Fluorescent Screen Research Articles

Designing boron-doped carbon dot-functionalized COFs for fluorescence screening and liquid chromatography tandem mass spectrometry detection of toxins

In recent years, mushroom poisoning has been one of the most important factors of food poisoning in China, timely identification of the toxins contained in mushrooms is crucial for the treatment of patients. In this study, boric acid carbon dots (BA-CDs) can undergo specific boron affinity reactions with amatoxins toxins containing o-dihydroxyl groups by means of boric acid groups. Functional covalent organic framework (COF) and BA-CDs were combined to design a adsorbent with boric acid group (COF@VBC@BA-CDs) was designed to meet the requirements of both fluorescent and pretreated materials for amatoxins. The mushrooms and urine samples were rapid screening using fluorescence detection, and then, for positive samples, the target analytes on the COF@VBC@BA-CDs are collected and eluted for next liquid chromatography tandem mass spectrometry (LC-MS/MS) detection. According to the fluorescence characteristics of COF@VBC@BA-CDs, the fluorescence quenching intensity was linearly correlated with the concentration (2-200 μg/L) and the detection limit was 1.2 μg/L. Meanwhile, the detection limit of LC-MS/MS was 0.5 μg/kg for musroom and 0.2μg /L for urine, as well as the recovery rate was 72.7-110.1%. This noval method meets the methodological requirements and can be used for actual sample analysis.

Discovery of 5-(Substituted Phenyl)-2-aryl Benzimidazole Derivatives as SIRT1 Activators: Their Design, in silico Studies, Synthesis, and in vitro Evaluation.

Silent information regulator two homologue one (SIRT1) is an emerging target for managing metabolic disorders. This study aimed to synthesize novel 5-(- substituted phenyl)-2-aryl benzimidazole derivatives and evaluate them for SIRT1 activation. The compounds were designed according to the findings of the QSAR models framed in our previous studies. Molecular docking and dynamics studies were also performed to explore the interactions of designed compounds with the active site of the SIRT1 enzyme using AutoDock Vina and Schrödinger Maestro version 11.8.012, respectively. Compounds with good binding affinity were synthesized by Suzuki-Miyaura cross-coupling and spectrally characterized. The molecules were evaluated for their in vitro SIRT1 activation properties using a fluorescent screening kit. Based on the results of in vitro assay, a structure-activity relationship was established. SwissADME was employed to calculate the pharmacokinetics characteristics of the synthesized molecules. The molecular docking studies revealed that all the activators were effectively docked in the catalytic active site. All compounds demonstrated interactions with important amino acids like Glu230 and Arg446. In molecular dynamics simulations, the root mean square deviation (RMSD) of compound 5m and protein SIRT1 remained stable, i.e., below 3mm. Compound 5m, 4-(2-(3,4-dihydroxy-5-nitrophenyl)-1H-benzo[d]imidazol- 5-yl)benzaldehyde, was the most potent compound with an EC50 value of 0.006 mM (±0.001) and maximum activation of 240.5%. All the synthesized compounds had acceptable theoretical ADME profiles, and drug-likeness properties complied with Lipinski's rule. According to the findings, synthesized compounds may be viable leads for SIRT1 activators and may be used to advance preclinical in vivo research utilizing animal models.

A High-Throughput Screening Platform for Engineering Poly(ethylene Terephthalate) Hydrolases.

