scholarly journals High-Throughput Phenotyping Toolkit for Characterizing Cellular Models of Hypertrophic Cardiomyopathy in Vitro

2019 ◽  
Vol 2 (4) ◽  
pp. 83 ◽  
Author(s):  
Mosqueira ◽  
Lis-Slimak ◽  
Denning
Keyword(s):  
Hypertrophic Cardiomyopathy ◽  
High Throughput ◽  
Molecular Mechanisms ◽  
Clinical Manifestations ◽  
Cellular Models ◽  
Molecular Features ◽  
High Throughput Drug Screening ◽  
High Throughput Phenotyping ◽  
Screening Approaches

Hypertrophic cardiomyopathy (HCM) is a prevalent and complex cardiovascular disease characterised by multifarious hallmarks, a heterogeneous set of clinical manifestations, and several molecular mechanisms. Various disease models have been developed to study this condition, but they often show contradictory results, due to technical constraints and/or model limitations. Therefore, new tools are needed to better investigate pathological features in an unbiased and technically refined approach, towards improving understanding of disease progression. Herein, we describe three simple protocols to phenotype cellular models of HCM in vitro, in a high-throughput manner where technical artefacts are minimized. These are aimed at investigating: (1) Hypertrophy, by measuring cell volume by flow cytometry; (2) HCM molecular features, through the analysis of a hypertrophic marker, multinucleation, and sarcomeric disarray by high-content imaging; and (3) mitochondrial respiration and content via the Seahorse™ platform. Collectively, these protocols comprise straightforward tools to evaluate molecular and functional parameters of HCM phenotypes in cardiomyocytes in vitro. These facilitate greater understanding of HCM and high-throughput drug screening approaches and are accessible to all researchers of cardiac disease modelling. Whilst HCM is used as an exemplar, the approaches described are applicable to other cellular models where the investigation of identical biological changes is paramount.

scholarly journals Investigating Molecular Mechanisms of Immunotoxicity and the Utility of ToxCast for Immunotoxicity Screening of Chemicals Added to Food

2021 ◽  
Vol 18 (7) ◽  
pp. 3332
Author(s):  
Olga V. Naidenko ◽  
David Q. Andrews ◽  
Alexis M. Temkin ◽  
Tasha Stoiber ◽  
Uloma Igara Uche ◽  
...  
Keyword(s):  
Immune System ◽  
High Throughput ◽  
Environmental Protection Agency ◽  
High Throughput Screening ◽  
Molecular Mechanisms ◽  
Specific Activity ◽  
Laboratory Animals ◽  
In Vitro Assays ◽  
Toxicological Studies

The development of high-throughput screening methodologies may decrease the need for laboratory animals for toxicity testing. Here, we investigate the potential of assessing immunotoxicity with high-throughput screening data from the U.S. Environmental Protection Agency ToxCast program. As case studies, we analyzed the most common chemicals added to food as well as per- and polyfluoroalkyl substances (PFAS) shown to migrate to food from packaging materials or processing equipment. The antioxidant preservative tert-butylhydroquinone (TBHQ) showed activity both in ToxCast assays and in classical immunological assays, suggesting that it may affect the immune response in people. From the PFAS group, we identified eight substances that can migrate from food contact materials and have ToxCast data. In epidemiological and toxicological studies, PFAS suppress the immune system and decrease the response to vaccination. However, most PFAS show weak or no activity in immune-related ToxCast assays. This lack of concordance between toxicological and high-throughput data for common PFAS indicates the current limitations of in vitro screening for analyzing immunotoxicity. High-throughput in vitro assays show promise for providing mechanistic data relevant for immune risk assessment. In contrast, the lack of immune-specific activity in the existing high-throughput assays cannot validate the safety of a chemical for the immune system.

scholarly journals Facilitated Cross-Bridge Interactions with Thin Filaments by Familial Hypertrophic Cardiomyopathy Mutations inα-Tropomyosin

2011 ◽  
Vol 2011 ◽  
pp. 1-12 ◽  
Author(s):  
Fang Wang ◽  
Nicolas M. Brunet ◽  
Justin R. Grubich ◽  
Ewa A. Bienkiewicz ◽  
Thomas M. Asbury ◽  
...  
Keyword(s):  
Hypertrophic Cardiomyopathy ◽  
Molecular Mechanisms ◽  
Helical Structure ◽  
Familial Hypertrophic Cardiomyopathy ◽  
Maximum Speed ◽  
Sliding Speed ◽  
Thin Filaments ◽  
In Vitro Motility ◽  
Cardiac Sarcomeres

