysteine conjugates is believed to be an important detoxification process. However, the resulting conjugates, 2-methyl-3-(glutathione-S-yl)-1,4-naphthoquinone (MQ-GS) and 2-methyl-3-(N-acetyl-l-cysteine-S-yl)-1,4-naphthoquinone (MQ-NAC), retain the ability to redox cycle and to arylate cellular nucleophiles. Although the nephrotoxicity and hepatotoxicity of MQ-thiol conjugates have been reported in vitro, methods for their determination in vivo have yet to be published. Herein, a highly sensitive, simple, and selective HPLC-chemiluminescence (HPLC-CL) coupled method is reported, allowing for the first time the simultaneous determination of MQ, MQ-GS, and MQ-NAC in rat plasma after MQ administration. Our method exploits the unique redox characteristics of MQ, MQ-GS, and MQ-NAC to react with dithiothreitol (DTT) to liberate reactive oxygen species (ROS) which are detected by a CL assay using luminol as a CL probe. To verify the proposed mechanism, MQ-GS and MQ-NAC were synthetically prepared. Specimen preparation involved solid-phase extraction on an Oasis HLB cartridge followed by isocratic elution on an ODS column. No interference from endogenous substances was detected. Linearity was observed in the range of 5-120 nM for MQ-GS and MQ-NAC and 10-240 nM for MQ, with detection limits (S/N of 3) of 1.4, 0.8, and 128 fmol for MQ-GS, MQ-NAC, and MQ, respectively. The application of our method reported here is the first to extensively study the stability and reversibility of thiol-quinones.

背景与目标： : 甲萘醌 (2-甲基-1,4-萘醌，MQ) 是具有抗出血，抗肿瘤和抗疟活性的多种维生素药物的成分，通常用于研究醌诱导的细胞毒性。通过Michael加成形成MQ缀合物与谷胱甘肽 (GSH) 并随后进行生物转化以产生N-乙酰基-l-半胱氨酸缀合物被认为是重要的解毒过程。然而，得到的共轭物，2-甲基-3-(谷胱甘肽-S-基)-1,4-萘醌 (mq-gs) 和2-甲基-3-(N-乙酰基-l-半胱氨酸-S-基)-1,4-萘醌 (mq-nac)，保留氧化还原循环和芳化细胞亲核试剂的能力。尽管已在体外报道了MQ-硫醇偶联物的肾毒性和肝毒性，但体内测定方法尚未公布。本文报道了一种高度灵敏、简单和选择性的HPLC-化学发光 (hplc-cl) 偶联方法，首次允许在MQ给药后同时测定大鼠血浆中的MQ、mq-gs和mq-nac。我们的方法利用MQ，mq-gs和mq-nac的独特氧化还原特性与二硫苏糖醇 (DTT) 反应释放活性氧 (ROS)，使用鲁米诺作为CL探针通过CL测定法检测到。为了验证所提出的机制，综合制备了mq-gs和mq-nac。标本制备涉及在Oasis HLB柱上进行固相萃取，然后在ODS柱上进行等度洗脱。未检测到内源性物质的干扰。对于mq-gs和mq-nac，在5-120 nM和10-240 nM范围内观察到线性，对于mq-gs、mq-nac和MQ，检出限 (S/N为3) 分别为1.4、0.8和128 fmol。本文报道的方法的应用是首次广泛研究硫醇-醌的稳定性和可逆性。

BACKGROUND & AIMS: :It has been well documented that long-term exposure to inorganic arsenic induces cancers and vascular diseases in a dose-response relationship. Nevertheless, arsenic has also demonstrated to have anticancer activity; thus, arsenic trioxide (ATO, As2O3) is an inorganic trivalent arsenic form, currently used in the treatment against acute promyelocytic leukaemia (APL). The open discussion about how arsenic compounds induce genotoxic damage has moved us to evaluate the mutational spectrum induced by ATO in mouse lymphoma cells. Thus, 49 Tk-/- mutant colonies obtained in the mouse lymphoma assay (MLA), after treatments lasting for 4h with 10microM ATO, and 49 spontaneous mutant colonies from independent untreated cultures, were used to analyse and to characterise the mutational spectrum induced by this arsenic compound, to understand its mechanism of action. RT-PCR analysis of Tk cDNA and PCR amplifications of eight selected microsatellite sequences, located on chromosome 11, were used to carry out this screening. Our results show that, in mouse lymphoma cells, ATO is a strong clastogenic compound inducing large deletions, at chromosomal level, covering the Tk gene, as well as other regions of chromosome 11.

