Assays for the Identification of Novel Antivirals against Bluetongue Virus

To identify potential antivirals against BTV, we have developed, optimized and validated three assays presented here. The CPE-based assay was the first assay developed to evaluate whether a compound showed any antiviral efficacy and have been used to screen large compound library. Meanwhile, cytotoxicity of antivirals could also be evaluated using the CPE-based assay. The dose-response assay was designed to determine the range of efficacy for the selected antiviral, i.e. 50% inhibitory concentration (IC₅₀) or effective concentration (EC₅₀), as well as its range of cytotoxicity (CC₅₀). The ToA assay was employed for the initial MoA study to determine the underlying mechanism of the novel antivirals during BTV viral lifecycle or the possible effect on host cellular machinery. These assays are vital for the evaluation of antiviral efficacy in cell culture system, and have been used for our recent researches leading to the identification of a number of novel antivirals against BTV.     

3D image-based dosimetry for Yttrium-90 radioembolization of hepatocellular carcinoma: Impact of imaging method on absorbed dose estimates

BackgroundTo improve therapy outcome of Yttrium-90 selective internal radiation therapy (⁹⁰Y SIRT), patient-specific post-therapeutic dosimetry is required. For this purpose, various dosimetric approaches based on different available imaging data have been reported. The aim of this work was to compare post-therapeutic 3D absorbed dose images using Technetium-99m (^99mTc) MAA SPECT/CT, Yttrium-90 (⁹⁰Y) bremsstrahlung (BRS) SPECT/CT, and ⁹⁰Y PET/CT.MethodsTen SIRTs of nine patients with unresectable hepatocellular carcinoma (HCC) were investigated. The ^99mTc SPECT/CT data, obtained from ^99mTc-MAA-based treatment simulation prior to ⁹⁰Y SIRT, were scaled with the administered ⁹⁰Y therapy activity. 3D absorbed dose images were generated by dose kernel convolution with scaled ^99mTc/⁹⁰Y SPECT/CT, ⁹⁰Y BRS SPECT/CT, and ⁹⁰Y PET/CT data of each patient. Absorbed dose estimates in tumor and healthy liver tissue obtained using the two SPECT/CT methods were compared against ⁹⁰Y PET/CT.ResultsThe percentage deviation of tumor absorbed dose estimates from ⁹⁰Y PET/CT values was on average −2 ± 18% for scaled ^99mTc/⁹⁰Y SPECT/CT, whereas estimates from ⁹⁰Y BRS SPECT/CT differed on average by −50 ± 13%. For healthy liver absorbed dose estimates, all three imaging methods revealed comparable values.ConclusionThe quantification capabilities of the imaging data influence ⁹⁰Y SIRT tumor dosimetry, while healthy liver absorbed dose values were comparable for all investigated imaging data. When no ⁹⁰Y PET/CT image data are available, the proposed scaled ^99mTc/⁹⁰Y SPECT/CT dosimetry method was found to be more appropriate for HCC tumor dosimetry than ⁹⁰Y BRS SPECT/CT based dosimetry.     

植物乳杆菌P-8对小鼠免疫功能的调节作用研究

摘要 目的：不同类型的益生菌株免疫调节功能各异。本文旨在评价植物乳杆菌P-8（Lactobacillus plantarum P-8）对小鼠免疫功能的调控作用及机制。方法：C57BL/6J小鼠每日灌胃给予不同剂量的植物乳杆菌P-8（0. 25 mg/kg、0.5 mg/kg、1.5 mg/kg）,连续30天,记录小鼠一般情况。给药结束后处死动物,测定小鼠脏器/体重比;小鼠碳廓清实验、小鼠腹腔巨噬细胞吞噬鸡红细胞实验评价各组小鼠的单核-巨噬细胞功能;血清溶血素测定、抗体生成细胞实验评价各组小鼠的体液免疫功能;脾淋巴细胞转化实验、迟发型变态反应实验评价各组小鼠的细胞免疫功能;NK细胞的活性测定实验评价小鼠的NK细胞活性。结果：与对照组相比,低、中、高剂量组植物乳杆菌P-8对小鼠脏器/体重比值差异无统计学意义（P>0.05）;且植物乳杆菌P-8可显著提高小鼠的碳廓清能力、小鼠腹腔巨噬细胞吞噬鸡红细胞能力、半数溶血值、二硝基氟苯诱导的小鼠迟发型变态反应及NK细胞活力（P均<0.05）。结论：植物乳杆菌P-8可通过提高单核-巨噬细胞功能、体液免疫功能、细胞免疫功能及NK细胞活力增强小鼠的免疫功能。     