Familial hypertrophic cardiomyopathy (FHC) is a disease of cardiac sarcomeres. To identify molecular mechanisms underlying FHC pathology, functional and structural differences in three FHC-related mutations in recombinantα-Tm (V95A, D175N, and E180G) were characterized using both conventional and modified in vitro motility assays and circular dichroism spectroscopy. Mutant Tm's exhibited reducedα-helical structure and increased unordered structure. When thin filaments were fully occupied by regulatory proteins, little or no motion was detected at pCa 9, and maximum speed (pCa 5) was similar for all tropomyosins. Ca2+-responsiveness of filament sliding speed was increased either by increasedpCa50(V95A), reduced cooperativityn(D175N), or both (E180G). When temperature was increased, thin filaments with E180G exhibited dysregulation at temperatures ~10°C lower, and much closer to body temperature, than WT. When HMM density was reduced, thin filaments with D175N required fewer motors to initiate sliding or achieve maximum sliding speed.

scholarly journals New Approach for High-Throughput Screening of Drug Activity on Plasmodium Liver Stages

2006 ◽  
Vol 50 (4) ◽  
pp. 1586-1589 ◽  
Author(s):  
Audrey Gego ◽  
Olivier Silvie ◽  
Jean-François Franetich ◽  
Khemaïs Farhati ◽  
Laurent Hannoun ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
High Throughput ◽  
High Throughput Screening ◽  
Scanning System ◽  
New Approach ◽  
Drug Activity ◽  
Hepatic Cells ◽  
High Throughput Drug Screening ◽  
Prophylactic Drugs

ABSTRACT Plasmodium liver stages represent potential targets for antimalarial prophylactic drugs. Nevertheless, there is a lack of molecules active on these stages. We have now developed a new approach for the high-throughput screening of drug activity on Plasmodium liver stages in vitro, based on an infrared fluorescence scanning system. This method allowed us to count automatically and rapidly Plasmodium-infected hepatocytes, using different hepatic cells and different Plasmodium species, including Plasmodium falciparum. This new technique is well adapted for high-throughput drug screening and should facilitate the identification of new antimalarial compounds active on Plasmodium liver stages.

scholarly journals DIPG-07. HIGH THROUGHPUT DRUG SCREENING IDENTIFIES POTENTIAL NEW THERAPIES FOR DIFFUSE INTRINSIC PONTINE GLIOMAS (DIPGs)

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii288-iii288
Author(s):  
Dannielle Upton ◽  
Santosh Valvi ◽  
Jie Liu ◽  
Nicole Yeung ◽  
Sandra George ◽  
...  
Keyword(s):  
High Throughput ◽  
Apoptotic Cell ◽  
Safety Data ◽  
Biologically Active ◽  
Orthotopic Model ◽  
High Throughput Drug Screening ◽  
Diffuse Intrinsic Pontine Gliomas ◽  
Almost All

Abstract DIPGs are the most devastating of all brain tumors. There are no effective treatments, hence almost all children will die of their tumor within 12 months. There is an urgent need for novel effective therapies for this aggressive tumor. We performed a high-throughput drug screen with over 3,500 biologically active, clinically approved compounds against a panel of neurosphere-forming DIPG cells. We identified 7 compounds- auranofin, fenretinide, ivermectin, lanatoside, parthenolide, SAHA and mefloquine- that were confirmed to have potent anti-tumor activity against a panel of DIPG-neurospheres, with minimal effect on normal cells. Using cytotoxicity and clonogenic assays, we found that these drugs were able to inhibit DIPG-neurosphere proliferation and colony formation in-vitro. To determine whether the in-vitro efficacy could be replicated in-vivo, we tested the activity of each of these compounds in an orthotopic DIPG model. Of the agents tested, fenretinide and SAHA were the most active anti-tumor agents, significantly enhancing the survival of tumor bearing animals. Mechanistic studies showed fenretinide enhancing apoptotic cell death of DIPG cells via inhibition of PDGFRa transcription and downregulation of the PI3K/AKT/MTOR pathway. We therefore examined the therapeutic efficacy of fenretinide using a second orthotopic model with PDGFRa amplification. We used two different Fenretinide formulations (LYM-X-Sorb and NanoMicelle) which were found to enhance survival. Fenretinide is clinically available with safety data in children. Validation of the activity of Fenretinide in PDGFRa-amplified or overexpressed DIPGs will lead to the development of a clinical trial, allowing the advancement of fenretinide as potentially the first active therapy for DIPG.

