A Novel In Vivo Functional Screening Method for the Candidate Polyphosphate Accumulating Organisms Isolation

The U.S. Food and Drug Administration’s (FDA′s) Center for Veterinary Medicine (CVM) has been investigating reports of pets becoming ill after consuming jerky pet treats since 2007. Renal failure accounted for 30% of reported cases. Jerky pet treats contain glycerin, which can be made from vegetable oil or as a byproduct of biodiesel production. Glycidyl esters (GEs) and 3-monochloropropanediol esters (3-MCPDEs) are food contaminants that can form in glycerin during the refining process. 3-MCPDEs and GEs pose food safety concerns, as they can release free 3-MCPD and glycidol in vivo. Evidence from studies in animals shows that 3-MCPDEs are potential toxins with kidneys as their main target. As renal failure accounted for 30% of reported pet illnesses after the consumption of jerky pet treats containing glycerin, there is a need to develop a screening method to detect 3-MCPDEs and GEs in glycerin. We describe the development of an ultra-high-pressure liquid chromatography/quadrupole time-of-flight (UHPLC/Q-TOF) method for screening glycerin for MCPDEs and GEs. Glycerin was extracted and directly analyzed without a solid-phase extraction procedure. An exact mass database, developed in-house, of MCPDEs and GEs formed with common fatty acids was used in the screening.

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Tumor Suppressive Role of miR-342-5p in Human Chondrosarcoma Cells and 3D Organoids

International Journal of Molecular Sciences ◽

10.3390/ijms22115590 ◽

2021 ◽

Vol 22 (11) ◽

pp. 5590

Author(s):

Clément Veys ◽

Abderrahim Benmoussa ◽

Romain Contentin ◽

Amandine Duchemin ◽

Emilie Brotin ◽

...

Keyword(s):

Luciferase Reporter ◽

Functional Screening ◽

Western Blots ◽

Egfr Expression ◽

Reporter Assays ◽

Direct Target ◽

Induced Apoptosis ◽

First Time

Chondrosarcomas are malignant bone tumors. Their abundant cartilage-like extracellular matrix and their hypoxic microenvironment contribute to their resistance to chemotherapy and radiotherapy, and no effective therapy is currently available. MicroRNAs (miRNAs) may be an interesting alternative in the development of therapeutic options. Here, for the first time in chondrosarcoma cells, we carried out high-throughput functional screening using impedancemetry, and identified five miRNAs with potential antiproliferative or chemosensitive effects on SW1353 chondrosarcoma cells. The cytotoxic effects of miR-342-5p and miR-491-5p were confirmed on three chondrosarcoma cell lines, using functional validation under normoxia and hypoxia. Both miRNAs induced apoptosis and miR-342-5p also induced autophagy. Western blots and luciferase reporter assays identified for the first time Bcl-2 as a direct target of miR-342-5p, and also Bcl-xL as a direct target of both miR-342-5p and miR-491-5p in chondrosarcoma cells. MiR-491-5p also inhibited EGFR expression. Finally, only miR-342-5p induced cell death on a relevant 3D chondrosarcoma organoid model under hypoxia that mimics the in vivo microenvironment. Altogether, our results revealed the tumor suppressive activity of miR-342-5p, and to a lesser extent of miR-491-5p, on chondrosarcoma lines. Through this study, we also confirmed the potential of Bcl-2 family members as therapeutic targets in chondrosarcomas.

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Therapeutic Potential of CUDC-907 (Fimepinostat) for Hepatocarcinoma Treatment Revealed by Tumor Spheroids-based Drug Screening

10.21203/rs.3.rs-125324/v1 ◽

2020 ◽

Author(s):

Wei Liao ◽

Wanren Yang ◽

Yue Zhang ◽

Fanhong Zeng ◽

Jiecheng Xu ◽

...

