scholarly journals Generation of High-Yield, Functional Oligodendrocytes from a c-myc Immortalized Neural Cell Line, Endowed with Staminal Properties

2021 ◽  
Vol 22 (3) ◽  
pp. 1124
Author(s):  
Mafalda Giovanna Reccia ◽  
Floriana Volpicelli ◽  
Eirkiur Benedikz ◽  
Åsa Fex Svenningsen ◽  
Luca Colucci-D’Amato
Keyword(s):  
Cell Line ◽  
Low Cost ◽  
Neural Cell ◽  
New Drugs ◽  
High Yield ◽  
Time Saving ◽  
Interacting Protein ◽  
Neuronal Marker ◽  
Differentiation Status

Neural stem cells represent a powerful tool to study molecules involved in pathophysiology of Nervous System and to discover new drugs. Although they can be cultured and expanded in vitro as a primary culture, their use is hampered by their heterogeneity and by the cost and time needed for their preparation. Here we report that mes-c-myc A1 cells (A1), a neural cell line, is endowed with staminal properties. Undifferentiated/proliferating and differentiated/non-proliferating A1 cells are able to generate neurospheres (Ns) in which gene expression parallels the original differentiation status. In fact, Ns derived from undifferentiated A1 cells express higher levels of Nestin, Kruppel-like factor 4 (Klf4) and glial fibrillary protein (GFAP), markers of stemness, while those obtained from differentiated A1 cells show higher levels of the neuronal marker beta III tubulin. Interestingly, Ns differentiation, by Epidermal Growth Factors (EGF) and Fibroblast Growth Factor 2 (bFGF) withdrawal, generates oligodendrocytes at high-yield as shown by the expression of markers, Galactosylceramidase (Gal-C) Neuron-Glial antigen 2 (NG2), Receptor-Interacting Protein (RIP) and Myelin Basic Protein (MBP). Finally, upon co-culture, Ns-A1-derived oligodendrocytes cause a redistribution of contactin-associated protein (Caspr/paranodin) protein on neuronal cells, as primary oligodendrocytes cultures, suggesting that they are able to form compact myelin. Thus, Ns-A1-derived oligodendrocytes may represent a time-saving and low-cost tool to study the pathophysiology of oligodendrocytes and to test new drugs.

scholarly journals Scoring functions for drug-effect similarity

2020 ◽  
Author(s):  
Stephan Struckmann ◽  
Mathias Ernst ◽  
Sarah Fischer ◽  
Nancy Mah ◽  
Georg Fuellen ◽  
...  
Keyword(s):  
Cell Line ◽  
Drug Effect ◽  
Pearson Correlation ◽  
Source Code ◽  
New Drugs ◽  
Supplementary Information ◽  
Disease Genes ◽  
Scoring Functions ◽  
Profile Changes

Abstract Motivation The difficulty to find new drugs and bring them to the market has led to an increased interest to find new applications for known compounds. Biological samples from many disease contexts have been extensively profiled by transcriptomics, and, intuitively, this motivates to search for compounds with a reversing effect on the expression of characteristic disease genes. However, disease effects may be cell line-specific and also depend on other factors, such as genetics and environment. Transcription profile changes between healthy and diseased cells relate in complex ways to profile changes gathered from cell lines upon stimulation with a drug. Despite these differences, we expect that there will be some similarity in the gene regulatory networks at play in both situations. The challenge is to match transcriptomes for both diseases and drugs alike, even though the exact molecular pathology/pharmacogenomics may not be known. Results We substitute the challenge to match a drug effect to a disease effect with the challenge to match a drug effect to the effect of the same drug at another concentration or in another cell line. This is welldefined, reproducible in vitro and in silico and extendable with external data. Based on the Connectivity Map (CMap) dataset, we combined 26 different similarity scores with six different heuristics to reduce the number of genes in the model. Such gene filters may also utilize external knowledge e.g. from biological networks. We found that no similarity score always outperforms all others for all drugs, but the Pearson correlation finds the same drug with the highest reliability. Results are improved by filtering for highly expressed genes and to a lesser degree for genes with large fold changes. Also a network-based reduction of contributing transcripts was beneficial, here implemented by the FocusHeuristics. We found no drop in prediction accuracy when reducing the whole transcriptome to the set of 1000 landmark genes of the CMap’s successor project Library of Integrated Network-based Cellular Signatures. All source code to re-analyze and extend the CMap data, the source code of heuristics, filters and their evaluation are available to propel the development of new methods for drug repurposing. Availability https://bitbucket.org/ibima/moldrugeffectsdb Contact [email protected] Supplementary information Supplementary data are available at Briefings in Bioinformatics online.

