Multiomic Metabolic Enrichment Network Analysis Reveals Metabolite–Protein Physical Interaction Subnetworks Altered in Cancer

Metabolism is recognized as an important driver of cancer progression and other complex diseases, but global metabolite profiling remains a challenge. Protein expression profiling is often a poor proxy since existing pathway enrichment models provide an incomplete mapping between the proteome and metabolism. To overcome these gaps, we introduce multiomic metabolic enrichment network analysis (MOMENTA), an integrative multiomic data analysis framework for more accurately deducing metabolic pathway changes from proteomics data alone in a gene set analysis context by leveraging protein interaction networks to extend annotated metabolic models. We apply MOMENTA to proteomic data from diverse cancer cell lines and human tumors to demonstrate its utility at revealing variation in metabolic pathway activity across cancer types, which we verify using independent metabolomics measurements. The novel metabolic networks we uncover in breast cancer and other tumors are linked to clinical outcomes, underscoring the pathophysiological relevance of the findings.     

严重急性呼吸综合征冠状病毒2膜蛋白对宿主细胞pre-mRNA 3"UTR加工的影响

目的 研究严重急性呼吸综合征冠状病毒2（SARS-CoV-2）膜蛋白对宿主细胞mRNA前体（pre-mRNA）3"非翻译区（UTR）加工的影响。方法 本研究以人肺上皮细胞系A549为模型，利用瞬时转染在细胞内过表达SARS-CoV-2膜蛋白；利用RNA-Seq测序技术及生物信息学分析方法，系统性描绘宿主细胞选择性多聚腺苷酸化（alternative polyadenylation，APA）事件；Metascape数据库对发生显著APA变化的基因进行功能富集分析；RT-qPCR验证靶基因3"UTR长度变化；蛋白质免疫印迹（Western blot）检测目的蛋白表达水平。结果 SARS-CoV-2膜蛋白外源表达后宿主细胞内共813个基因发生显著APA变化。GO和KEGG分析显示，差异APA基因广泛参与有丝分裂细胞周期、调节细胞应激等生物过程，涉及病毒感染和蛋白质加工等。从中进一步筛选出AKT1基因，在IGV软件中显示3"UTR延长；RT-qPCR验证AKT1基因的3"UTR长度变化趋势；Western blot结果显示AKT1蛋白磷酸化水平增加。结论 SARS-CoV-2膜蛋白潜在影响宿主pre-mRNA的3"UTR加工，其中参与多种病毒性生物过程的AKT1基因 3"UTR延长，且其编码的蛋白质功能在细胞内被激活。     

20-hydroxyecdysone accumulation and regulation in Ajuga lobata D. Don suspension culture

Suspension culture of Ajuga lobata D. Don cells provides a method of synthesis of the phytoecdysteroid 20-hydroxyecdysone (20E) which can regulate the molting process of larvae. We characterized the culture conditions to optimize 20E production. Growth of A. lobata D. Don cells fits the logistic equation curve with a growth cycle of 19 days. Medium conductivity was negatively correlated with dry cell weight and 20E accumulation, thus could be used to determine the optimal time for cell harvest. Continuous subculture reduced 20E synthesis, but supplementing medium with 20E precursors mevalonic (MVA), α-Pinene, and nitric oxide (NO) can significantly promote cell growth and influence 20E accumulation. Combination of α-Pinene, MVA, and SNP significantly elevated 20E accumulation, thus may synergistically enhance 20E synthesis in A. lobata D. Don. The optimal concentrations of α-Pinene, MVA, and NO donor SNP in suspension culture were 50 μL L^?1, 10 mg L^?1, and 80 μmol L^?1.     

