Spatiotemporal Quantification of In Vitro Cardiomyocyte Contraction Dynamics Using Video Microscopy-based Software Tool

Stereotactic targeting is a commonly used technique for performing injections in the brains of mice and other animals. The most common method for targeting stereoscopic injections uses the skull indentations bregma and lambda as reference points and is limited in its precision by factors such as skull curvature and individual variation, as well as an incomplete correspondence between skull landmarks and brain locations. In this software tool, a 3D laser scan of the mouse skull is taken in vitro and registered onto a reference skull using a point cloud matching algorithm, and the parameters of the transformation are used to position a glass pipette to place tracer injections. The software was capable of registering sample skulls with less than 100 micron error, and was able to target an injection in a mouse with error of roughly 500 microns. These results indicate that using skull scan registration has the potential to be widely applicable in automating stereotactic targeting of tracer injections.

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pGlycoQuant with a deep residual network for precise and minuscule-missing-value quantitative glycoproteomics enabling the functional exploration of site-specific glycosylation

10.1101/2021.11.15.468561 ◽

2021 ◽

Author(s):

Weiqian Cao ◽

Siyuan Kong ◽

Wenfeng Zeng ◽

Pengyun Gong ◽

Biyun Jiang ◽

...

Keyword(s):

Quantitative Analysis ◽

Large Scale ◽

Missing Values ◽

Software Tool ◽

Software Tools ◽

Residual Network ◽

Site Specific ◽

Carcinoma Cell Lines ◽

L1 Cell Adhesion Molecule

Interpreting large-scale glycoproteomic data for intact glycopeptide identification has been tremendously advanced by software tools. However, software tools for quantitative analysis of intact glycopeptides remain lagging behind, which greatly hinders exploring the differential expression and functions of site-specific glycosylation in organisms. Here, we report pGlycoQuant, a generic software tool for accurate and convenient quantitative intact glycopeptide analysis, supporting both primary and tandem mass spectrometry quantitation for multiple quantitative strategies. pGlycoQuant enables intact glycopeptide quantitation with very low missing values via a deep residual network, thus greatly expanding the quantitative function of several powerful search engines, currently including pGlyco 2.0, pGlyco3, Byonic and MSFragger-Glyco. The pGlycoQuant-based site-specific N-glycoproteomic study conducted here quantifies 6435 intact N-glycopeptides in three hepatocellular carcinoma cell lines with different metastatic potentials and, together with in vitro molecular biology experiments, illustrates core fucosylation at site 979 of the L1 cell adhesion molecule (L1CAM) as a potential regulator of HCC metastasis. pGlycoQuant is freely available at https://github.com/expellir-arma/pGlycoQuant/releases/. We have demonstrated pGlycoQuant to be a powerful tool for the quantitative analysis of site-specific glycosylation and the exploration of potential glycosylation-related biomarker candidates, and we expect further applications in glycoproteomic studies.

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IN VITRO EVALUATION OF NONTHROMBOGENIC POLYMERS OF DIFFERENT MOLECULAR DESIGNS USING EPIFLUORESCENT VIDEO MICROSCOPY

ASAIO Journal ◽

10.1097/00002480-199604000-00048 ◽

1996 ◽

Vol 42 (2) ◽

pp. 14

Author(s):

C. Nojiri ◽

M. Waki ◽

T. Kido ◽

N. Saito ◽

T. Sugiyama ◽

...

Keyword(s):

Video Microscopy ◽

In Vitro Evaluation

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UV-visible photodegradation of naproxen in water – Structural elucidation of photoproducts and potential toxicity

European Journal of Mass Spectrometry ◽

10.1177/1469066720973412 ◽

2020 ◽

Vol 26 (6) ◽

pp. 400-408

Author(s):

Noemi Cazzaniga ◽

Zsuzsanna Varga ◽

Edith Nicol ◽

Stéphane Bouchonnet

Keyword(s):

Mass Spectrometry ◽

High Performance ◽

Vibrio Fischeri ◽

Software Tool ◽

In Vitro Assays ◽

Naphthaleneacetic Acid ◽

Ion Cyclotron Resonance ◽

Potential Toxicity ◽

Uv Visible

The UV-visible photodegradation of Naproxen (6-methoxy-α-methyl-2-naphthaleneacetic acid, CAS: 22204-53-1), one of the most used and detected non-steroidal anti-inflammatory drugs (NSAIDs) in the world, and its ecotoxicological consequences were investigated in an aqueous medium. The photo-transformation products were analyzed and the structures of photoproducts were elucidated using gas chromatography coupled with tandem mass spectrometry (GC-MS/MS) and high-performance liquid chromatography coupled with ultrahigh-resolution Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR-MS). Seven photoproducts were detected and characterized, photo-transformation mechanisms have been postulated to rationalize their formation under irradiation. In silico Q.S.A.R. (Quantitative Structure-Activity Relationship) toxicity predictions were performed with the Toxicity Estimation Software Tool (T.E.S.T.) and in vitro assays were carried out on Vibrio fischeri bacteria. Some of the obtained photoproducts exhibit higher potential toxicity than Naproxen itself but the whole toxicity of the irradiated solution is not of major concern.

