scholarly journals Prolonged podocyte depletion in larval zebrafish resembles mammalian focal and segmental glomerulosclerosis

2019 ◽  
Author(s):  
Kerrin Ursula Ingeborg Hansen ◽  
Florian Siegerist ◽  
Sophie Daniel ◽  
Maximilian Schindler ◽  
Antje Blumenthal ◽  
...  
Keyword(s):  
Therapeutic Option ◽  
Screening Assay ◽  
Focal And Segmental Glomerulosclerosis ◽  
Larval Zebrafish ◽  
Zebrafish Larvae ◽  
High Throughput Drug Screening ◽  
Foot Process ◽  
Drug Screening Assay ◽  
Parietal Epithelial Cells ◽  
Glomerular Tuft

AbstractAlthough FSGS has been in the scientific focus for many years, it is still a massive burden for patients with no causal therapeutic option. In FSGS, podocytes are injured, parietal epithelial cells (PECs) are activated and engage in the formation of cellular lesions leading to progressive glomerular scarring. Herein we show that podocyte-depleted zebrafish larvae develop acute proteinuria, severe foot process effacement and activate PECs which create cellular lesions and deposit extracellular matrix on the glomerular tuft. We therefore propose that this model shows features of human FSGS and show its applicability for a high-throughput drug screening assay.

EXTH-67. THERAPEUTIC EFFICACY OF GC1118, A NOVEL ANTI-EGFR ANTIBODY, AGAINST GLIOBLASTOMA WITH HIGH EGFR AMPLIFICATION IN PATIENT-DERIVED XENOGRAFTS

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi178-vi178
Author(s):  
Seung Won Choi
Keyword(s):  
Therapeutic Efficacy ◽  
Growth Factor Receptor ◽  
Genomic Analysis ◽  
Screening Assay ◽  
Egfr Amplification ◽  
High Throughput Drug Screening ◽  
Number Variation ◽  
Drug Screening Assay ◽  
Pdx Models

Abstract We aimed to evaluate the preclinical efficacy of GC1118, a novel anti-epidermal growth factor receptor (EGFR) monoclonal antibody (mAb), against glioblastoma (GBM) tumors using patient-derived xenograft (PDX) models. A total of 15 distinct GBM PDX models were used to evaluate the therapeutic efficacy of GC1118. Genomic data derived from PDX models were analyzed to identify potential biomarkers associated with the anti-tumor efficacy of GC1118. A patient-derived cell-based high-throughput drug screening assay was performed to further validate the efficacy of GC1118. Compared to cetuximab, GC1118 exerted comparable growth inhibitory effects on the GBM tumors in the PDX models. We confirmed that GC1118 accumulated within the tumor by crossing the blood–brain barrier in in vivo specimens and observed the survival benefit in GC1118-treated intracranial models. Genomic analysis revealed high EGFR amplification as a potent biomarker for predicting the therapeutic efficacy of GC1118 in GBM tumors. In summary, GC1118 exerted a potent anti-tumor effect on GBM tumors in PDX models, and its therapeutic efficacy was especially pronounced in the tumors with high EGFR amplification. Our study supports the importance of patient stratification based on EGFR copy number variation in clinical trials for GBM. The superiority of GC1118 over other EGFR mAbs in GBM tumors should be assessed in future studies.

scholarly journals Comprehensive Analysis of Neurotoxin-Induced Ablation of Dopaminergic Neurons in Zebrafish Larvae

Biomedicines ◽  
2019 ◽  
Vol 8 (1) ◽  
pp. 1 ◽  
Author(s):  
Michael Kalyn ◽  
Khang Hua ◽  
Suzita Mohd Noor ◽  
Chee Ern David Wong ◽  
Marc Ekker
Keyword(s):  
Dopaminergic Neurons ◽  
Cell Loss ◽  
Motor Deficit ◽  
Transgenic Zebrafish ◽  
Larval Zebrafish ◽  
Zebrafish Larvae ◽  
High Throughput Drug Screening ◽  
Gene Expression Analyses ◽  
Substantial Degree ◽  
Ventral Diencephalon

Neurotoxin exposure of zebrafish larvae has been used to mimic a Parkinson’s disease (PD) phenotype and to facilitate high-throughput drug screening. However, the vulnerability of zebrafish to various neurotoxins was shown to be variable. Here, we provide a direct comparison of ablative effectiveness in order to identify the optimal neurotoxin-mediated dopaminergic (DAnergic) neuronal death in larval zebrafish. Transgenic zebrafish, Tg(dat:eGFP), were exposed to different concentrations of the neurotoxins MPTP, MPP+, paraquat, 6-OHDA, and rotenone for four days, starting at three days post-fertilization. The LC50 of each respective neurotoxin concentration was determined. Confocal live imaging on Tg(dat:eGFP) showed that MPTP, MPP+, and rotenone caused comparable DAnergic cell loss in the ventral diencephalon (vDC) region while, paraquat and 6-OHDA caused fewer losses of DAnergic cells. These results were further supported by respective gene expression analyses of dat, th, and p53. Importantly, the loss of DAnergic cells from exposure to MPTP, MPP+, and rotenone impacted larval locomotor function. MPTP induced the largest motor deficit, but this was accompanied by the most severe morphological impairment. We conclude that, of the tested neurotoxins, MPP+ recapitulates a substantial degree of DAnergic ablation and slight locomotor perturbations without systemic defects indicative of a Parkinsonian phenotype.