牵牛胁迫应答基因PnLOG2的克隆及功能验证

RING型E3泛素连接酶在植物应答非生物胁迫过程中发挥着重要功能。该研究从圆叶牵牛中克隆出RING型E3泛素连接酶基因PnLOG2,该基因序列号为XM_019321049.1。利用ORF Finder预测PnLOG2基因编码开放阅读框长度为912 bp (51~992 bp),编码313个氨基酸,蛋白分子质量34.38 kD,理论等电点为5.14。系统发育分析表明,PnLOG2基因与番茄亲缘关系最近。组织特异性分析表明,PnLOG2基因在牵牛不同组织均有表达,在老茎和新叶中表达量较高。qRT PCR分析结果表明,PnLOG2基因在圆叶牵牛根和叶中受干旱、盐碱胁迫诱导显著上调表达。通过异源表达PnLOG2基因于酵母细胞中,发现干旱、盐碱胁迫下PnLOG2基因提高了重组酵母的耐盐和耐旱能力,但降低了对碱的耐受性。该研究初步阐明了PnLOG2基因在干旱、盐碱胁迫下的功能,为进一步研究RING型E3泛素连接酶在非生物胁迫中的机理提供了理论依据。     

A practical tool to evaluate dose distributions using radiochromic film in radiation oncology

PurposeTriple channel algorithm and specific procedures make more reliable radiochromic dosimetry for treatment planning verification and quality assurance in radiation therapy. A tool to obtain radiochromic dose distributions and compare them with the ones resulting from a treatment planning system was developed and applied.MethodsThe tool was developed as Microsoft Excel macro; it builds dose calibration curves against net optical density of Gafchromic EBT3 film, produces axial, coronal and sagittal dose maps and allows to evaluate them against dose distributions calculated by the Varian treatment planning system Eclipse using gamma index and gamma angle.ResultsThe net optical density standard errors of estimate of calibration curves at 6 MV Varian DBX600 linac energy were 0.2%, 0.4% and 0.2% for the red, green and blue channels. Tests of these curves by means of three independent eight dose points measurement series, at 15 MV and 6 MV Varian 2100C linac and at 6 MV DBX600 linac energies, showed less than 2% of dose errors for the red channel and less than 3% for the green channel in the range 100–450 cGy. The comparisons between dose distributions from Gafchromic EBT3 triple channel algorithm and the ones from Eclipse analytic anisotropic algorithm (AAA) showed values of gamma index 95th percentile between 0.6 and 1.0.ConclusionThe obtained results encourage the application of this tool in radiation therapy quality assurance.     

A cine-EPID based method for jaw detection and quality assurance for tracking jaw in IMRT/VMAT treatments

A new tool with the potential to verify and track jaw position during delivery has been developed. The method should be suitable for independent quality assurance for jaw position during jaw tracking dynamic IMRT and VMAT treatments. The jaw detection and tracking algorithm developed consists of five main steps. Firstly, the image is enhanced by removing a normalised predicted EPID image (that does not include the collimator transmission) from each cine EPID image. Then, using a histogram clustering technique a global intensity threshold level was determined. This threshold level was used to classify each pixel of the image as either under the jaws or under the MLC. Additionally, the collimator angle was automatically detected and used to rotate the image to vertical direction. Finally, this rotation allows the jaw positions to be determined using vertical and horizontal projection profiles. Nine IMRT fields (with static jaws) and a single VMAT clinical field (with dynamic jaws) were tested by determining the root mean square difference between planned and detected jaw positions. The test results give a detection accuracy of ±1 mm RMS error for static jaw IMRT treatments and ±1.5 mm RMS error for the dynamic jaw VMAT treatment. This method is designed for quality assurance and verification in modern radiation therapy; to detect the position of static jaws or verify the position of tracking jaws in more complex treatments. This method uses only information extracted from EPID images and it is therefore independent from the linear accelerator.     

A possible role of lysophospholipids produced by calcium-independent phospholipase A2 in membrane-raft budding and fission