The ability of enzymes to hydrolyze the ubiquitous polyester, poly(ethylene terephthalate) (PET), has enabled the potential for bioindustrial recycling of this waste plastic. To date, many of these PET hydrolases have been engineered for improved catalytic activity and stability, but current screening methods have limitations in screening large libraries, including under high-temperature conditions. Here, we developed a platform that can simultaneously interrogate PET hydrolase libraries of 104-105 variants (per round) for protein solubility, thermostability, and activity via paired, plate-based split green fluorescent protein and model substrate screens. We then applied this platform to improve the performance of a benchmark PET hydrolase, leaf-branch compost cutinase, by directed evolution. Our engineered enzyme exhibited higher catalytic activity relative to the benchmark, LCC-ICCG, on amorphous PET film coupon substrates (∼9.4% crystallinity) in pH-controlled bioreactors at both 65 °C (8.5% higher conversion at 48 h and 38% higher maximum rate, at 2.9% substrate loading) and 68 °C (11.2% higher conversion at 48 h and 43% higher maximum rate, at 16.5% substrate loading), up to 48 h, highlighting the potential of this screening platform to accelerate enzyme development for PET recycling.

Characterization of extended defects in 2D materials using aperture-based dark-field STEM in SEM

Quantitative diffraction contrast analysis with defined diffraction vectors is a well-established method in TEM for studying defects in crystalline materials. A comparable transmission technique is however not available in the more widely used SEM platforms. In this work, we transfer the aperture-based dark-field imaging method from the TEM to the SEM, thus enabling quantitative diffraction contrast studies at lower voltages in SEM. This is achieved in STEM mode by inserting a custom-made aperture between the sample and the STEM detector and centering the hole on a desired reflection. To select individual reflections for dark-field imaging, we use our Low Energy Nanodiffraction (LEND) setup [Schweizer et al., Ultramicroscopy 213, 112956 (2020)], which captures transmission diffraction patterns from a fluorescent screen positioned below the sample. The aperture-based dark-field STEM method is particularly useful for studying extended defects in 2D materials, where (i) stronger diffraction at the lower voltages used in SEM is advantageous, but (ii) two-beam conditions cannot be established, making quantitative diffraction contrast analysis with standard bright-field and annular dark-field detectors impossible. We demonstrate the method by studying basal plane dislocations in bilayer graphene, which have attracted considerable research interest due to their exceptional structural and electronic properties. Direct comparison of results obtained on identical dislocations by the established TEM method and by the new aperture-based dark-field STEM method in SEM shows that a reliable Burgers vector analysis is possible by applying the well-known g·b=0 invisibility criterion. We further use the LEND setup to acquire 4D-STEM data and show that the virtual dark-field images match well with those in aperture-based dark-field STEM images for reliable Burgers vector analysis.

CRISPR-Mediated rDNA Integration and Fluorescence Screening for Pathway Optimization in Pichia pastoris

CRISPR-Mediated rDNA Integration and Fluorescence Screening for Pathway Optimization in <i>Pichia pastoris</i>

A homogeneous time-resolved fluorescence screen to identify SIRT2 deacetylase and defatty-acylase inhibitors.

Human sirtuin-2 (SIRT2) has emerged as an attractive drug target for a variety of diseases. The enzyme is a deacylase that can remove chemically different acyl modifications from protein lysine residues. Here, we developed a high-throughput screen based on a homogeneous time-resolved fluorescence (HTRF) binding assay to identify inhibitors of SIRT2's demyristoylase activity, which is uncommon among many ligands that only affect its deacetylase activity. From a test screen of 9600 compounds, we identified a small molecule that inhibited SIRT2's deacetylase activity (IC50 = 7 μM) as well as its demyristoylase activity (IC50 = 37 μM). The inhibitor was composed of two small fragments that independently inhibited SIRT2: a halogenated phenol fragment inhibited its deacetylase activity, and a tricyclic thiazolobenzimidazole fragment inhibited its demyristoylase activity. The high-throughput screen also detected multiple deacetylase-specific SIRT2 inhibitors.

Fluorescence Screening of DNA-AgNCs with Pulsed White Light Excitation.

