外源过氧化氢酶对液态发酵红茶色素的影响

为得到优质速溶红茶,本研究以低档绿茶为原料,对其水提液添加外源过氧化氢酶(CAT)进行液态发酵酶促转化成红茶茶汤,探讨液态发酵工艺对红茶色素生成量的影响,结果表明:参照优质红茶色素含量标准,CAT酶促转化的最优条件为:加酶量0.25 U/m L,反应温度45℃,反应时间2 h,p H6。CAT与多酚氧化酶(PPO)存在双酶协同作用,绿茶水提液先用PPO处理后(酶添加量0.05 U/m L,温度40℃,p H5,时间1.5 h)再用CAT处理,合成红茶色素的效果比单一酶源好。      

有氧运动训练和姜黄素对Ⅱ型糖尿病大鼠肝糖原及糖代谢信号通路的影响

为探讨有氧运动训练和姜黄素对Ⅱ型糖尿病大鼠肝糖原及糖代谢信号通路的影响,将大鼠分为对照组（C组）,模型组（D组）、运动组（S组）、姜黄素组（H组）和姜黄素+运动组（S+H组）,S组和S+H组大鼠给予8周有氧运动训练,H组和S+H组大鼠给予8周姜黄素灌胃干预,测定其空腹血糖、口服葡萄糖耐量实验（OGTT）后各时点血糖、胰岛素分泌、胰岛素敏感性、肝糖原水平及肝脏葡萄糖代谢及胰岛素信号分子mRNA表达的变化。结果表明:与D组相比,S+H组大鼠OGTT实验后各时刻血糖值均降低（p<0.05）;且胰岛素水平、胰岛素敏感指数及肝糖原水平均较S组和H组高（p<0.05）;肝脏葡萄糖激酶（GK）、肝细胞葡萄糖载体蛋白-2（GLUT2）、肝脏胰岛素样生长因子1（IGF-1）、胰岛素受体底物1（IRS-1）、胰岛素受体底物2（IRS-2）相对表达量均较S组和H组高（p<0.05）,而人葡萄糖激酶调节蛋白（GKRP）则较S组和H组降低（p<0.05）,且联合干预效果优于单独干预。结论表明有氧运动和姜黄素可有效降低血糖水平,改善胰岛素抵抗,促进肝糖原合成。      

多导体传输线瞬态响应研究

随着多导体传输线内各导体之间间距的减小, 导体之间的近邻效应对传输线的分布参数和传输特性的影响越来越大.为此, 我们针对三种典型的传输线结构, 分别建立了基于矢势有限元方法分析的多导体传输线的模型, 并分析了近邻效应对磁通密度和分布电感的影响.利用提出的方法计算了同轴传输线的单位长度分布电感, 并将它与采用解析方法得到的结果进行比较来证明该方法的正确性.计算双线传输线在不同间距时的单位长度电感, 与理论分析得到的结果相比较验证了导线间距越小, 近邻效应对单位长度电感的影响越大.最后, 计算考虑了近邻效应的耦合微带线的电感矩阵, 并将它与其他不考虑近邻效应的方法得到的结果相比较, 说明近邻效应对传输线电感矩阵的影响.     

A Simple and Efficient Method for the Substrate Identification of Amino Acid Decarboxylases

Amino acid decarboxylases convert amino acids into different biogenic amines which regulate diverse biological processes. Therefore, identifying the substrates of amino acid decarboxylases is critical for investigating the function of the decarboxylases, especially for the new genes predicted to be amino acid decarboxylases. In the present work, we have established a simple and efficient method to identify the substrates and enzymatic activity of amino acid decarboxylases based on LC-MS methods. We chose GAD65 and AADC as models to validate our method. GAD65 and AADC were expressed in HEK 293T cells and purified through immunoprecipitation. The purified amino acid decarboxylases were subjected to enzymatic reaction with different substrate mixtures in vitro. LC-MS analysis of the reaction mixture identified depleted or accumulated metabolites, which corresponded to candidate enzyme substrates and products, respectively. Our method successfully identified the substrates and products of known amino acid decarboxylases. In summary, our method can efficiently identify the substrates and products of amino acid decarboxylases, which will facilitate future amino acid decarboxylase studies.     

