scholarly journals Fluorescence‐Based Quantification of Messenger RNA and Plasmid DNA Decay Kinetics in Extracellular Biological Fluids and Cell Extracts

2020 ◽  
Vol 4 (5) ◽  
pp. 2000057 ◽  
Author(s):  
Heyang Zhang ◽  
Koen Rombouts ◽  
Laurens Raes ◽  
Ranhua Xiong ◽  
Stefaan C. De Smedt ◽  
...  
Keyword(s):  
Plasmid Dna ◽  
Messenger Rna ◽  
Biological Fluids ◽  
Decay Kinetics ◽  
Cell Extracts

The Role of microRNAs in Osteoporosis Diagnostics

2021 ◽  
Vol 30 (03) ◽  
pp. 222-229
Author(s):  
Matthias Hackl ◽  
Elisabeth Semmelrock ◽  
Johannes Grillari
Keyword(s):  
Bone Mass ◽  
Bone Metabolism ◽  
Messenger Rna ◽  
Biological Fluids ◽  
Clinical Diagnostics ◽  
Bone Architecture ◽  
Estrogen Metabolism ◽  
Age Related

AbstractMicroRNAs (miRNAs) are short (18–24 nucleotides) non-coding RNA sequences that regulate gene expression via binding of messenger RNA. It is estimated that miRNAs co-regulate the expression of more than 70% of all human genes, many of which fulfil important roles in bone metabolism and muscle function. In-vitro and in-vivo experiments have shown that the targeted loss of miRNAs in distinct bone cell types (osteoblasts and osteoclasts) results in altered bone mass and bone architecture. These results emphasize the biological relevance of miRNAs for bone health.MiRNAs are not only considered as novel bone biomarkers because of their biological importance to bone metabolism, but also on the basis of other favorable properties: 1) Secretion of miRNAs from cells enables “minimally invasive” detection in biological fluids such as serum. 2) High stability of miRNAs in serum enables the retrospective analysis of frozen blood specimens. 3) Quantification of miRNAs in the serum is based on the RT-PCR - a robust method that is considered as the gold standard for the analysis of nucleic acids in clinical diagnostics.With regard to osteoporosis, it has been shown that many of the known risk factors are characterized by distinct miRNA profiles in the affected tissues: i) age-related loss of bone mass, ii) sarcopenia, iii) changes in estrogen metabolism and related changes Loss of bone mass, and iv) diabetes. Therefore, numerous studies in recent years have dealt with the characterization of miRNAs in the serum of osteoporosis patients and healthy controls, and were able to identify recurring miRNA patterns that are characteristic of osteoporosis. These novel biomarkers have great potential for the diagnosis and prognosis of osteoporosis and its clinical outcomes.The aim of this article is to give a summary of the current state of knowledge on the research and application of miRNA biomarkers in osteoporosis.

scholarly journals Versatile Redox-Responsive Polyplexes for the Delivery of Plasmid DNA, Messenger RNA, and CRISPR-Cas9 Genome-Editing Machinery

2018 ◽  
Vol 10 (38) ◽  
pp. 31915-31927 ◽  
Author(s):  
Yuyuan Wang ◽  
Ben Ma ◽  
Amr A. Abdeen ◽  
Guojun Chen ◽  
Ruosen Xie ◽  
...  
Keyword(s):  
Genome Editing ◽  
Plasmid Dna ◽  
Messenger Rna ◽  
Redox Responsive

Intraocular exosomes in eye diseases

2021 ◽  
Vol 21 ◽  
Author(s):  
Hui Zhang ◽  
Xiaomin Zhang ◽  
Xiaorong Li
Keyword(s):  
Messenger Rna ◽  
Biological Fluids ◽  
Eye Diseases ◽  
Age Related Macular Degeneration ◽  
Pathological State ◽  
Barrier Dysfunction ◽  
Blood Retinal Barrier ◽  
Ecm Remodeling ◽  
Age Related ◽  
Eye Tissues