P0420PARTIAL PHARMACOGENETIC PODOCYTE DEPLETION IN A HIGH-THROUGHPUT ZEBRAFISH MODEL RESEMBLES HUMAN FOCAL AND SEGMENTAL GLOMERULOSCLEROSIS

2020 ◽  
Vol 35 (Supplement_3) ◽  
Author(s):  
Florian Siegerist ◽  
Kerrin Ursula Ingeborg Hansen ◽  
Sophie Daniel ◽  
Maximilian Schindler ◽  
Antje Blumenthal ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Epithelial Cells ◽  
High Throughput ◽  
Zebrafish Model ◽  
Matrix Deposition ◽  
Foot Process ◽  
Extracellular Matrix Deposition ◽  
Parietal Epithelial Cells ◽  
Glomerular Tuft

Abstract Background and Aims Although focal and segmental glomerulosclerosis (FSGS) has been in the scientific focus for many years, it is still a massive burden for patients with no causal therapeutic option. In FSGS, glomerular podocytes are injured, parietal epithelial cells (PECs) are activated and engage in the formation of cellular lesions leading to progressive glomerular scarring. Therefore, novel drug-screening assays are needed. Unfortunately, simple cellular in vitro-based screening assays are not ideal as glomerular architecture and crosstalk between glomerular cells is insufficiently modelled. Therefore, reliable animal models are still required for drug development, which unfortunately are not ideal for high-throughput applications. To date, due to its size, easy maintenance and breeding, zebrafish larvae are the simplest vertebrate model that are used in high-content screenings. Until today, it was unclear whether zebrafish can be used as a model for human FSGS. We therefore aimed to investigate whether partial podocyte-depletion in larval zebrafish leads to formation of FSGS-like disease and if the model can be used for screening purposes. Method We used a transgenic zebrafish model of pharmacogenetic podocyte depletion: In the Tg(nphs2:GAL4), Tg(UAS:Eco.nfsb-mCherry) strain, podocytes express the bacterial nitroreductase under control of the podocin promotor and can be dose-dependently ablated upon administration of metronidazole. Proteinuria was quantified using in vivo confocal laser scanning microscopy of intravenously administered high-molecular-weight fluorescent dextran. Plastic-embedded larvae where histologically and morphometrically assessed using HE, PAS and Jone’s silver staining after metronidazole washout. Glomerular ultrastructure was assessed using transmission electron microscopy of ultrathin sections. Immunofluorescence staining was carried out on kryosections to investigate extracellular matrix deposition (collagen-1, laminin), cellular proliferation (pcna) as well as parietal cell origin and activation (pax2a). Results To partially deplete podocytes, larvae where treated with 80 µM metronidazole from 4-6 days post fertilization, so that a subset of podocytes was depleted. In contrast to controls, podocyte-depleted larvae developed severe whole-body edema (Fig. A). Dynamic in vivo imaging of intravascular 500 kDa fluorescent dextran revealed massive leakage of the glomerular filtration barrier. Ultrastructural and immunofluorescent evaluation showed broad foot process effacement of remaining podocytes (Fig. D) and massive decrease of the slit diaphragm component podocin. Moreover, we found numerous sub-podocyte space pseudocysts (asterisk in Fig. D), microvillous transformation and formation of podocytic tight junctions as well as parietovisceral adhesions of the two layers of Bowman’s capsule. Parietal epithelial cells where activated, changed their phenotype towards a cuboidal shape, began to proliferate as demonstrated by pcna immunofluorescence and where recruited to cellular lesions on the glomerular tuft as demonstrated by the presence of cuboidal pax2a+ cells on the glomerular tuft (arrowheads Fig. B). Moreover, we found significant extracellular matrix deposition by the pax2a+ cells as demonstrated by Jone’s silver staining and laminin immunofluorescence (Fig. C). Conclusion Herein we show that upon podocyte-depletion, zebrafish larvae develop important functional and morphological features of human FSGS such as severe proteinuria and edema, podocyte foot process effacement, activation of parietal epithelial cells which contribute to cellular lesions and deposit extracellular matrix on the glomerular tuft. We conclude that this model resembles the human disease in important features and therefore propose its applicability for a high-throughput drug screening assay for FSGS.

scholarly journals Design, development, and validation of a high-throughput drug-screening assay for targeting of human leukemia

