Development of a single-step fluorogenic sirtuin assay and its applications for high-throughput screening

2022 ◽  
Author(s):  
Pan Wang ◽  
Di Chen ◽  
Jianxiong An ◽  
Shu-xian Lin ◽  
Ting Liu ◽  
...  
Keyword(s):  
High Throughput ◽  
Nicotinamide Adenine Dinucleotide ◽  
Histone Deacetylases ◽  
High Throughput Screening ◽  
Single Step ◽  
Nicotinamide Adenine

Sirtuins (SIRTs) are a class of nicotinamide adenine dinucleotide (NAD+)-dependent histone deacetylases. Since SIRTs have different subcellular locations and different preferences for deacylation activity, SIRTs are not only highly gaining...

scholarly journals Tryptophan plays an important role in yeast’s tolerance to isobutanol

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Hsien-Lin Liu ◽  
Christine H.-T. Wang ◽  
En-Pei Isabel Chiang ◽  
Chieh-Chen Huang ◽  
Wen-Hsiung Li
Keyword(s):  
Nicotinamide Adenine Dinucleotide ◽  
High Throughput Screening ◽  
Water Solubility ◽  
Energy Content ◽  
Enrichment Analysis ◽  
High Energy ◽  
Pentose Phosphate ◽  
Transcriptomic Analysis ◽  
Nicotinamide Adenine ◽  
Tryptophan Biosynthesis

Abstract Background Isobutanol is considered a potential biofuel, thanks to its high-energy content and octane value, limited water solubility, and compatibility with gasoline. As its biosynthesis pathway is known, a microorganism, such as Saccharomyces cerevisiae, that inherently produces isobutanol, can serve as a good engineering host. Isobutanol’s toxicity, however, is a major obstacle for bioproduction. This study is to understand how yeast tolerates isobutanol. Results A S. cerevisiae gene-deletion library with 5006 mutants was used to screen genes related to isobutanol tolerance. Image recognition was efficiently used for high-throughput screening via colony size on solid media. In enrichment analysis of the 161 isobutanol-sensitive clones identified, more genes than expected were mapped to tryptophan biosynthesis, ubiquitination, and the pentose phosphate pathway (PPP). Interestingly, adding exogenous tryptophan enabled both tryptophan biosynthesis and PPP mutant strains to overcome the stress. In transcriptomic analysis, cluster analysis of differentially expressed genes revealed the relationship between tryptophan and isobutanol stress through some specific cellular functions, such as biosynthesis and transportation of amino acids, PPP, tryptophan metabolism, nicotinate/nicotinamide metabolism (e.g., nicotinamide adenine dinucleotide biosynthesis), and fatty acid metabolism. Conclusions The importance of tryptophan in yeast’s tolerance to isobutanol was confirmed by the recovery of isobutanol tolerance in defective strains by adding exogenous tryptophan to the growth medium. Transcriptomic analysis showed that amino acid biosynthesis- and transportation-related genes in a tryptophan biosynthesis-defective host were up-regulated under conditions similar to nitrogen starvation. This may explain why ubiquitination was required for the protein turnover. PPP metabolites may serve as precursors and cofactors in tryptophan biosynthesis to enhance isobutanol tolerance. Furthermore, the tolerance mechanism may also be linked to tryptophan downstream metabolism, including the kynurenine pathway and nicotinamide adenine dinucleotide biosynthesis. Both pathways are responsible for cellular redox balance and anti-oxidative ability. Our study highlights the central role of tryptophan in yeast’s isobutanol tolerance and offers new clues for engineering a yeast host with strong isobutanol tolerance.

scholarly journals Identification of Selective Class II Histone Deacetylase Inhibitors Using a Novel Dual-Parameter Binding Assay Based on Fluorescence Anisotropy and Lifetime

2011 ◽  
Vol 16 (10) ◽  
pp. 1206-1216 ◽  
Author(s):  
Patricia Haus ◽  
Michael Korbus ◽  
Michael Schröder ◽  
Franz-Josef Meyer-Almes
Keyword(s):  
Histone Deacetylase ◽  
High Throughput ◽  
Fluorescence Anisotropy ◽  
Histone Deacetylases ◽  
High Throughput Screening ◽  
Cell Cycle Progression ◽  
Histone Deacetylase Inhibitors ◽  
Binding Assay ◽  
Cellular Functions ◽  
Isoform Selectivity

Histone deacetylases (HDACs) are important epigenetic factors regulating a variety of vital cellular functions such as cell cycle progression, differentiation, cell migration, and apoptosis. Consequently, HDACs have emerged as promising targets for cancer therapy. The drugability of HDACs has been shown by the discovery of several structural classes of inhibitors (HDACis), particularly by the recent approval of two HDACis, vorinostat (ZOLINZA) and romidepsin (Istodax), for the treatment of cutaneous T-cell lymphoma by the US Food and Drug Administration. The outstanding potential of HDACis, with a defined isoform selectivity profile as drugs against a plurality of diseases, vindicates increased effort in developing high-throughput capable assays for screening campaigns. In this study, a dual-competition assay exploiting changes in fluorescence anisotropy and lifetime was used to screen the LOPAC (Sigma-Aldrich, St Louis, MO) library against the bacterial histone deacetylase homologue HDAH from Bordetella, which shares 35% identity with the second deacetylase domain of HDAC6. The binding assay proved to be highly suitable for high-throughput screening campaigns. Several LOPAC compounds have been identified to inhibit HDAH in the lower micromolar range. Most interestingly, some of the hit compounds turned out to be weak but selective inhibitors of human class IIa and IIb HDACs.

