scholarly journals Activators of Alpha Synuclein Expression Identified by Reporter Cell Line-based High Throughput Drug Screen

2021 ◽  
Author(s):  
Fabian Stahl ◽  
Philip Denner ◽  
Dominik Piston ◽  
Bernd Evert ◽  
Ina Schmitt ◽  
...  
Keyword(s):  
Dopaminergic Neurons ◽  
Control Cell ◽  
Alpha Synuclein ◽  
Drug Screen ◽  
Reporter Cell Line ◽  
Reporter Cell ◽  
Protein Levels ◽  
Approved Drugs ◽  
And Control ◽  
Fda Approved Drugs

Abstract Multiplications, mutations and dysregulation of the alpha synuclein gene (SNCA) are associated with the demise of dopaminergic neurons and are considered to play important roles in the pathogenesis of familial and sporadic forms of Parkinson’s disease. Regulation of SNCA expression might thus be an appropriate target for treatment. We aimed to identify specific modulators of SNCA transcription, generated CRISPR/Cas9 modified SNCA-GFP-luciferase (LUC) genomic fusion- and control cell lines and screened a library of 1649 bioactive compounds, including the FDA approved drugs. We found no inhibitors but three selective activators which increased SNCA mRNA and protein levels.

scholarly journals Activators of alpha synuclein expression identified by reporter cell line-based high throughput drug screen

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Fabian Stahl ◽  
Philip Denner ◽  
Dominik Piston ◽  
Bernd O. Evert ◽  
Laura de Boni ◽  
...  
Keyword(s):  
Dopaminergic Neurons ◽  
Control Cell ◽  
Alpha Synuclein ◽  
Drug Screen ◽  
Reporter Cell Line ◽  
Reporter Cell ◽  
Protein Levels ◽  
Approved Drugs ◽  
And Control ◽  
Fda Approved Drugs

AbstractMultiplications, mutations and dysregulation of the alpha synuclein gene (SNCA) are associated with the demise of dopaminergic neurons and are considered to play important roles in the pathogenesis of familial and sporadic forms of Parkinson’s disease. Regulation of SNCA expression might thus be an appropriate target for treatment. We aimed to identify specific modulators of SNCA transcription, generated CRISPR/Cas9 modified SNCA-GFP-luciferase (LUC) genomic fusion- and control cell lines and screened a library of 1649 bioactive compounds, including the FDA approved drugs. We found no inhibitors but three selective activators which increased SNCA mRNA and protein levels.

scholarly journals A Drug Repositioning Approach Reveals that Streptococcus mutans Is Susceptible to a Diverse Range of Established Antimicrobials and Nonantibiotics

2017 ◽  
Vol 62 (1) ◽  
Author(s):  
S. Saputo ◽  
R. C. Faustoferri ◽  
R. G. Quivey
Keyword(s):  
Streptococcus Mutans ◽  
Biofilm Formation ◽  
Drug Repositioning ◽  
Drug Screen ◽  
Biofilm Inhibition ◽  
Diverse Range ◽  
Content Type ◽  
Multispecies Biofilm ◽  
Approved Drugs ◽  
Fda Approved Drugs

ABSTRACT Streptococcus mutans is the primary causative agent of dental caries and contributes to the multispecies biofilm known as dental plaque. An adenylate kinase-based assay was optimized for S. mutans to detect cell lysis when exposed to the Selleck library (Selleck Chemical, Houston, TX) of 853 FDA-approved drugs in, to our knowledge, the first high-throughput drug screen in S. mutans. We found 126 drugs with activity against S. mutans planktonic cultures, and they were classified into six categories: antibacterials (61), antineoplastics (23), ion channel effectors (9), other antimicrobials (7), antifungals (6), and other (20). These drugs were also tested for activity against S. mutans biofilm cultures, and 24 compounds were found to inhibit biofilm formation, 6 killed preexisting biofilms, 84 exhibited biofilm inhibition and killing activity, and 12 had no activity against biofilms. The activities of 9 selected compounds that exhibited antimicrobial activity were further characterized for their activity against S. mutans planktonic and biofilm cultures. Together, our results suggest that S. mutans exhibits a susceptibility profile to a diverse array of established and novel antibacterials.

scholarly journals Cellular alterations identified in pluripotent stem cell-derived midbrain spheroids generated from a female patient with progressive external ophthalmoplegia and parkinsonism who carries a novel variation (p.Q811R) in the POLG1 gene

