scholarly journals Microtubule inhibitors enhance DNA transfection efficiency through autophagy receptor p62/SQSTM1

2021 ◽  
Author(s):  
Megumi Tsuchiya ◽  
Hidesato Ogawa ◽  
Kento Watanabe ◽  
Takako Koujin ◽  
Chie Mori ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Transfection Efficiency ◽  
Dna Transfection ◽  
Compound Library ◽  
Autophagosome Formation ◽  
Microtubule Inhibitors ◽  
Positive Effects ◽  
Ectopic Gene Expression ◽  
Low Efficiency ◽  
Indispensable Tool

Ectopic gene expression is an indispensable tool in biology and medicine. However, it is often limited by the low efficiency of DNA transfection. It is known that depletion of p62/SQSTM1 enhances DNA transfection efficiency by preventing the degradation of transfected DNA. Therefore, p62 is a potential target of drugs to increase transfection efficiency. To identify drugs that enhance transfection efficiency, a non-biased high-throughput screening was applied to over 4,000 compounds from the Osaka University compound library, and their p62-dependency was evaluated. The top-scoring drugs were mostly microtubule inhibitors, such as colchicine and vinblastine, and all of them showed positive effects only in the presence of p62. To understand the mechanisms, the time of p62-dependent ubiquitination was examined using polystyrene beads that were introduced into cells as materials that mimicked transfected DNA. The microtubule inhibitors caused a delay in ubiquitination. Furthermore, the level of phosphorylated p62 at S405, which is required for ubiquitination during autophagosome formation, markedly decreased in the drug-treated cells. These results suggest that microtubule inhibitors inhibit p62-dependent autophagosome formation. Our findings provide new insights into the mechanisms of DNA transfection and also provide a solution to increase DNA transfection efficiency.

Opportunities for accelerating labor productivity growth: The role of small and medium enterprises

2020 ◽  
pp. 98-114
Author(s):  
Evguenia V. Bessonova ◽  
Alexander G. Morozov ◽  
Natalia A. Turdyeva ◽  
Anna N. Tsvetkova
Keyword(s):  
Labor Productivity ◽  
Productivity Growth ◽  
Small And Medium Enterprises ◽  
Growth Potential ◽  
Business Climate ◽  
Labor Productivity Growth ◽  
Positive Effects ◽  
Low Efficiency ◽  
Medium Enterprises ◽  
Medium Firms

The paper considers necessary conditions for acceleration of labor productivity growth in Russia. Based on micro data, as well as aggregate data, the paper quantifies the contribution of small and medium firms to labor productivity growth. It shows that mere increase of the number of small and medium enterprises is not as important for positive effects of these programs, as qualitative improvements: development of favorable environment for growth, which is largely determined by business climate. Accelerating productivity growth involves redistribution of labor and capital from inefficient to efficient enterprises. In particular, it is necessary to create conditions, which allow a firm to grow after it enters the market instead of stagnating as a small firm with low efficiency. At the same time, it is necessary for ineffective firms, which exhausted their growth potential, to have an opportunity to exit the market easily leaving resources including labor to fast-growing companies.

scholarly journals Development of a Simple Assay Protocol for High-Throughput Screening of Mycobacterium tuberculosis Glutamine Synthetase for the Identification of Novel Inhibitors

2005 ◽  
Vol 10 (7) ◽  
pp. 725-729 ◽  
Author(s):  
Upasana Singh ◽  
Vinita Panchanadikar ◽  
Dhiman Sarkar
Keyword(s):  
Escherichia Coli ◽  
Mycobacterium Tuberculosis ◽  
Glutamine Synthetase ◽  
Small Molecules ◽  
High Throughput ◽  
High Throughput Screening ◽  
Coli Strain ◽  
Compound Library ◽  
Essential Enzyme ◽  
Assay Protocol

