Maximizing intelligence obtained from higher-order DNA mixtures from volume crime samples

ABSTRACT

This work collates data from the analysis of complex mixtures analysed in STRmix during routine no-suspect volume crime work. It interrogates the upload rate for these types of mixtures and which component of the profile has been able to be interpreted for upload. The number of profiles giving multiple uploads and the amount of replicate PCR analysis has been collated.     

A DNA vaccine expressing an optimized secreted FAPα induces enhanced anti-tumor activity by altering the tumor microenvironment in a murine model of breast cancer

Cancer-associated fibroblasts (CAFs), major components of the tumor microenvironment (TME), promote tumor growth and metastasis and inhibit the anti-tumor immune response. We previously constructed a DNA vaccine expressing human FAPα, which is highly expressed by CAFs, to target these cells in the TME, and observed limited anti-tumor effects in the 4T1 breast cancer model. When the treatment time was delayed until tumor nodes formed, the anti-tumor effect of the vaccine completely disappeared. In this study, to improve the safety and efficacy, we constructed a new FAPα-targeted vaccine containing only the extracellular domain of human FAPα with a tissue plasminogen activator signal sequence for enhanced antigen secretion and immunogenicity. The number of CAFs was more effectively reduced by CD8⁺ T cells induced by the new vaccine. This resulted in decreases in CCL2 and CXCL12 expression, leading to a significant decrease in the ratio of myeloid-derived suppressor cells in the TME. Moreover, when mice were treated after the establishment of tumors, the vaccine could still delay tumor growth. To facilitate the future application of the vaccine in clinical trials, we further optimized the gene codons and reduced the homology between the vaccine and the original sequence, which may be convenient for evaluating the vaccine distribution in the human body. These results indicated that the new FAPα-targeted vaccine expressing an optimized secreted human FAPα induced enhanced anti-tumor activity by reducing the number of FAPα⁺ CAFs and enhancing the recruitment of effector T cells in the 4T1 tumor model mice.     

Genetic toxicity assessment using liver cell models: past,present, and future

ABSTRACT

Genotoxic compounds may be detoxified to non-genotoxic metabolites while many pro-carcinogens require metabolic activation to exert their genotoxicity in vivo. Standard genotoxicity assays were developed and utilized for risk assessment for over 40 years. Most of these assays are conducted in metabolically incompetent rodent or human cell lines. Deficient in normal metabolism and relying on exogenous metabolic activation systems, the current in vitro genotoxicity assays often have yielded high false positive rates, which trigger unnecessary and costly in vivo studies. Metabolically active cells such as hepatocytes have been recognized as a promising cell model in predicting genotoxicity of carcinogens in vivo. In recent years, significant advances in tissue culture and biological technologies provided new opportunities for using hepatocytes in genetic toxicology. This review encompasses published studies (both in vitro and in vivo) using hepatocytes for genotoxicity assessment. Findings from both standard and newly developed genotoxicity assays are summarized. Various liver cell models used for genotoxicity assessment are described, including the potential application of advanced liver cell models such as 3D spheroids, organoids, and engineered hepatocytes. An integrated strategy, that includes the use of human-based cells with enhanced biological relevance and throughput, and applying the quantitative analysis of data, may provide an approach for future genotoxicity risk assessment.     

DNA vaccine encoding the moonlighting protein Onchocerca volvulus glyceraldehyde-3-phosphate dehydrogenase (Ov-GAPDH) leads to partial protection in a mouse model of human filariasis