scholarly journals High-yield hydrogen production from biomass by in vitro metabolic engineering: Mixed sugars coutilization and kinetic modeling

2015 ◽  
Vol 112 (16) ◽  
pp. 4964-4969 ◽  
Author(s):  
Joseph A. Rollin ◽  
Julia Martin del Campo ◽  
Suwan Myung ◽  
Fangfang Sun ◽  
Chun You ◽  
...  
Keyword(s):  
New Technologies ◽  
Plant Biomass ◽  
Low Cost ◽  
Energy Conversion Efficiency ◽  
Green Energy ◽  
High Yield ◽  
Yield Parameters ◽  
Production Of Hydrogen ◽  
Nonlinear Kinetic

The use of hydrogen (H2) as a fuel offers enhanced energy conversion efficiency and tremendous potential to decrease greenhouse gas emissions, but producing it in a distributed, carbon-neutral, low-cost manner requires new technologies. Herein we demonstrate the complete conversion of glucose and xylose from plant biomass to H2 and CO2 based on an in vitro synthetic enzymatic pathway. Glucose and xylose were simultaneously converted to H2 with a yield of two H2 per carbon, the maximum possible yield. Parameters of a nonlinear kinetic model were fitted with experimental data using a genetic algorithm, and a global sensitivity analysis was used to identify the enzymes that have the greatest impact on reaction rate and yield. After optimizing enzyme loadings using this model, volumetric H2 productivity was increased 3-fold to 32 mmol H2⋅L−1⋅h−1. The productivity was further enhanced to 54 mmol H2⋅L−1⋅h−1 by increasing reaction temperature, substrate, and enzyme concentrations—an increase of 67-fold compared with the initial studies using this method. The production of hydrogen from locally produced biomass is a promising means to achieve global green energy production.

scholarly journals Inhibition of Protease-Resistant Prion Protein Formation in a Transformed Deer Cell Line Infected with Chronic Wasting Disease

2006 ◽  
Vol 80 (2) ◽  
pp. 596-604 ◽  
Author(s):  
Gregory J. Raymond ◽  
Emily A. Olsen ◽  
Kil Sun Lee ◽  
Lynne D. Raymond ◽  
P. Kruger Bryant ◽  
...  
Keyword(s):  
Cell Line ◽  
Prion Protein ◽  
Chronic Wasting Disease ◽  
Transmissible Spongiform Encephalopathy ◽  
Mule Deer ◽  
Simian Virus 40 ◽  
Spongiform Encephalopathy ◽  
Wasting Disease ◽  
Neuronal Marker

