Efficient Editing of an Adenoviral Vector Genome with CRISPR/Cas9

Immunotherapy based on genetic modification of T cells has played an important role in the treatment of tumors and viral infections. Moreover, adenoviral vectors engineered with improved safety due to their inability to integrate into the host genome have been key in the clinical application of T cell therapy. However, the commonly used adenoviral vector Ad5 exhibits low efficiency of infection of human T cells and the details of the intracellular trafficking pathway of adenoviral vectors in human primary T cells remains unclear. Resolution of these issues will depend on successful modification of the adenoviral vector. To this end, here we describe the successful establishment of a simple and efficient method for editing adenoviral vectors in vitro using the CRISPR-Cas9 gene editing system to target the adenoviral fiber gene. Electronic supplementary materialThe online version of this article (10.1007/s12088-020-00905-3) contains supplementary material, which is available to authorized users.     

Salidroside Inhibits Reactive Astrogliosis and Glial Scar Formation in Late Cerebral Ischemia via the Akt/GSK-3β Pathway

Neurochemical Research - Cerebral ischemia leads to reactive astrogliosis and glial scar formation. Glial scarring can impede functional restoration during the recovery phase of stroke. Salidroside...     

Identification and Characterization of a Novel Single Domain Antibody Against Ebola Virus

Virologica Sinica - Ebola virus (EBOV) belongs to the Filoviridae family and causes severe illnesses such as hemorrhagic fever with a high mortality rate up to 90%. Now two antibody drugs termed...     

联合TCGA和GEO数据库筛选乳腺癌易感基因

本研究是利用公共基因芯片数据库筛选乳腺癌的预后基因,预测和探索这些基因在乳腺癌进展中的可能机制和临床价值.首先,我们筛选了公共基因芯片数据库(gene expression omnibus,GEO)GSE22820和癌症基因组图谱(the cancer genome atlas,TCGA)乳腺癌数据库的重叠差异表达基因,联合R语言分析乳腺癌组织与癌旁正常组织差异表达的基因;其次,基于STRING数据库及Cytoscape软件构建蛋白质相互作用网络图,分析并识别了中枢基因和前3个模块;之后进行了更多的功能分析,包括基因本体(gene ontology,GO)和京都基因与基因组百科全书(kyoto encyclopedia of genes and genomes,KEGG)通路分析以及基因集富集分析(gene set enrichment analysis,GSEA),以研究这些基因的作用以及潜在的潜在机制;最后进行了Kaplan-Meier分析和Cox比例风险分析,以阐明这些基因的诊断和预后效果.相关数据分析表明15个基因的表达水平与生存预后相关,高表达基因患者的总生存时间短于低表达患者(P＜0.05);Cox比例风险分析表明UBE2T、ER-CC6L和RAD51这3个基因是预后生存的独立因素(P＜0.05);GSEA分析表明在UBE2T、ERCC6L和RAD51基因中细胞周期、基础转录因子和卵母细胞减数分裂明显富集.最终,我们得出结论,这3种基因标志物的高表达是乳腺癌预后不良因素,可作为预测乳腺癌患者转移和预后的有效生物标志物.     

Comprehensive landscape and interference of clonal haematopoiesis mutations for liquid biopsy: A Chinese pan-cancer cohort

Tumour-derived DNA found in the plasma of cancer patients provides the probability to detect somatic mutations from circulating cell-free DNA (cfDNA) in plasma samples. However, clonal hematopoiesis (CH) mutations affect the accuracy of liquid biopsy for cancer diagnosis and treatment. Here, we integrated landscape of CH mutations in 11,725 pan-cancer patients of Chinese and explored effects of CH on liquid biopsies in real-world. We first identified 5933 CHs based on panel sequencing of matched DNA of white blood cell and cfDNA on 301 genes for 5100 patients, in which CH number of patients had positive correlation with their diagnosis age. We observed that canonical genes related to CH, including DNMT3A, TET2, ASXL1, TP53, ATM, CHEK2 and SF3B1, were dominant in the Chinese cohort and 13.29% of CH mutations only appeared in the Chinese cohort compared with the Western cohort. Analysis of CH gene distribution bias indicated that CH tended to appear in genes with functions of tyrosine kinase regulation, PI3K-Akt signalling and TP53 activity, suggesting unfavourable effects of CH mutations in cancer patients. We further confirmed effect of driver genes carried by CH on somatic mutations in liquid biopsy of cancer patients. Forty-eight actionable somatic mutations in 17 driver genes were considered CH genes in 92 patients (1.80%) of the Chinese cohort, implying potential impacts of CH on clinical decision-making. Taken together, this study exhibits strong evidence that gene mutations from CH interfere accuracy of liquid biopsies using cfDNA in cancer diagnosis and treatment in real-world.     