Mycobacterium tuberculosis glutamine synthetase (GS) is an essential enzyme involved in the pathogenicity of the organism. The screening of a compound library using a robust high-throughput screening (HTS) assay is currently thought to be the most efficient way of getting lead molecules, which are potent inhibitors for this enzyme. The authors have purified the enzyme to a >90% level from the recombinant Escherichia coli strain YMC21E, and it was used for partial characterization as well as standardization experiments. The results indicated that the Kmof the enzyme for L-glutamine and hydroxylamine were 60 mM and 8.3 mM, respectively. The Km for ADP, arsenate, and Mn2+ were 2 [.proportional]M, 5 [.proportional]M, and 25 [.proportional]M, respectively. When the components were adjusted according to their Km values, the activity remained constant for at least 3 h at both 25° C and 37° C. The Z′ factor determined in microplate format indicated robustness of the assay. When the signal/noise ratios were determined for different assay volumes, it was observed that the 200-[.proportional]l volume was found to be optimum. The DMSO tolerance of the enzyme was checked up to 10%, with minimal inhibition. The IC50 value determined for L-methionine S-sulfoximine on the enzyme activity was 3 mM. Approximately 18,000 small molecules could be screened per day using this protocol by a Beckman Coulter HTS setup.

scholarly journals New Spiro[cycloalkane-pyridazinone] Derivatives with Favorable Fsp3 Character

Chemistry ◽  
2020 ◽  
Vol 2 (4) ◽  
pp. 837-848
Author(s):  
Csilla Sepsey Für ◽  
Hedvig Bölcskei
Keyword(s):  
High Throughput Screening ◽  
Pharmaceutical Companies ◽  
Compound Library ◽  
Aromatic Rings ◽  
Lead Discovery ◽  
Lipinski’S Rule ◽  
Design And Synthesis ◽  
Lipinski’S Rule Of Five ◽  
Pyridazinone Derivatives ◽  
New Compounds

The large originator pharmaceutical companies need more and more new compounds for their molecule banks, because high throughput screening (HTS) is still a widely used method to find new hits in the course of the lead discovery. In the design and synthesis of a new compound library, important points are in focus nowadays: Lipinski’s rule of five (RO5); the high Fsp3 character; the use of bioisosteric heterocycles instead of aromatic rings. With said aim in mind, we have synthesized a small compound library of new spiro[cycloalkane-pyridazinones] with 36 members. The compounds with this new scaffold may be useful in various drug discovery projects.

scholarly journals Efficiency Analysis of Official Development Assistance Provided by Korea

2018 ◽  
Vol 10 (8) ◽  
pp. 2697 ◽  
Author(s):  
Yun-Gi Hwang ◽  
Soohyun Park ◽  
Daecheol Kim
Keyword(s):  
Development Assistance ◽  
Efficiency Analysis ◽  
Official Development Assistance ◽  
Qualitative Assessment ◽  
Tobit Regression ◽  
Average Efficiency ◽  
Positive Effects ◽  
Dea Efficiency ◽  
Low Efficiency ◽  
Tobit Regression Analysis

Results of the CDI and QODA evaluation developed by OECD showed that Korea’s aid presented low efficiency compared to other aid countries. However, these methods represent a qualitative assessment of the effectiveness of each country’s aid and are not applicable to the evaluation of actual aid projects and the identification of causes of the inefficiency. Therefore, it is needed to grasp the reality of Korea’s aid and to identify the cause of aid inefficiency to set up a better ODA policy. The purpose of this study is to improve the effectiveness of Korea’s Official Development Assistance (ODA) provided to developing countries. To do this, we analyzed the efficiency of ODA provided to 33 recipient countries by Korea through data envelopment analysis method. The effects of three factors, illiteracy, integrity, and GDP, on efficiency were also investigated by utilizing a Tobit regression analysis. As a result of the DEA efficiency analysis, it was found that the average efficiency was about 65.74%. By region, the average efficiency scores of Asia, Africa, Central and South America, and Middle East and Eastern Europe were about 47.8%, 78.9%, 70.4%, and 62.7%, respectively. This indicates that Asian countries are inefficient compared to countries from other regions. It was also found that GDP, integrity, and illiteracy have positive effects on efficiency. The methodology proposed in this study can be used for other studies to create an aid policy that produces efficient results.

