scholarly journals High performance intensiometric direct- and inverse-response genetically encoded biosensors for citrate

2020 ◽  
Author(s):  
Yufeng Zhao ◽  
Yi Shen ◽  
Yurong Wen ◽  
Robert E. Campbell
Keyword(s):  
Conformational Changes ◽  
Mammalian Cells ◽  
High Performance ◽  
Catalytic Domain ◽  
Quantitative Imaging ◽  
Live Cells ◽  
Biological Processes ◽  
Direct Response ◽  
Qualitative And Quantitative ◽  
Inverse Response

AbstractMotivated by the growing recognition of citrate as a central metabolite in a variety of biological processes associated with healthy and diseased cellular states, we have developed a series of high-performance genetically encoded citrate biosensors suitable for imaging of citrate concentrations in mammalian cells. The design of these biosensors was guided by structural studies of the citrate-responsive sensor histidine kinase, and took advantage of the same conformational changes proposed to propagate from the binding domain to the catalytic domain. Following extensive engineering based on a combination of structure guided mutagenesis and directed evolution, we produced an inverse-response biosensor (ΔF/Fmin ~ 18) designated Citroff1 and a direct-response biosensor (ΔF/Fmin ~ 9) designated Citron1. We report the x-ray crystal structure of Citron1 and demonstrate the utility of both biosensors for qualitative and quantitative imaging of steady-state and pharmacologically-perturbed citrate concentrations in live cells.

scholarly journals Comparative assessment of fluorescent transgene methods for quantitative imaging in human cells

2014 ◽  
Vol 25 (22) ◽  
pp. 3610-3618 ◽  
Author(s):  
Robert Mahen ◽  
Birgit Koch ◽  
Malte Wachsmuth ◽  
Antonio Z. Politi ◽  
Alexis Perez-Gonzalez ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Function ◽  
Mammalian Cells ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Quantitative Imaging ◽  
Live Cells ◽  
Single Molecule Spectroscopy ◽  
Mitotic Kinase ◽  
Endogenous Proteins

Fluorescence tagging of proteins is a widely used tool to study protein function and dynamics in live cells. However, the extent to which different mammalian transgene methods faithfully report on the properties of endogenous proteins has not been studied comparatively. Here we use quantitative live-cell imaging and single-molecule spectroscopy to analyze how different transgene systems affect imaging of the functional properties of the mitotic kinase Aurora B. We show that the transgene method fundamentally influences level and variability of expression and can severely compromise the ability to report on endogenous binding and localization parameters, providing a guide for quantitative imaging studies in mammalian cells.

scholarly journals A High-Performance Genetically Encoded Fluorescent Biosensor for Imaging Physiological Peroxynitrite

2020 ◽  
Author(s):  
Zhijie Chen ◽  
Shen Zhang ◽  
Xinyu Li ◽  
Hui-wang Ai
Keyword(s):  
Hydrogen Peroxide ◽  
Amino Acid ◽  
Mammalian Cells ◽  
High Performance ◽  
Live Cells ◽  
Nitrogen Species ◽  
Fluorescent Biosensor ◽  
Turn On ◽  
Fluorescence Turn On ◽  
Noncanonical Amino Acid

SummaryPeroxynitrite is a highly reactive nitrogen species (RNS) that plays critical roles in signal transduction, stress response, and numerous human diseases. Advanced molecular tools that permit the selective, sensitive, and non-invasive detection of peroxynitrite is essential for understanding its pathophysiological functions. Here, we present pnGFP-Ultra, a high performance, reaction-based, genetically encodable biosensor for imaging peroxynitrite in live cells. pnGFP-Ultra features a p-boronophenylalanine-modified chromophore as the sensing moiety and exhibits a remarkable 123-fold fluorescence turn-on response towards peroxynitrite while displaying virtually no cross-reaction with other reactive oxygen/nitrogen species, including hydrogen peroxide. To facilitate the expression of pnGFP-Ultra in mammalian cells, we engineered a highly efficient noncanonical amino acid (ncAA) expression system that is broadly applicable to the mammalian expression of proteins containing various ncAAs. pnGFP-Ultra robustly detected peroxynitrite production during interferon γ and lipopolysaccharide-induced immune responses in macrophages, and in amyloid β-activated primary glial cells. Thus, pnGFP-Ultra fills an important technical gap and represents an important new addition to the molecular toolbox in probing RNS biology.In BriefChen et al. report pnGFP-Ultra, a high-performance fluorescent biosensor for minimally invasive and selective imaging of peroxynitrite production in live cells.HighlightspnGFP-Ultra is a genetically encoded peroxynitrite biosensor with a 123-fold fluorescence turn-on responsepnGFP-Ultra exhibits high selectivity toward peroxynitrite, with virtually no crossreaction with hydrogen peroxideAn optimized plasmid-based system increases noncanonical amino acid incorporation in mammalian cells by >10 foldpnGFP-Ultra robustly detects peroxynitrite production in macrophages and primary glial cells