Keyword(s):

Cell Lines ◽

Drug Screening ◽

Screening Method ◽

Screening Methods ◽

Spheroid Culture ◽

3D Print ◽

Culture Plate ◽

Hcc Cells

Abstract Background: Cancer is the second leading cause of death globally. However, most of the new anti-cancer agents screened by traditional drug screening methods fail in the clinic because of lack of efficacy. One of the reasons for this dilemma is that the two-dimensional (2D) culture cancer cell lines could not represent the in vivo cancer cells well. Fortunately, the development of a three-dimensional (3D) culture technique helps in this problem. Methods: The high-throughput spheroid culture plate was fabricated by using 3D print technique and agarose. 4 hepatocarcinoma (HCC) cell lines were 3D cultured to screen 19 small molecular agents based on the spheroid culture plate. 3D cultured primary HCC cells and tumor-bearing mice model were established to verify the candidate anti-hepatocarcinoma agent. Cell function experiments and western blotting were conducted to explore the anti-hepatocarcinoma mechanism of the candidate agent. Results: Based on the previous study, we established an in vitro 3D drug screening method by using our invented spheroid culture device and found that CUDC-907 can serve as a potent anti-hepatocarcinoma agent. The study data show that CUDC-907 (fimepinostat), a novel dual acting inhibitor of phosphoinositide 3-kinase (PI3K) and histone deacetylase (HDAC), has potent inhibitory effects on HCC cell lines and primary HCC cells in vitro, Animal studies have shown that CUDC-907 can also suppress HCC cells in vivo. Furthermore, we investigated the antitumor mechanism of CUDC-907 in HCC cells. We found that it inhibits the PI3K/AKT/mTOR pathway and downregulates the expression of c-Myc, leading to the suppression of HCC cells. Conclusion: Our results suggest that CUDC-907 can be a candidate anti-HCC drug, and the 3D in vitro drug screening method based on our novel spheroid culture device is promising for drug screening.

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Functional Screening and Biosafety Identify of the Potential Application of Let-7a in Injured Peripheral Nerve Regeneration

10.21203/rs.3.rs-1100490/v1 ◽

2021 ◽

Author(s):

Qianqian Chen ◽

Qianyan Liu ◽

Pan Wang ◽

Tianmei Qian ◽

Xinghui Wang ◽

...

Keyword(s):

Peripheral Nerve ◽

Potential Application ◽

Gene Expression Analysis ◽

Nerve Repair ◽

Peripheral Nerve Regeneration ◽

Functional Approach ◽

Cell Permeability ◽

Functional Screening ◽

The Core

Abstract Proper supporting factor can possess the ability to enhance neuron regeneration, for instance, neurotrophic effects especially nerve growth factor (NGF). However, the in vivo applications of NGF are largely limited by its intrinsic disadvantages. Considering that let-7 targets and regulates NGF, and let-7 is also the core and harbor regulators in peripheral nerve repair and regeneration, we evaluated the potential application in clinical. We firstly screened the let-7a as the most ideal let-7 family molecular by gene expression analysis and functional approach. We further evaluated the in vivo safety, the cell permeability of 3 main cells in regeneration micro-environment, and the morphological and functional indicators. Our study provides an essential basis for in vivo application of let-7 and pictured a vision for the clinical translation of miRNA as a prospective alternative for regenerative medicine.

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CRISPR/Cas9-mediated genome-edited mice reveal 10 testis-enriched genes are dispensable for male fecundity

Biology of Reproduction ◽

10.1093/biolre/ioaa084 ◽

2020 ◽

Vol 103 (2) ◽

pp. 195-204

Author(s):

Soojin Park ◽

Keisuke Shimada ◽

Yoshitaka Fujihara ◽

Zoulan Xu ◽

Kentaro Shimada ◽

...