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PTMProphet: Fast and Accurate Mass Modification Localization for the Trans-Proteomic Pipeline

10.1101/679845 ◽

2019 ◽

Cited By ~ 1

Author(s):

David D Shteynberg ◽

Eric W Deutsch ◽

David S Campbell ◽

Michael R Hoopmann ◽

Ulrike Kusebauch ◽

...

Keyword(s):

Cell Biology ◽

Software Tool ◽

Ground Truth ◽

Peptide Sequence ◽

Bayesian Mixture ◽

Downstream Analysis ◽

Bayesian Mixture Models ◽

Sequence Database Search

Spectral matching sequence database search engines commonly used on mass spectrometry-based proteomics experiments excel at identifying peptide sequence ions, and in addition, possible sequence ions carrying post-translational modifications (PTMs), but most do not provide confidence metrics for the exact localization of those PTMs when several possible sites are available. Localization is absolutely required for downstream molecular cell biology analysis of PTM function in vitro and in vivo. Therefore, we developed PTMProphet, a free and open-source software tool integrated into the Trans-Proteomic Pipeline, which reanalyzes identified spectra from any search engine for which pepXML output is available to provide localization confidence to enable appropriate further characterization of biologic events. Localization of any type of mass modification (e.g., phosphorylation) is supported. PTMProphet applies Bayesian mixture models to compute probabilities for each site/peptide spectrum match where a PTM has been identified. These probabilities can be combined to compute a global false localization rate at any threshold to guide downstream analysis. We describe the PTMProphet tool, its underlying algorithms and demonstrate its performance on ground-truth synthetic peptide reference datasets, one previously published small dataset, one new larger dataset, and also on a previously published phospho-enriched dataset where the correct sites of modification are unknown. Data have been deposited to ProteomeXchange with identifier PXD013210.

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Time-Lapse Video Microscopy and Single Cell Tracking to Study Neural Cell Behavior In Vitro

Imaging and Tracking Stem Cells - Methods in Molecular Biology ◽

10.1007/7651_2019_219 ◽

2019 ◽

pp. 183-194

Author(s):

Lucía Paniagua-Herranz ◽

Rosa Gómez-Villafuertes ◽

David de Agustín-Durán ◽

Sergio Gascón ◽

Raquel Pérez-Sen ◽

...

Keyword(s):

Single Cell ◽

Cell Tracking ◽

Time Lapse ◽

Neural Cell ◽

Video Microscopy ◽

Cell Behavior ◽

Time Lapse Video

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Effect of targeting of syndecan-1 by microRNA miR-10b on breast cancer cell motility and invasiveness via a rho-GTPase- and E-cadherin-dependent mechanism.

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.15_suppl.e21041 ◽

2012 ◽

Vol 30 (15_suppl) ◽

pp. e21041-e21041

Author(s):

Ludwig Kiesel ◽

Sherif Abdelaziz Ibrahim ◽

George W Yip ◽

Martin Gotte

Keyword(s):

Breast Cancer ◽

Cancer Cell ◽

Rho Gtpase ◽

Rho Kinase ◽

In Vitro Study ◽

Time Lapse ◽

Video Microscopy ◽

Migration And Invasion ◽

E Cadherin

e21041 Background: microRNAs are small endogenous non-coding RNAs, which posttranscriptionally regulate gene expression. In breast cancer, overexpression of the transmembrane heparan sulfate proteoglycan syndecan-1, a predicted target of the oncomiR miR-10b, correlates with poor clinical outcome. Here, we investigatet the potential functional relationship of miR-10b and syndecan-1 in an in vitro study. Methods: MDA-MB-231 and MCF-7 breast cancer cells were transiently transfected with pre-miR-10b, syndecan-1 siRNA or control reagents, respectively. Altered cell behaviour was monitored by proliferation, migration and invasion chamber assays, and time-lapse video microscopy. Results: miR-10b overexpression induced posttranscriptional downregulation of syndecan-1, as demonstrated by qPCR, flow cytometry, and 3’UTR luciferase assays, resulting in increased cancer cell migration and matrigel invasiveness. Syndecan-1 silencing generated a copy of this phenotype. Adhesion to fibronectin and laminin and basal cell proliferation were increased. Syndecan-1 co-immunoprecipitated with focal adhesion kinase, which showed increased activation upon syndecan-1 depletion. Affymetrix screening and confirmatory qPCR and Western blotting analysis of syndecan-1 deficient cells revealed upregulation of ATF-2, COX- 2, cadherin-11, vinculin, ACTG2, MYL9, transgelin-1, RhoA/C, MMP2 and heparanase, and downregulation of AML1/RUNX1, E-cadherin, CLDN1, p21WAF/CIP, Cdk6, TLR-4, PAI1/2, Collagen1alpha1, JHDM1D, Mpp4, MMP9, matrilin-2 and ANXA3/A10. Video microscopy demonstrated massively increased Rho kinase-dependent motility of syndecan-1-depleted cells, which displayed increased filopodia formation. Conclusions: We conclude that syndecan-1 is a novel target of the oncomiR miR-10b. Rho-GTPase dependent modulation of cytoskeletal function and downregulation of E-cadherin expression are identified as relevant effectors of the miR-10b-syndecan-1 axis, which emerges as a promising target for the development of new therapeutic approaches for breast cancer.

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