背景与目标： : 有充分的证据表明，长期暴露于无机砷会以剂量-反应关系诱发癌症和血管疾病。尽管如此，砷也已证明具有抗癌活性; 因此，三氧化二砷 (ATO，As2O3) 是一种无机三价砷形式，目前用于治疗急性早幼粒细胞白血病 (APL)。关于砷化合物如何诱导遗传毒性损伤的公开讨论已使我们能够评估ATO在小鼠淋巴瘤细胞中诱导的突变谱。因此，在用10microM ATO治疗4小时后，在小鼠淋巴瘤试验 (MLA) 中获得的49个Tk-/-突变菌落和来自独立未经处理的培养物的49个自发突变菌落被用来分析和表征这种砷化合物诱导的突变谱，了解它的作用机制。Tk cDNA的rt-pcr分析和位于11号染色体上的八个选定的微卫星序列的PCR扩增用于进行此筛选。我们的结果表明，在小鼠淋巴瘤细胞中，ATO是一种强的致残性化合物，可在染色体水平上诱导大量缺失，覆盖Tk基因以及11号染色体的其他区域。

BACKGROUND & AIMS: :Chikungunya and dengue, two arboviral infections are common in South-East Asia and their early clinical manifestations are very similar hence it is important to discriminate between them as early as possible for better clinical management. The aim of this study was to design a rapid, sensitive and specific method for the differential diagnosis of these two viruses simultaneously. A rapid one-tube duplex RT-PCR assay was developed that requires 110 min including RNA extraction, RT-PCR and agarose gel electrophoresis by using a novel Taq polymerase with high processivity. This one-tube duplex RT-PCR system with primers designed from the conserved regions of the genome allowed discrimination between the two viral groups. Bioinformatics analysis of the DNA sequences from PCR amplified products confirmed that this method was very specific and accurate. The time required for this duplex RT-PCR was comparable to the standard IgM capture ELISA method. This novel approach would help to diagnose specifically and accurately these two closely related arboviruses and enable early detection from blood. This method could be applied in resource limited settings, for surveillance in endemic regions or for routine epidemiological screening.

背景与目标： : 基孔肯雅热和登革热，两种虫媒病毒感染在东南亚很常见，它们的早期临床表现非常相似，因此尽早区分它们以更好的临床管理很重要。这项研究的目的是设计一种快速，灵敏和特异的方法来同时鉴别诊断疾病这两种病毒。开发了一种需要110分钟的快速单管双链rt-pcr分析，包括RNA提取、rt-pcr和琼脂糖凝胶电泳，通过使用具有高加工性的新型Taq聚合酶。这种单管双链rt-pcr系统具有从基因组保守区域设计的引物，可以区分两个病毒组。对PCR扩增产物DNA序列的生物信息学分析证实，该方法非常特异性和准确性。这种双重rt-pcr所需的时间与标准IgM捕获ELISA方法相当。这种新颖的方法将有助于特异性和准确地诊断这两种密切相关的虫媒病毒，并能够从血液中早期发现。此方法可用于资源有限的环境，流行地区的监测或常规流行病学筛查。

BACKGROUND & AIMS: :The HET-MN assay (hen's egg test for micronucleus induction) is different from other in vitro genotoxicity assays in that it includes toxicologically important features such as absorption, distribution, metabolic activation, and excretion of the test compound. As a promising follow-up to complement existing in vitro test batteries for genotoxicity, the HET-MN is currently undergoing a formal validation. To optimize the validation, the present study describes a critical analysis of previously obtained HET-MN data to check the experimental design and to identify the most appropriate statistical procedure to evaluate treatment effects. Six statistical challenges (I-VI) of general relevance were identified, and remedies were provided which can be transferred to similarly designed test methods: a Williams-type trend test is proposed for overdispersed counts (II) by means of a square-root transformation which is robust for small sample sizes (I), variance heterogeneity (III), and possible downturn effects at high doses (IV). Due to near-to-zero or even zero-count data occurring in the negative control (V), a conditional comparison of the treatment groups against the mean of the historical controls (VI) instead of the concurrent control was proposed, which is in accordance with US-FDA recommendations. For the modified Williams-type tests, the power can be estimated depending on the magnitude and shape of the trend, the number of dose groups, and the magnitude of the MN counts in the negative control. The experimental design used previously (i.e. six eggs per dose group, scoring of 1000 cells per egg) was confirmed. The proposed approaches are easily available in the statistical computing environment R, and the corresponding R-codes are provided.