Non-isotopic dual parameter competition assay suitable for high-throughput screening of histone deacetylases

2009 ◽  
Vol 19 (13) ◽  
pp. 3651-3656 ◽  
Author(s):  
Daniel Riester ◽  
Christian Hildmann ◽  
Patricia Haus ◽  
Antonia Galetovic ◽  
Andreas Schober ◽  
...  
Keyword(s):  
High Throughput ◽  
Histone Deacetylases ◽  
High Throughput Screening ◽  
Competition Assay

scholarly journals Development and validation of a rapid, single-step reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) system potentially to be used for reliable and high-throughput screening of COVID-19

2020 ◽  
Author(s):  
Minghua Jiang ◽  
Weihua Pan ◽  
Amir Aratehfar ◽  
Wenjie Fang ◽  
Liyan Ling ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
High Throughput ◽  
Predictive Value ◽  
High Throughput Screening ◽  
Lamp Assay ◽  
Single Step ◽  
Clinical Samples ◽  
Qrt Pcr ◽  
Pcr Assays ◽  
Development And Validation

AbstractObjectivesDevelopment and validation of a single-step and accurate reverse transcriptase loop-mediated isothermal amplification technique (RT-LAMP) for rapid identification of SARS COV-2 relative to commercial quantitative reverse transcriptase real-time PCR (qRT-PCR) assays to allow prompt initiation of proper medical care and containment of virus spread.MethodsPrimers showing optimal in-silico features were subjected to analytical sensitivity and specificity to assess the limit of detection (LOD) and cross-reaction with closely- and distantly-related viral species, and clinically prominent bacterial and fungal species. In order to evaluate the clinical utility, our RT-LAMP was subjected to a large number of clinical samples, including 213 negative and 47 positive patients, relative to two commercial quantitative RT-PCR assays.ResultsThe analytical specificity and sensitivity of our assay was 100% and 500 copies/ml when serial dilution performed in both water and sputum. Subjecting our RT-LAMP assay to clinical samples showed a high degree of specificity (99.5%), sensitivity (91.4%), positive predictive value (97.7%), and negative predictive value (98.1%) when used relative to qRT-PCR. Our RT-LAMP assay was two times faster than qRT-PCR and is storable at room temperature. A suspected case that later became positive tested positive using both our RT-LAMP and the two qRT-PCR assays, which shows the capability of our assay for screening purposes.ConclusionsWe present a rapid RT-LAMP assay that could extend the capacity of laboratories to process two times more clinical samples relative to qRT-PCR and potentially could be used for high-throughput screening purposes when demand is increasing at critical situations.

High-Throughput Screening-Compatible Single-Step Protocol to Differentiate Embryonic Stem Cells in Neurons

2008 ◽  
Vol 17 (3) ◽  
pp. 573-584 ◽  
Author(s):  
Annalisa Fico ◽  
Genesia Manganelli ◽  
Marino Simeone ◽  
Stefano Guido ◽  
Gabriella Minchiotti ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
High Throughput ◽  
High Throughput Screening ◽  
Embryonic Stem ◽  
Single Step

Effect of Niacin on Mitochondria of Allium Cepa Root Cells

1967 ◽  
Vol 25 ◽  
pp. 78-79
Author(s):  
M. Arif Hayat
Keyword(s):  
Nicotinamide Adenine Dinucleotide ◽  
Hydrogen Transfer ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Nicotinamide Adenine ◽  
Root Tips ◽  
Root Cells ◽  
Transfer Processes ◽  
Standard Procedures ◽  
A Cell ◽  
Critical Interest

Although it is recognized that niacin (pyridine-3-carboxylic acid), incorporated as the amide in nicotinamide adenine dinucleotide (NAD) or in nicotinamide adenine dinucleotide phosphate (NADP), is a cofactor in hydrogen transfer in numerous enzyme reactions in all organisms studied, virtually no information is available on the effect of this vitamin on a cell at the submicroscopic level. Since mitochondria act as sites for many hydrogen transfer processes, the possible response of mitochondria to niacin treatment is, therefore, of critical interest.Onion bulbs were placed on vials filled with double distilled water in the dark at 25°C. After two days the bulbs and newly developed root system were transferred to vials containing 0.1% niacin. Root tips were collected at ¼, ½, 1, 2, 4, and 8 hr. intervals after treatment. The tissues were fixed in glutaraldehyde-OsO4 as well as in 2% KMnO4 according to standard procedures. In both cases, the tissues were dehydrated in an acetone series and embedded in Reynolds' lead citrate for 3-10 minutes.

High-throughput screening program for botanical extracts

Planta Medica ◽  
2012 ◽  
Vol 78 (11) ◽  
Author(s):  
L Hingorani ◽  
NP Seeram ◽  
B Ebersole
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Screening Program ◽  
Botanical Extracts
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