A scalable filtration method for high throughput screening based on cell deformability

Lab on a Chip ◽  
2019 ◽  
Vol 19 (2) ◽  
pp. 343-357 ◽  
Author(s):  
Navjot Kaur Gill ◽  
Chau Ly ◽  
Kendra D. Nyberg ◽  
Linus Lee ◽  
Dongping Qi ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Cell Deformability ◽  
Filtration Method ◽  
High Throughput Method

We demonstrate a scalable, high throughput method to screen cells based on their ability to deform through micron-scale gaps.

scholarly journals High-Throughput Liberation of Water-Soluble Yeast Content by Irreversible Electropermeation (HT-irEP)

2007 ◽  
Vol 12 (2) ◽  
pp. 267-275 ◽  
Author(s):  
Maxim Zakhartsev ◽  
Carmen Momeu ◽  
Valentina Ganeva
Keyword(s):  
Recombinant Protein ◽  
High Throughput ◽  
High Throughput Screening ◽  
Enzyme Assay ◽  
Water Soluble ◽  
Extracellular Expression ◽  
Yeast Saccharomyces Cerevisiae ◽  
Soluble Cell ◽  
High Throughput Method ◽  
Intracellular Expression

The article describes a high-throughput method for the liberation of water-soluble cell contents by exploiting the phenomenon of irreversible membrane electropermeation (HT-irEP). The method is exemplified in recombinant proteins and plasmid liberation from yeast Saccharomyces cerevisiae on the detectable level. Obtained extracts are pure enough to be readily applied for further analytical analysis such as enzyme assay, PCR, and so on. From the same HT-irEP extract, one can measure activity of the target protein and perform amplification of the corresponding gene from the DNA vector by PCR for recombinant protein with intracellular expression. Therefore, the method is suitable for the high-throughput screening (HTS) of yeast libraries where extracellular expression of recombinant protein is problematic. The method can be easily automated and integrated into existing HTS systems.

Macro-/mesoporous titania thin films: analysing the effect of pore architecture on photocatalytic activity using high-throughput screening

2015 ◽  
Vol 3 (48) ◽  
pp. 24557-24567 ◽  
Author(s):  
Natalita M. Nursam ◽  
Xingdong Wang ◽  
Rachel A. Caruso
Keyword(s):  
Thin Films ◽  
Photocatalytic Activity ◽  
High Throughput ◽  
High Throughput Screening ◽  
Mesoporous Titania ◽  
Pore Architecture ◽  
High Throughput Method

The photocatalytic behaviour of titania thin films was studied using a high-throughput method to correlate the effect of pore and thickness modification.

scholarly journals A High-Throughput RNA Displacement Assay for Screening SARS-CoV-2 nsp10-nsp16 Complex toward Developing Therapeutics for COVID-19

2021 ◽  
pp. 247255522098504
Author(s):  
Sumera Perveen ◽  
Aliakbar Khalili Yazdi ◽  
Kanchan Devkota ◽  
Fengling Li ◽  
Pegah Ghiabi ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Nonstructural Protein ◽  
Cost Effective ◽  
Titration Calorimetry ◽  
Human Immune System ◽  
High Throughput Method ◽  
Displacement Assay ◽  
Human Immune ◽  
Rna Capping

SARS-CoV-2, the coronavirus that causes COVID-19, evades the human immune system by capping its RNA. This process protects the viral RNA and is essential for its replication. Multiple viral proteins are involved in this RNA capping process, including the nonstructural protein 16 (nsp16), which is an S-adenosyl-l-methionine (SAM)-dependent 2′- O-methyltransferase. Nsp16 is significantly active when in complex with another nonstructural protein, nsp10, which plays a key role in its stability and activity. Here we report the development of a fluorescence polarization (FP)-based RNA displacement assay for nsp10-nsp16 complex in a 384-well format with a Z′ factor of 0.6, suitable for high-throughput screening. In this process, we purified the nsp10-nsp16 complex to higher than 95% purity and confirmed its binding to the methyl donor SAM, the product of the reaction, S-adenosyl-l-homocysteine (SAH), and a common methyltransferase inhibitor, sinefungin, using isothermal titration calorimetry (ITC). The assay was further validated by screening a library of 1124 drug-like compounds. This assay provides a cost-effective high-throughput method for screening the nsp10-nsp16 complex for RNA competitive inhibitors toward developing COVID-19 therapeutics.

A Novel High-Throughput Method for Evaluating deoxy-Hb S Solubility.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3782-3782
Author(s):  
Osheiza Abdulmalik ◽  
Patrick Lec ◽  
Jisheng Yang ◽  
Toshio Asakura ◽  
Kwaku Ohene-Frempong ◽  
...  
Keyword(s):  
Sickle Cell ◽  
High Throughput ◽  
Phosphate Buffer ◽  
High Throughput Screening ◽  
Gelling Agent ◽  
High Reproducibility ◽  
Cell Therapies ◽  
High Throughput Method ◽  
Hb S

