scholarly journals Molecular Beacons: Powerful Tools for Imaging RNA in Living Cells

2011 ◽  
Vol 2011 ◽  
pp. 1-15 ◽  
Author(s):  
Ricardo Monroy-Contreras ◽  
Luis Vaca
Keyword(s):  
Real Time ◽  
Imaging Techniques ◽  
Spatial Separation ◽  
Living Cells ◽  
Molecular Beacons ◽  
Cell Physiology ◽  
Complementary Sequence ◽  
Stem Loop ◽  
Recent Advances

Recent advances in RNA functional studies highlights the pivotal role of these molecules in cell physiology. Diverse methods have been implemented to measure the expression levels of various RNA species, using either purified RNA or fixed cells. Despite the fact that fixed cells offer the possibility to observe the spatial distribution of RNA, assays with capability to real-time monitoring RNA transport into living cells are needed to further understand the role of RNA dynamics in cellular functions. Molecular beacons (MBs) are stem-loop hairpin-structured oligonucleotides equipped with a fluorescence quencher at one end and a fluorescent dye (also called reporter or fluorophore) at the opposite end. This structure permits that MB in the absence of their target complementary sequence do not fluoresce. Upon binding to targets, MBs emit fluorescence, due to the spatial separation of the quencher and the reporter. Molecular beacons are promising probes for the development of RNA imaging techniques; nevertheless much work remains to be done in order to obtain a robust technology for imaging various RNA molecules together in real time and in living cells. The present work concentrates on the different requirements needed to use successfully MB for cellular studies, summarizing recent advances in this area.

scholarly journals Construction of a Nanosensor for Non-Invasive Imaging of Hydrogen Peroxide Levels in Living Cells

Biology ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 430
Author(s):  
Amreen ◽  
Hayssam M. Ali ◽  
Mohammad Ahmad ◽  
Mohamed Z. M. Salem ◽  
Altaf Ahmad
Keyword(s):  
Hydrogen Peroxide ◽  
Real Time ◽  
Mammalian Cells ◽  
Dynamic Range ◽  
Resonance Energy ◽  
Living Cells ◽  
Stress Conditions ◽  
Live Cells ◽  
Cell Physiology ◽  
Broad Dynamic Range

Hydrogen peroxide (H2O2) serves fundamental regulatory functions in metabolism beyond the role as damage signal. During stress conditions, the level of H2O2 increases in the cells and causes oxidative stress, which interferes with normal cell growth in plants and animals. The H2O2 also acts as a central signaling molecule and regulates numerous pathways in living cells. To better understand the generation of H2O2 in environmental responses and its role in cellular signaling, there is a need to study the flux of H2O2 at high spatio–temporal resolution in a real-time fashion. Herein, we developed a genetically encoded Fluorescence Resonance Energy Transfer (FRET)-based nanosensor (FLIP-H2O2) by sandwiching the regulatory domain (RD) of OxyR between two fluorescent moieties, namely ECFP and mVenus. This nanosensor was pH stable, highly selective to H2O2, and showed insensitivity to other oxidants like superoxide anions, nitric oxide, and peroxynitrite. The FLIP-H2O2 demonstrated a broad dynamic range and having a binding affinity (Kd) of 247 µM. Expression of sensor protein in living bacterial, yeast, and mammalian cells showed the localization of the sensor in the cytosol. The flux of H2O2 was measured in these live cells using the FLIP-H2O2 under stress conditions or by externally providing the ligand. Time-dependent FRET-ratio changes were recorded, which correspond to the presence of H2O2. Using this sensor, real-time information of the H2O2 level can be obtained non-invasively. Thus, this nanosensor would help to understand the adverse effect of H2O2 on cell physiology and its role in redox signaling.

Real-Time Investigation of Engineered Nanomaterials Cytotoxicity in Living Alveolar Epithelia with Hopping Probe Ion Conductance Microscopy

2013 ◽  
Vol 651 ◽  
pp. 24-28 ◽  
Author(s):  
Xiao Liu ◽  
Hui Zhu ◽  
Hu Jie Lu ◽  
Ying Li ◽  
Jian Ning Zhang ◽  
...  
Keyword(s):  
Real Time ◽  
Imaging Techniques ◽  
Living Cells ◽  
Alveolar Epithelial Cells ◽  
Engineered Nanomaterials ◽  
Ion Conductance ◽  
Physiological Conditions ◽  
Alveolar Epithelial ◽  
Ideal Tool ◽  
Topographic Imaging

Widely used engineered nanomaterials (NMs) display unique properties that may have impact on human health, and thus require a reliable evaluation of their potential cytotoxicity. There is a continuing need for real-time imaging techniques capable of studying the interactions between NMs and living alveolar epithelial cells under physiological conditions. A new developed noninvasive HPICM is designed for continuous high-resolution topographic imaging of living cells, which makes it an ideal tool to study NMs cytotoxicity in living alveolar epithelia by performing reliable repetitive scanning. In this review, we concisely introduced the operation principle of HPICM and its applications to real-time investigation of engineered NMs cytotoxicity in living alveolar epithelia. Published results demonstrate that non-contact HPICM combined with patch-clamp has the potential to become a powerful microscopy for real-time studies of NM-cell interactions under physiological conditions.