Fluorescent potentiometric dyes for membrane-potential imaging

1994 ◽  
Vol 52 ◽  
pp. 166-167
Author(s):  
Leslie M. Loew
Keyword(s):  
Membrane Potential ◽  
Single Cells ◽  
Quantitative Imaging ◽  
Optical Recording ◽  
Biological Processes ◽  
Major Focus ◽  
The Past ◽  
Excitable Systems ◽  
Major Application ◽  
The Impact

A major application of potentiometric dyes has been the multisite optical recording of electrical activity in excitable systems. After being championed by L.B. Cohen and his colleagues for the past 20 years, the impact of this technology is rapidly being felt and is spreading to an increasing number of neuroscience laboratories. A second class of experiments involves using dyes to image membrane potential distributions in single cells by digital imaging microscopy - a major focus of this lab. These studies usually do not require the temporal resolution of multisite optical recording, being primarily focussed on slow cell biological processes, and therefore can achieve much higher spatial resolution. We have developed 2 methods for quantitative imaging of membrane potential. One method uses dual wavelength imaging of membrane-staining dyes and the other uses quantitative 3D imaging of a fluorescent lipophilic cation; the dyes used in each case were synthesized for this purpose in this laboratory.

Apoptosis and development

2003 ◽  
Vol 39 ◽  
pp. 11-24 ◽  
Author(s):  
Justin V McCarthy
Keyword(s):  
Drosophila Melanogaster ◽  
Mammalian Cells ◽  
Cell Number ◽  
Model Organisms ◽  
Biological Processes ◽  
Nematode Caenorhabditis Elegans ◽  
Apoptotic Pathway ◽  
Genetic Pathways ◽  
Evolutionarily Conserved ◽  
Multicellular Organisms

Apoptosis is an evolutionarily conserved process used by multicellular organisms to developmentally regulate cell number or to eliminate cells that are potentially detrimental to the organism. The large diversity of regulators of apoptosis in mammalian cells and their numerous interactions complicate the analysis of their individual functions, particularly in development. The remarkable conservation of apoptotic mechanisms across species has allowed the genetic pathways of apoptosis determined in lower species, such as the nematode Caenorhabditis elegans and the fruitfly Drosophila melanogaster, to act as models for understanding the biology of apoptosis in mammalian cells. Though many components of the apoptotic pathway are conserved between species, the use of additional model organisms has revealed several important differences and supports the use of model organisms in deciphering complex biological processes such as apoptosis.

High Performance Thin Layer Chromatography (HPTLC) for the Investigation of Medicinal Plants

2020 ◽  
Vol 16 ◽  
Author(s):  
Alper Gökbulut
Keyword(s):  
Medicinal Plants ◽  
High Performance ◽  
Separation Efficiency ◽  
Rapid Development ◽  
Herbal Remedies ◽  
Qualitative And Quantitative ◽  
Powerful Analytical Tool ◽  
Quantitative Examination ◽  
Data Acquisition And Processing ◽  
Layer Chromatography

