激光捕获显微切割结合RNA线性扩增技术在膀胱移行细胞癌相关基因研究中的应用

目的当对有间质细胞混杂的膀胱移行上皮细胞与膀胱移行细胞癌进行基因差异表达分析时,激光捕获显微切割技术(lasercap-turemicrodissection,LCM)是不可缺少的,然而从LCM所得的细胞中获取足够RNA量相当困难。本文首次将LCM与RNA线性扩增结合用于膀胱癌相关基因研究,目的是确定一条切实可行的技术路线,将膀胱移行上皮细胞从膀胱粘膜中分离出来,将膀胱癌细胞从肿瘤间质细胞中分离出来,并对其进行RNA体外线性扩增,以备进一步研究。方法采用LCM技术分别从正常膀胱粘膜及膀胱癌组织冰冻切片中获取膀胱移行上皮细胞及膀胱癌细胞,提取RNA,并对微量RNA进行体外线性扩增。然后用RT-PCR验证扩增前、后RNA中β-actin基因表达水平。结果对照实验I证实经LCM后RNA完整性较好;经对照实验Ⅱ初步确定设定条件下LCMshooting次数与可获得RNA量间对应关系。从膀胱粘膜种捕获膀胱移行上皮细胞25万shootings;从膀胱癌组织中获取癌细胞20万shootings。产物RNA扩增后获得片段大小为0.5-2.5kh的aRNA,且β-actin基因表达表达完整。结论LCM结合RNA体外线性扩增技术能成功地获取较为单一的研究目的细胞,扩增后RNA完整性较好,能用于进一步研究中。     

A Unique Robust Dual-Promoter-Driven and Dual-Reporter-Expressing SARS-CoV-2 Replicon: Construction and Characterization

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2, SARS2) remains a great global health threat and demands identification of more effective and SARS2-targeted antiviral drugs, even with successful development of anti-SARS2 vaccines. Viral replicons have proven to be a rapid, safe, and readily scalable platform for high-throughput screening, identification, and evaluation of antiviral drugs against positive-stranded RNA viruses. In the study, we report a unique robust HIV long terminal repeat (LTR)/T7 dual-promoter-driven and dual-reporter firefly luciferase (fLuc) and green fluorescent protein (GFP)-expressing SARS2 replicon. The genomic organization of the replicon was designed with quite a few features that were to ensure the replication fidelity of the replicon, to maximize the expression of the full-length replicon, and to offer the monitoring flexibility of the replicon replication. We showed the success of the construction of the replicon and expression of reporter genes fLuc and GFP and SARS structural N from the replicon DNA or the RNA that was in vitro transcribed from the replicon DNA. We also showed detection of the negative-stranded genomic RNA (gRNA) and subgenomic RNA (sgRNA) intermediates, a hallmark of replication of positive-stranded RNA viruses from the replicon. Lastly, we showed that expression of the reporter genes, N gene, gRNA, and sgRNA from the replicon was sensitive to inhibition by Remdesivir. Taken together, our results support use of the replicon for identification of anti-SARS2 drugs and development of new anti-SARS strategies targeted at the step of virus replication.     

Human Retrovirus Genomic RNA Packaging

Two non-covalently linked copies of the retrovirus genome are specifically recruited to the site of virus particle assembly and packaged into released particles. Retroviral RNA packaging requires RNA export of the unspliced genomic RNA from the nucleus, translocation of the genome to virus assembly sites, and specific interaction with Gag, the main viral structural protein. While some aspects of the RNA packaging process are understood, many others remain poorly understood. In this review, we provide an update on recent advancements in understanding the mechanism of RNA packaging for retroviruses that cause disease in humans, i.e., HIV-1, HIV-2, and HTLV-1, as well as advances in the understanding of the details of genomic RNA nuclear export, genome translocation to virus assembly sites, and genomic RNA dimerization.     