Genipin Attenuates Tau Phosphorylation and Aβ Levels in Cellular Models of Alzheimer's Disease

2021 ◽  
Author(s):  
Meiting Li ◽  
Nan Cai ◽  
Liang Gu ◽  
Lijun Yao ◽  
Decheng Bi ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Signaling Pathway ◽  
Molecular Mechanisms ◽  
Tau Phosphorylation ◽  
Brain Disorder ◽  
Cellular Models ◽  
Aβ Generation ◽  
Aβ Production

Abstract Alzheimer’s disease (AD) is a devastating brain disorder characterized by neurofibrillary tangles and amyloid plaques. Inhibiting Tau protein and amyloid-beta (Aβ) production or removing these molecules are considered potential therapeutic strategies for AD. Genipin is an aglycone and is isolated from the extract of Gardenia jasminoides Ellis fruit. In this study, the effect and molecular mechanisms of genipin on the inhibition of Tau aggregation and Aβ generation were investigated. The results showed that genipin bound to Tau and protected against heparin-induced Tau fibril formation. Moreover, genipin suppressed Tau phosphorylation probably by downregulating the expression of CDK5 and GSK-3β, and activated mTOR-dependent autophagy via the SIRT1/LKB1/AMPK signaling pathway in Tau-overexpressing cells. In addition, genipin decreased Aβ production by inhibiting BACE1 expression through the PERK/eIF2α signaling pathway in N2a/SweAPP cells. These data indicated that genipin could effectively lead to a significant reduction of phosphorylated Tau level and Aβ generation in vitro, suggesting that genipin might be developed into an effective therapeutic complement or a potential nutraceutical for preventing AD.

scholarly journals Challenges and perspectives for structural biology of lncRNAs—the example of the Xist lncRNA A-repeats

2019 ◽  
Vol 11 (10) ◽  
pp. 845-859 ◽  
Author(s):  
Alisha N Jones ◽  
Michael Sattler
Keyword(s):  
High Resolution ◽  
Structural Biology ◽  
Recent Progress ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Structural Features ◽  
Molecular Features

Abstract Following the discovery of numerous long non-coding RNA (lncRNA) transcripts in the human genome, their important roles in biology and human disease are emerging. Recent progress in experimental methods has enabled the identification of structural features of lncRNAs. However, determining high-resolution structures is challenging as lncRNAs are expected to be dynamic and adopt multiple conformations, which may be modulated by interaction with protein binding partners. The X-inactive specific transcript (Xist) is necessary for X inactivation during dosage compensation in female placental mammals and one of the best-studied lncRNAs. Recent progress has provided new insights into the domain organization, molecular features, and RNA binding proteins that interact with distinct regions of Xist. The A-repeats located at the 5′ end of the transcript are of particular interest as they are essential for mediating silencing of the inactive X chromosome. Here, we discuss recent progress with elucidating structural features of the Xist lncRNA, focusing on the A-repeats. We discuss the experimental and computational approaches employed that have led to distinct structural models, likely reflecting the intrinsic dynamics of this RNA. The presence of multiple dynamic conformations may also play an important role in the formation of the associated RNPs, thus influencing the molecular mechanism underlying the biological function of the Xist A-repeats. We propose that integrative approaches that combine biochemical experiments and high-resolution structural biology in vitro with chemical probing and functional studies in vivo are required to unravel the molecular mechanisms of lncRNAs.