DNA-stabilized silver nanoclusters (DNA-AgNCs) are a class of fluorophores with interesting photophysical properties dominated by the choice of DNA sequence. Screening methods with ultraviolet excitation and steady state well plate readers have previously been used for deepening the understanding between DNA sequence and emission color of the resulting DNA-AgNCs. Here, we present a new method for screening DNA-AgNCs by using pulsed white light excitation (λex ≈ 490-900 nm). By subtraction and time gating we are able to circumvent the dominating scatter of the white excitation light and extract both temporally and spectrally resolved emission of DNA-AgNCs over the visible to near-infrared range. Additionally, we are able to identify weak long-lived emission, which is often buried underneath the intense nanosecond fluorescence. This new approach will be useful for future screening of DNA-AgNCs (or other novel emissive materials) and aid machine-learning models by providing a richer training data set.

Development of new steroid-based hydrazide and (thio)semicarbazone compounds with anticancer properties

Most breast and prostate cancers are caused by abnormal production or action of steroidal hormones. Hormonal drugs based on steroid scaffolds represent a significant class of chemotherapeutics that are routinely used in chemotherapy. In this study, the synthesis of new 17a-homo lactone and 17α-(pyridine-2-ylmethyl) androstane derivatives with hydrazide and semicarbazone motifs is presented. All compounds were screened for their effect on cell viability against a panel of five cancer cell lines and one healthy cell line. Two compounds showed significant cytotoxicity against cancer cells, with low toxicity against healthy cells. The relative binding affinities of compounds for the ligand-binding domains of estrogen receptor α, estrogen receptor β, androgen receptor and glucocorticoid receptor were tested using a fluorescence screen in yeast. Potential for inhibition of aldo-keto reductase 1C3 and 1C4 activity was measured in vitro. Experimental results are analyzed in the context of molecular docking simulations. Our results could help guide design of steroid compounds with improved anticancer properties against androgen- and estrogen-dependent cancers.

Development of single-cell ROS regulome profiles of CD8+ T cells

Abstract Cells under oxidative stress regulate their functions, but current methods for detecting this stress mostly use mass spectrometry or fluorescent probes sensitive to reactive oxygen species (ROS). While valuable, these methods can't simultaneously characterize specific ROS-mediated pathways at the single-cell level. Thus, we developed an approach to characterize the ROS regulome of single-cells together with their phenotypic identity, termed single-cell ROS regulome profiling (scROP). To accomplish this, we first assessed over 100 commercially available antibodies by fluorescent cell barcoding screening and then selected a subset of ROS antibodies (n = 25). We combine our method with in vitro-activated OTI-CD8+ T cells to quantify the proteins that regulate the activity of the ROS pathway, it provides a more detailed sequential progression of how ROS regulome response through oxidative and redox pathways in different CD8+ T cell states that lead to exhaustion. Moreover, our results show that T cell exhaustion rates differ under hypoxic conditions or in the presence of antioxidants. We confirmed our findings using clinical samples, such as blood from hemodialysis patients, CD8+ T cells from CAR-T patients, and various tumor sections from hepatic carcinoma patients. Overall, our novel single-cell ROS regulome profiling approach revealed a comprehensive molecular roadmap of CD8+ T cells in response to sustained stimulations.

Theranostic Bottle-Brush Polymers Tailored for Universal Solid-Tumor Targeting.

Designing an efficient nanocarrier to target multiple types of cancer remains a major challenge in the development of cancer nanomedicines. The majority of systemically administered nanoparticles (NPs) are rapidly cleared by the liver, resulting in poor tumor-targeting efficiency and severe side effects. Here, we present a delicately tailored design and synthesis of fluorescent bottle-brush polymers and screen nine derived NPs, each varying in size and surface coatings, for tumor imaging and targeted delivery. Our optimized polymer bearing (oligo(ethylene glycol) methyl ether methacrylate) in the side chains shows reduced macrophage uptake, prolonged blood-circulation time (up to 27 h), and exceptionally high accumulation in the tumor compared to the liver, elucidating an immune-evasion-induced tumor-targeting mechanism. High tumor accumulation significantly improved the antitumor efficacy. The outstanding tumor-targeting ability has been further validated across five distinct tumor models, including orthotopic glioblastoma and pancreatic cancer, which demonstrate the universality of our polymeric nanocarrier for tumor-targeting delivery.