Branched Amphipathic Peptide Capsules: Different Ratios of the Two Constituent Peptides Direct Distinct Bilayer Structures, Sizes, and DNA Transfection Efficiency

Langmuir ◽  
2017 ◽  
Vol 33 (28) ◽  
pp. 7096-7104 ◽  
Author(s):  
Sheila de M. Barros ◽  
L. Adriana Avila ◽  
Susan K. Whitaker ◽  
Kayla E. Wilkinson ◽  
Pinakin Sukthankar ◽  
...  
Keyword(s):  
Transfection Efficiency ◽  
Dna Transfection ◽  
Amphipathic Peptide ◽  
Peptide Capsules

scholarly journals On-chip multiplexed single-cell patterning and controllable intracellular delivery

2020 ◽  
Vol 6 (1) ◽  
Author(s):  
Zaizai Dong ◽  
Yanli Jiao ◽  
Bingteng Xie ◽  
Yongcun Hao ◽  
Pan Wang ◽  
...  
Keyword(s):  
Single Cell ◽  
Low Voltage ◽  
Transfection Efficiency ◽  
Cell Damage ◽  
Intracellular Delivery ◽  
Cell Manipulation ◽  
Expression Vectors ◽  
Vacuum System ◽  
Low Efficiency ◽  
On Chip

Abstract Conventional electroporation approaches show limitations in the delivery of macromolecules in vitro and in vivo. These limitations include low efficiency, noticeable cell damage and nonuniform delivery of cells. Here, we present a simple 3D electroporation platform that enables massively parallel single-cell manipulation and the intracellular delivery of macromolecules and small molecules. A pyramid pit micropore array chip was fabricated based on a silicon wet-etching method. A controllable vacuum system was adopted to trap a single cell on each micropore. Using this chip, safe single-cell electroporation was performed at low voltage. Cargoes of various sizes ranging from oligonucleotides (molecular beacons, 22 bp) to plasmid DNA (CRISPR-Cas9 expression vectors, >9 kb) were delivered into targeted cells with a significantly higher transfection efficiency than that of multiple benchmark methods (e.g., commercial electroporation devices and Lipofectamine). The delivered dose of the chemotherapeutic drug could be controlled by adjusting the applied voltage. By using CRISPR-Cas9 transfection with this system, the p62 gene and CXCR7 gene were knocked out in tumor cells, which effectively inhibited their cellular activity. Overall, this vacuum-assisted micropore array platform provides a simple, efficient, high-throughput intracellular delivery method that may facilitate on-chip cell manipulation, intracellular investigation and cancer therapy.

scholarly journals High-Throughput Screening of a 100,000-Compound Library for Inhibitors of Influenza A Virus (H3N2)

2008 ◽  
Vol 13 (9) ◽  
pp. 879-887 ◽  
Author(s):  
William E. Severson ◽  
Michael McDowell ◽  
Subramaniam Ananthan ◽  
Dong-Hoon Chung ◽  
Lynn Rasmussen ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Influenza A ◽  
Moderate Activity ◽  
Compound Library ◽  
Highly Active ◽  
Virus Life Cycle ◽  
Influenza Activity ◽  
Compound Concentration ◽  
Viral Cytopathic Effect

Using a highly reproducible and robust cell-based high-throughput screening (HTS) assay, the authors screened a 100,000-compound library at 14- and 114-µM compound concentration against influenza strain A/Udorn/72 (H3N2). The “hit” rates (>50% inhibition of the viral cytopathic effect) from the 14- and 114-µM screens were 0.022% and 0.38%, respectively. The hits were evaluated for their antiviral activity, cell toxicity, and selectivity in dose-response experiments. The screen at the lower concentration yielded 3 compounds, which displayed moderate activity (SI50 = 10-49). Intriguingly, the screen at the higher concentration revealed several additional hits. Two of these hits were highly active with an SI50 > 50. Time of addition experiments revealed 1 compound that inhibited early and 4 other compounds that inhibited late in the virus life cycle, suggesting they affect entry and replication, respectively. The active compounds represent several different classes of molecules such as carboxanilides, 1-benzoyl-3-arylthioureas, sulfonamides, and benzothiazinones, which have not been previously identified as having antiviral/anti-influenza activity. ( Journal of Biomolecular Screening 2008:879-887)