Characterization Of FLT3/ITD Resistant Cells and Identification Of Novel Therapeutic Targets Utilizing High Throughput Drug Screening

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2522-2522
Author(s):  
Katherine Tarlock ◽  
C. Anthony Blau ◽  
Timothy Martins ◽  
Soheil Meshinchi
Keyword(s):  
Cell Lines ◽  
High Throughput ◽  
Drug Screening ◽  
Therapeutic Options ◽  
High Throughput Drug Screening ◽  
Mutational Profiling ◽  
Flt3 Inhibitors ◽  
Resistant Cells

Abstract The overall survival (OS) of pediatric acute myeloid leukemia (AML) exceeds 60%, however for high risk (HR) patients, including high allelic ratio FLT3/ITD+, survival remains poor. FLT3/ITD is one of the first genomic alterations in AML to be exploited for therapeutic benefit as it has greater sensitivity to the pro-apoptotic effects of FLT3-inhibitors. Children’s Oncology Group (COG) phase III AML trial AAML1031 is investigating the role of sorafenib in combination with chemotherapy in HR FLT3/ITD+ patients. In vitro and in vivo studies indicate that resistance to FLT3-inhibitors can develop through varying mechanisms including up-regulation of FLT3 receptor, acquisition of secondary mutations, or activation of alternate survival mechanisms leading to apoptotic escape. For FLT3/ITD+ patients who relapse despite treatment with FLT3-inhibitors, there are often no therapeutic options and survival is very poor. In evaluation of therapeutic options for those who relapse on sorafenib, we developed an in vitro resistance model using the FLT3/ITD+ cell line MV4-11. Resistance was induced thru long-term exposure to incrementally increasing doses of sorafenib. Two distinct cell lines with resistance at 10 and 100 fold above the IC50 of naïve MV4-11 were generated for experimental evaluation. Genotypic and phenotypic characterization of the resistant cells was conducted by multidimensional flow cytometry (MDF), conventional karyotyping, and mutational profiling. MDF revealed an overall similar immunophenotype, however the resistant cells were significantly more homogeneous for expression of HLA-DR and had significantly higher CD11b expression compared to their naïve counterparts. CD135 expression was minimally increased in the resistant cells. In comparison of the karyotypes, the resistant cells were a more homogenous population with emergence of one dominant clone and disappearance of a number of pre-existing sub-clones. Mutational profiling by Sanger sequencing revealed a novel N841Y mutation in activation loop, an area implicated in TKI-resistance. Using a high throughput drug screening assay, we explored sensitivity profiles of the naïve and resistant MV4-11 cells to 163 oncology agents, including 45 FDA approved and 118 investigational agents that target a number of key pathways regulating cell growth, differentiation, and survival. The naïve MV4-11s had a sorafenib IC50 of 1.3 nM (published 1-5nM) and resistant cells had IC50 of approximately 2-log folds above the naïve, which was consistent to what we had seen in our lab-based validations. We initially assessed whether resistance to sorafenib induces cross-resistance to other TKIs. Agents in the panel with previously demonstrated efficacy for FLT3/ITD included quizartinib (AC-220), tandutinib, ponatinib, sunitinib, and midostaurin, and in all cases sorafenib-resistant cells were also more resistant to these agents. We then examined whether we could identify agents with efficacy in the resistant cells. We identified 5 novel agents to which the resistant cells retained sensitivity. Two bcl-2 inhibitors tested maintained sensitivity in the resistant cells with IC50s in the 20-100nM range. In addition, YM-155, a survivin inhibitor, also maintained sensitivity in the resistant cells with IC50s of approximately 25-50nM across the cell lines. Survivin over-expression is associated with AML stem progenitor cells and decreased OS in adults, and transcription regulation has been linked to the FLT3/STAT5 pathway. Two CRM inhibitors, a novel class of agents which inhibit nuclear export to restore tumor suppressor function, also maintained sensitivity in the resistant cell lines with an approximate 3-fold increase in IC50 from 12nM in the naïve to 32-40nM in the resistant cells. Experience with the use of directed therapy to target specific somatic events has provided evidence that leukemic evolution can continue under this selection pressure and therapeutic options for patients with emergent disease is often insufficient. Using the high throughput drug assay in a FLT3/ITD+ cell line as an in vitro model for sorafenib-resistant FLT3/ITD patients, we identified classes of targeted agents that maintain sensitivity in resistant cells. Further validation of the targets in specimens from those with resistance to such TKIs can inform on the class of agents that can be used to treat or prevent refractory disease FLT3/ITD+ patients. Disclosures: No relevant conflicts of interest to declare.

