scholarly journals Genetically Encoded Sensor Cells for the Screening of Glucocorticoid Receptor (GR) Effectors in Herbal Extracts

Biosensors ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 341
Author(s):  
Chungwon Kang ◽  
Soyoun Kim ◽  
Euiyeon Lee ◽  
Jeahee Ryu ◽  
Minhyeong Lee ◽  
...  
Keyword(s):  
Drug Screening ◽  
Low Cost ◽  
Biologically Active ◽  
Drug Candidate ◽  
Fluorescence Signal ◽  
Biologically Relevant ◽  
Biological Targets ◽  
Sensing Technology ◽  
Medicinal Plant Extracts

Although in vitro sensors provide facile low-cost ways to screen for biologically active targets, their results may not accurately represent the molecular interactions in biological systems. Cell-based sensors have emerged as promising platforms to screen targets in biologically relevant environments. However, there are few examples where cell-based sensors have been practically applied for drug screening. Here, we used engineered cortisol-detecting sensor cells to screen for natural mimetics of cortisol. The sensor cells were designed to report the presence of a target through signal peptide activation and subsequent fluorescence signal translocation. The developed sensor cells were able to detect known biological targets from human-derived analytes as well as natural product extracts, such as deer antlers and ginseng. The multi-use capability and versatility to screen in different cellular environments were also demonstrated. The sensor cells were used to identify novel GR effectors from medicinal plant extracts. Our results suggest that decursin from dongquai had the GR effector function as a selective GR agonist (SEGRA), making it a potent drug candidate with anti-inflammatory activity. We demonstrated the superiority of cell-based sensing technology over in vitro screening, proving its potential for practical drug screening applications that leads to the function-based discovery of target molecules.

scholarly journals Paper-based Sensing of Fucosylated Biological Compounds

2020 ◽  
Author(s):  
Fatima Enam ◽  
Emily Kramer ◽  
Frederick Robinson ◽  
Andrea Alvarez-Acosta ◽  
Rebecca Cademartiri ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Low Cost ◽  
Analytical Techniques ◽  
Rapid Analysis ◽  
Biologically Relevant ◽  
Diagnostic Assays ◽  
Sensing Technology ◽  
Biological Compounds ◽  
Wax Printing

SummaryAdvances in sensing technology have enabled rapid analysis of various biomolecules including complex carbohydrates. However, glycan analysis is limited by the throughput and complexity of assays for quantifying them. We describe a simple, low-cost enzymatic assay for the rapid analysis of fucosylation, down to linkage specificity, and its application to high-throughput screening of biologically relevant fucosylated compounds, to facilitate simple and straightforward analytical techniques. Paper-based devices integrate biosensor platforms and other diagnostic assays by fusing them with wax printing technology, making their fabrication even more inexpensive and simple. The specificity of the assay is established by linkage-specific glycosidic enzymes and the colorimetric output is visible to the naked eye, with costs that are lower than fluorescence/luminescence-based assays ($0.02/reaction). This platform was further improved by enhancing storage stability to retain analytical performance over time using desiccation and freeze-drying techniques. The assay platform allows analysis of hundreds of samples in minutes and we anticipate that this rapid and simple analytical method will be extended towards developing a universal glyco-barcoding platform for high throughput screening of glycosylation.

scholarly journals In Silico Driven Prediction of MAPK14 Off-Targets Reveals Unrelated Proteins with High Accuracy

2020 ◽  
Author(s):  
Florian Kaiser ◽  
Maximilian G. Plach ◽  
Christoph Leberecht ◽  
Thomas Schubert ◽  
V. Joachim Haupt
Keyword(s):  
Side Effects ◽  
In Silico ◽  
Target Identification ◽  
Low Cost ◽  
Time Frame ◽  
New Drugs ◽  
Drug Candidate ◽  
Delivery Times ◽  
Target Screening

During the discovery and development of new drugs, candidates with undesired and potentially harmful side-effects can arise at all stages, which poses significant scientific and economic risks. Most of such phenotypic side-effects can be attributed to binding of the drug candidate to unintended proteins, so-called off-targets. The early identification of potential off-targets is therefore of utmost importance to mitigate any downstream risks. We showcase how the combination of knowledge-based in silico off-target screening and state-of-the-art biophysics can be applied to rapidly identify off-targets for a MAPK14 inhibitor. Out of 13 predicted off-targets, six proteins were confirmed to interact with the inhibitor in vitro, which translates to an exceptional hit rate of 46%. For two proteins, affinities in the lower micromolar range were obtained: The kinase IRE1 and the Hematopoietic Prostaglandin D Synthase, which is entirely unrelated to MAPK14 and is involved in different cell-regulatory processes. The whole off-target identification/validation pipeline can be completed as fast as within two months, excluding delivery times of proteins. These results emphasize how computational off-target screening in combination with MicroScale Thermophoresis can effectively reduce downstream development risks in a very competitive time frame and at low cost.