Transgenerationally Transmitted DNA Demethylation of a Spontaneous Epialleles Using CRISPR/dCas9-TET1cd Targeted Epigenetic Editing in Arabidopsis

CRISPR/dCas9 is an important DNA modification tool in which a disarmed Cas9 protein with no nuclease activity is fused with a specific DNA modifying enzyme. A previous study reported that overexpression of the TET1 catalytic domain (TET1cd) reduces genome-wide methylation in Arabidopsis. A spontaneous naturally occurring methylation region (NMR19-4) was identified in the promoter region of the PPH (Pheophytin Pheophorbide Hydrolase) gene, which encodes an enzyme that can degrade chlorophyll and accelerate leaf senescence. The methylation status of NMR19-4 is associated with PPH expression and leaf senescence in Arabidopsis natural accessions. In this study, we show that the CRISPR/dCas9-TET1cd system can be used to target the methylation of hypermethylated NMR19-4 region to reduce the level of methylation, thereby increasing the expression of PPH and accelerating leaf senescence. Furthermore, hybridization between transgenic demethylated plants and hypermethylated ecotypes showed that the demethylation status of edited NMR19-4, along with the enhanced PPH expression and accelerated leaf senescence, showed Mendelian inheritance in F1 and F2 progeny, indicating that spontaneous epialleles are stably transmitted trans-generationally after demethylation editing. Our results provide a rational approach for future editing of spontaneously mutated epialleles and provide insights into the epigenetic mechanisms that control plant leaf senescence.     

Novel Roles of RNA m6A Methylation Regulators in the Occurrence of Alzheimer’s Disease and the Subtype Classification

Alzheimer’s disease (AD) is one of the most common forms of dementia, closely related to epigenetic factors. N6-methyladenosine (m6A) is the most abundant RNA modification, affecting the pathogenesis and development of neurodegenerative diseases. This study was the first exploration of the combined role of 25 common m6A RNA methylation regulators in AD through the integrated bioinformatics approaches. The 14 m6A regulators related to AD were selected by analyzing differences between AD patients and normal controls. Based on the selected m6A regulators, AD patients could be well classified into two m6A models using consensus clustering. The two clusters of patients had different immune profiles, and m6A regulators were associated with the components of immune cells. Additionally, there were 19 key AD genes obtained by screening differential genes through weighted gene co-expression network and least absolute shrinkage and selection operator regression analysis, which were highly associated with important m6A regulators during the occurrence of AD. More interestingly, NOTCH2 and NME1 could be potential targets for m6A regulation of AD. Taken together, these findings indicate that dysregulation of m6A methylation affects the occurrence of AD and is vital for the subtype classification and immune infiltration of AD.     

Targeting PSEN1 by lnc-CYP3A43-2/miR-29b-2-5p to Reduce β Amyloid Plaque Formation and Improve Cognition Function

Presenilin-1 (PSEN1) is a crucial subunit within the γ-secretase complex and regulates β-amyloid (Aβ) production. Accumulated evidence indicates that n-butylidenephthalide (BP) acts effectively to reduce Aβ levels in neuronal cells that are derived from trisomy 21 (Ts21) induced pluripotent stem cells (iPSCs). However, the mechanism underlying this effect remains unclear. This article aims to investigate the possible mechanisms through which BP ameliorates the development of Alzheimer’s disease (AD) and verify the effectiveness of BP through animal experiments. Results from RNA microarray analysis showed that BP treatment in Ts21 iPSC-derived neuronal cells reduced long noncoding RNA (lncRNA) CYP3A43-2 levels and increased microRNA (miR)-29b-2-5p levels. Bioinformatics tool prediction analysis, biotin-labeled miR-29b-2-5p pull-down assay, and dual-luciferase reporter assay confirmed a direct negative regulatory effect for miRNA29b-2-5p on lnc-RNA-CYP3A43-2 and PSEN1. Moreover, BP administration improved short-term memory and significantly reduced Aβ accumulation in the hippocampus and cortex of 3xTg-AD mice but failed in miR-29b-2-5p mutant mice generated by CRISP/Cas9 technology. In addition, analysis of brain samples from patients with AD showed a decrease in microRNA-29b-2-5p expression in the frontal cortex region. Our results provide evidence that the LncCYP3A43-2/miR29-2-5p/PSEN1 network might be involved in the molecular mechanisms underlying BP-induced Aβ reduction.     

