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[Methods in Molecular Biology] Calcium Signalling Volume 1925 (Methods and Protocols) || High-Throughput Screening Using Photoluminescence Probe to Measure Intracellular Calcium Levels
摘要： Aequorin, a 22 kDa protein produced by the jellyfish Aequorea victoria, was the first probe used to measure Ca2+ concentrations ([Ca2+]) of specific intracellular organelles in intact cells. After the binding of Ca2+ to three high-affinity binding sites, an irreversible reaction occurs leading to the emission of photons that is proportional to [Ca2+]. While native aequorin is suitable for measuring cytosolic [Ca2+] after cell stimulation in a range from 0.5 to 10 μM, it cannot be used in organelles where [Ca2+] is much higher, such as in the lumen of endoplasmic/sarcoplasmic reticulum (ER/SR) and mitochondria. However, some modifications made on aequorin itself or on coelenterazine, its lipophilic prosthetic luminophore, and the addition of targeting sequences or the fusion with resident proteins allowed the specific organelle localization and the measurements of intra-organelle Ca2+ levels. In the last years, the development of multiwell plate readers has opened the possibility to perform aequorin-based high-throughput screenings and has overcome some limitation of the standard method. Here we present the procedure for expressing, targeting, and reconstituting aequorin in intact cells and for measuring Ca2+ in the bulk cytosol, mitochondria, and ER by a high-throughput screening system.
关键词： Cytosol,Calcium probes,Calcium,High-throughput screening,Aequorin,Mitochondria,ER
更新于2025-11-21 11:20:42
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Peptic Fluorescent “Signal-On” and “Signal-Off” Sensors Utilized for the Detection Protein Post-Translational Modifications
摘要： Protein post-translational modifications (PTMs) are typically enzyme-catalyzed events generating functional diversi?cation of proteome; thus, multiple PTM enzymes have been validated as potential drug targets. We have previously introduced energy-transfer-based signal-modulation method called quenching resonance energy transfer (QRET), and utilize it to monitor PTM addition or removal using the developed peptide-break technology. Now we have reinvented the QRET technology, and as a model, we introduced the tunable ?uorescent “signal-on” and “signal-o?” detection scheme in the peptide-break PTM detection. Taking the advantage of time-resolved ?uorescence-based single-label detection technology, we were able to select the signal direction upon PTM addition or removal by simply introducing di?erent soluble Eu3+-signal-modulating molecule. This enables the selection of positive signal change upon measurable event, without any additional labeling steps, changes in assay condition or Eu3+-reporter. The concept functionality was demonstrated with four Eu3+-signal modulators in a high-throughput compatible kinase and phosphatase assays using signal-on and signal-o? readout at 615 nm or time-resolved Fo?rster resonance energy transfer at 665 nm. Our data suggest that the introduced signal modulation methodology provides a transitional ?uorescence-based single-label detection concept not limited only to PTM detection.
关键词： time-resolved fluorescence,signal-off,high-throughput screening,peptide-break technology,protein post-translational modifications,FRET,signal-on
更新于2025-11-19 16:56:35
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Identification of Small-Molecule Modulators of Diguanylate Cyclase by FRET-Based High-Throughput Screening
摘要： The bacterial second messenger cyclic diguanosine monophosphate (c-di-GMP) is a key regulator of cellular motility, the cell cycle, and biofilm formation with its resultant antibiotic tolerance, which can make chronic infections difficult to treat. Therefore, diguanylate cyclases, which regulate the spatiotemporal production of c-di-GMP, might be attractive drug targets for control of biofilm formation that is part of chronic infections. We present a FRET-based biochemical high-throughput screening approach coupled with detailed structure–activity studies to identify synthetic small-molecule modulators of the diguanylate cyclase DgcA from Caulobacter crescentus. We identified a set of seven small molecules that regulate DgcA enzymatic activity in the low-micromolar range. Subsequent structure–activity studies on selected scaffolds revealed a remarkable diversity of modulatory behavior, including slight chemical substitutions that reverse the effects from allosteric enzyme inhibition to activation. The compounds identified represent new chemotypes and are potentially developable into chemical genetic tools for the dissection of c-di-GMP signaling networks and alteration of c-di-GMP-associated phenotypes. In sum, our studies underline the importance of detailed mechanism-of-action studies for inhibitors of c-di-GMP signaling and demonstrate the complex interplay between synthetic small molecules and the regulatory mechanisms that control the activity of diguanylate cyclases.