2019 ◽  
Vol 7 (1) ◽  
Author(s):  
Margarita Chumarina ◽  
Kaspar Russ ◽  
Carla Azevedo ◽  
Andreas Heuer ◽  
Maria Pihl ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Female Patient ◽  
Dopaminergic Neurons ◽  
Alpha Synuclein ◽  
Genetic Identification ◽  
Progressive External Ophthalmoplegia ◽  
Gene Encoding ◽  
Mitochondrial Dna Copy Number ◽  
And Control ◽  
Cellular Alterations

AbstractVariations in the POLG1 gene encoding the catalytic subunit of the mitochondrial DNA polymerase gamma, have recently been associated with Parkinson’s disease (PD), especially in patients diagnosed with progressive external ophthalmoplegia (PEO). However, the majority of the studies reporting this association mainly focused on the genetic identification of the variation in POLG1 in PD patient primary cells, and determination of mitochondrial DNA copy number, providing little information about the cellular alterations existing in patient brain cells, in particular dopaminergic neurons. Therefore, through the use of induced pluripotent stem cells (iPSCs), we assessed cellular alterations in novel p.Q811R POLG1 (POLG1Q811R) variant midbrain dopaminergic neuron-containing spheroids (MDNS) from a female patient who developed early-onset PD, and compared them to cultures derived from a healthy control of the same gender. Both POLG1 variant and control MDNS contained functional midbrain regionalized TH/FOXA2-positive dopaminergic neurons, capable of releasing dopamine. Western blot analysis identified the presence of high molecular weight oligomeric alpha-synuclein in POLG1Q811R MDNS compared to control cultures. In order to assess POLG1Q811R-related cellular alterations within the MDNS, we applied mass-spectrometry based quantitative proteomic analysis. In total, 6749 proteins were identified, with 61 significantly differentially expressed between POLG1Q811R and control samples. Pro- and anti-inflammatory signaling and pathways involved in energy metabolism were altered. Notably, increased glycolysis in POLG1Q811R MDNS was suggested by the increase in PFKM and LDHA levels and confirmed using functional analysis of glycolytic rate and oxygen consumption levels. Our results validate the use of iPSCs to assess cellular alterations in relation to PD pathogenesis, in a unique PD patient carrying a novel p.Q811R variation in POLG1, and identify several altered pathways that may be relevant to PD pathogenesis.

scholarly journals Large-Scale Drug Screen Identifies FDA-Approved Drugs for Repurposing in Sickle-Cell Disease

2020 ◽  
Vol 9 (7) ◽  
pp. 2276
Author(s):  
Matthew Cannon ◽  
Hannah Phillips ◽  
Sidney Smith ◽  
Katie Williams ◽  
Lindsey Brinton ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Large Scale ◽  
Treatment Options ◽  
Fetal Hemoglobin ◽  
Erythroid Cell ◽  
Drug Screen ◽  
Cell Disease ◽  
Approved Drugs ◽  
Fda Approved Drugs

Sickle-cell disease (SCD) is a debilitating hematological disorder with very few approved treatment options. Therapeutic reactivation of fetal hemoglobin (HbF) is one of the most pursued methods for ameliorating the systemic manifestations of SCD. Despite this, very few pharmacological agents have advanced to clinical trials or marketing for use. In this study, we report the development of an HbF in situ intracellular immunoblot assay coupled to a high-throughput drug screen to identify Food and Drug Administration (FDA) approved drugs that can be repurposed clinically for treatment of SCD. Using this assay we evaluated the National Institute of Health (NIH) Clinical Collection (NCC), a publicly available library of 725 small molecules, and found nine candidates that can significantly re-express HbF in erythroid cell lines as well as primary erythroblasts derived from SCD patients. Furthermore, we show the strong effects on HbF expression of these candidates to occur with minimal cytotoxicity in 7 of the 9 drugs. Given these data and their proven history of use for other indications, we hypothesize that several of these candidate drugs warrant further investigation for use in SCD.

scholarly journals Gene-corrected Parkinson’s disease neurons show the A30P alpha-synuclein point mutation leads to reduced neuronal branching and function

2020 ◽  
Author(s):  
Peter A. Barbuti ◽  
Bruno FR. Santos ◽  
Paul M. Antony ◽  
Francois Massart ◽  
Gérald Cruciani ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Cell Lines ◽  
Dopaminergic Neurons ◽  
Cell Model ◽  
Control Cell ◽  
Electrode Array ◽  
Alpha Synuclein ◽  
Functionally Impaired ◽  
Mitochondrial Toxin