Knockout of Akt1/2 suppresses the metastasis of human prostate cancer cells CWR22rv1 in vitro and in vivo

Although primary androgen deprivation therapy resulted in tumour regression, unfortunately, majority of prostate cancer progress to a lethal castration-resistant prostate cancer, finally die to metastasis. The mutual feedback between AKT and AR pathways plays a vital role in the progression and metastasis of prostate cancer. Therefore, the treatment of a single factor will eventually inevitably lead to failure. Therefore, better understanding of the molecular mechanisms underlying metastasis is critical to the development of new and more effective therapeutic agents. In this study, we created prostate cancer CWR22rv1 cells with the double knockout of Akt1 and Akt2 genes through CRISPR/Cas9 method to investigate the effect of Akt in metastasis of prostate cancer. It was found that knockout of Akt1/2 resulted in markedly reduced metastasis in vitro and in vivo, and appeared to interfere AR nuclear translocation through regulating downstream regulatory factor, FOXO proteins. It suggests that some downstream regulatory factors in the AKT and AR interaction network play a vital role in prostate cancer metastasis and are potential targeting molecules for prostate cancer metastasis treatment.     

Breast milk contains probiotics with anti-infantile diarrhoea effects that may protect infants as they change to solid foods

Infants often experience complementary food-induced diarrhoea (CFID), which occurs when infants switch from breast milk to solid foods. The relative abundances of Prevotella and Rothia were higher in stools of infants with CFID, while the relative abundances of Enterococcus and Escherichia were higher in healthy infants. The abundance of Lactobacillus spp. normally found in breast milk fed to infants with CFID was significantly reduced, and Enterococcus spp. were less abundant when diarrhoea occurred. Furthermore, Lactobacillus and Enterococcus were present as shared bacteria in both mother and infant, and they were considered potential anti-CFID probiotics as their relative abundances in breast milk were negatively correlated to infant CFID. Kyoto encyclopedia of genes and genomes (KEGG) functional analysis showed that the function of amino acid metabolism differed between infants with CFID and healthy infants. Therefore, CFID might be related to the decomposition of proteins in food supplements. The screening revealed seven hydrolytic casein and five hydrolytic casein and rice protein isolates from 320 suspected Lactobacillus and Enterococcus isolates. The animal experiments demonstrated that a mixture of five isolates effectively hydrolysed the casein and rice protein and prevented diarrhoea in young rats. Thus, the occurrence of CFID was found to be closely related to the intestinal and breast milk microbiota, and bacteria that could assist in the digestion of cereal proteins were involved in CFID.     

Protective potential of Chinese herbal extracts against microsporidian Nosema ceranae,an emergent pathogen of western honey bees,Apis mellifera L.

Nosema ceranae, a newly emergent parasite invading western honey bees (Apis mellifera L.), is indicated to threaten honey bee health at both individual and colony levels. However, the efficient and environmentally-friendly treatments are quite limited at present. To find alternative medicine to control Nosema diseases, the effect of 8 types of herbal extracts against N. ceranae infection were screened under laboratory condition. Of which, 1% Andrographis paniculata (A. paniculata) decoction was found to significantly decrease N. ceranae spore numbers on 7 days post infection (dpi) and 13 dpi. Then, our results further revealed that A. paniculata decoction at doses ranging from 1% to 7% displayed significant efficient inhibition of Nosema spore proliferation and improved the infected bees' survival rates in a dose-dependent manner. A. paniculata decoction was found to protect the gut tissues of infected workers from damage cause by N. ceranae, which might be due to the regulation of the expression of certain genes in Wnt and JNK pathways, including armadillo, basket, frizzled2 and groucho. Additionally, our study suggested that A. paniculata decoction performed this Nosema spore-reducing potential over its two monomers, andrographolide and dehydrographolide. Taken together, this work enables us to better understand A. paniculata decoction's potential to inhibit N. ceranae infection, thus providing a new guidance for developing applicable drugs to control Nosema diseases.     

Crystal structure of Drosophila melanogaster tryptophan 2,3-dioxygenase reveals insights into substrate recognition and catalytic mechanism

Tryptophan 2,3-dioxygenase (TDO) catalyzes the oxidative cleavage of the indole ring of l-tryptophan to N-formylkynurenine in the kynurenine pathway, and is considered as a drug target for cancer immunotherapy. Here, we report the first crystal structure of a eukaryotic TDO from Drosophila melanogaster (DmTDO) in complex with heme at 2.7 Å resolution. DmTDO consists of an N-terminal segment, a large domain and a small domain, and assumes a tetrameric architecture. Compared with prokaryotic TDOs, DmTDO contains two major insertion sequences: one forms part of the heme-binding site and the other forms a large portion of the small domain. The small domain which is unique to eukaryotic TDOs, interacts with the active site of an adjacent monomer and plays a role in the catalysis. Molecular modeling and dynamics simulation of DmTDO-heme-Trp suggest that like prokaryotic TDOs, DmTDO adopts an induced-fit mechanism to bind l-Trp; in particular, two conserved but flexible loops undergo conformational changes, converting the active site from an open conformation to a closed conformation. The functional roles of the key residues involved in recognition and binding of the heme and the substrate are verified by mutagenesis and kinetic studies. In addition, a modeling study of DmTDO in complex with the competitive inhibitor LM10 provides useful information for further inhibitor design. These findings reveal insights into the substrate recognition and the catalysis of DmTDO and possibly other eukaryotic TDOs and shed lights on the development of effective anti-TDO inhibitors.