Adaptation to Endoplasmic Reticulum Stress Enhances Resistance of Oral Cancer Cells to Cisplatin by Up-Regulating Polymerase η and Increasing DNA Repair Efficiency

Endoplasmic reticulum (ER) stress response is an adaptive program to cope with cellular stress that disturbs the function and homeostasis of ER, which commonly occurs during cancer progression to late stage. Late-stage cancers, mostly requiring chemotherapy, often develop treatment resistance. Chemoresistance has been linked to ER stress response; however, most of the evidence has come from studies that correlate the expression of stress markers with poor prognosis or demonstrate proapoptosis by the knockdown of stress-responsive genes. Since ER stress in cancers usually persists and is essentially not induced by genetic manipulations, we used low doses of ER stress inducers at levels that allowed cell adaptation to occur in order to investigate the effect of stress response on chemoresistance. We found that prolonged tolerable ER stress promotes mesenchymal–epithelial transition, slows cell-cycle progression, and delays the S-phase exit. Consequently, cisplatin-induced apoptosis was significantly decreased in stress-adapted cells, implying their acquisition of cisplatin resistance. Molecularly, we found that proliferating cell nuclear antigen (PCNA) ubiquitination and the expression of polymerase η, the main polymerase responsible for translesion synthesis across cisplatin-DNA damage, were up-regulated in ER stress-adaptive cells, and their enhanced cisplatin resistance was abrogated by the knockout of polymerase η. We also found that a fraction of p53 in stress-adapted cells was translocated to the nucleus, and that these cells exhibited a significant decline in the level of cisplatin-DNA damage. Consistently, we showed that the nuclear p53 coincided with strong positivity of glucose-related protein 78 (GRP78) on immunostaining of clinical biopsies, and the cisplatin-based chemotherapy was less effective for patients with high levels of ER stress. Taken together, this study uncovers that adaptation to ER stress enhances DNA repair and damage tolerance, with which stressed cells gain resistance to chemotherapeutics.     

Radiation- and Photo-Induced Oxidation Pathways of Methionine in Model Peptide Backbone under Anoxic Conditions

Within the reactive oxygen species (ROS) generated by cellular metabolisms, hydroxyl radicals (HO^•) play an important role, being the most aggressive towards biomolecules. The reactions of HO^• with methionine residues (Met) in peptides and proteins have been intensively studied, but some fundamental aspects remain unsolved. In the present study we examined the biomimetic model made of Ac-Met-OMe, as the simplest model peptide backbone, and of HO^• generated by ionizing radiation in aqueous solutions under anoxic conditions. We performed the identification and quantification of transient species by pulse radiolysis and of final products by LC-MS and high-resolution MS/MS after γ-radiolysis. By parallel photochemical experiments, using 3-carboxybenzophenone (CB) triplet with the model peptide, we compared the outcomes in terms of short-lived intermediates and stable product identification. The result is a detailed mechanistic scheme of Met oxidation by HO^•, and by CB triplets allowed for assigning transient species to the pathways of products formation.     

菌酶协同改善花生粕的饲用品质

花生粕是重要的蛋白饲料原料，但由于其氨基酸不平衡，特别是精氨酸与赖氨酸比例严重失衡（精氨酸与赖氨酸含量比值在3~4，理想的精氨酸与赖氨酸含量比值为1.0），限制了其在动物养殖中的应用。研究了复合酶预处理结合乳酸菌发酵花生粕对其品质的改善。结果表明：经菌酶协同处理后，花生粕粗蛋白质含量由46.4%提高至506%，大分子蛋白明显降解为小分子蛋白，酸溶蛋白质含量由2.3%提高至17.8%，多肽含量由1.6%提高至15.7%，蛋氨酸和赖氨酸含量分别提高了77.1%和42.0%，精氨酸降解率为18.7%，精氨酸与赖氨酸含量比值从3.7降低至2.1，总酸含量由06%提高到4.7%，其中乳酸含量由0.64 mg/g提高至14.63 mg/g。菌酶协同处理后的花生粕抗氧化性明显增强，其中每克菌酶协同处理后的花生粕对羟自由基的清除能力与171.6 mg VC相当，比花生粕（与47.6 mg VC相当）提高了2.6倍。     