ABSTRACT Chronic wasting disease (CWD) is an emerging transmissible spongiform encephalopathy (prion disease) of North American cervids, i.e., mule deer, white-tailed deer, and elk (wapiti). To facilitate in vitro studies of CWD, we have developed a transformed deer cell line that is persistently infected with CWD. Primary cultures derived from uninfected mule deer brain tissue were transformed by transfection with a plasmid containing the simian virus 40 genome. A transformed cell line (MDB) was exposed to microsomes prepared from the brainstem of a CWD-affected mule deer. CWD-associated, protease-resistant prion protein (PrPCWD) was used as an indicator of CWD infection. Although no PrPCWD was detected in any of these cultures after two passes, dilution cloning of cells yielded one PrPCWD-positive clone out of 51. This clone, designated MDBCWD, has maintained stable PrPCWD production through 32 serial passes thus far. A second round of dilution cloning yielded 20 PrPCWD-positive subclones out of 30, one of which was designated MDBCWD2. The MDBCWD2 cell line was positive for fibronectin and negative for microtubule-associated protein 2 (a neuronal marker) and glial fibrillary acidic protein (an activated astrocyte marker), consistent with derivation from brain fibroblasts (e.g., meningeal fibroblasts). Two inhibitors of rodent scrapie protease-resistant PrP accumulation, pentosan polysulfate and a porphyrin compound, indium (III) meso-tetra(4-sulfonatophenyl)porphine chloride, potently blocked PrPCWD accumulation in MDBCWD cells. This demonstrates the utility of these cells in a rapid in vitro screening assay for PrPCWD inhibitors and suggests that these compounds have potential to be active against CWD in vivo.

scholarly journals Synthesis, Spectral Characterization, andIn VitroCytotoxicity of N-2′-Hydroxyethyl-Substituted Azacholestanes Prepared from 6-Oxocholestanes by Modified Schmidt Reaction

2013 ◽  
Vol 2013 ◽  
pp. 1-7 ◽  
Author(s):  
Shahab A. A. Nami ◽  
Suraiya Khan ◽  
Mahboob Alam ◽  
M. Mushfiq ◽  
Dong-Ung Lee ◽  
...  
Keyword(s):  
Cell Line ◽  
Carcinoma Cell ◽  
Human Colon ◽  
Spectroscopic Characterization ◽  
Carcinoma Cell Line ◽  
High Yield ◽  
Spectroscopic Techniques ◽  
Human Colon Carcinoma Cell ◽  
Schmidt Reaction

The present paper reports the synthesis and spectroscopic characterization of few N-2′-hydroxyethyl-substituted azacholestanes using BF3-OEt2, TiCl4, SnCl4, and H2SO4as catalysts in moderate yields by a modified version of Schmidt reaction. A notable feature is the passivity of SnCl4in case of 3β-acetoxy-N-2′-hydroxyethyl-6-aza-B-homo-5α-cholestan-7-one and 3β-chloro-N-2′-hydroxyethyl-6-aza-B-homo-5α-cholestan-7-one. However, the reaction was unsuccessful in case of N-2′-Hydroxyethyl-6-aza-B-homo-5α-cholestan-7-one. Another striking aspect is the attainment of high yield in case of H2SO4as catalyst. The semisolid compounds are characterized using various spectroscopic techniques such as FT-IR,1H-NMR and mass spectra, and microanalytical data. A reaction mechanism has been proposed on the basis of previous studies. Moreover, the compounds have also been screened for theirin vitrocytotoxicity against human colon carcinoma cell line, HCT116, and human liver hepatocellular carcinoma cell line, HepG2, using doxorubicin as standard. On the basis of IC50values, 3β-chloro-N-2′-hydroxyethyl-6-aza-B-homo-5α-cholestan-7-one (5) was found to inhibit the cancer cells most effectively.

scholarly journals Rapid generation of conditional knockout mice using the CRISPR-Cas9 system and electroporation for neuroscience research

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Hirofumi Nishizono ◽  
Yuki Hayano ◽  
Yoshihisa Nakahata ◽  
Yasuhito Ishigaki ◽  
Ryohei Yasuda
Keyword(s):  
Function Analysis ◽  
Low Cost ◽  
Conditional Knockout ◽  
Specific Gene ◽  
Time Saving ◽  
Vitro Fertilization ◽  
Short Period ◽  
Genome Analyses ◽  
Time Specific

