A rapid method for measuring drug enrichment in epidermis

Summary A rapid and simple method is described for measuring the enrichment of small molecules in epidermal tissue. To measure such an enrichment, a small tissue sample (2–10 mg) is allowed to equilibrate with a buffered solution of a labelled substance for periods of 12–36 h. The concentration of the radioactive molecule in the tissue is measured as a decrease of radioactivity in the solution. Concentration measurements in the tissue itself can be performed, but are not required to detect enrichment in the tissue or to assess its magnitude.The specific density of appendage free human epidermis has been determined and was found to be 1.20 g/cm³. Using this value, tissue weight can be translated into volume and concentration changes in the solution can be recalculated to yield the concentration of the substance in the tissue itself. Close agreement was found between the calculated tissue concentration and the values actually measured, following digestion of the epidermis with NaOH and measuring the activity in the tissue digest.The enrichment of five substances in human epidermis was measured: -estradiol, thiopyronine, 8-methoxypsoralen (8-MOP), 5-methoxypsoralen (5-MOP), and theophylline. Of these substances, the first four are concentrated by human epidermis and the concentrations reached within the tissue are 10–500 times higher than the concentration of the same substance in the surrounding buffer. The enrichment data has been analysed in an attempt to distinguish between reversible affinity binding to specific tissue sites and partitioning of the substances between buffer and tissue components (lipids, membranes, etc.). In the case of thiopyronine and 8-MOP, reversible binding is indicated with dissociation constants of 10^-7 M and 10^-5 M, respectively, while partitioning distribution could account for the behavior of 5-MOP and -estradiol. The method can be used either as a rapid screening method or as a quantitative analysis for the characterization of tissue enrichment with specific drugs.This work is part of the Ph. D. thesis to be submitted by A. M. to the Technical University of Berlin     

Mitigating the effects of reference sequence bias in single-multiplex massively parallel sequencing of the mitochondrial DNA control region

Sequence analysis of the mitochondrial DNA (mtDNA) control region can provide forensically useful information, particularly in challenging samples where autosomal DNA profiling fails. Sub-division of the 1122-bp region into shorter PCR fragments improves data recovery, and such fragments can be analysed together via massively parallel sequencing (MPS). Here, we generate mtDNA data using the prototype PowerSeq™ Auto/Mito/Y System (Promega) MPS assay, in which a single PCR reaction amplifies ten overlapping amplicons of the control region, in a set of 101 highly diverse samples representing most major clades of the mtDNA phylogeny. The overlapping multiplex design leads to non-uniform coverage in the regions of overlap, where it is further increased by short amplicons generated alongside the intended products. Primer sequences in targeted amplification libraries are a potential source of reference sequence bias and thus should be removed, but the proprietary nature of the primers in commercial kits necessitates an alternative approach that minimises data loss: here, we introduce the bioinformatic selection of sequencing reads spanning putative primer sites (Overarching Read Enrichment Option, OREO). While OREO performs well in mitigating the effects of primer sequences at the ends of sequence reads, we still find evidence of the internalisation of primer-derived sequences by overlap extension, which may compromise the ability to call variants or to measure heteroplasmy in primer-binding regions. The commercially available PowerSeq™ CRM Nested System design prevents primer internalisation, as shown in a reanalysis of a subset of 57 samples that contain possible heteroplasmies. In combination with OREO, the CRM Nested kit mitigates reference sequence bias, allowing heteroplasmic variants to be estimated down to a 5% threshold. Provided appropriate steps are taken in data processing, single-reaction multiplex assays represent robust tools to analyse mtDNA control region variation. The OREO approach will allow users to bypass the effects of unknown primer sequences in any single-reaction tiled multiplex and eliminate primer-derived bias in overlapping amplicon sequencing studies, in both forensic and non-forensic settings.     

Levels of Heavy Metals Pollution in Different Types of Soil of Central Greece

This investigation was performed in order to clarify the degree of heavy metals pollution in forest, agricultural and industrial surface soil samples in relation to pre-anthropogenic soils of Almyros region, in Central Greece. In 2004 and 2005 soil samples were collected and analysed for available (DTPA method) and total (Aqua Regia method) Cd, Cr, Cu, Pb, Zn and Ni concentrations. For each metal the enrichment factor with respect to the levels in pre-anthropogenic soils was calculated. All the types of soils appeared to be less polluted than in other investigations. In agricultural and industrial soils the available Cd concentration was higher than the other metals studied. The enrichment factor of Cu in relation to total concentration has the maximum value of the metals studied. Professor Mitsios is deceased.     

基于网络药理学预测苦参治疗肝癌的分子机制

目的:运用网络药理学的方法,探讨苦参治疗肝癌的作用机制.方法:通过中医药系统药理学数据库分析平台(TC-MSP)筛选出苦参活性成分及作用靶点;在OMIM和Genecards中获取疾病靶点.通过R语言对基因进行匹配并绘制韦恩图;在String网站构建活性成分-蛋白质-蛋白质相互作用网络图;Cytoscape 3.6.1软...     