Background: Chromatographic techniques such as TLC basically and, HPLC, GC, HPTLC equipped with various detectors are most frequently used for the qualitative and quantitative examination of herbals. Method: An overview of the recent literature concerning the usage of HPTLC for the analysis of medicinal plants has been reviewed. Results: During the last decade/s, HPTLC, a modern, sophisticated and automatized TLC technique with better and advanced separation efficiency, detection limit, data acquisition and processing, has been used for the analysis of herbal materials and preparations since the rapid development of technology in chromatography world. HPTLC with various detectors is a powerful analytical tool especially for the phytochemical applications such as herbal drug quantification and fingerprint analysis. Conclusion: In this review, a latest perspective has been established and some of the previous studies were summarized for the usage of HPTLC in the analysis of herbal remedies, dietary supplements and nutraceuticals.

scholarly journals Incorporating radiomics into clinical trials: expert consensus on considerations for data-driven compared to biologically driven quantitative biomarkers

2021 ◽  
Author(s):  
Laure Fournier ◽  
Lena Costaridou ◽  
Luc Bidaut ◽  
Nicolas Michoux ◽  
Frederic E. Lecouvet ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Quantitative Imaging ◽  
A Priori ◽  
External Validation ◽  
Data Driven ◽  
Imaging Biomarkers ◽  
Clinical Validation ◽  
Biological Processes ◽  
Imaging Data ◽  
Selection Of

Abstract Existing quantitative imaging biomarkers (QIBs) are associated with known biological tissue characteristics and follow a well-understood path of technical, biological and clinical validation before incorporation into clinical trials. In radiomics, novel data-driven processes extract numerous visually imperceptible statistical features from the imaging data with no a priori assumptions on their correlation with biological processes. The selection of relevant features (radiomic signature) and incorporation into clinical trials therefore requires additional considerations to ensure meaningful imaging endpoints. Also, the number of radiomic features tested means that power calculations would result in sample sizes impossible to achieve within clinical trials. This article examines how the process of standardising and validating data-driven imaging biomarkers differs from those based on biological associations. Radiomic signatures are best developed initially on datasets that represent diversity of acquisition protocols as well as diversity of disease and of normal findings, rather than within clinical trials with standardised and optimised protocols as this would risk the selection of radiomic features being linked to the imaging process rather than the pathology. Normalisation through discretisation and feature harmonisation are essential pre-processing steps. Biological correlation may be performed after the technical and clinical validity of a radiomic signature is established, but is not mandatory. Feature selection may be part of discovery within a radiomics-specific trial or represent exploratory endpoints within an established trial; a previously validated radiomic signature may even be used as a primary/secondary endpoint, particularly if associations are demonstrated with specific biological processes and pathways being targeted within clinical trials. Key Points • Data-driven processes like radiomics risk false discoveries due to high-dimensionality of the dataset compared to sample size, making adequate diversity of the data, cross-validation and external validation essential to mitigate the risks of spurious associations and overfitting. • Use of radiomic signatures within clinical trials requires multistep standardisation of image acquisition, image analysis and data mining processes. • Biological correlation may be established after clinical validation but is not mandatory.

scholarly journals High-performance chemical- and light-inducible recombinases in mammalian cells and mice

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Benjamin H. Weinberg ◽  
Jang Hwan Cho ◽  
Yash Agarwal ◽  
N. T. Hang Pham ◽  
Leidy D. Caraballo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
High Performance ◽  
Genome Engineering ◽  
Genetic Circuits ◽  
Control Of Gene Expression ◽  
Expression Control ◽  
Gene Expression Control ◽  
High Performance Systems ◽  
Spatiotemporal Control

Abstract Site-specific DNA recombinases are important genome engineering tools. Chemical- and light-inducible recombinases, in particular, enable spatiotemporal control of gene expression. However, inducible recombinases are scarce due to the challenge of engineering high performance systems, thus constraining the sophistication of genetic circuits and animal models that can be created. Here we present a library of >20 orthogonal inducible split recombinases that can be activated by small molecules, light and temperature in mammalian cells and mice. Furthermore, we engineer inducible split Cre systems with better performance than existing systems. Using our orthogonal inducible recombinases, we create a genetic switchboard that can independently regulate the expression of 3 different cytokines in the same cell, a tripartite inducible Flp, and a 4-input AND gate. We quantitatively characterize the inducible recombinases for benchmarking their performances, including computation of distinguishability of outputs. This library expands capabilities for multiplexed mammalian gene expression control.

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