scholarly journals MOATAI-VIR - an AI algorithm that predicts severe adverse events and molecular features for COVID-19’s complications

2021 ◽  
Author(s):  
Courtney Astore ◽  
Hongyi Zhou ◽  
Joshy Jacob ◽  
Jeffrey Skolnick
Keyword(s):  
Adverse Events ◽  
Molecular Mechanisms ◽  
Chromosomal Abnormalities ◽  
Clinical Manifestations ◽  
Host Responses ◽  
Molecular Features ◽  
Disease Protein ◽  
Human Proteins ◽  
Severe Adverse Events ◽  
Patients Experience

AbstractFollowing SARS-CoV-2 infection, some COVID-19 patients experience severe adverse events caused by pathogenic host responses. To treat these complications, their underlying etiology must be identified. Thus, a novel AI-based methodology, MOATAI-VIR, which predicts disease-protein-pathway relationships for 22 clinical manifestations attributed to COVID-19 was developed. SARS-CoV-2 interacting human proteins and GWAS identified respiratory failure associated risk genes provide the input from which the mode-of-action (MOA) proteins/pathways of the resulting disease comorbidities are predicted. These comorbidities are then mapped to their clinical manifestations. Three uncharacterized manifestation categories are found: neoplasms, mental and behavioral disorders, and congenital malformations, deformations, and chromosomal abnormalities. The prevalence of neoplasms suggests a possible association between COVID-19 and cancer, whether by shared molecular mechanisms between oncogenesis and viral replication, or perhaps, SARS-CoV-2 is an oncovirus. To assess the molecular basis of each manifestation, the proteins shared across each group of comorbidities were prioritized and subject to global pathway analysis. From these most frequent pathways, the molecular features associated with hallmark COVID-19 phenotypes, such as loss of sense of smell/taste, unusual neurological symptoms, cytokine storm, and blood clots were explored. Results of MOATAI-VIR are available for academic users at: http://pwp.gatech.edu/cssb/MOATAI-VIR/.

scholarly journals Probing the VIPR2 Microduplication Linkage to Schizophrenia in Animal and Cellular Models

2021 ◽  
Vol 15 ◽  
Author(s):  
Yukio Ago ◽  
Satoshi Asano ◽  
Hitoshi Hashimoto ◽  
James A. Waschek
Keyword(s):  
Synaptic Plasticity ◽  
Brain Development ◽  
Molecular Mechanisms ◽  
Human Genetics ◽  
Autism Spectrum ◽  
Cellular Models ◽  
Pituitary Adenylate Cyclase ◽  
Innervation Patterns ◽  
The Brain

Pituitary adenylate cyclase-activating polypeptide (PACAP, gene name ADCYAP1) is a multifunctional neuropeptide involved in brain development and synaptic plasticity. With respect to PACAP function, most attention has been given to that mediated by its specific receptor PAC1 (ADCYAP1R1). However, PACAP also binds tightly to the high affinity receptors for vasoactive intestinal peptide (VIP, VIP), called VPAC1 and VPAC2 (VIPR1 and VIPR2, respectively). Depending on innervation patterns, PACAP can thus interact physiologically with any of these receptors. VPAC2 receptors, the focus of this review, are known to have a pivotal role in regulating circadian rhythms and to affect multiple other processes in the brain, including those involved in fear cognition. Accumulating evidence in human genetics indicates that microduplications at 7q36.3, containing VIPR2 gene, are linked to schizophrenia and possibly autism spectrum disorder. Although detailed molecular mechanisms have not been fully elucidated, recent studies in animal models suggest that overactivation of the VPAC2 receptor disrupts cortical circuit maturation. The VIPR2 linkage can thus be potentially explained by inappropriate control of receptor signaling at a time when neural circuits involved in cognition and social behavior are being established. Alternatively, or in addition, VPAC2 receptor overactivity may disrupt ongoing synaptic plasticity during processes of learning and memory. Finally, in vitro data indicate that PACAP and VIP have differential activities on the maturation of neurons via their distinct signaling pathways. Thus perturbations in the balance of VPAC2, VPAC1, and PAC1 receptors and their ligands may have important consequences in brain development and plasticity.

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