关键词： c-di-GMP,structure–activity relationships,FRET,diguanylate cyclase inhibitors,high-throughput screening
更新于2025-09-23 15:23:52
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PhenoFly Planning Tool: flight planning for high-resolution optical remote sensing with unmanned areal systems
摘要： Background: Driven by a huge improvement in automation, unmanned areal systems (UAS) are increasingly used for field observations and high-throughput phenotyping. Today, the bottleneck does not lie in the ability to fly a drone anymore, but rather in the appropriate flight planning to capture images with sufficient quality. Proper flight preparation for photography with digital frame cameras should include relevant concepts such as view, sharpness and exposure calculations. Additionally, if mapping areas with UASs, one has to consider concepts related to ground control points (GCPs), viewing geometry and way-point flights. Unfortunately, non of the available flight planning tools covers all these aspects. Results: We give an overview of concepts related to flight preparation, present the newly developed open source software PhenoFly Planning Tool, and evaluate other recent flight planning tools. We find that current flight planning and mapping tools strongly focus on vendor-specific solutions and mostly ignore basic photographic properties—our comparison shows, for example, that only two out of thirteen evaluated tools consider motion blur restrictions, and none of them depth of field limits. In contrast, PhenoFly Planning Tool enhances recent sophisticated UAS and autopilot systems with an optical remote sensing workflow that respects photographic concepts. The tool can assist in selecting the right equipment for your needs, experimenting with different flight settings to test the performance of the resulting imagery, preparing the field and GCP setup, and generating a flight path that can be exported as waypoints to be uploaded to an UAS. Conclusion: By considering the introduced concepts, uncertainty in UAS-based remote sensing and high-throughput phenotyping may be considerably reduced. The presented software PhenoFly Planning Tool (https://shiny.usys.ethz.ch/PhenoFlyPlanningTool) helps users to comprehend and apply these concepts.
关键词： Flight planning,Ground control point (GCP),High-throughput phenotyping,Viewing geometry,Low-altitude remote sensing,Mapping from imagery
更新于2025-09-23 15:23:52
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High-throughput design of functional materials using materials genome approach
摘要： High-throughput computational materials design provides one efficient solution to accelerate the discovery and development of functional materials. Its core concept is to build a large quantum materials repository and to search for target materials with desired properties via appropriate materials descriptors in a high-throughput fashion, which shares the same idea with the materials genome approach. This article reviews recent progress of discovering and developing new functional materials using high-throughput computational materials design approach. Emphasis is placed on the rational design of high-throughput screening procedure and the development of appropriate materials descriptors, concentrating on the electronic and magnetic properties of functional materials for various types of industrial applications in nanoelectronics.
关键词： first-principles,high-throughput,functional materials,materials genome
更新于2025-09-23 15:23:52
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Signal enhancement in laser diode thermal desorption-triple quadrupole mass spectrometry analysis using microwell surface coatings
摘要： Laser diode thermal desorption (LDTD) is an ionization source usually coupled to triple quadrupole mass spectrometry (QqQMS) and specifically designed for laboratories requiring high-throughput analysis. It has been observed that surface coatings on LDTD microwell plates can improve the sensitivity of the analysis of small polar molecules. The objective of the present study is to understand and quantify the effect of microwell surface coatings on signal intensity of small organic molecules of clinical, environmental and forensic interest. Experiments showed that the peak areas of diclofenac, chloramphenicol, salicylic acid and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol obtained by LDTD-QqQMS increased by up to 3 orders of magnitude when using microwells coated with ethylenediaminetetraacetic acid (EDTA). Tests with different chelating agents and polytetrafluoroethylene as microwell surface coatings showed that nitrilotriacetic acid gave significantly higher peak areas for five out of the nine compounds that showed signal enhancement using chelating agents as coatings. Scanning electron microscopy studies of EDTA-coated and uncoated microwells showed that analytes deposited in the former formed more uniform and thinner films than in the latter. The enhancement effect of surface coatings in LDTD-QqQMS was explained mainly by the formation of homogenous and thinner layers of nanocrystals of analytes that are easier to desorb thermally than the layers formed when the analytes dry in direct contact with the bare stainless steel surface. Chemisorption of some analytes to the stainless steel surface of the microwell plate appeared to be a minor factor. Surface coatings widen the number of compounds analyzable by LDTD-QqQMS and can also improve sensitivity and limits of detection.
