One hundred years of high-throughput Drosophila research

From the beginning, Drosophila was a high-throughput model organism. Unbiased and genome-wide efforts ranging from Morgan's search for spontaneous mutations and subsequent saturating loss-of-function and gain-of-function screens up to more recent techniques such as microarrays, proteomics and cellular assays have been and will continue to be the backbone of Drosophila research. Integrating these large datasets is one of the next challenges. However, once achieved, a plethora of information far exceeding the information content of the singular experiments will be revealed. Several high-throughput techniques and experimental strategies highlighting the unbiased and integrative nature of Drosophila research during the last century will be discussed.     

活体荧光成像技术评价X射线对小鼠乳腺癌移植瘤的治疗效果

目的 建立荧光素酶标记的小鼠乳腺癌细胞爪垫淋巴结转移模型,监测肿瘤早期淋巴结转移,并用荧光成像评价X射线局部治疗效果。方法 将表达荧光素酶的小鼠乳腺癌细胞系4T1-Luc接种至裸鼠爪垫皮下,建立爪垫皮下淋巴结转移模型。通过裸鼠活体荧光成像系统连续观察肿瘤细胞在淋巴结中的转移情况,并将有早期淋巴转移的荷瘤裸鼠按随机数字表法分为对照组和治疗组,活体荧光成像系统观察治疗效果,HE染色观察评价病理形态学变化。结果 成功建立了小鼠乳腺癌淋巴结转移模型,爪垫原发灶肿瘤体积与荧光光子数呈正相关性（r=0.958,P<0.001）,在肿瘤接种的第24天,治疗组爪垫肿瘤和腘窝处肿瘤部位的荧光光子数较对照组呈显著性降低（t=32.58,P<0.05）;用荧光光子数计算抑瘤率高达85%以上。HE染色观察到治疗组较对照组移植瘤坏死明显。结论 运用活体生物发光成像技术能够动态、客观、灵敏、可视化地评估X射线对小鼠乳腺癌肿瘤的抑瘤效应。     

Multicenter evaluation of a new quantitative highly sensitive D-dimer assay, the Hemosil® D-dimer HS 500, in patients with clinically suspected venous thromboembolism

Introduction

D-dimer testing is widely used in conjunction with clinical pretest probability (PTP) for venous thromboembolism (VTE) exclusion. We report on a multicenter evaluation of a new, automated, latex enhanced turbidimetric immunoassay [HemosIL® D-Dimer HS 500, Instrumentation Laboratory (IL)].

Materials and Methods

747 consecutive outpatients with suspected proximal deep vein thrombosis (DVT, n = 401) or pulmonary embolism (PE, n = 346) were evaluated at four university hospitals in a management study with a 3 month follow-up. Samples were tested at each center using the new D-dimer assay on an automated coagulation analyzer [ACL TOP (IL)], with clinical cut-off for VTE at 500 ng/mL (FEU).

Results

The sensitivity and negative predictive value (NPV) were 100% for all PTP subgroups (no false negative results); for both sensitivity and NPV the lower limit of the 95% CI in patients with moderate/low PTP was higher than 95%. The overall specificity was 45.1% (95%CI: 41.1-49.3%). Higher specificity value was recorded in the low PTP subgroup [49.2% (95%CI: 41.7-56.7)]. No significant differences were found between patients suspected of having DVT or PE; sensitivity and NPV were 100%. The reproducibility of the assay was good, being the total CVs% less than 10% for D-dimer concentration near the clinical cut-off.

Conclusions

The new, highly sensitive D-dimer assay proved to be accurate when used for VTE diagnostic work-up in outpatients. Based on 100% sensitivity and NPV and lower limit of the 95% CI higher than 95%, the assay can be used as a stand-alone test in patients with non high PTP.     

Actin sliding velocity on pure myosin isoforms from dystrophic mouse muscles

Duchenne muscular dystrophy (DMD) is a genetic disease characterized by skeletal muscle wasting and atrophy. Recent evidence suggests that the impaired skeletal muscle performance in DMD is not solely dependent on a loss of contractile muscle mass. In this study the myosin motor function of mdx and control (wildtype, WT) mice was compared using pure myosin isoforms in an “in vitro motility assay” (IVMA). Actin sliding velocity (Vf) on myosin 2B extracted from single muscle fibers of gastrocnemius muscles was significantly lower in mdx mice (3.48 ± 0.13 μm/s, n = 18) than in WT mice (4.02 ± 0.19 μm/s, n = 10). No difference in Vf was found between myosin 1 extracted from soleus muscles of mdx (0.84 ± 0.04 μm/s, n = 13) and of WT (0.89 ± 0.04 μm/s, n = 10). The results suggest that the dystrophic process alters myosin molecular function, and this contributes to the functional impairment in dystrophic muscles. Muscle Nerve 40: 249–256, 2009     

嗅鞘细胞及其表达的NGF和BDNF对神经干细胞增殖的影响

目的 研究嗅鞘细胞及其表达的神经生长因子(NGF)和脑源性神经营养因子(BDNF)对神经干细胞增殖的影响.方法 采用共培养液培养以及NGF或BDNF抗体封闭的方法,观察嗅鞘细胞对神经干细胞增殖的影响.免疫组化和RT.PCR半定量分析嗅鞘细胞表达的细胞因子及其受体的情况.结果 共培养液培养4d后,神经干细胞数量明显增多(P<0.05).免疫组化和RT-PCR半定量分析结果显示,嗅鞘细胞表达多种细胞因子及受体,共培养液培养2d后嗅鞘细胞表达NGF和BDNF mRNA的相对值明显高于对照组(P<0.05).抗体封闭培养液中的NGF或BDNF后,没有影响神经干细胞的增殖.结论 嗅鞘细胞表达大量细胞因子作用于神经干细胞,促进其增殖.在本研究条件下,神经干细胞的增殖可能与嗅鞘细胞表达的碱性成纤维生长因子(bFGF)和表皮生长因子(EGF)有关,而与NGF和BDNF无关.     