scholarly journals The Quest to Enhance the Efficacy of Berberine for Type-2 Diabetes and Associated Diseases: Physicochemical Modification Approaches

Biomedicines ◽  
2020 ◽  
Vol 8 (4) ◽  
pp. 90
Author(s):  
Solomon Habtemariam
Keyword(s):  
Type 2 Diabetes ◽  
Ex Vivo ◽  
Isoquinoline Alkaloid ◽  
Biologically Active ◽  
Specific Reference ◽  
Biological Targets ◽  
Associated Diseases ◽  
Systems Research

Berberine is a quaternary isoquinoline alkaloid that has been isolated from numerous plants which are still in use today as medicine and herbal supplements. The great deal of enthusiasm for intense research on berberine to date is based on its diverse pharmacological effects via action on multiple biological targets. Its poor bioavailability resulting from low intestinal absorption coupled with its efflux by the action of P-glycoprotein is, however, the major limitation. In this communication, the chemical approach of improving berberine’s bioavailability and pharmacological efficacy is scrutinised with specific reference to type-2 diabetes and associated diseases such as hyperlipidaemia and obesity. The application of modern delivery systems, research from combination studies to preparation of berberine structural hybrids with known biologically active compounds (antidiabetic, antihyperlipidaemic and antioxidant), as well as synthesis approaches of berberine derivative are presented. Improvement of bioavailability and efficacy through in vitro and ex vivo transport studies, as well as animal models of bioavailability/efficacy in lipid metabolism and diabetes targets are discussed.

scholarly journals A simple, fast and reliable scan-based technique as a novel approach to quantify intracellular bacteria

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Meysam Sarshar ◽  
Daniela Scribano ◽  
Giulia Tranquilli ◽  
Marisa Di Pietro ◽  
Simone Filardo ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
High Speed ◽  
Low Cost ◽  
Shigella Flexneri ◽  
Intracellular Bacteria ◽  
Fluorescence Signal ◽  
Chronic Infections ◽  
Novel Approach ◽  
Infection Models

Abstract Background Quantification of intracellular bacteria is fundamental in many areas of cellular and clinical microbiology to study acute and chronic infections. Therefore, rapid, accurate and low-cost methods represent valuable tools in determining bacterial ability to persist and proliferate within eukaryotic cells. Results Herein, we present the first application of the immunofluorescence In-Cell Western (ICW) assay aimed at quantifying intracellular bacteria in in vitro infection models. The performance of this new approach was evaluated in cell culture infection models using three microorganisms with different lifestyles. Two facultative intracellular bacteria, the fast-growing Shigella flexneri and a persistent strain of Escherichia coli, as well as the obligate intracellular bacterium Chlamydia trachomatis were chosen as bacterial models. The ICW assay was performed in parallel with conventional quantification methods, i.e. colony forming units (CFUs) and inclusion forming units (IFUs). The fluorescence signal intensity values from the ICW assay were highly correlated to CFU/IFUs counting and showed coefficients of determination (R2), ranging from 0,92 to 0,99. Conclusions The ICW assay offers several advantages including sensitivity, reproducibility, high speed, operator-independent data acquisition and overtime stability of fluorescence signals. All these features, together with the simplicity in performance, make this assay particularly suitable for high-throughput screening and diagnostic approaches.

scholarly journals Pentoxifylline, a drug with antiviral, anti-inflammatory and bronchodilatory effects may be a possible drug candidate for SARS-CoV-2

2020 ◽  
Author(s):  
Morteza Ghasemnejad-Berenji ◽  
Sarvin Pashapour ◽  
Sonia Sadeghpour
Keyword(s):  
Viral Infections ◽  
Immune Cell ◽  
Low Cost ◽  
Elevated Serum ◽  
Phosphodiesterase Inhibitor ◽  
Adenosine Monophosphate ◽  
Serum Levels ◽  
Drug Candidate ◽  
Anti Inflammatory

