权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Novel Separation Methods for exRNA Carriers: Extracellular Vesicles, Lipoprotein Particles, and Protein Aggregates

exRNA 载体的新型分离方法：细胞外囊泡、脂蛋白颗粒和蛋白质聚集体

基本信息

批准号：
10483185
负责人：
Kenneth W Witwer
金额：
$ 54.9万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-08-01 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10483185
关键词：
Affinity Age Analytical Chemistry Antibodies Attention Binding Biochemical Biological Biological Factors Biology Catalogs Cells Centrifugation Characteristics Charge Data Set Detection Development Diet Disease Engineering Ensure Field Flow Fractionation Gold Health High Density Lipoproteins Higher Order Chromatin Structure Immobilization Intervention Studies Libraries Lipid Bilayers Lipoproteins Liquid substance Location Low-Density Lipoproteins MLLT2 gene Measures Methodology Methods MicroRNAs Modification Molecular Morphologic artifacts Phase Process Proteomics RNA Resolution Ribonucleases Ribonucleoproteins Sampling Speed Surface Techniques Technology Testing Therapeutic Time Tissues Validation base biological sex density design extracellular extracellular vesicles improved inhibitor lipidomics liquid biopsy member molecular carrier novel particle preservation protein aggregation sample collection screening sensor sex validation studies

项目摘要

ABSTRACT Extracellular RNA (exRNA) is a particularly attractive molecular component of liquid biopsy because RNA species can be specifically amplified. Of the three major classes of exRNA vehicle—extracellular vesicles (EVs), lipoprotein particles (LPPs), and free ribonucleoproteins (RNPs)—EVs have so far received the most attention. Within each class, there is also tremendous diversity by physical characteristics of size, density, and surface charge. Indeed, to our knowledge, no study to date has profiled exRNA in multiple members of the three carrier classes that have been isolated rigorously from the same samples. There is a strong need to develop new strategies and controls to ensure that comparisons of exRNA carriers are not confounded by co- isolation (different classes of carriers present in the same fraction) or contamination (detection of uncomplexed and/or foreign RNAs introduced during sample collection and processing). To this end, we assemble a team of experts on EVs, LPPs, and RNPs, along with experts in cutting-edge separation and characterization methods. In an initial UG3 phase, we will first (Aim 1) use a combination of state-of-the-field physical and biochemical separation methods to separate a library of eight subtypes of EVs, LPPs, and RNPs from the same biological samples and with the best achievable purity. “Gold standard” proteomic, lipidomic, glycomic, and RNomic datasets will be generated. Carefully designed “process” controls will for the first time establish an across-the- board baseline of contaminants and other artifacts that may complicate interpretation. In Aim 2, we will test asymmetric field-flow fractionation (AF4) and affinity capture platforms including the ExoView platform and sensitive electrochemical sensors as superior alternatives to the most commonly used legacy method, differential centrifugation. We will seek gains in speed, resolution, and purity compared with legacy techniques. If go/no-go criteria are met by the end of the second year (UG3), we will proceed to a UH3 phase. This phase will include an Aim 3, validating results in multiple locations and with approximately 6 times the original sample numbers to account for influence of sex and age. The AF4 method will be further developed with additional modifications based on our engineering and analytical chemistry expertise, while ExoView technology will be harnessed to screen antibodies and other affinity materials for rapid isolations and to detect abundant RNA species directly in immobilized exRNA carriers. Finally, an Aim 4 will assess the biological factor of diet with valuable samples from intervention studies, along with the possible desirability of collecting samples in RNase inhibitors to preserve more fragile RNA species. Overall, we hypothesize that 1) AF4, on its own or with methodologic modifications, as well as 2) novel affinity separation approaches will improve substantially on ultracentrifuge-based methods in ease and purity and on current state-of-the-art but tedious and lengthy exRNA carrier separation methods.

摘要

项目成果

期刊论文数量（11）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Circulating SNORD57 rather than piR-54265 is a promising biomarker for colorectal cancer: common pitfalls in the study of somatic piRNAs in cancer.

DOI：
10.1261/rna.078444.120
发表时间：
2021-04
期刊：
RNA (New York, N.Y.)
影响因子：
0
作者：
Tosar JP;García-Silva MR;Cayota A
通讯作者：
Cayota A

Letter by Halushka and Witwer Regarding Article, "Circulating MicroRNA-122-5p Is Associated With a Lack of Improvement in Left Ventricular Function After Transcatheter Aortic Valve Replacement and Regulates Viability of Cardiomyocytes Through Extracellula

Halushka 和 Witwer 关于文章“循环 MicroRNA-122-5p 与经导管主动脉瓣置换术后左心室功能缺乏改善有关，并通过细胞外调节心肌细胞的活力”的信函

DOI：
10.1161/circulationaha.122.061834
发表时间：
2023
期刊：
Circulation
影响因子：
37.8
作者：
Halushka,MarcK;Witwer,KennethW
通讯作者：
Witwer,KennethW

RI-SEC-seq: Comprehensive Profiling of Nonvesicular Extracellular RNAs with Different Stabilities.

RI-SEC-seq：具有不同稳定性的非囊泡细胞外 RNA 的综合分析。

DOI：
10.21769/bioprotoc.3918
发表时间：
2021
期刊：
Bio-protocol
影响因子：
0.8
作者：
Tosar,JuanPablo;Gámbaro,Fabiana;Castellano,Mauricio;Cayota,Alfonso
通讯作者：
Cayota,Alfonso

Lipid-Based Nutrient Supplementation Increases High-Density Lipoprotein (HDL) Cholesterol Efflux Capacity and Is Associated with Changes in the HDL Glycoproteome in Children.

DOI：
10.1021/acsomega.1c04811
发表时间：
2021-11-30
期刊：
ACS omega
影响因子：
4.1
作者：
Hong BV;Zhu C;Wong M;Sacchi R;Rhodes CH;Kang JW;Arnold CD;Adu-Afarwuah S;Lartey A;Oaks BM;Lebrilla CB;Dewey KG;Zivkovic AM
通讯作者：
Zivkovic AM

Urinary extracellular vesicles contain mature transcriptome enriched in circular and long noncoding RNAs with functional significance in prostate cancer.