改进Trizol法提取白色念珠菌总RNA初探

目的:比较Trizol法、改进Trizol法和超声粉碎法提取白色念珠菌总RNA产量、纯度和完整性的差异。方法:对生长至静止期的白色念珠菌标准株ATCC10231分别采用Trizol法、改进Trizol法和超声粉碎法提取总RNA,用紫外分光光度计测量其A260,A280的值,并进行琼脂糖凝胶电泳,对总RNA的产量、纯度和完整性进行比较。结果:三种方法提取白色念珠菌总RNA产量分别为1.904μg、7.208μg和5.000μg,A260/A280比值分别为1.896、1.415和1.612。电泳结果显示改进Trizol法提取的总RNA呈现28 s rRNA,18 s rRNA和5 s rRNA三条清晰的条带,而Trizol法和超声粉碎法提取的总RNA均有降解。结论:改进Trizol法提取的白色念珠菌总RNA产量及纯度均高于Trizol法和超声粉碎法,且完整性更好。该法优于Trizol法和超声粉碎法。     

益气活血冲剂对创伤骨折致肾脏损伤的保护作用

目的：探讨益气活血冲剂对创伤骨折所致肾损伤的保护作用。方法：将大鼠或小鼠分为空白组、对照组和实验组。分别于术后不同时期处死动物,测定三组的肾指数、肾中柠檬酸、DNA、RNA含量及^3H-TdR ^3H-UR参入率。结果：创伤骨折引起大鼠肾指数明显下降,^3H-TdR 参入受抑制,DNA含量降低,^3H-UR参入率和RNA含量升高;应用益气活血冲剂可以改善这些变化、促进DNA和RNA的合成。结论：创伤骨折后应用益气活血冲剂可以改善创伤引起的肾脏的变化。     

An indirect treatment comparison of the efficacy of patisiran and tafamidis for the treatment of hereditary transthyretin-mediated amyloidosis with polyneuropathy

Background: Hereditary transthyretin-mediated amyloidosis (hATTR amyloidosis) is a progressive, life-threatening disease. Until recently, tafamidis was the only approved pharmacotherapy. Patisiran significantly improved polyneuropathy and quality of life (QoL) in the phase III APOLLO trial. In the absence of direct comparisons, this analysis aimed to evaluate the comparative efficacy of tafamidis and patisiran in hATTR amyloidosis with polyneuropathy.

Research design and methods: Randomized controlled trial evidence for tafamidis was identified by systematic literature review. Indirect treatment comparisons were performed using the standard pairwise Bucher method for endpoints used in both APOLLO and the tafamidis Fx-005 trial: change from baseline in Neuropathy Impairment Score-lower limbs (NIS-LL), Norfolk QoL-Diabetic Neuropathy questionnaire (QoL-DN), NIS-LL response, and mBMI vs. placebo. Inter-trial population differences were assessed by sensitivity analysis.

Results: The base-case analysis (FAP Stage 1 APOLLO patients vs. intent-to-treat Fx-005 population) suggested patisiran had a greater treatment effect vs. tafamidis for all endpoints, with significant improvements in mean change in NIS-LL (–5.49) and QoL-DN (–13.10) from baseline to Month 18. Similar trends were observed in all sensitivity analyses.

Conclusions: In the absence of direct comparisons, this analysis suggests patisiran has a greater treatment effect than tafamidis in patients with hATTR amyloidosis with polyneuropathy.     

Altered Regulation of mRNA and miRNA Expression in Epithelial and Stromal Tissue of Keratoconus Corneas

PurposeEvaluation of mRNA and microRNA (miRNA) expression in epithelium and stroma of patients with keratoconus.MethodsThe epithelium and stroma of eight corneas of eight patients with keratoconus and eight corneas of eight non-keratoconus healthy controls were studied separately. RNA was extracted, and mRNA and miRNA analyses were performed using microarrays. Differentially expressed mRNAs and miRNAs in epithelial and stromal keratoconus samples compared to healthy controls were identified. Selected genes and miRNAs were further validated using RT-qPCR.ResultsWe discovered 170 epithelial and 1498 stromal deregulated protein-coding mRNAs in KC samples. In addition, in epithelial samples 180 miRNAs and in stromal samples 379 miRNAs were significantly deregulated more than twofold compared to controls. Pathway analysis revealed enrichment of metabolic and axon guidance pathways for epithelial cells and enrichment of metabolic, mitogen-activated protein kinase (MAPK), and focal adhesion pathways for stromal cells.ConclusionsThis study demonstrates significant differences in the expression and regulation of mRNAs and miRNAs in the epithelium and stroma of Patients with KC. Also, in addition to the well-known target candidates, we were able to identify further genes and miRNAs that may be associated with keratoconus. Signaling pathways influencing metabolic changes and cell contacts are affected in epithelial and stromal cells of patients with keratoconus.