The emperor's new glass: The introduction of Carolingian wood ash glass in North Sea trade

AbstractMicro‐X‐ray fluorescence (XRF) screening of 244 glass sherds from Ribe, Denmark, identified 23 wood ash glasses. The closely dated finds pinpoint the arrival of the earliest wood ash glass produced in Western Europe in this important North Sea trading hub. This glass type was absent among glass from 700 to 790 CE but feature strongly from 790 to 810 CE. Electron microprobe analysis (EMPA) and laser ablation inductively coupled plasma mass spectrometry (LA‐ICP‐MS) analysis of 24 glasses show the presence of four types. We show the strengths of classifying wood ash glass based on the glass‐producing sands and conclude that the earliest production took place on a considerable scale and had a major impact on glass circulation in Northern Europe in the ninth century.

Genetic Knock-Ins of Endogenous Fluorescent Tags in RAW 264.7 Murine Macrophages Using CRISPR/Cas9 Genome Editing.

CRISPR/Cas9 genome editing is a widely used tool for creating genetic knock-ins, which allow for endogenous tagging of genes. This is in contrast with random insertion using viral vectors, where expression of the inserted transgene changes the total copy number of a gene in a cell and does not reflect the endogenous chromatin environment or any trans-acting regulation experienced at a locus. There are very few protocols for endogenous fluorescent tagging in macrophages. Here, we describe a protocol to design and test CRISPR guide RNAs and donor plasmids, to transfect them into RAW 264.7 mouse macrophage-like cells using the Neon transfection system and to grow up clonal populations of cells containing the endogenous knock-in at various loci. We have used this protocol to create endogenous fluorescent knock-ins in at least six loci, including both endogenously tagging genes and inserting transgenes in the Rosa26 and Tigre safe harbor loci. This protocol uses circular plasmid DNA as the donor template and delivers the sgRNA and Cas9 as an all-in-one expression plasmid. We designed this protocol for fluorescent protein knock-ins; it is best used when positive clones can be identified by fluorescence. However, it may be possible to adapt the protocol for non-fluorescent knock-ins. This protocol allows for the fairly straightforward creation of clonal populations of macrophages with tags at the endogenous loci of genes. We also describe how to set up imaging experiments in 24-well plates to track fluorescence in the edited cells over time. Key features • CRISPR knock-in of fluorescent proteins in RAW 264.7 mouse macrophages at diverse genomic loci. • This protocol is optimized for the use of the Neon transfection system. • Includes instructions for growing up edited clonal populations from single cells with one single-cell sorting step and efficient growth in conditioned media after cell sorting. • Designed for knocking in fluorescent proteins and screening transfected cells by FACS, but modification for non-fluorescent knock-ins may be possible.

Discovery of RORγ Allosteric Fluorescent Probes and Their Application: Fluorescence Polarization, Screening, and Bioimaging.

Retinoic acid receptor-related orphan receptor γ (RORγ) acts as a crucial transcription factor in Th17 cells and is involved in diverse autoimmune disorders. RORγ allosteric inhibitors have gained significant research focus as a novel strategy to inhibit RORγ transcriptional activity. Leveraging the high affinity and selectivity of RORγ allosteric inhibitor MRL-871 (1), this study presents the design, synthesis, and characterization of 11 allosteric fluorescent probes. Utilizing the preferred probe 12h, we established an efficient and cost-effective fluorescence polarization-based affinity assay for screening RORγ allosteric binders. By employing virtual screening in conjunction with this assay, 10 novel RORγ allosteric inhibitors were identified. The initial SAR studies focusing on the hit compound G381-0087 are also presented. The encouraging outcomes indicate that probe 12h possesses the potential to function as a powerful tool in facilitating the exploration of RORγ allosteric inhibitors and furthering understanding of RORγ function.