AbstractParkinson’s disease is characterised by the degeneration of A9 dopaminergic neurons and the pathological accumulation of alpha-synuclein. In a patient-derived stem cell model, we have generated dopaminergic neurons from an individual harbouring the p.A30P SNCA mutation and compared those neurons against gene-corrected isogenic control cell lines. We have used confocal microscopy to assess the neuronal network, specifically segmenting dopaminergic neurons and have identified image-based phenotypes showing axonal impairment and reduced neurite branching. We show using multi-electrode array (MEA) technology that the neurons carrying the endogenous p.A30P alpha-synuclein mutation are functionally impaired and identified mitochondrial dysfunction as a pathogenic cellular phenotype. We report that against gene-corrected isogenic control cell lines the neurons carrying the p.A30P SNCA mutation have a deficit and are susceptible to the mitochondrial toxin and environmental pesticide Rotenone. Our data supports the use of isogenic cell lines in identifying image-based pathological phenotypes that can serve as an entry point for future disease modifying compound screenings and drug discovery strategies.

scholarly journals Homozygous pathogenic variant in BRAT1 associated with nonprogressive cerebellar ataxia

2019 ◽  
Vol 5 (5) ◽  
pp. e359 ◽  
Author(s):  
Areej Mahjoub ◽  
Zuzana Cihlarova ◽  
Martine Tétreault ◽  
Lauren MacNeil ◽  
Neal Sondheimer ◽  
...  
Keyword(s):  
Oxygen Consumption ◽  
Cerebellar Ataxia ◽  
Cell Lines ◽  
Western Blotting ◽  
Control Cell ◽  
Cerebellar Atrophy ◽  
Atm Kinase ◽  
Protein Levels ◽  
And Control ◽  
The Impact

ObjectiveTo investigate the pathogenicity of a novel homozygous BRAT1 variant in 2 siblings with nonprogressive cerebellar ataxia (NPCA) through functional studies on primary and immortalized patient cell lines.MethodsBRAT1 protein levels and ataxia-telangiectasia mutated (ATM) kinase activity in patient-derived and control cell lines were assessed by Western blotting. The impact of the novel BRAT1 variants on mitochondrial function was also assessed, by comparing patient and control cell lines for rates of oxygen consumption and for phosphorylation (S293) of the E1⍺ subunit of pyruvate dehydrogenase (PDH).ResultsTwo male siblings with NPCA, mild intellectual disability, and isolated cerebellar atrophy were found to be homozygous for a c.185T>A (p.Val62Glu) variant in BRAT1 by whole exome sequencing. Western blotting revealed markedly decreased BRAT1 protein levels in lymphocytes and/or fibroblast cells from both affected siblings compared to control cell lines. There were no differences between the patient and control cells in ATM kinase activation, following ionizing radiation. Mitochondrial studies were initially suggestive of a defect in regulation of PDH activity, but there was no evidence of increased phosphorylation of the E1⍺ subunit of the PDH complex. Measurement of oxygen consumption rates similarly failed to identify differences between patient and control cells.ConclusionsBiallelic pathogenic variants in BRAT1 can be associated with NPCA, a phenotype considerably milder than previously reported. Surprisingly, despite the molecular role currently proposed for BRAT1 in ATM regulation, this disorder is unlikely to result from defective ATM kinase or mitochondrial dysfunction.

scholarly journals In silico drug repositioning of FDA-approved drugs to predict new inhibitors for alpha-synuclein aggregation

2020 ◽  
Vol 88 ◽  
pp. 107308
Author(s):  
Sedighe Sadat Jafaripour ◽  
Sajjad Gharaghani ◽  
Elmira Nazarshodeh ◽  
Shozeb Haider ◽  
Ali Akbar Saboury
Keyword(s):  
In Silico ◽  
Drug Repositioning ◽  
Alpha Synuclein ◽  
Approved Drugs ◽  
Alpha Synuclein Aggregation ◽  
Fda Approved Drugs

Conjugated Linoleic Acid Causes a Marked Increase in Liver α-Tocopherol and Liver α-Tocopherol Transfer Protein in C57BL/6 J Mice