: Exosomes, nanosized extracellular vesicles with a size of 30–150nm, contain many biological materials, such as messenger RNA (mRNA), microRNA (miRNA), proteins, and transcription factors. It has been identified in all biological fluids and recognized as an important part of intercellular communication. While the role of exosomes in cancer has been studied in-depth, our understanding of their relevance for ocular tissues has just begun to evolve. Intraocular fluids, including aqueous humor and vitreous humor, play a role in nourishing eye tissues and in expelling metabolites. In the pathological state, intraocular exosomes can mediate pathological processes such as ECM remodeling, retinal inflammation, and blood-retinal barrier dysfunction. Herein, we reviewed the latest advances of intraocular exosomes in the research of several eye diseases, including glaucoma, age-related macular degeneration, myopia, and ocular tumors, and discuss how intraocular exosomes contribute to the pathogenesis and progression of multiple eye diseases.

scholarly journals Nanomedicines to Deliver mRNA: State of the Art and Future Perspectives

Nanomaterials ◽  
2020 ◽  
Vol 10 (2) ◽  
pp. 364 ◽  
Author(s):  
Itziar Gómez-Aguado ◽  
Julen Rodríguez-Castejón ◽  
Mónica Vicente-Pascual ◽  
Alicia Rodríguez-Gascón ◽  
María Ángeles Solinís ◽  
...  
Keyword(s):  
Immune Responses ◽  
Plasmid Dna ◽  
Messenger Rna ◽  
Insertional Mutagenesis ◽  
Gene Editing ◽  
State Of The Art ◽  
Translational Efficiency ◽  
Future Perspectives ◽  
Therapeutic Tool

The use of messenger RNA (mRNA) in gene therapy is increasing in recent years, due to its unique features compared to plasmid DNA: Transient expression, no need to enter into the nucleus and no risk of insertional mutagenesis. Nevertheless, the clinical application of mRNA as a therapeutic tool is limited by its instability and ability to activate immune responses; hence, mRNA chemical modifications together with the design of suitable vehicles result essential. This manuscript includes a revision of the strategies employed to enhance in vitro transcribed (IVT) mRNA functionality and efficacy, including the optimization of its stability and translational efficiency, as well as the regulation of its immunostimulatory properties. An overview of the nanosystems designed to protect the mRNA and to overcome the intra and extracellular barriers for successful delivery is also included. Finally, the present and future applications of mRNA nanomedicines for immunization against infectious diseases and cancer, protein replacement, gene editing, and regenerative medicine are highlighted.

In vitro recombination of bacteriophage T7 DNA: Further characterization of the reaction using plasmid DNA

1985 ◽  
Vol 63 (4) ◽  
pp. 243-248 ◽  
Author(s):  
Donald Lee ◽  
Dan Vetter ◽  
Linda Beatty ◽  
Paul Sadowski
Keyword(s):  
Infected Cell ◽  
Plasmid Dna ◽  
Recombination Event ◽  
Bacteriophage T7 ◽  
In Vitro Recombination ◽  
Cell Extracts ◽  
Phage T7 ◽  
General Recombination

We have used a plasmid which contains a cloned fragment of T7 DNA to study the properties of general recombination of phage T7 in vitro. It was shown that T7-infected cell extracts promote recombination by the exchange of double strands of DNA. While both products of these double-strand exchanges were detected, we were unable to show that they were formed during a single recombination event.

scholarly journals In vitro cell cycle arrest induced by using artificial DNA templates

1992 ◽  
Vol 12 (7) ◽  
pp. 3216-3223
Author(s):  
S Kornbluth ◽  
C Smythe ◽  
J W Newport
Keyword(s):  
Cell Cycle ◽  
Tyrosine Kinase ◽  
Plasmid Dna ◽  
Signal Transduction Pathway ◽  
Dna Templates ◽  
Single Stranded Dna ◽  
Cell Extracts ◽  
Artificial Dna ◽  
Dna Template

