High-Throughput Measurement of Mitochondrial RNA Turnover in Human Cultured Cells

2020 ◽  
pp. 133-146
Author(s):  
Anna V. Kotrys ◽  
Lukasz S. Borowski ◽  
Roman J. Szczesny
Keyword(s):  
High Throughput ◽  
Cultured Cells ◽  
Mitochondrial Rna ◽  
Rna Turnover

scholarly journals Suppressor Analysis of Mutations in the 5′-Untranslated Region of COB mRNA Identifies Components of General Pathways for Mitochondrial mRNA Processing and Decay in Saccharomyces cerevisiae

Genetics ◽  
1999 ◽  
Vol 151 (4) ◽  
pp. 1315-1325
Author(s):  
Wei Chen ◽  
Maria A Islas-Osuna ◽  
Carol L Dieckmann
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Untranslated Region ◽  
Mitochondrial Rna ◽  
Rna Turnover ◽  
Single Nucleotide ◽  
Mrna Stabilization ◽  
Recessive Mutations ◽  
A Subunit ◽  
Suppressor Analysis ◽  
Dominant Suppressor

Abstract The cytochrome b gene in Saccharomyces cerevisiae, COB, is encoded by the mitochondrial genome. Nuclear-encoded Cbp1 protein is required specifically for COB mRNA stabilization. Cbp1 interacts with a CCG element in a 64-nucleotide sequence in the 5′-untranslated region of COB mRNA. Mutation of any nucleotide in the CCG causes the same phenotype as cbp1 mutations, i.e., destabilization of both COB precursor and mature message. In this study, eleven nuclear suppressors of single-nucleotide mutations in CCG were isolated and characterized. One dominant suppressor is in CBP1, while the other 10 semidominant suppressors define five distinct linkage groups. One group of four mutations is in PET127, which is required for 5′ end processing of several mitochondrial mRNAs. Another mutation is linked to DSS1, which is a subunit of mitochondrial 3′ → 5′ exoribonuclease. A mutation linked to the SOC1 gene, previously defined by recessive mutations that suppress cbp1 ts alleles and stabilize many mitochondrial mRNAs, was also isolated. We hypothesize that the products of the two uncharacterized genes also affect mitochondrial RNA turnover.

Automatic 3D Cell Analysis in High-Throughput Microarray Using Micropillar and Microwell Chips

2015 ◽  
Vol 20 (9) ◽  
pp. 1178-1184 ◽  
Author(s):  
Dong Woo Lee ◽  
Moo-Yeal Lee ◽  
Bosung Ku ◽  
Do-Hyun Nam
Keyword(s):  
Standard Deviation ◽  
High Throughput ◽  
High Throughput Screening ◽  
Doubling Time ◽  
Cultured Cells ◽  
Measurement Methods ◽  
Cell Analysis ◽  
Response Curves ◽  
U251 Cell ◽  
Doubling Times

Area-based and intensity-based 3D cell viability measurement methods are compared in high-throughput screening in order to analyze their effects on the assay results (doubling time and IC50) and their repeatability. Many other 3D cell-based high-throughput screening platforms had been previously introduced, but these had not clearly addressed the effects of the two methods on the assay results and assay repeatability. In this study, the optimal way to analyze 3D cultured cells is achieved by comparing day-to-day data of doubling times and IC50 values obtained from the two methods. In experiments, the U251 cell line is grown in chips. The doubling time, based on the area of the 3D cells, was 27.8 ± 1.8 h (standard deviation: 6.6%) and 27.8 ± 3.8 h (standard deviation: 13.7%) based on the intensity of the 3D cells. The doubling time calculated by area shows a smaller standard deviation than one calculated by intensity. IC50 values calculated by both methods are very similar. The standard deviations of IC50 values for the two methods were within ±3-fold. The IC50 variations of the 12 compounds were similar regardless of the viability measurement methods and were highly related to the shape of the dose–response curves.

scholarly journals High Throughput Mechanobiological Screens Enable Mechanical Priming of Pluripotency in Mouse Fibroblasts

2018 ◽  
Author(s):  
Jason Lee ◽  
Miguel Ochoa ◽  
Pablo Maceda ◽  
Eun Yoon ◽  
Lara Samarneh ◽  
...  
Keyword(s):  
Small Molecules ◽  
High Throughput ◽  
Cultured Cells ◽  
Somatic Cells ◽  
Tumor Formation ◽  
Mechanical Forces ◽  
Mouse Fibroblasts ◽  
Cell Culture Systems ◽  
Ipsc Generation ◽  
Induced Pluripotent