Keyword(s):

Protein Function ◽

Male Fertility ◽

System Analysis ◽

In Silico Analysis ◽

Male Reproduction ◽

Hormonal Contraceptives ◽

World Population ◽

Functional Screening ◽

Mouse Lines

Abstract As the world population continues to increase to unsustainable levels, the importance of birth control and the development of new contraceptives are emerging. To date, male contraceptive options have been lagging behind those available to women, and those few options available are not satisfactory to everyone. To solve this problem, we have been searching for new candidate target proteins for non-hormonal contraceptives. Testis-specific proteins are appealing targets for male contraceptives because they are more likely to be involved in male reproduction and their targeting by small molecules is predicted to have no on-target harmful effects on other organs. Using in silico analysis, we identified Erich2, Glt6d1, Prss58, Slfnl1, Sppl2c, Stpg3, Tex33, and Tex36 as testis-abundant genes in both mouse and human. The genes, 4930402F06Rik and 4930568D16Rik, are testis-abundant paralogs of Glt6d1 that we also discovered in mice but not in human, and were also included in our studies to eliminate the potential compensation. We generated knockout (KO) mouse lines of all listed genes using the CRISPR/Cas9 system. Analysis of all of the individual KO mouse lines as well as Glt6d1/4930402F06Rik/4930568D16Rik TKO mouse lines revealed that they are male fertile with no observable defects in reproductive organs, suggesting that these 10 genes are not required for male fertility nor play redundant roles in the case of the 3 Glt6D1 paralogs. Further studies are needed to uncover protein function(s), but in vivo functional screening using the CRISPR/Cas9 system is a fast and accurate way to find genes essential for male fertility, which may apply to studies of genes expressed elsewhere. In this study, although we could not find any potential protein targets for non-hormonal male contraceptives, our findings help to streamline efforts to find and focus on only the essential genes.

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In vivo drug screening method of radiosensitizers using tumor-bearing chick embryo

Chick Chorioallantoic Membrane Model and Precision Cancer Therapy - The Enzymes ◽

10.1016/bs.enz.2019.08.008 ◽

2019 ◽

pp. 113-127

Author(s):

Yoshihiro Uto ◽

Chiaki Abe ◽

Mana Futawaka ◽

Hisatsugu Yamada ◽

Masahide Tominaga ◽

...

Keyword(s):

Chick Embryo ◽

Drug Screening ◽

Screening Method ◽

Tumor Bearing

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Cell-based 3D bionic screening by mimicking the drug–receptor interaction environment in vivo

Journal of Materials Chemistry B ◽

10.1039/d0tb02661a ◽

2021 ◽

Author(s):

Fen Wei ◽

Xiaxi Zhang ◽

Ping Cui ◽

Xilan Gou ◽

Sicen Wang

Keyword(s):

Screening Method ◽

Receptor Interaction ◽

Lead Target ◽

Receptor Cells ◽

Target Capture ◽

Drug Lead ◽

A Cell ◽

Drug Receptor ◽

Drug Receptor Interaction

A cell-based screening method for drug lead target capture using a microcarrier covered with highly expressed receptor cells as a 3D biomimetic framework.

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New Anti-Cancer Strategy to Suppress Colorectal Cancer Growth Through Inhibition of ATG4B and Lysosome Function

Cancers ◽

10.3390/cancers12061523 ◽

2020 ◽

Vol 12 (6) ◽

pp. 1523 ◽

Cited By ~ 1

Author(s):

Yuanyuan Fu ◽

Qianqian Gu ◽

Li Luo ◽

Jiecheng Xu ◽

Yuping Luo ◽

...

Keyword(s):