基于GIS单线图的配网停电单模拟操作应用研究

配电网停电会造成电力系统供配电可靠性以及服务质量下降,研究基于地理信息系统(GIS)单线图的配网停电单模拟操作应用。利用网格长度作为基本单位建立坐标系,以选取起始点与终止点为基础,通过四参数法将GIS坐标映射至图纸网格内,实现配网内设备初步布局,将杆塔、站房和整体均匀分布作为优化目标,设置多目标优化目标函数实现GIS单线图最终优化。选取某电力公司配网作为单模拟操作应用对象,模拟结果表明,单模拟操作配网停电后,该配网各负荷点年故障率、次平均停电时间以及年停电时间均有所减少,可有效提升配网的供配电可靠性。     

In Vivo Simultaneous Analysis of Gene Expression by Dual-Color Luciferases in Caenorhabditis elegans

Both fluorescent and luminescent observation are widely used to examine real-time gene expression patterns in living organisms. Several fluuorescent and luminescent proteins with specific optical properties have been developed and applied for simultaneous, multi-color observation of more than two gene expression profiles. Compared to fluorescent proteins, however, the application of multi-color luminescent imaging in living organisms is still limited. In this study, we introduced two-color luciferases into the soil nematode C. elegans and performed simultaneous analysis of two gene expression profiles. Using a green-emitting luciferase Eluc (emerald luciferase) and red-emitting luciferase SLR (stable luciferase red), the expression patterns of two genes were simultaneously observed in single animals from embryonic to adult stages over its whole life span. In addition, dual gene activities were observed at the single embryo level, with the simultaneous observation of morphological changes. These are the first application of a two-color luciferase system into a whole animal and suggest that precise relationship of expression patterns of multiple genes of interest can be analyzed over the whole life of the animal, dependent on the changes in genetic and/or environmental conditions.     

Human DDX17 Unwinds Rift Valley Fever Virus Non-Coding RNAs

Rift Valley fever virus (RVFV) is a mosquito-transmitted virus from the Bunyaviridae family that causes high rates of mortality and morbidity in humans and ruminant animals. Previous studies indicated that DEAD-box helicase 17 (DDX17) restricts RVFV replication by recognizing two primary non-coding RNAs in the S-segment of the genome: the intergenic region (IGR) and 5′ non-coding region (NCR). However, we lack molecular insights into the direct binding of DDX17 with RVFV non-coding RNAs and information on the unwinding of both non-coding RNAs by DDX17. Therefore, we performed an extensive biophysical analysis of the DDX17 helicase domain (DDX17_135–555) and RVFV non-coding RNAs, IGR and 5’ NCR. The homogeneity studies using analytical ultracentrifugation indicated that DDX17_135–555, IGR, and 5’ NCR are pure. Next, we performed small-angle X-ray scattering (SAXS) experiments, which suggested that DDX17 and both RNAs are homogenous as well. SAXS analysis also demonstrated that DDX17 is globular to an extent, whereas the RNAs adopt an extended conformation in solution. Subsequently, microscale thermophoresis (MST) experiments were performed to investigate the direct binding of DDX17 to the non-coding RNAs. The MST experiments demonstrated that DDX17 binds with the IGR and 5’ NCR with a dissociation constant of 5.77 ± 0.15 µM and 9.85 ± 0.11 µM, respectively. As DDX17_135–555 is an RNA helicase, we next determined if it could unwind IGR and NCR. We developed a helicase assay using MST and fluorescently-labeled oligos, which suggested DDX17_135–555 can unwind both RNAs. Overall, our study provides direct evidence of DDX17_135–555 interacting with and unwinding RVFV non-coding regions.