Transgenic methods for direct reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) are effective in cell culture systems but ultimately limit the utility of iPSCs due to concerns of mutagenesis and tumor formation. Recent studies have suggested that some transgenes can be eliminated by using small molecules as an alternative to transgenic methods of iPSC generation. We developed a high throughput platform for applying complex dynamic mechanical forces to cultured cells. Using this system, we screened for optimized conditions to stimulate the activation of Oct-4 and other transcription factors to prime the development of pluripotency in mouse fibroblasts. Using high throughput mechanobiological screening assays, we identified small molecules that can synergistically enhance the priming of pluripotency of mouse fibroblasts in combination with mechanical loading. Taken together, our findings demonstrate the ability of mechanical forces to induce reprograming factors and support that biophysical conditioning can act cooperatively with small molecules to priming the induction pluripotency in somatic cells.

scholarly journals Recombinant Lassa Virus Expressing Green Fluorescent Protein as a Tool for High-Throughput Drug Screens and Neutralizing Antibody Assays

Viruses ◽  
2018 ◽  
Vol 10 (11) ◽  
pp. 655 ◽  
Author(s):  
Yíngyún Caì ◽  
Masaharu Iwasaki ◽  
Brett Beitzel ◽  
Shuīqìng Yú ◽  
Elena Postnikova ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
High Throughput ◽  
Reverse Genetics ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Neutralizing Antibody ◽  
Quantitative Detection ◽  
Lassa Virus ◽  
Wild Type ◽  
Green Fluorescent

Lassa virus (LASV), a mammarenavirus, infects an estimated 100,000–300,000 individuals yearly in western Africa and frequently causes lethal disease. Currently, no LASV-specific antivirals or vaccines are commercially available for prevention or treatment of Lassa fever, the disease caused by LASV. The development of medical countermeasure screening platforms is a crucial step to yield licensable products. Using reverse genetics, we generated a recombinant wild-type LASV (rLASV-WT) and a modified version thereof encoding a cleavable green fluorescent protein (GFP) as a reporter for rapid and quantitative detection of infection (rLASV-GFP). Both rLASV-WT and wild-type LASV exhibited similar growth kinetics in cultured cells, whereas growth of rLASV-GFP was slightly impaired. GFP reporter expression by rLASV-GFP remained stable over several serial passages in Vero cells. Using two well-characterized broad-spectrum antivirals known to inhibit LASV infection, favipiravir and ribavirin, we demonstrate that rLASV-GFP is a suitable screening tool for the identification of LASV infection inhibitors. Building on these findings, we established a rLASV-GFP-based high-throughput drug discovery screen and an rLASV-GFP-based antibody neutralization assay. Both platforms, now available as a standard tool at the IRF-Frederick (an international resource), will accelerate anti-LASV medical countermeasure discovery and reduce costs of antiviral screens in maximum containment laboratories.

scholarly journals CAENORHABDITIS ELEGANS AS A MODEL OF AIR POLLUTION TOXICITY DURING DEVELOPMENT AND LIFESPAN

2019 ◽  
Vol 3 (Supplement_1) ◽  
pp. S97-S97
Author(s):  
Amin Haghani ◽  
Hans M Dalton ◽  
Nikoo Safi ◽  
Farimah Shirmohammadi ◽  
Constantinos Sioutas ◽  
...  
Keyword(s):  
Air Pollution ◽  
Caenorhabditis Elegans ◽  
Mouse Models ◽  
High Throughput ◽  
Molecular Mechanisms ◽  
Cultured Cells ◽  
Exposure Model ◽  
C Elegans ◽  
Biological Responses ◽  
Short Term Exposure

Abstract Air pollution (AirPoll) is among the leading human mortality risk factors and yet little is known about the molecular mechanisms of this global environmental toxin. Our recent studies using mouse models even showed genetic variation and sex can alter biological responses to air pollution. To expand genetic studies of AirPoll toxicity throughout the lifespan, we introduced Caenorhabditis elegans as a new AirPoll exposure model which has a short lifespan, high throughput capabilities and shared longevity pathways with mammals. Acute exposure of C. elegans to airborne nanosized AirPoll matter (nPM) caused similar gene expression changes to our prior findings in cell culture and mouse models. Initial C. elegans responses to nPM included antioxidant, inflammatory and Alzheimer homolog genes. The magnitude of changes was dependent on the developmental stage of the worms. Even short term exposure of C. elegans to nPM altered developmental and lifespan hormetic effects, with pathways that included skn-1/Nrf family antioxidant responses. We propose C. elegans as a new and complementary model for mouse and cultured cells to study AirPoll across the lifespan. Future chronic nPM exposure and high throughput genetic screening of C. elegans can identify other major regulators of the developmental and lifespan effects of air pollution. This work was supported by grants R01AG051521 (CEF); R21AG05020 (CEF); Cure Alzheimer’s Fund (CEF); R01GM109028 (SPC), F31AG051382 (HMD) and T32AG000037 (HMD), T32AG052374 (AH).