银川市冬、春季大气PM2.5中12种元素浓度差异及来源分析

目的 通过对银川市空气污染高发的冬、春季大气PM2.5中12种元素含量进行分析,研究冬、春季空气污染物来源特点,为银川市空气污染治理提供针对性的建议。方法 2017年11月至2018年5月每月定期采集大气PM2.5,采用称重法和电感耦合等离子质谱法测定大气PM2.5及其12种元素的质量浓度,分析季节差异,采用富集因子分析和主成分分析判断污染物来源。结果 银川市冬季大气PM2.5浓度及元素Pb和Tl的浓度分别为77μg/m3和50.40ng/m3、0.71ng/m3,显著高于春季,存在统计学差异,P＜0.05。无论冬、春季,元素Sb、Cd、Hg、Pb、Se和Tl的富集程度均较高,且主成分分析显示这些元素载荷较大,主要来自人为源,如燃料的燃烧、交通源、工业源和垃圾燃烧等。结论 银川市冬季大气污染较春季严重,冬季与春季有共同的污染物来源,但冬季受供暖的影响,污染较春季严重。     

速效救心丸治疗心肌缺血疾病的网络药理学研究

目的：借助网络药理学分析方法,研究速效救心丸主要活性成分对心肌缺血疾病的网络调控作用。方法：预测速效救心丸中生物利用度较好的化合物,整合PharmMapper和Mas等公共数据库提供的成分、靶点、通路信息,使用Cytoscape 3.1.0软件构建速效救心丸成分-靶点-通路的药理学网络。运用蛋白互作分析、子簇聚类算法和功能富集分析,来发现蛋白互作网络中的功能模块及其所参与的生物学过程。结果：速效 救心丸的化学成分-靶点-通路网络图包括145个点和374条边。MCODE算法提取出得分≥3的5个子簇经功能富集分析（BinGo）显示,这5个子簇参与的生物学过程主要有：脂质和固醇荷尔蒙的相关代谢、谷胱甘肽和氨 基酸的相关代谢、炎症反应和免疫功能、心肌功能和能量供应及血压调节等方面。结论：本研究运用网络药理学的方法和技术,从分子网络层面阐释了速效救心丸主要活性成分通过调节心脏能量供应、脂质紊乱、免疫反 应等方面发挥了多成分、多靶点、多途径相互协同治疗疾病的机制。     

基于PPI网络预测和验证结直肠癌相关Hub基因

目的:通过PPI网络分析确定CRC相关Hub基因,为CRC靶向治疗提供一定参考。方法:从OpenTargets数据库获得CRC相关基因,使用Metascape对基因集进行富集分析,确定基因集富集术语。采用STRING数据库及CytoScape构建PPI网络,采用MCODE寻找PPI网络中的功能模块（子网）。根据功能模块中节点的网络属性确定Hub基因,通过文献法和GEPIA数据库对Hub基因进行验证。结果:共得到CXCL8、ERBB2、CYCS 3个Hub基因,它们均与CRC的发生和发展有一定关系,。它们均在CRC组织与正常组织中存在差异表达,并与CRC患者的总体生存时间相关。结论:基于PPI的网络分析可准确预测CRC相关Hub基因和为CRC靶向治疗提供参考。     

基于网络药理学探讨止嗽散治疗喉源性咳嗽的作用机制

目的：利用网络药理学探讨止嗽散治疗喉源性咳嗽的作用机制。方法：运用中药系统药理学数据库与分析平台(TCMSP)对止嗽散的主要成分及作用靶点进行筛选和挖掘;利用Cytoscape 3.7.2软件,构建中药成分-靶点网络图;以GeneCards平台挖掘喉源性咳嗽的相关靶点;运用STRING数据库构建止嗽散治疗喉源性咳嗽的蛋白质-蛋白质相互作用(PPI)网络图;进行拓扑分析并筛选出止嗽散治疗喉源性咳嗽的核心靶点;利用David数据库对药物、疾病交集靶点进行富集分析。结果：止嗽散药物-成分-靶点-疾病网络图包含152个有效成分,相对应靶点155个;喉源性咳嗽相关靶点1 176个。其中β-谷甾醇、槲皮素、木犀草素、豆甾醇、山柰酚等可能是止嗽散治疗喉源性咳嗽的关键成分,涉及TP53、VEGFA、IL6、MAPK、JUN、TNF、EGF等14个核心靶点。基因本体(GO)生物过程分析涉及一氧化氮生物合成过程的正调控、细胞外间隙、基因表达的正调控、细胞外区、脂多糖介导的信号通路等。京都基因和基因组百科全书(KEGG)通路富集分析提示这些靶点参与TNF信号通路、NOD样受体信号通路、FoxO信号通路、To...     

基于网络稳健性的和血明目片潜在适应证预测

目的：基于药物干扰疾病网络稳健性的方法来预测和血明目片潜在的适应证。方法：使用中药系统药理学数据库与分析平台(TCMSP)、中医药综合数据库(TCMID)等数据库搜集和血明目片中的活性成分,利用BATMAN-TCM预测成分的潜在靶点;通过DisGeNET和HPO数据库获取相应的疾病蛋白靶标,基于靶点移除前后整体网络拓扑特征的变化评价药物靶点对于网络稳定性的影响,实现和血明目片干预不同疾病药效的预测。通过建立“中药成分-药物靶点-疾病蛋白”关联网络,从中筛选出潜在药物作用靶点并对其进行富集分析,考察和血明目片干预疾病可能的生物学过程和信号通路,最终从体外细胞实验的角度初步对药效进行验证。结果：搜集整理后得到和血明目片所含化学成分946个,预测高可信度靶点761个。通过考察药物扰动疾病网络稳健性,发现和血明目片对翼状胬肉、角膜炎、高眼压症、角膜新生血管化的疾病网络有较强的扰动作用。通过构建和血明目片干预不同疾病的“中药成分-药物靶点-疾病蛋白”异质网络,获得相应的潜在药物作用靶点,多项富集分析结果显示和血明目片可能通过干预血管新生、白细胞迁移、脂多糖(LPS)应答、核因子κB转录调控、凋亡...