Engineered Immature Testicular Tissue by Electrospun Mats for Prepubertal Fertility Preservation in a Bioluminescence Imaging Transgenic Mouse Model

Prepubertal boys with cancer may suffer from reduced fertility and maturity following gonadotoxic chemoradiotherapy. Thus, a viable method of immature testicular tissue (ITT) preservation is required in this cohort. In this study, we used poly-L-lactic acid electrospun scaffolds with two levels of fineness to support the development of ITT transplanted from transgenic donors to wild-type recipient mice. The purpose of this study was to evaluate the potential of ITT transplantation and spermatogenesis after using the two scaffolds, employing bioluminescence imaging for evaluation. The results suggest that ITT from 4-week-old mice possessed the most potential in spermatogenesis on the 70th day, together with the fine electrospun scaffolds. Moreover, bioluminescent imaging intensity was observed in recipient mice for up to 107 days, approximately six times more than the coarse electrospun scaffold and the control group. This occurs since the fine scaffold is more akin to the microenvironment of native testicular tissue as it reduces stiffness resulting from micronization and body fluid infiltration. The thermal analysis also exhibited recrystallization during the biodegradation process, which can lead to a more stable microenvironment. Overall, these findings present the prospect of fertility preservation in prepubertal males and could serve as a framework for future applications.     

Comprehensive Molecular Characterization of the Mitochondrial Genome of the Takin Lungworm Varestrongylus eleguneniensis (Strongylida: Protostrongylidae)

The takin lungworm Varestrongylus eleguneniensis (Strongylida: Protostrongylidae) causes lethal bronchopneumonia and represents severe threats to captive and wild populations. However, until now there has been very limited information available concerning the molecular epidemiology and evolutionary biology of V. eleguneniensis. Mitochondrial genomes (mtDNAs) can provide resources for investigations in these areas and, therefore, can assist with the surveillance and control of this lungworm. Herein, the complete mtDNA of V. eleguneniensis was sequenced and characterized with Illumina pipeline analyses. This circular genome (13,625 bp) encoded twelve protein-coding genes (PCGs), two rRNAs, and twenty-two tRNAs, with notable levels of AT and GC skews. Comparative genomics revealed a purifying selection among PCGs, with cox1 and nad6 having the lowest and the highest evolutionary rate, respectively. Genome-wide phylogenies showed a close relationship between V. eleguneniensis and Protostrongylus rufescens in Strongylida. Single gene (PCGs or rRNAs)-based phylogenies indicated that cox1 and nad5 genes shared the same family-level topology with that inferred from genomic datasets, suggesting that both genes could be suitable genetic markers for evolutionary and phylogenetic studies of Strongylida species. This was the first mtDNA of any member of the genus Varestrongylus, and its comprehensive molecular characterization represents a new resource for systematic, population genetic and evolutionary biological studies of Varestrongylus lungworms in wildlife.     

2cChIP-seq and 2cMeDIP-seq: The Carrier-Assisted Methods for Epigenomic Profiling of Small Cell Numbers or Single Cells

Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) can profile genome-wide epigenetic marks associated with regulatory genomic elements. However, conventional ChIP-seq is challenging when examining limited numbers of cells. Here, we developed a new technique by supplementing carrier materials of both chemically modified mimics with epigenetic marks and dUTP-containing DNA fragments during conventional ChIP procedures (hereafter referred to as 2cChIP-seq), thus dramatically improving immunoprecipitation efficiency and reducing DNA loss of low-input ChIP-seq samples. Using this strategy, we generated high-quality epigenomic profiles of histone modifications or DNA methylation in 10–1000 cells. By introducing Tn5 transposase-assisted fragmentation, 2cChIP-seq reliably captured genomic regions with histone modification at the single-cell level in about 100 cells. Moreover, we characterized the methylome of 100 differentiated female germline stem cells (FGSCs) and observed a particular DNA methylation signature potentially involved in the differentiation of mouse germline stem cells. Hence, we provided a reliable and robust epigenomic profiling approach for small cell numbers and single cells.