Microcarrier-based fluorescent yeast estrogen screen assay for fast determination of endocrine disrupting compounds

The presence of endocrine-disrupting compounds (EDCs) in water poses a significant threat to human and animal health, as recognized by regulatory agencies throughout the world. The Yeast Estrogen Screen (YES) assay is an excellent method to evaluate the presence of these compounds in water due to its simplicity and capacity to assess the bioaccessible forms/fractions of these compounds. In the presence of a compound with estrogenic activity, Saccharomyces cerevisiae cells, containing a lacZ reporter gene encoding the enzyme β-galactosidase, are induced, the enzyme is synthesised, and released to the extracellular medium. In this work, a YES-based approach encompassing the use of a lacZ reporter gene modified strain of S. cerevisiae, microcarriers as solid support, and a fluorescent substrate, fluorescein di-β-d-galactopyranoside, is proposed, allowing for the assessment of EDCs’ presence after only 2 h of incubation. The proposed method provided an EC50 of 0.17 ± 0.03 nM and an LLOQ of 0.03 nM, expressed as 17β-estradiol. The assessment of different EDCs provided EC50 values between 0.16 and 1.2 × 103 nM. After application to wastewaters, similar results were obtained for EDCs screening, much faster, compared to the conventional 45 h spectrophotometric procedure using a commercial kit, showing potential for onsite high-throughput screening of environmental contamination.

Genetic screens in Saccharomyces cerevisiae identify a role for 40S ribosome recycling factors Tma20 and Tma22 in nonsense-mediated decay.

The decay of messenger RNA with a premature termination codon by nonsense-mediated decay (NMD) is an important regulatory pathway for eukaryotes and an essential pathway in mammals. NMD is typically triggered by the ribosome terminating at a stop codon that is aberrantly distant from the poly-A tail. Here, we use a fluorescence screen to identify factors involved in NMD in Saccharomyces cerevisiae. In addition to the known NMD factors, including the entire UPF family (UPF1, UPF2, and UPF3), as well as NMD4 and EBS1, we identify factors known to function in posttermination recycling and characterize their contribution to NMD. These observations in S. cerevisiae expand on data in mammals indicating that the 60S recycling factor ABCE1 is important for NMD by showing that perturbations in factors implicated in 40S recycling also correlate with a loss of NMD.

Design and study of cavity quadrupole moment and energy spread monitor

A nondestructive method to measure beam energy spread using the quadrupole modes within a microwave cavity is proposed. Compared with a button beam position monitor (BBPM) or a stripline beam position monitor (SBPM), the cavity monitor is a narrow band pickup and therefore has better signal-to-noise ratio (SNR) and resolution. In this study, a rectangular cavity monitor is designed. TM220 mode operating at 4.76 GHz in the cavity reflects the quadrupole moment of the beam. The cavity plans to be installed behind a bending magnet in Dalian Coherent Light Source (DCLS), an extreme ultraviolet FEL facility. In this position, the beam has a larger dispersion, which is beneficial to measure the energy spread. A quadrupole magnet, a fluorescent screen, and a SBPM with eight electrodes is installed near the cavity for calibration and comparison. The systematic framework and simulation results are also discussed in this paper.

Detection of Esophageal Cancer Lesions Based on CBAM Faster R-CNN

Esophageal cancer is a common malignant tumor in daily life, which seriously affects human health. Esophageal cancer in China. The incidence rate is among the highest in the world, and there are a large number of new cases of esophageal cancer every year. At present, the diagnosis of esophageal cancer is mainly based on the use of electronic gastroscopy, which reflects the observed situation on the fluorescent screen and conducts detection through the fluorescent screen. With the increasing number of patients, the pressure and intensity of the doctor's work are getting greater and greater[1]. From the perspective of molecular level, the occurrence and development of esophageal cancer, like other cancers, are related to the activation of proto-oncogenes and the inhibition of anti-apoptosis genes. CBAM Faster R-CNN is proposed to solve the problems such as the esophageal region is not obvious and the background region occupies a large proportion in the feature map obtained by the backbone network of Faster R-CNN in barium meal imaging. CBAM is added to the convolutional attention module CBAM on the basis of the original Faster R-CNN model. To enhance the saliency of the features of the esophageal region in the feature map. CBAM Faster R-CNN model was used to train the training set after data enhancement, and Recall, Precision and AP values were used for evaluation and analysis.