scholarly journals Balance between Transcription and RNA Degradation Is Vital forSaccharomyces cerevisiaeMitochondria: Reduced Transcription Rescues the Phenotype of Deficient RNA Degradation

2006 ◽  
Vol 17 (3) ◽  
pp. 1184-1193 ◽  
Author(s):  
Agata T. Rogowska ◽  
Olga Puchta ◽  
Anna M. Czarnecka ◽  
Aneta Kaniak ◽  
Piotr P. Stepien ◽  
...  
Keyword(s):  
Mitochondrial Gene ◽  
Rna Degradation ◽  
Transcription Rate ◽  
Mitochondrial Rna ◽  
Rna Turnover ◽  
Loss Of Function ◽  
Mitochondrial Rna Polymerase ◽  
Genes Encoding ◽  
Mitochondrial Introns ◽  
Synthesis And Degradation

The Saccharomyces cerevisiae SUV3 gene encodes the helicase component of the mitochondrial degradosome (mtEXO), the principal 3′-to-5′ exoribonuclease of yeast mitochondria responsible for RNA turnover and surveillance. Inactivation of SUV3 (suv3Δ) causes multiple defects related to overaccumulation of aberrant transcripts and precursors, leading to a disruption of mitochondrial gene expression and loss of respiratory function. We isolated spontaneous suppressors that partially restore mitochondrial function in suv3Δ strains devoid of mitochondrial introns and found that they correspond to partial loss-of-function mutations in genes encoding the two subunits of the mitochondrial RNA polymerase (Rpo41p and Mtf1p) that severely reduce the transcription rate in mitochondria. These results show that reducing the transcription rate rescues defects in RNA turnover and demonstrates directly the vital importance of maintaining the balance between RNA synthesis and degradation.

Effective Automatic Recognition of Cultured Cells in Bright Field Images Using Fisher’s Linear Discriminant Preprocessing

2004 ◽  
Author(s):  
Xi Long ◽  
W. Louis Cleveland ◽  
Y. Lawrence Yao
Keyword(s):  
High Throughput ◽  
Cultured Cells ◽  
Single Cells ◽  
Molecular Genetic ◽  
Molecular Genetic Analysis ◽  
Automatic Recognition ◽  
Robotic Systems ◽  
Human Observer ◽  
Bright Field ◽  
Linear Discriminant

Recent progress in the development of methods for molecular genetic analysis (e.g. RT-PCR, microarrays) has brought sensitivities to the level where single cells can be analyzed [1]. To carry out single-cell-level assays on significant numbers of cells, high throughput robotic systems are needed. These systems require identification of cultured cells (often in bright field images) for micromanipulation and subsequent molecular analysis. Given the variability of cell size and morphology, the presence of “trash,” as well as variations in microscope parameters, such as focus and illumination, identification of cultured cells in bright field images is a difficult task that, traditionally, is done by an experienced human observer. However, the use of human observers represents a severe impediment to the development of high throughput robotic systems. Therefore, there is a major need for algorithms that permit automatic recognition of cells in bright field images.

scholarly journals A high throughput screening system for studying the effects of applied mechanical forces on reprogramming factor expression

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Jason Lee ◽  
Miguel Armenta Ochoa ◽  
Pablo Maceda ◽  
Eun Yoon ◽  
Lara Samarneh ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Cultured Cells ◽  
Mechanical Load ◽  
Mechanical Stretch ◽  
Mechanical Forces ◽  
Mouse Fibroblasts ◽  
Factor Expression ◽  
Physiological Homeostasis ◽  
Transcription Factor Expression

Abstract Mechanical forces are important in the regulation of physiological homeostasis and the development of disease. The application of mechanical forces to cultured cells is often performed using specialized systems that lack the flexibility and throughput of other biological techniques. In this study, we developed a high throughput platform for applying complex dynamic mechanical forces to cultured cells. We validated the system for its ability to accurately apply parallel mechanical stretch in a 96 well plate format in 576 well simultaneously. Using this system, we screened for optimized conditions to stimulate increases in Oct-4 and other transcription factor expression in mouse fibroblasts. Using high throughput mechanobiological screening assays, we identified small molecules that can synergistically enhance the increase in reprograming-related gene expression in mouse fibroblasts when combined with mechanical loading. Taken together, our findings demonstrate a new powerful tool for investigating the mechanobiological mechanisms of disease and performing drug screening in the presence of applied mechanical load.

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