2010 ◽  
Vol 80 (1) ◽  
pp. 65-73 ◽  
Author(s):  
Pei-Min Chao ◽  
Wan-Hsuan Chen ◽  
Chun-Huei Liao ◽  
Huey-Mei Shaw
Keyword(s):  
Antioxidant Enzymes ◽  
Linoleic Acid ◽  
Conjugated Linoleic Acid ◽  
Thiobarbituric Acid ◽  
Control Group ◽  
Thiobarbituric Acid Reactive Substances ◽  
Control Groups ◽  
Protein Levels ◽  
Transfer Protein ◽  
And Control

Conjugated linoleic acid (CLA) is a collective term for the positional and geometric isomers of a conjugated diene of linoleic acid (C18:2, n-6). The aims of the present study were to evaluate whether levels of hepatic α-tocopherol, α-tocopherol transfer protein (α-TTP), and antioxidant enzymes in mice were affected by a CLA-supplemented diet. C57BL/6 J mice were divided into the CLA and control groups, which were fed, respectively, a 5 % fat diet with or without 1 g/100 g of CLA (1:1 mixture of cis-9, trans-11 and trans-10, cis-12) for four weeks. α-Tocopherol levels in plasma and liver were significantly higher in the CLA group than in the control group. Liver α-TTP levels were also significantly increased in the CLA group, the α-TTP/β-actin ratio being 2.5-fold higher than that in control mice (p<0.01). Thiobarbituric acid-reactive substances were significantly decreased in the CLA group (p<0.01). There were no significant differences between the two groups in levels of three antioxidant enzymes (superoxide dismutase, glutathione peroxidase, and catalase). The accumulation of liver α-tocopherol seen with the CLA diet can be attributed to the antioxidant potential of CLA and the ability of α-TTP induction. The lack of changes in antioxidant enzyme protein levels and the reduced lipid peroxidation in the liver of CLA mice are due to α-tocopherol accumulation.

Comparing the Metabolome of a Caribbean Sponge to 420 FDA Approved Drugs, a Molecular Networking Approach

Planta Medica ◽  
2013 ◽  
Vol 79 (10) ◽  
Author(s):  
H Houson ◽  
J Schlesser ◽  
J Beverage ◽  
V Macherla ◽  
E Esquenazi
Keyword(s):  
Molecular Networking ◽  
Approved Drugs ◽  
Fda Approved Drugs

Pengaruh Lama Pengasinan Terhadap Kadar Protein Putih Telur Itik

2014 ◽  
Vol 1 (1) ◽  
pp. 36 ◽  
Author(s):  
Siti Fatimah ◽  
Muji Rahayu ◽  
Siti Aminah
Keyword(s):  
Protein Level ◽  
Egg White ◽  
Special Treatment ◽  
Egg White Protein ◽  
Protein Levels ◽  
Duck Egg ◽  
Egg Period ◽  
Graph Presentation ◽  
And Control ◽  
The Impact

Background : Egg  is one of the animal protein source, which has delicious taste, easy to digest and highly nutritious. Besides its affordable price, its supply availability is unquestionable as well. However, due to its short storability, it requires special treatment, such as preserving, to store it for long period. One way to preserve the egg is by pickling egg, which generally requires seven to ten days of marinating. During the process of marinating, there will be a visual change of egg white and yolk. Their structures  will be more solid (the occurrence of thickening process) because salinization will lead to protein denaturalization. Consequently, it has an influence as well towards the content of egg white protein of duck egg. This study is aimed to explore the impact of various time of pickling egg towards egg white protein of duck egg. Method  : The study where takes place in a laboratories, is a true experimental study for the reason that the researcher must provide intervention, hence all of potentially confounding variables are manageable. Samples that had been used in this study are duck eggs which were bought from North Brebes. This study is expected to generate data from four various time of pickling egg and control (no treatment). Since there are four samples, accordingly the number of data resulted are twenty. The resulted data will be descriptively presented in table, graph, presentation, and narration. Result  : Protein level examination within duck white egg shows changes  in protein levels that occurs in every variation of pickling egg time, where the average results of the assay of duck egg white protein is 14.94% without treatment (control), in five days of pickling time is 13.68%, in seven days of pickling time is 13.29%, in nine days of pickling time is 12.87% and eleven days of pickling time is 12.78%. Conclusion  : There is a significant impact among the period of pickling time to the protein level degradation of duck white egg. Keywords : Duck egg, period of pickling time, level protein of duck white egg.

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