背景与目标： : HET-MN测定 (用于微核诱导的鸡蛋测试) 与其他体外遗传毒性测定的不同之处在于，它包括毒理学上重要的特征，例如吸收，分布，代谢活化和测试化合物的排泄。作为补充现有的体外遗传毒性测试电池的有希望的后续行动，HET-MN目前正在进行正式验证。为了优化验证，本研究描述了对先前获得的het-mn数据的严格分析，以检查实验设计并确定最合适的统计程序来评估治疗效果。确定了六个具有一般相关性的统计挑战 (i-vi)，并提供了可以转移到类似设计的测试方法的补救措施: 通过平方根变换，针对过度分散的计数 (II) 提出了威廉姆斯型趋势检验，该方法对于小样本量 (I)，方差异质性 (III) 以及高剂量下可能的下降效应 (IV) 具有鲁棒性。由于阴性对照 (V) 中出现接近零甚至零计数的数据，因此提出了治疗组与历史对照 (VI) 的平均值的条件比较，而不是并发对照，这是根据US-FDA的建议。对于改良的Williams型测试，可以根据趋势的大小和形状，剂量组的数量以及阴性对照中MN计数的大小来估计功率。确认了先前使用的实验设计 (即每个剂量组六个卵，每个卵1000个细胞的评分)。所提出的方法在统计计算环境R中很容易获得，并提供了相应的R代码。

BACKGROUND & AIMS: :The hydrolysis of triglycerides in triglyceride-rich lipoproteins by LPL is critical for the delivery of triglyceride-derived fatty acids to tissues, including heart, skeletal muscle, and adipose tissues. Physiologically active LPL is normally bound to the endothelial cell protein glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1 (GPIHBP1), which transports LPL across endothelial cells, anchors LPL to the vascular wall, and stabilizes LPL activity. Disruption of LPL-GPIHBP1 binding significantly alters triglyceride metabolism and lipid partitioning. In this study, we modified the NanoLuc® Binary Technology split-luciferase system to develop a novel assay that monitors the binding of LPL to GPIHBP1 on endothelial cells in real time. We validated the specificity and sensitivity of the assay using endothelial lipase and a mutant version of LPL and found that this assay reliably and specifically detected the interaction between LPL and GPIHBP1. We then interrogated various endogenous and exogenous inhibitors of LPL-mediated lipolysis for their ability to disrupt the binding of LPL to GPIHBP1. We found that angiopoietin-like (ANGPTL)4 and ANGPTL3-ANGPTL8 complexes disrupted the interactions of LPL and GPIHBP1, whereas the exogenous LPL blockers we tested (tyloxapol, poloxamer-407, and tetrahydrolipstatin) did not. We also found that chylomicrons could dissociate LPL from GPIHBP1 and found evidence that this dissociation was mediated in part by the fatty acids produced by lipolysis. These results demonstrate the ability of this assay to monitor LPL-GPIHBP1 binding and to probe how various agents influence this important complex.

背景与目标： : LPL水解富含甘油三酸酯的脂蛋白中的甘油三酸酯对于将甘油三酸酯衍生的脂肪酸递送到包括心脏，骨骼肌和脂肪组织在内的组织至关重要。生理活性LPL通常与内皮细胞蛋白糖基磷脂酰肌醇锚定的高密度脂蛋白结合蛋白1 (GPIHBP1) 结合，该蛋白可跨内皮细胞运输LPL，将LPL锚定在血管壁上，并稳定LPL活性。LPL-GPIHBP1结合的破坏显著改变甘油三酯代谢和脂质分配。在这项研究中，我们修改了NanoLuc®二元技术裂解荧光素酶系统开发了一种新的检测方法，可实时监测LPL与内皮细胞GPIHBP1的结合。我们使用内皮脂肪酶和LPL的突变版本验证了该分析的特异性和敏感性，并发现该分析可靠且特异性地检测了LPL与gpihbp1之间的相互作用。然后，我们询问了LPL介导的脂解的各种内源性和外源性抑制剂是否具有破坏LPL与gpihbp1结合的能力。我们发现血管生成素样 (ANGPTL)4和ANGPTL3-ANGPTL8复合物破坏了LPL和GPIHBP1的相互作用，而我们测试的外源性LPL阻滞剂 (tyloxapol，poloxamer-407和四氢脂抑素) 则没有。我们还发现乳糜微粒可以将LPL与GPIHBP1解离，并发现证据表明这种解离部分是由脂解产生的脂肪酸介导的。这些结果证明了该测定法监测LPL-GPIHBP1结合和探测各种试剂如何影响该重要复合物的能力。