A novel collapse strategy of zeolitic imidazole frameworks shell triggered by p-benzoquinone for the fluorescence monitoring α-glucosidase activity and screening natural anti-diabetes drug

Luminescent materials encapsulated into zeolite imidazole framework-8 (ZIF-8) as fluorescent probes are widely applied to detect the various analytes. Notably, these analytes react with Zn2+ through the coordination, oxidation, or acidification of 2-methylimidazole (2-MIM) to cause the degradation of ZIF-8 shell, which enormously constraints the improvement of the sensor sensitivity and selectivity. Herein, we have rationally designed the new way to collapse the shell of ZIF-8 entrapped with copper nanoclusters (CuNCs@ZIF-8) triggered by p-benzoquinone. It is worth noting that p-benzoquinone reactions with 2-MIM through hydrogen bonding interaction. As a proof-of-concept, a sensitive method for the detection of hydroquinone (HQ) is developed based on the degradation of CuNCs@ZIF-8 because HQ is easily oxidized to p-benzoquinone. The limit of detection (LOD) is estimated as low as 76 nM. Importantly, this method is highly selective for HQ over other dihydroxybenzene isomers. Inspired by the generation of HQ from hydrolysis of α-arbutin as a substrate for α-glucosidase (α-Glu), we have further proposed a straightforward α-Glu activity assay. LOD is down to 0.003 U/mL, which is lower than most of other α-Glu activity assays. Moreover, the inhibition effect of acarbose and three kinds of natural flavonoids including baicalein, silibinin, and apigenin on α-Glu activity is measured with IC50 values of 37, 155, 1940, and 2930 nM, respectively. The synergistic and antagonistic inhibition effect of α-Glu activity is also evaluated by binary combination with three types of flavonoids. This approach provides a promising strategy in clinical diagnosis and anti-diabetic drug screening.

Single-Cell Secretion Analysis via Microfluidic Cell Membrane Immunosorbent Assay for Immune Profiling.

Single-cell multiplexed phenotypic analysis expands the biomarkers for diagnosis, heralding a new era of precision medicine. Cell secretions are the primary measures of immune function, but single-cell screening remains challenging. Here, a novel cell membrane-based assay was developed using cholesterol-linked antibodies (CLAbs), integrating immunosorbent assays and droplet microfluidics to develop a flexible high-throughput single-cell secretion assay for multiplexed phenotyping. CLAb-grafted single cells were encapsulated in water-in-oil droplets to capture their own secretions. Subsequently, the cells were extracted from droplets for fluorescence labeling and screening. Multiple secretions and surface proteins were simultaneously measured from single cells by flow cytometry. To validate the approach, THP-1 cells, THP-1-derived M1 macrophages, and dendritic cells were assayed, indicating the differentiation efficiency of THP-1 cells under different chemical stimulations. Moreover, peripheral blood mononuclear cells from healthy donors under various stimuli showed varied active immune cell populations (6.62-47.14%). The peripheral blood mononuclear cells (PBMCs) of nasopharyngeal carcinoma patients were analyzed to identify a higher percentage of actively cytokine-secreted single cells in the basal state (2.82 ± 1.48%), compared with that in the health donors (0.70 ± 0.29%).

Nanoporous Pyrene-Based Metal–Organic Frameworks for Fluorescence Screening and Discrimination of Sulfonamide Antibiotics

Nanoporous Pyrene-Based Metal–Organic Frameworks for Fluorescence Screening and Discrimination of Sulfonamide Antibiotics