AbstractThe Cre/LoxP-based conditional knockout technology is a powerful tool for gene function analysis that allows region- and time-specific gene manipulation. However, inserting a pair of LoxP cassettes to generate conditional knockout can be technically challenging and thus time- and resource-consuming. This study proposes an efficient, low-cost method to generate floxed mice using in vitro fertilization and the CRISPR-Cas9 system over two consecutive generations. This method allowed us to produce floxed mice targeting exons 5 and 6 of CaMK1 in a short period of 125 days, using only 16 mice. In addition, we directly edited the genome of fertilized eggs of mice with our target genetic background, C57BL/6 N, to eliminate additional backcrossing steps. We confirmed that the genome of the generated floxed mice was responsive to the Cre protein. This low-cost, time-saving method for generating conditional knockout will facilitate comprehensive, tissue-specific genome analyses.

scholarly journals In Vitro Inhibitory Effect of Recombinant Human Calprotectin on Nalm6 Leukemia Cell Line

2020 ◽  
Vol 20 (8) ◽  
pp. 951-962
Author(s):  
Samira Charkhizadeh ◽  
Mehdi Imani ◽  
Nematollah Gheibi ◽  
Fateme Shabaani ◽  
Akbar Nikpajouh ◽  
...  
Keyword(s):  
Cell Line ◽  
Leukemia Cell ◽  
Annexin V ◽  
Inhibitory Effect ◽  
New Drugs ◽  
Leukemia Cell Line ◽  
Cytotoxic Effects ◽  
Tumor Expression ◽  
Induced Apoptosis

Background & Purpose: In evaluating new drugs for the treatment of various types of cancer, investigations have been made to discover a variety of anti-tumor compounds with less side effects on normal cells. Investigations have shown that the heterodimers S100A8 and S100A9 inhibit the enzyme casein kinase 2 and then prevent the activation of the E7 oncoprotein. Therefore, the aim of this study was to evaluate the effect of calprotectin as an antitumor compound on the Nalm6 (B cell precursor leukemia cell line). Material & Methods: Transformation of genes encoding S100A8 and S100A9 human, designed in the pQE32 plasmid, was performed by the thermal shock method into E. coli M15 bacteria. After bacterial growth in LB medium, the expression of two S100A8 and S100A9 subunits, the solubility of the protein by SDS-PAGE method was determined. Finally, the S100A8 / A9 complex was equally placed in the microtube. In the next step, the cytotoxic effects of calprotectin produced on the Nalm6 cell line were evaluated using the wst1 test. Then, the apoptosis in these cells was measured using flow cytometry methods with Annexin-V coloration. Results: In the current study, the results showed that the cytotoxic effects of Calprotectin are time and concentration- dependent. Therefore, it can reduce the tumor expression and had a beneficial effect by induced apoptosis in Nalm6 cell line. Conclusion: Calprotectin has an anti-tumor effect on the Nalm6 cell line by increasing apoptosis.

scholarly journals Chagas Disease Drug Discovery

2014 ◽  
Vol 20 (1) ◽  
pp. 22-35 ◽  
Author(s):  
Eric Chatelain
Keyword(s):  
Chagas Disease ◽  
New Technologies ◽  
Low Cost ◽  
Screening Strategy ◽  
New Drugs ◽  
In Vivo Models ◽  
The Right ◽  
The Impact

American trypanosomiasis, or Chagas disease, is the result of infection by the Trypanosoma cruzi parasite. Endemic in Latin America where it is the major cause of death from cardiomyopathy, the impact of the disease is reaching global proportions through migrating populations. New drugs that are safe, efficacious, low cost, and adapted to the field are critically needed. Over the past five years, there has been increased interest in the disease and a surge in activities within various organizations. However, recent clinical trials with azoles, specifically posaconazole and the ravuconazole prodrug E1224, were disappointing, with treatment failure in Chagas patients reaching 70% to 90%, as opposed to 6% to 30% failure for benznidazole-treated patients. The lack of translation from in vitro and in vivo models to the clinic observed for the azoles raises several questions. There is a scientific requirement to review and challenge whether we are indeed using the right tools and decision-making processes to progress compounds forward for the treatment of this disease. New developments in the Chagas field, including new technologies and tools now available, will be discussed, and a redesign of the current screening strategy during the discovery process is proposed.