River blindness, caused by the filarial parasite Onchocerca volvulus, is a major socio-economic and public health problem in Sub-Saharan Africa. In January 2015, The Onchocerciasis Vaccine for Africa (TOVA) Initiative has been launched with the aim of providing new tools to complement mass drug administration (MDA) of ivermectin, thereby promoting elimination of onchocerciasis in Africa. In this context we here present Onchocerca volvulus glyceraldehyde-3-phosphate dehydrogenase (Ov-GAPDH) as a possible DNA vaccine candidate. We report that in a laboratory model for filariasis, immunization with Ov-GAPDH led to a significant reduction of adult worm load and microfilaraemia in BALB/c mice after challenge infection with the filarial parasite Litomosoides sigmodontis. Mice were either vaccinated with Ov-GAPDH.DNA plasmid (Ov-pGAPDH.DNA) alone or in combination with recombinantly expressed Ov-GAPDH protein (Ov-rGAPDH). During the following challenge infection of immunized and control mice with L. sigmodontis, those formulations which included the DNA plasmid, led to a significant reduction of adult worm loads (up to 57% median reduction) and microfilaraemia (up to 94% reduction) in immunized animals. In a further experiment, immunization with a mixture of four overlapping, synthetic Ov-GAPDH peptides (Ov-GAPDHpept), with alum as adjuvant, did not significantly reduce worm loads. Our results indicate that DNA vaccination with Ov-GAPDH has protective potential against filarial challenge infection in the mouse model. This suggests a transfer of the approach into the cattle Onchocerca ochengi model, where it is possible to investigate the effects of this vaccination in the context of a natural host–parasite relationship.     

前S1蛋白抗原测定在乙型肝炎诊疗中的应用评价

目的进一步评价前S1蛋白抗原测定在乙型肝炎诊断疗效和预后等方面的作用.方法通过ELISA方法对380例急慢性乙型肝炎患者进行PreS1Ag测定,同时用ELISA法测定HBV病毒血清标志物和用PCR法测定HBV-DNA.结果 "大三阳"患者PreS1Ag阳性率为90.09%,"小三阳"患者PreS1Ag阳性率为50%,PreS1Ag与HBV-DNA、HBeAg相一致.PreS1Ag随着HBV的清除而消长.对23例经拉米夫定治疗的患者进行动态观察,PreS1Ag消除率达85%.结论前S1蛋白抗原测定在乙型肝炎诊疗中有很大的应用价值.     

Erythematous and reticular forms of oral lichen planus and oral lichenoid reactions differ in pathological features related to disease activity

BACKGROUND: Common clinical forms of oral lichen planus (OLP) and oral lichenoid reactions (OLR) are erythematous (ERY) or reticular (RET). The purpose of this study was to find histopathological changes that differ between these forms. METHODS: Epithelial thickness, epithelial proliferation rate, apoptosis, and HLA-DR expression were compared among 10 reticular and 12 erythematous lesions, and 11 normal oral mucosa samples (NOM). RESULTS: The epithelium in ERY was thinner than in NOM, whereas RET showed values between ERY and NOM. Cell proliferation increased significantly in ERY as compared with RET and NOM, with no difference between RET and NOM. Relative numbers of epithelial cell nuclei displaying visible chromatin condensation were reduced in ERY form. CONCLUSIONS: The markedly increased cell proliferation in ERY supports the notion that this form displays a higher disease activity as compared to RET. It can therefore be important to study each disease form separately.     

DNA-Methylierung von monohalogenierten Methanen in F-344 Ratten

Monohalogenated methanes (methyl chloride, methyl bromide and methyl iodide) are mutagenic and carcinogenic. The possible mechanism of these effects, DNA methylation, was studied. DNA adducts from orgnas of F344 rats exposed to these chemicals were separated and identified with high performance liquid chromatography (HPLC) and gaschro-matography/massspectrometry (GC/ MS). DNA adducts, 7-methylguanine (7-MeG) and O⁶-Methylguanine(0⁸-MeG), incorporation of¹⁴C into de novo synthesis of nucleobases could be observed in enzymatic DNA hydrolysates by HPLC and determination of the radioactivity in the fractions. The formation of DNA add,ue,ts in the studied organs was only quantitatively different. The formation of O⁶-MeG was further pioved by analysing the acidic hydrolysates using HPLC with non-radioactive O⁶MeG as internal standard. 7-MeG and 3-MeA were identified with GC/MS analysis.     