Phospholipase A₂ (PLA₂) not only plays a role in the membrane vesiculation system but also mediates membrane-raft budding and fission in artificial giant liposomes. This study aimed to demonstrate the same effects in living cells. Differentiated Caco-2 cells were cultured on filter membranes. MDCK cells were challenged with Influenza virus. The MDCK cultures were harvested for virus titration with a plaque assay. Alkaline phosphatase (ALP), a membrane-raft associated glycosylphosphatidylinositol (GPI)-anchored protein, was 70% released by adding 0.2 mmol/l lysophosphatidylcholine, which was abolished by treatment with a membrane-raft disrupter, methyl-β-cyclodextrin. Activation of calcium-independent PLA₂ (iPLA₂) by brefeldin A increased the apical release of ALP by approximately 1.5-fold (p < 0.01), which was blocked by PLA₂ inhibitor bromoenol lactone (BEL). BEL also reduced Influenza virus production into the media (< 10%) in the MDCK culture. These results suggest that cells utilize inverted corn-shaped lysophospholipids generated by PLA₂ to modulate plasma membrane structure and assist the budding of raft-associated plasma membrane particles, which virus utilizes for its budding. Brush borders are enriched with membrane-rafts and undergo rapid turnover; thus, PLA₂ may be involved in the regulatory mechanism in membrane dynamism. Further, iPLA₂ may provide a therapeutic target for viral infections.     

Alterations of the Plasma Membrane Caused by Murine Polyomavirus Proliferation: An Electrorotation Study

In this report we investigate the alterations of the dielectric properties of the plasma membrane caused by the infection of cultured fibroblasts with murine polyomavirus. The approach consists in a well-established dielectric spectroscopy technique, electrorotation, which has been successfully used in our laboratory to study the alterations of the plasma membrane of cells exposed to various forms of stress. The response to viral proliferation was time dependent as shown by evaluation of the de novo synthesis of viral DNA. This response was paralleled by gradual damage of the membrane evidenced by alteration of the dielectric parameters, specific capacitance and conductance. The electrorotation results show a reduced effect on the dielectric properties of the membrane when infection is carried out in the presence of a natural oil (MEX). In this case a drastic reduction in viral DNA synthesis was also monitored, thus indicating an antiviral action of this product.     

Adaptive dose-finding: Proof of concept with type I error control

We consider an adaptive dose‐finding study with two stages. The doses for the second stage will be chosen based on the first stage results. Instead of considering pairwise comparisons with placebo, we apply one test to show an upward trend across doses. This is a possibility according to the ICH‐guideline for dose‐finding studies (ICH‐E4). In this article, we are interested in trend tests based on a single contrast or on the maximum of multiple contrasts. We are interested in flexibly choosing the Stage 2 doses including the possibility to add doses. If certain requirements for the interim decision rules are fulfilled, the final trend test that ignores the adaptive nature of the trial (naïve test) can control the type I error. However, for the more common case that these requirements are not fulfilled, we need to take the adaptivity into account and discuss a method for type I error control. We apply the general conditional error approach to adaptive dose‐finding and discuss special issues appearing in this application. We call the test based on this approach Adaptive Multiple Contrast Test. For an example, we illustrate the theory discussed before and compare the performance of several tests for the adaptive design in a simulation study.     

Controlling False Discoveries in Multidimensional Directional Decisions,with Applications to Gene Expression Data on Ordered Categories

Summary Microarray gene expression studies over ordered categories are routinely conducted to gain insights into biological functions of genes and the underlying biological processes. Some common experiments are time‐course/dose‐response experiments where a tissue or cell line is exposed to different doses and/or durations of time to a chemical. A goal of such studies is to identify gene expression patterns/profiles over the ordered categories. This problem can be formulated as a multiple testing problem where for each gene the null hypothesis of no difference between the successive mean gene expressions is tested and further directional decisions are made if it is rejected. Much of the existing multiple testing procedures are devised for controlling the usual false discovery rate (FDR) rather than the mixed directional FDR (mdFDR), the expected proportion of Type I and directional errors among all rejections. Benjamini and Yekutieli (2005, Journal of the American Statistical Association 100, 71–93) proved that an augmentation of the usual Benjamini–Hochberg (BH) procedure can control the mdFDR while testing simple null hypotheses against two‐sided alternatives in terms of one‐dimensional parameters. In this article, we consider the problem of controlling the mdFDR involving multidimensional parameters. To deal with this problem, we develop a procedure extending that of Benjamini and Yekutieli based on the Bonferroni test for each gene. A proof is given for its mdFDR control when the underlying test statistics are independent across the genes. The results of a simulation study evaluating its performance under independence as well as under dependence of the underlying test statistics across the genes relative to other relevant procedures are reported. Finally, the proposed methodology is applied to a time‐course microarray data obtained by Lobenhofer et al. (2002, Molecular Endocrinology 16, 1215–1229). We identified several important cell‐cycle genes, such as DNA replication/repair gene MCM4 and replication factor subunit C2, which were not identified by the previous analyses of the same data by Lobenhofer et al. (2002) and Peddada et al. (2003, Bioinformatics 19, 834–841). Although some of our findings overlap with previous findings, we identify several other genes that complement the results of Lobenhofer et al. (2002) .