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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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Screening of quantum-confined Stark effect in nitride laser diodes and superluminescent diodes
摘要： In the present work we report on the observation of carrier-induced screening of built-in electric fields in (Al,In)GaN laser diodes and superluminescent diodes. We use the emission peak energy as a measure of the quantum-confined Stark effect and its screening by free carriers. For superluminescent diodes we observe a steady increase of screening up to the current density of 10 kA/cm2. This shows that the lasing in nitride laser diodes occurs under high electric fields, far from the flat band conditions.
关键词： nitride laser diodes,electric fields,carrier screening,quantum-confined Stark effect,superluminescent diodes
更新于2025-11-14 15:24:45
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Red Fluorescent Chlamydia trachomatis Applied to Live Cell Imaging and Screening for Antibacterial Agents
摘要： In this study, we describe the application of a transformed Chlamydia trachomatis strain constitutively expressing the red fluorescent protein mCherry, to allow real-time monitoring of the infection cycle and screening for agents that block replication of C. trachomatis. The red fluorescent C. trachomatis strain was detected autonomously without antibody staining and was equally susceptible to doxycycline as the wild type strain. A high-throughput screening assay was developed using the transformed strain and automated fluorescence microscopy. The assay was used in a pilot screen of a 349 compound library containing natural products from Australian flora and fauna. Compounds with anti-chlamydial activity were tested for dose response and toxicity to host cells and two non-toxic compounds had 50% effective concentration (EC50) values in the low micromolar range. Natural products are valuable sources for drug discovery and the identified Chlamydia growth inhibition may be starting points for future drug development. Live cell imaging was used to visualize growth of the red fluorescent C. trachomatis strain over time. The screening assay reduced workload and reagents compared to an assay requiring immunostaining and could further be used to monitor the development of Chlamydia inclusions and anti-chlamydial effect in real time.
关键词： mCherry,antibacterial,Chlamydia trachomatis,Australian natural products,high content screening,fluorescence-based screening,anti-chlamydial
更新于2025-09-23 15:23:52
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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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Changes in urine autofluorescence in ovarian cancer patients
摘要： Ovarian cancer is the type of cancer with the highest mortality rate among gynaecologic malignancies. Due to lack of screening tools, this disease is mainly diagnosed at a progressed stage, when it is too late to adequate therapy. Despite many attempts, enough sensitive and specific biomarker was not still uncovered. Fluorescence spectroscopy has proven to be a useful diagnostic tool with high efficiency. Fluorescence detection has three major advantages over other light-based investigation methods: high sensitivity, high speed, and reliability. Biological materials consist of a number of intrinsic fluorescent compounds – autofluorophores, which are associated with cardinal metabolic pathways. It is well known, that cancerous tissue metabolism is altered compared to healthy one, what influence also intrinsic fluorophores composition of bodily fluids. Urine is one of the biological fluids that could be obtained most easily and displays a blue – green fluorescence that can change in case of pathological process. Analysis of urine autofluorescence is non invasive and simple technique. Using fluorescent spectroscopy, ovarian cancer patients and healthy control group were discerned with high significance, so we predict that fluorescence analysis of urine could be a potential means of ovarian cancer screening.
关键词： fluorescence,cancer screening,urine,ovarian cancer
更新于2025-09-23 15:23:52
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Improved Cloud Screening Method for the Analysis of Sky Radiometer Measurements and Application to Asian Dust Detection
摘要： A cloud screening method employing two successive procedures of variability test and coarse mode test was developed, aiming at better elimination of cloud-contaminated data in the sky radiometer retrievals. The performance of the new cloud screening method was evaluated by examining statistical features with cloud coverage observations and lidar measurements. The variability test appeared to effectively eliminate data contaminated by relatively thick low-level clouds, whereas the coarse mode test appeared to eliminate data likely contaminated by thin cirrus-type clouds. Overall, the new method was considered to improve the current Sky Radiometer Network (SKYNET) data. The cloud screening method was then applied to dust detection from sky radiometer measurements. The detection performance was evaluated using surface synoptic observations (SYNOP) dust reports and the yellow sand index from NIES lidar measurements. It was shown that the new method helped to detect dust, effectively eliminating cloud-contaminated signals that were similar to those of the dust.