Noninvasive visualization of tumor growth in a human colorectal liver metastases xenograft model using bioluminescence in vivo imaging

Background

Bioluminescence imaging (BLI) is an ideal tool for noninvasive, quantitative monitoring of tumor progression/regression in animal models. The effectiveness of different treatment strategies is displayed by an altered intensity of bioluminescence, demonstrating a change of the tumor burden. The aim of this study was to establish a reliable, reproducible colorectal hepatic metastases cancer animal model.

Methods

Cells of the human colon carcinoma cell line HCT-116 Luc^pos expressing the firefly luciferase enzyme gene were used. HCT-116 Luc^pos cells (2.5 × 10⁶) were injected through the portal vein into the liver of immunoincompetent nude mice. BLI was used to analyze intrahepatic tumor burden and growth kinetic.

Results

HCT-116 Luc^pos cells demonstrated a progressive and reproducible growth in the liver after intraportal injection. Four days after injection, the animals were analyzed for tumor growth by BLI, and mice without or too low bioluminescence signals were excluded (between 10% and 20% animals). HCT-116 Luc^pos intrahepatic tumors responded successfully to different dosages (5 and 10 mg/kg) of 5-fluorouracil.

Conclusions

BLI is an important tool with many potential advantages for investigators. The measurement of intrahepatic tumor growth by imaging luciferase activity noninvasively provides valuable information on tumor burden and effectiveness of therapy. Thus, the presented intrahepatic metastases model based on the growth of HCT-116 Luc^pos cells is suitable for in vivo testing of different cancer therapy strategies.     

Role for interferon-gamma release assays in latent tuberculosis screening before TNF-α antagonist therapy

TNF-α antagonist therapy is associated with a risk of severe, extrapulmonary, disseminated tuberculosis, which is fatal in 10% of cases. The risk of tuberculosis is increased four-fold in patients on TNF-α antagonist therapy. The main risk factors are a history of untreated or inadequately treated primary tuberculosis, recent contact with a tuberculosis patient, and residence in or travel to a high-endemicity region. Infection surveillance agencies throughout the world have issued recommendations to ensure the detection and treatment of latent tuberculosis before TNF-α antagonist initiation. These recommendations have returned the incidence of tuberculosis to the level seen before the introduction of TNF-α antagonists. Nevertheless, there is still room for improvement. Recommendations about latent tuberculosis screening include the use of tuberculin skin tests. However, these tests are positive in individuals vaccinated with the BCG vaccine, which leads to overuse of tuberculosis chemoprophylaxis and, therefore, to unnecessary patient exposure to hepatotoxic effects. Furthermore, tuberculin skin tests may be falsely negative in immunosuppressed patients, leading to underuse of tuberculosis prophylaxis. These shortcomings of tuberculin skin tests have generated interest in interferon-gamma release assays (IGRAs). In patients with overt tuberculosis, IGRAs are more sensitive and more specific than tuberculin skin tests. However, the accuracy of IGRAs for diagnosing latent tuberculosis remains unknown, because no reference standard is available. In addition, patients taking immunosuppressant agents to treat systemic disease may exhibit anergia, which complicates the interpretation of IGRAs. Until additional data become available, caution requires that IGRAs be used only when a positive or negative result, as assessed on a case-by-case basis, will help to decide whether tuberculosis chemoprophylaxis is in order.     

裸鼠原位和异位脑肿瘤生物发光信号成像实验研究

目的应用生物发光成像技术,非侵入性地连续检测活体裸鼠原位和异位脑肿瘤发展演进过程。方法用SMPU-R-MND-luc载体转染人脑肿瘤U87MG细胞系,形成具有高荧光素酶活性的细胞克隆。在裸鼠脑内和胁腰部皮下植入持续表达荧光素酶的肿瘤细胞,建立原位和异位脑肿瘤模型,用影像学资料显示肿瘤部位。用光子发射定量分析动态监测肿瘤生长情况。结果成功地建立了表达荧光素酶活性的原位和异位脑肿瘤动物模型。采集反映肿瘤生长的生物发光信号,肿瘤细胞植入后不同时间点的发光信号值呈显著正相关,而且原位和异位脑肿瘤间存在明显差异。但生物发光脑肿瘤生物发光信号值在第4 d和第14 d时无显著差异。结论体内生物发光成像可以非侵入性地动态检测活体内脑肿瘤演进过程,为研究肿瘤发展机制及最佳治疗策略的选择提供了新的手段和工具。     

结核分枝杆菌感染的免疫学诊断:进展与争议

近年来,全球结核病疫情出现再度加重趋势,我国结核病负担在全球居于前列.控制结核病依赖于早期诊断和早期治疗,而诊断水平的落后将直接影响临床疗效和进一步的疫情控制.目前结核病确诊的主要依据仍然是细菌学检查(包括培养和涂片),因此发展快速易行的检测方法尤为重要.免疫学诊断是结核病诊断的重要部分,近期取得了诸多进展.目前发展的以T细胞为基础的γ-干扰素释放试验(IGRAs),相比于目前最常用的结核菌素试验(TST,又称PPD试验)有更高的敏感性与特异性.