The Influence of Physicochemical Parameters on the Efficacy of Non-Viral DNA Transfection Complexes: A Comparative Study

2006 ◽  
Vol 6 (9) ◽  
pp. 2776-2782 ◽  
Author(s):  
Carsten Kneuer ◽  
Carsten Ehrhardt ◽  
Heike Bakowsky ◽  
M. N. V. Ravi Kumar ◽  
Volker Oberle ◽  
...  
Keyword(s):  
Zeta Potential ◽  
Mammalian Cells ◽  
Transfection Efficiency ◽  
Dna Delivery ◽  
Dna Transfection ◽  
Dna Complexes ◽  
General Validity ◽  
Delivery Vehicles ◽  
Foreign Dna ◽  
Pronounced Reduction

Various polycationic vehicles have been developed to facilitate the transfer of foreign DNA into mammalian cells. Structure-activity studies suggested that biophysical properties, such as size, charge, and morphology of the resulting DNA complexes determine transfection efficiency within one class of vector. To investigate the general validity of these criteria, we studied the efficacy of a variety of DNA delivery vehicles including liposomes (DOTAP, SAINT2) with and without helper lipid (DOPE), the polymer polyethyleneimine (PEI), and cationic nanoparticles (Si26H, PLGA/chitosan) in a comparative manner. Sizes of the DNA complexes varied between 100 and 500 nm for PEI polyplexes and DOTAP/DOPE lipoplexes, respectively. The zeta potential was positive for PEI, Si26H, and DOTAP based complexes, while it was neutral for SAINT2-DNA complexes and negative for PLGA/chitosan-DNA complexes. The latter finding was elucidated by AFM, showing a layer of DNA adsorbed onto the nanoparticles. Transfection activity was negligible for PLGA/chitosan nanospheres, moderate for Si26H nanospheres and high for all other complexes, PEI being the most active carrier. The liposomal preparations were of low (DOTAP) or moderate (SAINT2) stability in serum, resulting in a pronounced reduction of gene expression, which was partially restored by the addition of chloroquine. In conclusion, transfection efficiency (i) seems to require a positive or neutral zeta potential, (ii) is depending on size, e.g., is higher for smaller particles, and (iii) requires a vector that is stable in serum.

DNA Transfection Efficiency of Antimicrobial Peptide as Revealed by Molecular Dynamics Simulation

2013 ◽  
Vol 1569 ◽  
pp. 109-114
Author(s):  
Namsrai Javkhlantugs ◽  
Janlav Munkhtsetseg ◽  
Chimed Ganzorig ◽  
Kazuyoshi Ueda
Keyword(s):  
Molecular Dynamics ◽  
Molecular Dynamics Simulation ◽  
Transfection Efficiency ◽  
Dynamics Simulation ◽  
Amino Acid Residues ◽  
Dna Transfection ◽  
Amphipathic Peptide ◽  
Endosomal Membrane ◽  
N Terminus ◽  
Dna Complex

ABSTRACTThe peptide-DNA complex was investigated by using molecular dynamics simulation to analyze the transfection efficiency of cationic amphipathic peptide. Previously, the cationic peptide, LFampinB, with positively charged amino acid residues of Lysines was used to investigate the orientation and interaction energies for entering the cell though disruption of the endosomal membrane. The same interactions were obtained for N-terminus of the LFampinB peptide with membrane and with plasmid DNA. The N-terminus of LFampinB can bind at minor groove of DNA to make complexation of the peptide with DNA.

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