In cell extracts of Xenopus eggs which oscillate between S and M phases of the cell cycle, the onset of mitosis is blocked by the presence of incompletely replicated DNA. In this report, we show that several artificial DNA templates (M13 single-stranded DNA and double-stranded plasmid DNA) can trigger this feedback pathway, which inhibits mitosis. Single-stranded M13 DNA is much more effective than double-stranded plasmid DNA at inhibiting the onset of mitosis. Furthermore, we have shown that low levels of M13 single-stranded DNA and high levels of double-stranded plasmid DNA can elevate the tyrosine kinase activity responsible for phosphorylating p34cdc2, thereby inactivating maturation-promoting factor and inhibiting entry into mitosis. This constitutes a simplified system with which to study the signal transduction pathway from the DNA template to the tyrosine kinase responsible for inhibiting p34cdc2 activity.

A MONOCLONAL BASED ELISA FOR HUMAN u-PA QUANTIFICATION

1987 ◽  
Author(s):  
M Camacho ◽  
A Fabra ◽  
F Carretero ◽  
M Borrell ◽  
I Millet ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibody ◽  
Conditioned Medium ◽  
Human Plasma ◽  
Biological Fluids ◽  
Elisa Assay ◽  
Additional Band ◽  
Cell Extracts ◽  
Set Up ◽  
Immunoblotting Technique

An ELISA has been developped for quantifiing the anti gen levels of u-PA present in human plasma, tissue and cell extracts, conditioned medium and others biological fluids.The assay was set up using PVC plates coated with rabbit anti u-PA IgG and a monoclonal antibody against human u-PA as second antibody (UKM23 obtained in our laboratory as previously described).Detection was performed with a rabbit anti-mouse IgG conjugated with horseradish peroxidase.By immunoblotting technique the monoclonal antibody used UKM23 , recognizes all human molecular weight spe cies and an additional band of 81 KD in human plasma. Also recognizes the u-PA present in conditioned medium from HT-1080 cell line.The detection limit of ELISA assay is 0,1 ng of total u-PA.The first assay in human plasma from healty volun ters, shows u-PA levels of 4,39 ± 0,94 ng/ml.This study was supported by grants from the CAICYTn9 1258/81 and 3628/86.

scholarly journals Nanoparticle-based local translation reveals mRNA as a translation-coupled scaffold with anchoring function

2019 ◽  
Vol 116 (27) ◽  
pp. 13346-13351 ◽  
Author(s):  
Shunnichi Kashida ◽  
Dan Ohtan Wang ◽  
Hirohide Saito ◽  
Zoher Gueroui
Keyword(s):  
Messenger Rna ◽  
Protein Localization ◽  
Continuous Production ◽  
Protein Domains ◽  
Mrna Translation ◽  
Actin Binding ◽  
Local Translation ◽  
Local Formation ◽  
Complex Processes ◽  
Cell Extracts

The spatial regulation of messenger RNA (mRNA) translation is central to cellular functions and relies on numerous complex processes. Biomimetic approaches could bypass these endogenous complex processes, improve our comprehension of the regulation, and allow for controlling local translation regulations and functions. However, the causality between local translation and nascent protein function remains elusive. Here, we developed a nanoparticle (NP)-based strategy to magnetically control mRNA spatial patterns in mammalian cell extracts and investigate how local translation impacts nascent protein localization and function. By monitoring the translation of the magnetically localized mRNAs, we show that mRNA–NP complexes operate as a source for the continuous production of proteins from defined positions. By applying this approach to actin-binding proteins, we triggered the local formation of actin cytoskeletons and identified the minimal requirements for spatial control of the actin filament network. In addition, our bottom-up approach identified a role for mRNA as a translation-coupled scaffold for the function of nascent N-terminal protein domains. Our approach will serve as a platform for regulating mRNA localization and investigating the function of nascent protein domains during translation.

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