scholarly journals In silico prospection of antineoplastic molecules from the Artemisia annua species

2021 ◽  
Vol 7 (19) ◽  
Author(s):  
Isabela Sacienti Lavezo ◽  
Juracy Cirino de Souza Neto ◽  
Túlio Nunes Pinto ◽  
Leonardo Luiz Borges
Keyword(s):  
In Silico ◽  
Artemisia Annua ◽  
Biological Effects ◽  
Low Cost ◽  
Target Prediction ◽  
Molecular Target ◽  
New Drugs ◽  
Antineoplastic Activity

Lung cancer kills the most men and the second that kills the most women (behind only breast cancer). The in silico study makes it possible to search for new drugs at low cost, with a greater possibility of rapid manufacturing and a lower future cost for their manufacture. The objective of this study was to analyze an antineoplastic activity of the compounds of Artemisia annua to obtain an active substance that can reach the molecular target of the cancer cells. Compounds with antineoplastic effects were selected using Scielo, PubMed, and ScienceDirect platforms. Afterward, the first screening of compound compounds was performed with a high ability to predict biological and pharmacological activity through the PASS Prediction, Pubchem, and Swiss ADME platforms. After the current screening, we determined the toxicological and molecular target prediction by the Portox II and Swiss Target Prediction platforms. As a final part, molecular docking and redocking were performed for a compound using the PDB server and the GOLD Suite 5.7.0 program. For another, we completed the pharmacophoric mapping using the Binding DB and PharmaGist database. The compounds scopoletin and caffeic acid were the most promising structures in silico models capable of interacting with EGFR (epidermal growth factor) and MM-9 (metalloproteinase type 9), respectively. The results obtained that these structures are promising to be tested in in vitro and in vivo tests about the antineoplastic activity. In addition, in silico analyses help to understand the biological effects of A. annua extracts regarding antineoplastic evidence.

Synthesis, Characterization, Biological Evaluation and Molecular Docking Studies of New Oxoacrylate and Acetamide on heLa Cancer Cell Lines

2020 ◽  
Vol 16 ◽  
Author(s):  
Nevin Çankaya ◽  
Mehmetcan İzdal ◽  
Serap Yalçin Azarkan
Keyword(s):  
Molecular Docking ◽  
Hela Cell ◽  
Anticancer Activity ◽  
Cell Line ◽  
Critical Role ◽  
Biological Evaluation ◽  
New Drugs ◽  
Hela Cell Line ◽  
Molecular Docking Study

Background: In recent years, discovery and development of new drugs play a critical role in cancer therapy. Objective: In this study, the effect of MPAEA and p-acetamide on cellular toxicity and on silico in HeLa cancer cells have been investigated. Methods: In this study, 2-choloro-N-(4-methoxyphenyl)acetamide (p-acetamide) and 2-(4-methoxyphenylamino)-2- oxoethyl acrylate (MPAEA) have been synthesized and characterized by FTIR, 1H, and 13C-NMR. Cytotoxicity of pacetamide and MPAEA have been investigated by XTT cell proliferation assay on the HeLa cell line. IC50 values of pacetamide and MPAEA have been identified on the HeLa cell line. Further, molecular docking study was carried out by Autodock Vina using BCL-2 (PDB ID: 4MAN), BCL-W (PDB ID: 2Y6W), MCl-1 (PDB ID: 5FDO) AKT (PDB ID: 4GV1) and BRAF (PDB ID: 5VAM) as a possible apoptotic target for anticancer activity. Results: According to the obtained results, MPAEA and p-acetamide were successfully synthesized and characterized. The interactions between ligands and anti-apoptotic proteins were evaluated by molecular docking and their free energy of binding were calculated and used as descriptor. Conclusion: In vitro and in silico the results demonstrated that MPAEA had potent anticancer activity on HeLa cell line.

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