Cancer ◽  
2013 ◽  
Vol 120 (4) ◽  
pp. 589-602 ◽  
Author(s):  
Katja Karjalainen ◽  
Renata Pasqualini ◽  
Jorge E. Cortes ◽  
Steven M. Kornblau ◽  
Benjamin Lichtiger ◽  
...  
Keyword(s):  
High Throughput ◽  
Drug Screening ◽  
Human Leukemia ◽  
Design Development ◽  
Screening Assay ◽  
High Throughput Drug Screening ◽  
Drug Screening Assay ◽  
Development And Validation

scholarly journals Therapeutic Efficacy of GC1118, a Novel Anti-EGFR Antibody, against Glioblastoma with High EGFR Amplification in Patient-Derived Xenografts

Cancers ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 3210
Author(s):  
Kyoungmin Lee ◽  
Harim Koo ◽  
Yejin Kim ◽  
Donggeon Kim ◽  
Eunju Son ◽  
...  
Keyword(s):  
Therapeutic Efficacy ◽  
Growth Factor Receptor ◽  
Genomic Analysis ◽  
Screening Assay ◽  
Egfr Amplification ◽  
High Throughput Drug Screening ◽  
Number Variation ◽  
Drug Screening Assay ◽  
Pdx Models

We aimed to evaluate the preclinical efficacy of GC1118, a novel anti-epidermal growth factor receptor (EGFR) monoclonal antibody (mAb), against glioblastoma (GBM) tumors using patient-derived xenograft (PDX) models. A total of 15 distinct GBM PDX models were used to evaluate the therapeutic efficacy of GC1118. Genomic data derived from PDX models were analyzed to identify potential biomarkers associated with the anti-tumor efficacy of GC1118. A patient-derived cell-based high-throughput drug screening assay was performed to further validate the efficacy of GC1118. Compared to cetuximab, GC1118 exerted comparable growth inhibitory effects on the GBM tumors in the PDX models. We confirmed that GC1118 accumulated within the tumor by crossing the blood–brain barrier in in vivo specimens and observed the survival benefit in GC1118-treated intracranial models. Genomic analysis revealed high EGFR amplification as a potent biomarker for predicting the therapeutic efficacy of GC1118 in GBM tumors. In summary, GC1118 exerted a potent anti-tumor effect on GBM tumors in PDX models, and its therapeutic efficacy was especially pronounced in the tumors with high EGFR amplification. Our study supports the importance of patient stratification based on EGFR copy number variation in clinical trials for GBM. The superiority of GC1118 over other EGFR mAbs in GBM tumors should be assessed in future studies.

Development of a High Throughput Drug Screening Assay for the Detection of Changes in Tau Levels - Proof of Concept with HSP90 inhibitors

2005 ◽  
Vol 2 (2) ◽  
pp. 231-238 ◽  
Author(s):  
Chad Dickey ◽  
Jason Eriksen ◽  
Adeela Kamal ◽  
Francis Burrows ◽  
Srinivas Kasibhatla ◽  
...  
Keyword(s):  
High Throughput ◽  
Drug Screening ◽  
Proof Of Concept ◽  
Hsp90 Inhibitors ◽  
Screening Assay ◽  
High Throughput Drug Screening ◽  
Drug Screening Assay

scholarly journals Investigation of the Antimicrobial Activity of Soy Peptides by Developing a High Throughput Drug Screening Assay

2016 ◽  
Author(s):  
Rekha Dhayakaran ◽  
Suresh Neethirajan ◽  
Xuan Weng
Keyword(s):  
Antimicrobial Activity ◽  
Raman Spectroscopy ◽  
Optical Microscopy ◽  
High Throughput ◽  
Drug Screening ◽  
Rapid Screening ◽  
Screening Assay ◽  
High Throughput Drug Screening ◽  
Soy Peptides ◽  
Drug Screening Assay

Background: Antimicrobial resistance is a great concern in the medical community, as well as food industry. Soy peptides were tested against bacterial biofilms for their antimicrobial activity. A high throughput drug screening assay was developed using microfluidic technology, RAMAN spectroscopy, and optical microscopy for rapid screening of antimicrobials and rapid identification of pathogens. Methods: Synthesized PGTAVFK and IKAFKEATKVDKVVVLWTA soy peptides were tested against Pseudomonas aeruginosa and Listeria monocytogenes using a microdilution assay. Microfluidic technology in combination with Surface Enhanced RAMAN Spectroscopy (SERS) and optical microscopy was used for rapid screening of soy peptides, pathogen identification, and to visualize the impact of selected peptides. Results: The PGTAVFK peptide did not significantly affect P. aeruginosa, although it had an inhibitory effect on L. monocytogenes above a concentration of 625 uM. IKAFKEATKVDKVVVLWTA was effective against both P. aeruginosa and L. monocytogenes above a concentration of 37.2 uM. High throughput drug screening assays were able to reduce the screening and bacterial detection time to 4 h. SERS spectra was used to distinguish the two bacterial species.

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