BACKGROUND & AIMS: :The SCO7222 protein and ActR are two of approximately 150 TetR-like transcription factors encoded in the Streptomyces coelicolor genome. Using bioluminescence as a readout, we have developed Escherichia coli-based biosensors that accurately report the regulatory activity of these proteins and used it to investigate their interactions with DNA and small-molecule ligands. We found that the SCO7222 protein and ActR repress the expression of their putative target genes, SCO7223 and actII-ORF2 (actA), respectively, by interacting with operator sequence in the promoters. The operators recognized by the two proteins are related such that O(7223) (an operator for SCO7223) could be bound by both the SCO7222 protein and ActR with similar affinities. In contrast, O(act) (an operator for actII-ORF2) was bound tightly by ActR and more weakly by the SCO7222 protein. We demonstrated ligand specificity of these proteins by showing that while TetR (but not ActR or the SCO7222 protein) interacts with tetracyclines, ActR (but not TetR or the SCO7222 protein) interacts with actinorhodin and related molecules. Through operator-targeted mutagenesis, we found that at least two nucleotide changes in O(7223) were required to disrupt its interaction with SCO7222 protein, while ActR was more sensitive to changes on O(act). Most importantly, we found that the interaction of each protein with wild-type and mutant operator sequences in vivo and in vitro correlated perfectly. Our data suggest that E. coli-based biosensors of this type should be broadly applicable to TetR-like transcription factors.

背景与目标： : SCO7222蛋白和ActR是coelicolor链霉菌基因组中编码的大约150个TetR样转录因子中的两个。使用生物发光作为读数，我们开发了基于大肠杆菌的生物传感器，可准确报告这些蛋白质的调节活性，并将其用于研究它们与DNA和小分子配体的相互作用。我们发现，SCO7222蛋白和ActR通过与启动子中的操纵子序列相互作用，分别抑制了其假定的靶基因SCO7223和actII-ORF2 (actA) 的表达。由两种蛋白质识别的算子相关，使得O(7223) (SCO7223的算子) 可以被SCO7222蛋白和ActR结合，具有相似的亲和力。相反，O(act) (actII-ORF2的操作员) 被ActR紧密结合，而被SCO7222蛋白更弱地结合。我们通过显示TetR (但不是ActR或SCO7222蛋白) 与四环素相互作用，ActR (但不是TetR或SCO7222蛋白) 与放线菌素和相关分子相互作用，证明了这些蛋白的配体特异性。通过操作员靶向诱变，我们发现至少需要两个核苷酸变化O(7223) 来破坏其与SCO7222蛋白的相互作用，而ActR对O(act) 的变化更敏感。最重要的是，我们发现每种蛋白质在体内和体外与野生型和突变算子序列的相互作用完全相关。我们的数据表明，这种类型的基于大肠杆菌的生物传感器应广泛适用于TetR样转录因子。

BACKGROUND & AIMS: BACKGROUND:Postoperative parathyroid gland function after total thyroidectomy (TT) has traditionally been monitored by the measurement of serum calcium concentrations. The purpose of this study is to determine whether measurement of parathyroid hormone (PTH) concentrations in the early postoperative period accurately predicts patients at risk of developing hypocalcaemia. METHODS:A prospective cohort study of patients undergoing TT was carried out. PTH concentrations were measured preoperatively and at 4 and 23 h postoperatively. Serum calcium concentration was measured preoperatively and twice daily for 48 h after surgery. RESULTS:One hundred patients undergoing TT were recruited into the study in the period June 2004 to July 2005. Benign multinodular goitre was the most common indication for surgery (77%). The incidence of temporary hypocalcaemia (Ca < 2.0 mmol/L) was 18%. The mean PTH concentration at 4 h after surgery was 22.3 ng/L and was not significantly different from the 23-h concentration of 23.2 ng/L (P = 0.18). A PTH concentration of < or = 3 ng/L measured at 4 h after surgery had a sensitivity, specificity and likelihood ratio of 0.71, 0.94 and 11.3, respectively, for predicting postoperative hypocalcaemia. The accuracy of a single PTH concentration at 4 h was good for predicting hypocalcaemia (area under receiver-operator characteristic curve 0.90; confidence interval 0.81-0.96). There was no significant difference in accuracy between the 4- and 24-h PTH concentrations (P = 0.14). CONCLUSIONS:A single measurement of PTH concentration in the early postoperative period after TT reliably predicts patients who are likely to develop hypocalcaemia. This approach facilitates early discharge and may decrease the need for multiple postoperative blood tests.