关键词： dust,cloud screening,aerosol,SKYNET
更新于2025-09-23 15:23:52
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-doped two-dimensional semiconductor quantum wells
摘要： We present a theoretical study of photoabsorption in n-doped two-dimensional (2D) and quasi-2D semiconductors that takes into account the interaction of the photocreated exciton with Fermi-sea (FS) electrons through (i) Pauli blocking, (ii) Coulomb screening, and (iii) excitation of FS electron-hole pairs—that we here restrict to one. The system we tackle is thus made of one exciton plus zero or one FS electron-hole pair. At low doping, the system ground state is predominantly made of a 'trion-hole'—a trion (two opposite-spin electrons plus a valence hole) weakly bound to a FS hole—with a small exciton component. As the trion is poorly coupled to photon, the intensity of the lowest absorption peak is weak; it increases with doping, thanks to the growing exciton component, due to a larger coupling between two-particle and four-particle states. Under a further doping increase, the trion-hole complex is less bound because of Pauli blocking by FS electrons, and its energy increases. The lower peak then becomes predominantly due to an exciton dressed by FS electron-hole pairs, that is, an exciton-polaron. As a result, the absorption spectra of n-doped semiconductor quantum wells show two prominent peaks, the nature of the lowest peak turning from trion-hole to exciton-polaron under a doping increase. Our work also nails down the physical mechanism behind the increase with doping of the energy separation between the trion-hole peak and the exciton-polaron peak, even before the anticrossing, as experimentally observed.
关键词： Fermi sea,trion-hole complex,exciton-polaron,photoabsorption,Pauli blocking,n-doped semiconductors,Coulomb screening
更新于2025-09-23 15:23:52
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Detection of visual field defects by opticians - with Damato Multifixation Campimetry Online
摘要： Purpose: To study Damato Multifixation Campimetry Online (DMCO), an inexpensive online visual field test, used for screening at optician shops in Denmark. Methods: This was an evaluation of a diagnostic test and customers were included if minimum 50 years old, with a visual acuity of minimum 0.5 and with less myopia than 6 D. Standard equipment was a computer, a wireless mouse and a computer monitor. We used the 'DMCO STANDARD 4,5' algorithm for screening. DMCO results were electronically transmitted to the authors. Customers with a positive DMCO test received an eye examination including the reference standard Humphrey Visual Field Analyzer 30–2 SITA Fast test. A subset of control participants with a negative DMCO test received the same examination in order to estimate the number of false and true negatives. DMCO specificity and sensitivity were estimated according to five reference standards with 95% confidence intervals. Results: The population comprised 627 individuals, 381 women and 246 men, from 13 optician shops. Mean age was 62 years (SD 7.4). DMCO was positive in 32 individuals and 27 individuals were classified as 'true positives' with diagnoses such as glaucoma, cataract and neurological visual field defects. Of the 595 individuals with a negative DMCO test, 110 were examined and nine individuals were classified as 'false negatives' according to diagnoses. Depending on reference the specificity was 97–99% and sensitivity was 14–69%. Conclusion: Screening with DMCO demonstrated high specificity, whereas sensitivity was unsatisfactory. Future studies with enough power to estimate the true sensitivity is needed.
关键词： Humphrey Field Analyzer,glaucoma,screening test,Damato Multifixation Campimetry Online,specificity,opticians/optometrists
更新于2025-09-23 15:23:52
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Multidimensional screening yields channelrhodopsin variants having improved photocurrent and order-of-magnitude reductions in calcium and proton currents
摘要： Channelrhodopsins (ChRs) are light-gated ion channels in widespread use in neuroscience for mediating the genetically targetable optical control of neurons (optogenetics). Wild-type ChRs pass multiple kinds of ions, and although nonspecific ChR-mediated conductance is not an issue in many neuroscience studies, conductance of calcium and protons, which can mediate diverse cellular signals, may be undesirable in some instances. We here turned our attention to the creation of ChRs that have high cation photocurrent, but pass fewer calcium ions and protons. We developed an automated, time-resolved screening method capable of rapidly phenotyping channelrhodopsin-2 (ChR2) variants. We found substitution mutations throughout ChR2 that could boost current while altering ion selectivity, and also observed that the mutations that reduced calcium or proton conductance have additive effects. By combining four mutations, we obtained a ChR, ChromeQ, that possesses improved photocurrent and order-of-magnitude reductions in calcium and proton conductance and high fidelity in driving repetitive action potentials in neurons. The approach presented here offers a viable pathway toward customization of complex physiological properties of optogenetic tools. We propose that our screening method not only enables elucidation of new ChR variants that affect microbial opsin performance, but may also reveal new principles of optogenetic protein engineering.
关键词： neurobiology,calcium,protein engineering,physiology,optogenetics,proton,Channelrhodopsin,screening
更新于2025-09-23 15:23:52