In December 2019, a new coronavirus, named SARS-CoV-2, has emerged from China causing pneumonia outbreaks first in the Wuhan region and have now spread worldwide but there are still no “specific drug” available. In the difficulty where new synthesized drug cannot be applied immediately to patients, “conventional drug in new use” becomes a feasible solution. Chloroquine, remdesivir, favipiravi, lopinavir, ribavirin or ritonavir have shown efficacy to inhibit coronavirus in vitro. Pentoxifylline, a drug with anti-inflammatory, immunomodulatory and bronchodilatory effects, previously showed efficacy to inhibit various viral infections. Immunological studies have shown that most patients with severe COVID-19 exhibit substantially elevated serum levels of pro-inflammatory cytokines. Pentoxifylline is a phosphodiesterase inhibitor that increases cyclic adenosine monophosphate levels, which in turn activate protein kinase, leading to a reduction in the synthesis of proinflammatory cytokines and immune cell migration. Here we propose pentoxifylline, a drug with low cost and toxicity, as a possible treatment for COVID-19 in basis of its interesting properties.

Properties and Biotechnological Application of mutant Derivatives of Mini-intein PRP8 from Penicillium chrysogenum with Improved Control of C-terminal Processing

2019 ◽  
Vol 35 (6) ◽  
pp. 91-101
Author(s):  
F.A. Klebanov ◽  
S.E. Cheperegin ◽  
D.G. Kozlov
Keyword(s):  
Penicillium Chrysogenum ◽  
Protein Denaturation ◽  
Biologically Active ◽  
Cultivation Temperature ◽  
Target Proteins ◽  
E Coli ◽  
Complete Inactivation ◽  
Target Molecules ◽  
Proteins And Peptides

Mutant variants of mini-intein PRP8 from Penicillium chrysogenum (Int4b) with improved control of C-terminal processing were characterized. The presented variants can serve as a basis for self-removed polypeptide tags capable of carrying an affine label and allowing to optimize the process of obtaining target proteins and peptides in E. coli cells. They allow to synthesize target molecules in the composition of soluble and insoluble hybrid proteins (fusions), provide their afnne purification, autocatalytic processing and obtaining mature target products. The presented variants have a number of features in comparison with the known prototypes. In particular the mutant mini-intein Int4bPRO, containing the L93P mutation, has temperature-dependent properties. At cultivation temperature below 30 °C it allows the production of target molecules as part of soluble fusions, but after increasing of cultivation temperature to 37 °C it directs the most of synthesized fusions into insoluble intracellular aggregates. The transition of Int4bPRO into insoluble form is accompanied by complete inactivation of C-terminal processing. Further application of standard protein denaturation-renaturation procedures enable efficiently reactivate Int4bPRO and to carry out processing of its fusions in vitro. Two other variants, Int4b56 and Int4b36, containing a point mutation T62N or combination of mutations D144N and L146T respectively, have a reduced rate of C-terminal processing. Their use in E. coli cells allows to optimize the biosynthesis of biologically active target proteins and peptides in the composition of soluble fusions, suitable for afnne purification and subsequent intein-dependent processing without the use of protein denaturation-renaturation procedures. intein, fusion, processing, processing rate, gelonin The work was supported within the framework of the State Assignment no. 595-00003-19 PR.

Metabolomics of Healthy Berry Fruits

2019 ◽  
Vol 25 (37) ◽  
pp. 4888-4902 ◽  
Author(s):  
Gilda D'Urso ◽  
Sonia Piacente ◽  
Cosimo Pizza ◽  
Paola Montoro
Keyword(s):  
Biological Activities ◽  
Biologically Active ◽  
In Vivo Studies ◽  
Bioactive Metabolites ◽  
Active Metabolites ◽  
Positive Effects ◽  
Cell Functions ◽  
Berry Fruits

The consumption of berry-type fruits has become very popular in recent years because of their positive effects on human health. Berries are in fact widely known for their health-promoting benefits, including prevention of chronic disease, cardiovascular disease and cancer. Berries are a rich source of bioactive metabolites, such as vitamins, minerals, and phenolic compounds, mainly anthocyanins. Numerous in vitro and in vivo studies recognized the health effects of berries and their function as bioactive modulators of various cell functions associated with oxidative stress. Plants have one of the largest metabolome databases, with over 1200 papers on plant metabolomics published only in the last decade. Mass spectrometry (MS) and NMR (Nuclear Magnetic Resonance) are the most important analytical technologies on which the emerging ''omics'' approaches are based. They may provide detection and quantization of thousands of biologically active metabolites from a tissue, working in a ''global'' or ''targeted'' manner, down to ultra-trace levels. In the present review, we highlighted the use of MS and NMR-based strategies and Multivariate Data Analysis for the valorization of berries known for their biological activities, important as food and often used in the preparation of nutraceutical formulations.