Molecular Beacons for Sensitive Gene Detection in Living Cells

2003 ◽  
Author(s):  
Andrew Tsurkas ◽  
Gang Bao
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Molecular Beacon ◽  
Point Mutations ◽  
Living Cells ◽  
Molecular Beacons ◽  
High Signal ◽  
Gene Detection ◽  
Real Time Imaging ◽  
Background Ratio

Real-time imaging of gene expression in living cells has the potential to significantly impact clinical and laboratory studies of cancer, including cancer diagnosis and analysis. Molecular beacons (MBs) provide a simple and promising tool for the detection of target mRNA as tumor markers due to their high signal-to-background ratio, and their improved specificity in detecting point mutations. However, the harsh intracellular environment does limit the sensitivity of MB-based gene detection. Specifically, MBs bound to target mRNAs cannot be distinguished from those degraded by nucleases, or opened due to non-specific interactions. To overcome this difficulty, we have developed a novel dual FRET molecular beacons approach in which a pair of molecular beacons, one with a donor fluorophore and a second with an acceptor fluorophore, hybridize to adjacent regions on the same target resulting in fluorescence resonance energy transfer (FRET). The detection of a FRET signal leads to a substantially increased signal-to-background ratio compared with that in single molecular beacon assays and enables discrimination between fluorescence due to specific probe/target hybridization and a variety of false-positive events. We have performed systematic in-solution and cellular studies of dual FRET molecular beacon and demonstrated that this new approach allows for real-time imaging of gene expression in living cells.

“REAL TIME” IMAGE ANALYSIS OF THE CHANGES OF THE SHAPE AND/OR SIZE OF LIVING CELLS AS A METHOD FOR STUDYING CELL PHYSIOLOGY

1991 ◽  
Vol 73 (2-3) ◽  
pp. 31a-31a
Author(s):  
Pierre TRAVO ◽  
Delphine LEES ◽  
Philippe BODIN ◽  
Denis PUGNERE ◽  
Amaud CASTEL ◽  
...  
Keyword(s):  
Image Analysis ◽  
Real Time ◽  
Living Cells ◽  
Cell Physiology ◽  
Real Time Image ◽  
Time Image

scholarly journals Mismatch Repair: From Preserving Genome Stability to Enabling Mutation Studies in Real-Time Single Cells

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1535
Author(s):  
Marina Elez
Keyword(s):  
Real Time ◽  
Mismatch Repair ◽  
Genetic Information ◽  
Genome Stability ◽  
Direct Evidence ◽  
Single Cells ◽  
Living Cells ◽  
Spontaneous Mutations ◽  
Mismatch Recognition

Mismatch Repair (MMR) is an important and conserved keeper of the maintenance of genetic information. Miroslav Radman’s contributions to the field of MMR are multiple and tremendous. One of the most notable was to provide, along with Bob Wagner and Matthew Meselson, the first direct evidence for the existence of the methyl-directed MMR. The purpose of this review is to outline several aspects and biological implications of MMR that his work has helped unveil, including the role of MMR during replication and recombination editing, and the current understanding of its mechanism. The review also summarizes recent discoveries related to the visualization of MMR components and discusses how it has helped shape our understanding of the coupling of mismatch recognition to replication. Finally, the author explains how visualization of MMR components has paved the way to the study of spontaneous mutations in living cells in real time.

A thousand words about the imaging in cardiac amyloidosis

2019 ◽  
Vol 88 (2) ◽  
pp. 123-128
Author(s):  
Rafał Dankowski ◽  
Marek Leszniewski ◽  
Małgorzata Pyda ◽  
Andrzej Szyszka
Keyword(s):  
Cardiac Magnetic Resonance ◽  
Magnetic Resonance ◽  
Early Diagnosis ◽  
Cardiac Amyloidosis ◽  
Imaging Techniques ◽  
Restrictive Cardiomyopathy ◽  
Recent Advances

Cardiac amyloidosis is an infiltrative disease which usually occurs under the form of restrictive cardiomyopathy. Advances in the treatment have changed the unfavorable outcomes of this disease. Thus, early diagnosis is essential. Recent advances in echocardiography and cardiac magnetic resonance provided new modalities for the detection of cardiac amyloidosis. This summary discusses the role of imaging techniques in the diagnosis of cardiac amyloidosis.

scholarly journals CRISPR-based Live Imaging of Epigenetic Modification-Mediated Genome Reorganization