中国汉坦病毒H82O5株G2糖蛋白基因的克隆及在真核细胞中的瞬时表达

从感染病毒乳鼠脑组织提取总RNA，采用RT－PCR和分子克隆技术将扩增到的G2糖蛋白基因插入含CMV启动子的pcDNA3.1/His质粒载体中，通过脂质体介导转染COS－7细胞，用SDS－PAGE、Western-blot及IFIA方法分别测定表达产物的相对分子量及特异性。结果证明获得正向插入的G2－pcDNA3.1/His重组表达质粒，表达产物的相对分子量为56ku，与理论预期大小一致，并且可与汉坦病毒H8205株的腹水抗体起特异反应。表明构建的G2－pcDNA3.1/His重组质粒所表达的蛋白为中国汉坦病毒株特有，能在哺乳动物细胞中表达并具有抗原性，重组质粒可应用于汉坦病毒的DNA疫苗研究。     

Sequence-dependent Termination of Mammalian DNA Polymerase Reaction by a New Platinum Compound, (–)-(R)-2-Aminomethylpyrrolidine(1,1-cyclobutane-dicarboxylato)-2-platinum(II) Monohydrate

We examined the mechanisms of the inhibition of DNA synthesis by a new platinum compound, (-)-( R )-2-aminomethylpyrrolidine(1,1-cyclobutane-dicarboxylato)-2-platinum(II) monohydrate (DWA-2114R), a derivative of the antitumor drug cis- diamminedichloroplatinum(II) (CDDP), using prokaryotic and eukaryotic DNA polymerases. Preincubating activated DNA with CDDP or DWA-2114R reduced its template activity for prokaryotic and eukaryotic DNA polymerases in a dose-dependent manner. DWA2114R required six times greater drug concentration and two times longer incubation time to show the same decrease of the template activity compared to CDDP. Treatment of primed pUC118 ssDNA templates with the two drugs followed by second-strand synthesis by prokaryotic and eukaryotic DNA polymerases revealed that DWA2114R bound to DNA in a similar manner to CDDP and these adducts blocked DNA elongation by DNA polymerases of eukaryotes as well as of prokaryotes. With these two drugs, the elongations by E. coli DNA polymerase I (Klenow fragment), T7 DNA polymerase and calf thymus DNA polymerase α were strongly arrested at guanine-guanine sequences (GG). Stop bands were also observed at adenine-guanine sequences (AG) guanine-adenine-guanine sequences (GAG) and mono-guanine sequence (G). Calf testis DNA polymerase β was also arrested efficiently at AG, GAG and G, but much more weakly at GG. This pattern was common to DWA2114R and CDDP.     

二乙酰二脱水卫矛醇对小鼠白血病L1210细胞增殖的影响

目的　研究二乙酰二脱水卫矛醇 (1 ,2 :5 ,6 dianhydro 3 ,4 diacetylgalactitol,DADAG)的抗脑白血病作用及机制。方法　用小鼠脑内移植瘤模型、MTT法、DNA掺入法、流式细胞仪和Westernblot法 ,观察DADAG对小鼠脑内移植瘤和体外白血病L1 2 1 0 细胞的作用 ,并探讨作用机制。结果　DADAG对DBA/ 2小鼠脑内移植白血病L1 2 1 0 有明显的抑制作用 ;对体外白血病L1 2 1 0 细胞同样有很强的抗增殖作用 ,其IC50 值为 2 4 6mg·L- 1 。DADAG不可逆地抑制L1 2 1 0 细胞内DNA的生物合成。DADAG 2 4mg·L- 1 处理L1 2 1 0 细胞 6h后 ,细胞发生G2 /M周期阻滞 ,2 4h后达最高峰。细胞周期素B1 蛋白水平在DADAG处理 2 4h后开始下降 ,而磷酸化的细胞周期依赖性激酶CDK1在DADAG处理 6h后开始上调 ,并呈时间依赖性。结论　DADAG的抗脑白血病作用与其抑制白血病细胞的增殖密切相关