Reactivation and Dedifferentiation of Differentiated Murine Erythroleukemic Cell Nuclei

A murine erythroleukemic cell line, 745 A4-TG, deficient in hypoxanthine-guanine-phosphoribosyl transferase, can be induced with 3 mM hexamethylene bisacetamide to yield at least 50% of cells undergoing irreversible erythroid differentiation and finally losing capacity for cell divisions. The effects of such induced differentiation of 745 A4-TG on its ability to form viable and proliferating hybrids when fused with 3T3 1T22 fibroblasts were investigated. We found that when the induced 745 A4-TG cells were used, more continuously proliferating hybrids were obtained than could be accounted for by the residual uninduced cells which remained in these induced preparations. This suggests that some of the induced 745 A4-TG cells, when fused with 3T3 1T22 reverted from the induced phenotype of a limited capacity for cell proliferation to an uninduced state of continuous proliferation. This observation was further confirmed with the use of fully differentiated 745 A4-TG cells, which were obtained after selection with a bromodeoxyuridine suicide treatment to eliminate the uninduced and the partially differentiated cells in the preparations. When these selected, fully differentiated cells, as characterized by their lack of proliferation capacity and thymidine kinase activity, were fused with 3T3 1T22 (also deficient in thymidine kinase), it was found that not only were viable hybrid colonies obtained in a selection medium, which precluded the proliferation of either parental cells, but these hybrids continued to proliferate for more than two months in selection medium. These data thus confirmed that some fully differentiated erythroleukemic nucleus components in the hybrids were reactivated to regain capacity for cell proliferation and to dedifferentiate to synthesize thymidine kinase for survival in the selection medium. The lack of hemoglobin synthesis by these hybrids also indicates dedifferention of these murine erythroleukemic components in the hybrids.     

The protective effects of MSC‐EXO against pulmonary hypertension through regulating Wnt5a/BMP signalling pathway

The aim of the study was to explore the mechanism of mesenchymal stem cell‐derived exosomes (MSC‐EXO) to protect against experimentally induced pulmonary hypertension (PH). Monocrotaline (MCT)‐induced rat model of PH was successfully established by a single intraperitoneal injection of 50 mg/kg MCT, 3 weeks later the animals were treated with MSC‐EXO via tail vein injection. Post‐operation, our results showed that MSC‐EXO could significantly reduce right ventricular systolic pressure (RVSP) and the right ventricular hypertrophy index, attenuate pulmonary vascular remodelling and lung fibrosis in vivo. In vitro experiment, the hypoxia models of pulmonary artery endothelial cell (PAEC) and pulmonary vascular smooth muscle cell (PASMC) were used. We found that the expression levels of Wnt5a, Wnt11, BMPR2, BMP4 and BMP9 were increased, but β‐catenin, cyclin D1 and TGF‐β1 were decreased in MSC‐EXO group as compared with MCT or hypoxia group in vivo or vitro. However, these increased could be blocked when cells were transfected with Wnt5a siRNA in vitro. Taken together, these results suggested that the mechanism of MSC‐EXO to prevent PH vascular remodelling may be via regulation of Wnt5a/BMP signalling pathway.     

Nitrosothiol Formation and Protection against Fenton Chemistry by Nitric Oxide-induced Dinitrosyliron Complex Formation from Anoxia-initiated Cellular Chelatable Iron Increase

Dinitrosyliron complexes (DNIC) have been found in a variety of pathological settings associated with ^•NO. However, the iron source of cellular DNIC is unknown. Previous studies on this question using prolonged ^•NO exposure could be misleading due to the movement of intracellular iron among different sources. We here report that brief ^•NO exposure results in only barely detectable DNIC, but levels increase dramatically after 1–2 h of anoxia. This increase is similar quantitatively and temporally with increases in the chelatable iron, and brief ^•NO treatment prevents detection of this anoxia-induced increased chelatable iron by deferoxamine. DNIC formation is so rapid that it is limited by the availability of ^•NO and chelatable iron. We utilize this ability to selectively manipulate cellular chelatable iron levels and provide evidence for two cellular functions of endogenous DNIC formation, protection against anoxia-induced reactive oxygen chemistry from the Fenton reaction and formation by transnitrosation of protein nitrosothiols (RSNO). The levels of RSNO under these high chelatable iron levels are comparable with DNIC levels and suggest that under these conditions, both DNIC and RSNO are the most abundant cellular adducts of ^•NO.