Edible Mushrooms: Novel Medicinal Agents to Combat Metabolic Syndrome and Associated Diseases

2020 ◽  
Vol 26 (39) ◽  
pp. 4970-4981
Author(s):  
Yu-Tang Tung ◽  
Chun-Hsu Pan ◽  
Yi-Wen Chien ◽  
Hui-Yu Huang
Keyword(s):  
Metabolic Syndrome ◽  
Unsaturated Fatty Acids ◽  
Edible Mushroom ◽  
Biologically Active ◽  
Edible Mushrooms ◽  
Beta Glucan ◽  
Medicinal Mushrooms ◽  
Associated Diseases ◽  
Increased Risk

Metabolic syndrome is an aggregation of conditions and associated with an increased risk of developing diabetes, obesity and cardiovascular diseases (CVD). Edible mushrooms are widely consumed in many countries and are valuable components of the diet because of their attractive taste, aroma, and nutritional value. Medicinal mushrooms are higher fungi with additional nutraceutical attributes having low-fat content and a transisomer of unsaturated fatty acids along with high fiber content, biologically active compounds such as polysaccharides or polysaccharide β-glucans, alkaloids, steroids, polyphenols and terpenoids. In vitro experiments, animal models, and even human studies have demonstrated not only fresh edible mushroom but also mushroom extract that has great therapeutic applications in human health as they possess many properties such as antiobesity, cardioprotective and anti-diabetic effect. They are considered as the unmatched source of healthy foods and drugs. The focus of this report was to provide a concise and complete review of the novel medicinal properties of fresh or dry mushroom and extracts, fruiting body or mycelium and its extracts, fiber, polysaccharides, beta-glucan, triterpenes, fucoidan, ergothioneine from edible mushrooms that may help to prevent or treat metabolic syndrome and associated diseases.

Identification of 2-Fluoropalmitic Acid as a Potential Therapeutic Agent Against Glioblastoma

2020 ◽  
Vol 26 (36) ◽  
pp. 4675-4684 ◽  
Author(s):  
Shabierjiang Jiapaer ◽  
Takuya Furuta ◽  
Yu Dong ◽  
Tomohiro Kitabayashi ◽  
Hemragul Sabit ◽  
...  
Keyword(s):  
Combination Therapy ◽  
Drug Screening ◽  
Therapeutic Agent ◽  
Gelatin Zymography ◽  
Therapeutic Agents ◽  
Drug Candidate ◽  
Potential Therapeutic Agent ◽  
Sphere Formation ◽  
Improve Patient

Background: Glioblastomas (GBMs) are aggressive malignant brain tumors. Although chemotherapy with temozolomide (TMZ) can extend patient survival, most patients eventually demonstrate resistance. Therefore, novel therapeutic agents that overcome TMZ chemoresistance are required to improve patient outcomes. Purpose: Drug screening is an efficient method to find new therapeutic agents from existing drugs. In this study, we explored a novel anti-glioma agent by drug screening and analyzed its function with respect to GBM treatment for future clinical applications. Methods: Drug libraries containing 1,301 diverse chemical compounds were screened against two glioma stem cell (GSC) lines for drug candidate selection. The effect of selected agents on GSCs and glioma was estimated through viability, proliferation, sphere formation, and invasion assays. Combination therapy was performed to assess its ability to enhance TMZ cytotoxicity against GBM. To clarify the mechanism of action, we performed methylation-specific polymerase chain reaction, gelatin zymography, and western blot analysis. Results: The acyl-CoA synthetase inhibitor 2-fluoropalmitic acid (2-FPA) was selected as a candidate anti-glioma agent. 2-FPA suppressed the viability and stem-like phenotype of GSCs. It also inhibited proliferation and invasion of glioma cell lines. Combination therapy of 2-FPA with TMZ synergistically enhanced the efficacy of TMZ. 2-FPA suppressed the expression of phosphor-ERK, CD133, and SOX-2; reduced MMP-2 activity; and increased methylation of the MGMT promoter. Conclusion: 2-FPA was identified as a potential therapeutic agent against GBM. To extend these findings, physiological studies are required to examine the efficacy of 2-FPA against GBM in vivo.

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