2020 ◽  
Author(s):  
Ying Feng ◽  
Yao Wang ◽  
Chen Yang ◽  
Ardalan Naseri ◽  
Thoru Pederson ◽  
...  
Keyword(s):  
Genome Organization ◽  
Essential Role ◽  
Epigenetic Modification ◽  
Imaging Techniques ◽  
Living Cells ◽  
Genome Reorganization ◽  
Genomic Regions ◽  
Single Living Cells ◽  
Single Living

Epigenetic modifications play an essential role in chromatin architecture and dynamics. The role of epigenetic modification in chromatin organization has been studied by Hi-C from population cells, but imaging techniques to study their correlation and regulation in single living cells are lacking. Here we develop a CRISPR-based EpiGo (Epigenetic perturbation induced Genome organization) system to track epigenetic modification-mediated relocation, interaction or reorganization of genomic regions in living cells. EpiGo-KRAB is sufficient to induce the relocation of genomic loci to HP1α condensates and trigger genomic interactions. EpiGo-KRAB also triggers the induction of H3K9me3 at large genomic regions, which decorate on the surface of HP1α condensates possibly driven by phase separation.

Role of imaging techniques in liver veno-occlusive disease diagnosis: recent advances and literature review

2019 ◽  
Vol 13 (5) ◽  
pp. 463-484 ◽  
Author(s):  
Federico Ravaioli ◽  
Antonio Colecchia ◽  
Luigina Vanessa Alemanni ◽  
Amanda Vestito ◽  
Elton Dajti ◽  
...  
Keyword(s):  
Literature Review ◽  
Imaging Techniques ◽  
Disease Diagnosis ◽  
Occlusive Disease ◽  
Recent Advances

Advances in the Treatment of Localized Prostate Cancer: The Role of Anatomic and Functional Imaging in Men Managed With Radiotherapy

2007 ◽  
Vol 25 (8) ◽  
pp. 987-995 ◽  
Author(s):  
Joycelyn L. Speight ◽  
Mack Roach
Keyword(s):  
Prostate Cancer ◽  
Real Time ◽  
Functional Imaging ◽  
Imaging Techniques ◽  
Three Dimensional ◽  
Intensity Modulated Radiotherapy ◽  
Conformal Radiotherapy ◽  
Three Dimensional Conformal Radiotherapy ◽  
Cure Rates

Radiation therapy is an active modality in the management of local and regional prostate cancer, but can be curative only if all existing disease is encompassed within the treatment portal. In addition to the ability to deliver sufficient radiation dose, accurate targeting is critical to achieve better treatment outcomes. Failure to accommodate daily variations in setup and organ motion potentially limits the efficacy of sophisticated conformal techniques (three-dimensional conformal radiotherapy and intensity-modulated radiotherapy). Increased use of various online and real-time imaging techniques is an important step toward enhancing treatment accuracy. The incorporation of functional imaging techniques into treatment planning is another important step. The addition of biologic and metabolic information regarding the location and extent of disease combined with real-time online imaging will allow us to better determine where, how, and with what to treat appropriate targets and improve cure rates.

scholarly journals Design Of A Rapid And Reversible Fluorescence Assay To Detect Covid-19 And Other Pathogens

2020 ◽  
Author(s):  
V. Siddartha Yerramilli ◽  
Suzanne Scarlata
Keyword(s):  
Internal Control ◽  
Fold Increase ◽  
Fluorescent Sensors ◽  
Molecular Beacons ◽  
Native Form ◽  
Complementary Sequence ◽  
Stem Loop ◽  
Loop Region ◽  
Target Rna ◽  
Biophysical Method

AbstractWe describe a rapid and reusable biophysical method to assay COVID-19 and other pathogens. The method uses fluorescent sensors (i.e. molecular beacons) designed to detect COVID-19 RNA or any RNA of interest, concurrent with an internal control without the need for amplification. The molecular beacons are stem-loop structures in which a ∼10 nucleotide loop region has the complementary sequence of a region of the target RNA, and a fluorophore and quencher are placed on the 5’ and 3’ ends of the stem. The energy of hybridization of the loop with its target is designed to be greater than the hybridization energy of the energy of the stem so that when the beacon encounters its target RNA, the structure opens resulting in dequenching of the fluorophore. Here, we designed a COVID-19 beacon that is completely quenched in its native form and undergoes a 50-fold increase in fluorescence when exposed to nanomolar amounts of synthetic viral oligonucleotide. No changes in intensity are seen when control RNA is added. A control beacon to a human GAPDH RNA, chosen for its high levels in saliva, behaved similar to the COVID-19 beacon. This increase in fluorescence with beacon opening can be completely reversed upon addition of single stranded DNA complementary to COVID-19 beacon loop region. Beacons can be attached to an insert matrix allowing their use in concentrated form and can be made from morphilino oligonucleotides that are resistant to RNases. We present an analysis of the parameters that will allow the development of test strips to detect virus in aerosol, body fluids and community waste.Statement of significanceA platform for reusable and rapid detection of COVID-19 RNA and other pathogenic RNAs without the need for amplification or sophisticated instrumentation in a complex environment is described.

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