关键词： pharmaceuticals,high-throughput,hormones,pesticides,x-ray photoelectron spectroscopy,scanning electron microscopy,stainless steel,small organic molecules,chelating agents
更新于2025-09-23 15:23:52
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Modification of AlphaLISA Excitation Wavelength Leads to Improved Assay Sensitivity for Photosynthetic Tissue Samples
摘要： For the purposes of high-throughput immunoassay screening, PerkinElmer’s AlphaLISA technology offers many benefits over traditional enzyme-linked immunosorbant assay (ELISA). However, its 680 nm excitation wavelength coincides with a wavelength of peak photosynthetic pigment absorbance, hindering the technology’s utility within the plant biotechnology industry. In assays containing photosynthetic matrices, it is proposed that excitation of chlorophyll leads to the production of singlet oxygen, which initiates a pigment-associated background signal, reducing assay sensitivity. A customized donor bead, modified for excitation outside the range of photosystem absorbance, was tested for its capacity to improve assay sensitivity with extracts containing photosynthetic pigments. In three assays designed against crystalline domain insecticidal protein targets, use of the customized donor bead along with its altered excitation wavelength led to the elimination of pigment-associated signal and improved separation between target-positive and null samples. Reduction in null photosynthetic extract signal led to a 16× sensitivity improvement in a quantitative assay. The customized donor bead was also found to be photostable under ambient laboratory lighting, potentially improving the overall utility of AlphaLISA technology. The customized donor bead enables sensitive, high-throughput immunoassay screening of photosynthetic tissues within the plant biotechnology industry using a convenient, photostable protocol.
关键词： homogenous,HTS,photosynthetic,Alpha,plant,high-throughput screening,automated biology
更新于2025-09-23 15:22:29
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High-Throughput Combinatorial Synthesis of Stimuli-Responsive Materials
摘要： Stimuli-responsive materials find wide applications in different biological and medical settings. Traditionally, stimuli-responsive materials are synthesized and evaluated individually one-by-one. The drawback of this approach is the scarceness of possible combinations that can be practically tested for the purpose of saving time, consumables, and manpower. High-throughput methods are therefore important to accelerate the discovery of stimuli-responsive materials and to screen for biological interactions of interest in parallel. The objective of this article is to provide an overview of the successful employment of combinatorial high-throughput synthesis and screening of stimuli-responsive materials. In particular, these include thermoresponsive and hydroresponsive materials. Advantages of a combinatorial approach as well as of utilizing high-throughput methodologies in the development of stimuli-responsive materials are reviewed. Possible evolution trends of stimuli-responsive materials, advanced by high-throughput methodologies, are discussed.
关键词： combinatorial libraries,material microarrays,smart materials,stimuli-responsive,high-throughput synthesis
更新于2025-09-23 15:22:29
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Possibilities for serial femtosecond crystallography sample delivery at future light sourcesa)
摘要： Serial femtosecond crystallography (SFX) uses X-ray pulses from free-electron laser (FEL) sources that can outrun radiation damage and thereby overcome long-standing limits in the structure determination of macromolecular crystals. Intense X-ray FEL pulses of sufficiently short duration allow the collection of damage-free data at room temperature and give the opportunity to study irreversible time-resolved events. SFX may open the way to determine the structure of biological molecules that fail to crystallize readily into large well-diffracting crystals. Taking advantage of FELs with high pulse repetition rates could lead to short measurement times of just minutes. Automated delivery of sample suspensions for SFX experiments could potentially give rise to a much higher rate of obtaining complete measurements than at today’s third generation synchrotron radiation facilities, as no crystal alignment or complex robotic motions are required. This capability will also open up extensive time-resolved structural studies. New challenges arise from the resulting high rate of data collection, and in providing reliable sample delivery. Various developments for fully automated high-throughput SFX experiments are being considered for evaluation, including new implementations for a reliable yet flexible sample environment setup. Here, we review the different methods developed so far that best achieve sample delivery for X-ray FEL experiments and present some considerations towards the goal of high-throughput structure determination with X-ray FELs.
关键词： automation,serial femtosecond crystallography,X-ray free-electron laser,sample delivery,high-throughput
更新于2025-09-23 15:22:29
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Thermodynamic Stability Landscape of Halide Double Perovskites via High-Throughput Computing and Machine Learning
摘要： Formability and stability issues are of core importance and difficulty in current research and applications of perovskites. Nevertheless, over the past century, determination of the formability and stability of perovskites has relied on semi empirical models derived from physics intuition, such as the commonly used Goldschmidt tolerance factor, t. Here, through high-throughput density functional theory (DFT) calculations, a database containing the decomposition energies, considered to be closely related to the thermodynamic stability of 354 halide perovskite candidates, is established. To map the underlying relationship between the structure and chemistry features and the decomposition energies, a well-functioned machine learning (ML) model is trained over this theory-based database and further validated by experimental observations of perovskite formability (F1 score, 95.9%) of 246 A2B(I)B(III)X6 compounds that are not present in the training database; the model performs a lot better than empirical descriptors such as tolerance factor t (F1 score, 77.5%). This work demonstrates that the experimental engineering of stable perovskites by ML could solely rely on training data derived from high-throughput DFT computing, which is much more economical and efficient than experimental attempts at materials synthesis.
关键词： halide double perovskite,stability,machine learning,high-throughput
更新于2025-09-23 15:22:29