Abstract The distortion of erythrocytes in sickle cell disease (SCD) is a direct consequence of self-assembly of deoxy-Hb S heterotetramers into low-solubility polymers. Despite the broad recognition that Hb S solubility can be used to predict the efficacy of potential antipolymerization (antisickling) therapies, currently available methods require significant time and effort that detract from their utility by limiting assay as well as the reliability of any relevant results. To this end, we have developed an uncomplicated, rapid, and highly reproducible high-throughput assay for measuring the solubility of deoxy-Hb S in vitro. In contrast to existing methods that remove insoluble deoxy-Hb S polymers by centrifugation, we utilize a highly efficient filtration approach. Briefly, the solubility of deoxy-Hb S is determined in a defined phosphate buffer by filtering insoluble polymers and determining the concentration of filtered Hb spectrophotometrically. We tested the utility of this method by assessing the effects of four parameters known to affect deoxy-Hb S polymerization in vitro, including the concentration of Hb S, the molarity of the phosphate buffer, and the presence of antipolymerization agents including Hb F and the previously reported anti-gelling agent INN 312. Eight different concentrations of Hb S (0.01 – 0.07 g/dL) were prepared in each of three phosphate buffers (1.60 M, 1.65 M, and 1.70 M; pH 7.4). Each reaction was prepared in quadruplicate to facilitate evaluation of assay reproducibility. Soluble oxy-Hbs were prepared in a 96-well 0.45 μm Multiscreen® Solvinert filter plate, sealed with oxygen-impervious optically inert mineral oil, then deoxygenated by supplementation with 30 mM sodium dithionite. The filter plate was incubated at 30°C for 60 min and then vacuum-filtered into a collection plate where the filtrate was maintained under deoxygenated conditions. The absorbances of the filtrates (corresponding to soluble heterotetrameric Hb) were determined at 556 nm. Parallel analyses were carried out using Hb S samples containing 5, 10, 15, or 20% Hb F; 10, 20, 30, or 40% Hb A; or 0.5, 1.0, or 2.0 mM of the known anti-gelling agent INN 312. We observed that the solubility of Hb S in the filtrate varied in direct proportion to the concentration of phosphate buffer, Hb F concentration, or the concentration of INN 312, consistent with the known effect of each parameter thus validating the utility of the method. In addition, the assay demonstrated high reproducibility with a standard deviation of ±3.2% for all samples. Consequently, the reliability, rapidity (< 2 h), and high reproducibility of this novel assay make it a suitable option for high-throughput screening of potential anti-polymerization agents. To further validate this assay, we tested hemolysates from eight de-identified blood samples obtained from patients with SCD or sickle trait. The solubility of the samples determined by our novel high-throughput method correlated exactly with the globin phenotypes of each sample determined by HPLC. These results confirm the potential utility of the microfilter assay as a tool for assessing the solubility of deoxy-Hb S under a variety of experimental and clinical conditions. We conclude that this simple, rapid, and highly reliable high-throughput method—, which may be adapted to automated systems—, may be highly valuable for the primary screening of sickle cell therapies that act by inhibiting the polymerization of deoxy-Hb S.

scholarly journals A high throughput RNA displacement assay for screening SARS-CoV-2 nsp10-nsp16 complex towards developing therapeutics for COVID-19

2020 ◽  
Author(s):  
Sumera Perveen ◽  
Aliakbar Khalili Yazdi ◽  
Kanchan Devkota ◽  
Fengling Li ◽  
Pegah Ghiabi ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Nonstructural Protein ◽  
Cost Effective ◽  
Titration Calorimetry ◽  
Human Immune System ◽  
High Throughput Method ◽  
Displacement Assay ◽  
Human Immune ◽  
Rna Capping

AbstractSARS-CoV-2, the coronavirus that causes COVID-19, evades the human immune system by capping its RNA. This process protects the viral RNA and is essential for its replication. Multiple viral proteins are involved in this RNA capping process including the nonstructural protein 16 (nsp16) which is an S-adenosyl-L-methionine (SAM)-dependent 2’-O-methyltransferase. Nsp16 is significantly active when in complex with another nonstructural protein, nsp10, which plays a key role in its stability and activity. Here we report the development of a fluorescence polarization (FP)-based RNA displacement assay for nsp10-nsp16 complex in 384-well format with a Z′-Factor of 0.6, suitable for high throughput screening. In this process, we purified the nsp10-nsp16 complex to higher than 95% purity and confirmed its binding to the methyl donor SAM, product of the reaction, SAH, and a common methyltransferase inhibitor, sinefungin using Isothermal Titration Calorimetry (ITC). The assay was further validated by screening a library of 1124 drug-like compounds. This assay provides a cost-effective high throughput method for screening nsp10-nsp16 complex for RNA-competitive inhibitors towards developing COVID-19 therapeutics.

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

High throughput screening of microbial biodiversity for the discovery of novel cosmeceutical agents

Planta Medica ◽  
2015 ◽  
Vol 81 (16) ◽  
Author(s):  
K Georgousaki ◽  
N DePedro ◽  
AM Chinchilla ◽  
N Aliagiannis ◽  
F Vicente ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Microbial Biodiversity

A Fluorescent High Throughput Method To Identify Potential Skin Sensitizers

Planta Medica ◽  
2016 ◽  
Vol 82 (05) ◽  
Author(s):  
C Avonto ◽  
AG Chittiboyina ◽  
D Rua ◽  
IA Khan
Keyword(s):  
High Throughput ◽  
High Throughput Method
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