Colorectal Cancer ◽

Cancer Therapy ◽

Therapeutic Strategy ◽

Screening Method ◽

Cancer Drug ◽

Autophagic Flux ◽

Single Target ◽

Anti Cancer

Autophagy inhibition has been proposed to be a potential therapeutic strategy for cancer, however, few autophagy inhibitors have been developed. Recent studies have indicated that lysosome and autophagy related 4B cysteine peptidase (ATG4B) are two promising targets in autophagy for cancer therapy. Although some inhibitors of either lysosome or ATG4B were reported, there are limitations in the use of these single target compounds. Considering multi-functional drugs have advantages, such as high efficacy and low toxicity, we first screened and validated a batch of compounds designed and synthesized in our laboratory by combining the screening method of ATG4B inhibitors and the identification method of lysosome inhibitors. ATG4B activity was effectively inhibited in vitro. Moreover, 163N inhibited autophagic flux and caused the accumulation of autolysosomes. Further studies demonstrated that 163N could not affect the autophagosome-lysosome fusion but could cause lysosome dysfunction. In addition, 163N diminished tumor cell viability and impaired the development of colorectal cancer in vivo. The current study findings indicate that the dual effect inhibitor 163N offers an attractive new anti-cancer drug and compounds having a combination of lysosome inhibition and ATG4B inhibition are a promising therapeutic strategy for colorectal cancer therapy.

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The Identification of Naturally Occurring Neoruscogenin as a Bioavailable, Potent, and High-Affinity Agonist of the Nuclear Receptor RORα (NR1F1)

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057113497095 ◽

2013 ◽

Vol 19 (3) ◽

pp. 399-406 ◽

Cited By ~ 14

Author(s):

Stéphane Helleboid ◽

Christian Haug ◽

Kai Lamottke ◽

Yijun Zhou ◽

Jianbing Wei ◽

...

Keyword(s):

Nuclear Receptor ◽

Screening Method ◽

Structural Diversity ◽

Orphan Receptor ◽

Compound Identification ◽

High Affinity ◽

Specific Selectivity ◽

Reporter Assays

Plants represent a tremendous structural diversity of natural compounds that bind to many different human disease targets and are potentially useful as starting points for medicinal chemistry programs. This resource is, however, still underexploited due to technical difficulties with the identification of minute quantities of active ingredients in complex mixtures of structurally diverse compounds upon raw phytomass extraction. In this work, we describe the successful identification of a novel class of potent RAR-related orphan receptor alpha (RORα or nuclear receptor NR1F1) agonists from a library of 12,000 plant extract fractions by using an optimized, robust high-throughput cell-free screening method, as well as an innovative hit compound identification procedure through further extract deconvolution and subsequent structural elucidation of the active natural compound(s). In particular, we demonstrate that neoruscogenin, a member of the steroidal sapogenin family, is a potent and high-affinity RORα agonist, as shown by its activity in RORα reporter assays and from its effect on RORα target gene expression in vitro and in vivo. Neoruscogenin represents a universal pharmacological tool for RORα research due to its specific selectivity profile versus other nuclear receptors, its excellent microsomal stability, good bioavailability, and significant peripheral exposure in mouse.

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Small-Molecule Screen Identifies ROS as Key Regulators of Actin Dynamics in Neutrophils

Blood ◽

10.1182/blood.v112.11.4641.4641 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4641-4641

Author(s):

Hidenori Hattori ◽

Kulandayan K Subramanian ◽

Hongbo R. Luo

Keyword(s):

Small Molecule ◽

Actin Polymerization ◽

Neutrophil Migration ◽

Physiological Role ◽

Leading Edge ◽

Actin Dynamics ◽

Functional Screening ◽

Cellular Processes

Abstract Precise spatial and temporal control of actin polymerization and depolymerization is essential for mediating various cellular processes such as migration, phagocytosis, vesicle trafficking and adhesion. In this study, we used a small-molecule functional screening approach to identify novel regulators of actin dynamics during neutrophil migration. Here we show that NADPH-oxidase dependent Reactive Oxygen Species act as negative regulators of actin polymerization. Neutrophils with pharmacologically inhibited oxidase or isolated from Chronic Granulomatous Disease (CGD) patient and mice displayed enhanced F-actin polymerization, multiple pseudopods formation and impaired chemotaxis. ROS localized to pseudopodia and inhibited actin polymerization by driving actin glutathionylation at the leading edge of migrating cells. Consistent with these in vitro results, adoptively transferred CGD murine neutrophils also showed impaired in vivo recruitment to sites of inflammation. Together, these results present a novel physiological role for ROS in regulation of action polymerization and shed new light on the pathogenesis of CGD.

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