Development of an Automated and Sensitive Microfluidic Device for Capturing and Characterizing Circulating Tumor Cells (CTCs) from Clinical Blood Samples

Current analysis of circulating tumor cells (CTCs) is hindered by sub-optimal sensitivity and specificity of devices or assays as well as lack of capability of characterization of CTCs with clinical biomarkers. Here, we validate a novel technology to enrich and characterize CTCs from blood samples of patients with metastatic breast, prostate and colorectal cancers using a microfluidic chip which is processed by using an automated staining and scanning system from sample preparation to image processing. The Celsee system allowed for the detection of CTCs with apparent high sensitivity and specificity (94% sensitivity and 100% specificity). Moreover, the system facilitated rapid capture of CTCs from blood samples and also allowed for downstream characterization of the captured cells by immunohistochemistry, DNA and mRNA fluorescence in-situ hybridization (FISH). In a subset of patients with prostate cancer we compared the technology with a FDA-approved CTC device, CellSearch and found a higher degree of sensitivity with the Celsee instrument. In conclusion, the integrated Celsee system represents a promising CTC technology for enumeration and molecular characterization.     

Characterization of polymorphic microsatellites in the castration parasite plant‐ant Allomerus octoarticulatus cf. demerarae (Formicidae: Myrmicinae)

Fifteen microsatellite loci were isolated from the Peruvian tropical plant‐ant Allomerus octoarticulatus cf. demerarae (Hymenoptera: Myrmicinae) and their polymorphism was characterized. High levels of within‐population variation were observed at most loci, with number of alleles ranging from one to 21, and heterozygosity from 0 to 1 per population sample. Cross‐species amplification of these loci was also tested in one other species of the ant genus Allomerus (Allomerus decemarticulatus), displaying similar life history.     

流感病毒抑制剂的研究进展

流感病毒(Influenza Virus,IVs)属于正黏病毒科(Orthomyyxoviridae),单股负链RNA包膜病毒,由于其传播和变异速度快,且致病力和致死率高,严重威胁人类的健康和生命.理论上,抑制流感病毒生命周期的任何一个阶段,都可以有效地抑制病毒的复制和传播.目前FDA已批准的药物主要作用于子代病毒的释放阶段,随着流感病毒耐药问题的日益严重,不同作用机制的抑制剂不断被发现,部分已经进入临床研究阶段.新技术、新思路持续推进着流感病毒抑制剂的研究,本文将对最新的流感病毒抑制剂研究进展进行综述,旨在为新型的流感病毒抑制剂的设计和研发提供参考和思路.     

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。     

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.     

Conformational Heterogeneity of Cyclosporin A in Cyclophilin 18 Binding

The immunosuppressive drug cyclosporin A (CsA) binds to its receptor protein cyclophilin 18 (Cyp18) in two distinct kinetic phases, while the mechanism remains elusive. Stopped-flow measurements coupled with titration and competition experiments were used to investigate the puzzling two-phase process of CsA and Cyp18 interaction. This study leads to the dissection of different conformational fractions of either direct fast binding or slow binding with rate-limiting conformational inter-conversion and the real-time measurement of k_on value (8.34 ± 0.22 x10⁶ M^-1s^-1) in solution. Furthermore, our study indicates that the structure of CsA during dissociation from the protein possesses a distribution of conformations different from those in solution under equilibrium condition.     

Characterization of Cre recombinase mouse lines enabling cell type-specific targeting of postnatal intervertebral discs

Cre/loxP technology is an important tool for studying cell type-specific gene functions. Cre recombinase mouse lines, including Agc1-CreER^T2, Col2a1-Cre; Col2a1-CreER^T2, Shh-Cre, Shh-CreER^T2, and Osx-Cre, have been proven to be valuable tools to elucidate the biology of long bones, yet the information for their activity in postnatal intervertebral disc (IVD) tissues was very limited. In this study, we used R26-mTmG fluorescent reporter to systematically analyze cell specificity and targeting efficiency of these six mouse lines in IVD tissues at postnatal growing and adult stages. We found that Agc1-CreER^T2 is effective to direct recombination in all components of IVDs, including annulus fibrosus (AF), nucleus pulposus (NP), and cartilaginous endplate (CEP), upon tamoxifen induction at either 2 weeks or 2 months of ages. Moreover, Col2a1-Cre targets most of the cells in IVDs, except for some cells in the outer AF (OAF) and NP. In contrast, the activity of Col2a1-CreER^T2 is mainly limited to the IAF of IVD tissues at either stage of tamoxifen injection. Similarly, Shh-Cre directs recombination specifically in all NP cells, whereas Shh-CreER^T2 is active only in a few NP cells when tamoxifen is administered at either stage. Finally, Osx-Cre targets cells in the CEP, but not in the NP or AF of IVDs tissues at these two stages. Thus, our data demonstrated that all these Cre lines can direct recombination in IVD tissues at postnatal stages with different cell type specificity and/or targeting efficiency, and can, therefore, serve as valuable tools to dissect cell type-specific gene functions in IVD development and homeostasis.