背景与目标：

BACKGROUND & AIMS: :We examined 73 children with respiratory infections for Chlamydophila (Chlamydia) pneumoniae and Mycoplasma pneumoniae using real-time PCR assay and serological tests. C. pneumoniae and M. pneumoniae infections were found in 11 (15.1%) and 6 (8.2%) cases, respectively. The sensitivities and specificities of real-time PCR versus definite diagnosis of acute infection were 63.6% and 100% for C. pneumoniae, and 100% and 100% for M. pneumoniae, respectively. C. pneumoniae PCR-negative results appeared to be due to poor growth of the organism. The sensitivity and specificity of ImmunoCard tests were 33.3% and 82.1%, respectively, indicating that the efficacy of rapid diagnosis was disputable. The present results suggest that real-time PCR is suitable for rapid diagnosis as a first screening test to determine first-line antibacterial agents to be used against these infectious diseases.

背景与目标： : 我们使用实时PCR分析和血清学测试检查了73名呼吸道感染儿童的肺炎衣原体 (衣原体) 和肺炎支原体。分别在11例 (15.1% 例) 和6例 (8.2% 例) 中发现了肺炎支原体和肺炎支原体感染。分别63.6% 和100% 了肺炎支原体的实时PCR与明确诊断的急性感染的敏感性和特异性，以及肺炎支原体的100% 和100%。肺炎链球菌PCR阴性结果似乎是由于生物体生长不良所致。免疫卡试验的敏感性和特异性分别33.3% 和82.1%，表明快速诊断的疗效存在争议。目前的结果表明，real-time PCR适用于快速诊断，作为确定用于这些传染病的一线抗菌剂的首次筛选测试。

BACKGROUND & AIMS: :The evaluation of a recently introduced kit (Cortipac (R)) for measuring plasma cortisol by competitive protein binding technique without prior extraction of steroids from plasma, is presented. The blank value was less than 2.5 mug/100 ml, and the lowest measurable value was 2.5 mug/100 ml. The coefficients of variation within- and between-batch determinations were 8.64% and 9.21% respectively. By adding known amounts of cortisol to pre-extracted plasma a linear correlation between calculated and measured cortisol was obtained. In comparison with the double isotope derivate technique, a linear correlation was found. A high degree of cross-reaction was found with nearly all of 11 tested steroids. Due to the considerably higher plasma cortisol concentration in most clinical cond-tions, the lack of specificity may be of minor importance, but must be considered in evaluating increased plasma cortisol values. The mean plasma cortisol concentration in normal subjects (20.3 mug/100 ml) was equal to or slightly higher than found by other methods. The kit was found appropriate for routine clinical application, due to the easy, rapid and inespensive performance with an acceptable recovery and reliability, but it cannot entirely replace more specific methods.

背景与目标： : 介绍了最近推出的试剂盒 (Cortipac (R)) 的评估，该试剂盒通过竞争性蛋白质结合技术测量血浆皮质醇，而无需事先从血浆中提取类固醇。空白值小于2.5马克杯/100毫升，最低可测值为2.5马克杯/100毫升。批次内和批次间测定的变异系数分别为8.64% 和9.21%。通过将已知量的皮质醇添加到预提取的血浆中，可以获得计算的皮质醇与测量的皮质醇之间的线性相关性。与双同位素衍生技术相比，发现了线性相关性。几乎所有11种测试的类固醇都发现高度交叉反应。由于在大多数临床条件下血浆皮质醇浓度相当高，缺乏特异性可能不太重要，但在评估血浆皮质醇值增加时必须考虑这一点。正常受试者 (20.3杯/100毫升) 的平均血浆皮质醇浓度等于或略高于通过其他方法发现的。由于易于，快速和快速的性能以及可接受的恢复和可靠性，该试剂盒被认为适合常规临床应用，但不能完全替代更具体的方法。