Fluorescence Competitive Assays Research Articles

N-Adamantyl-1-alkyl-4-oxo-1,4-dihydroquinoline-3-carboxamide Derivatives as Fluorescent Probes to Detect Microglia Activation through the Imaging of Cannabinoid Receptor Subtype 2 (CB2R).

Cannabinoid receptor subtype 2 (CB2R) is emerging as a pivotal biomarker to identify the first steps of inflammation-based diseases such as cancer and neurodegeneration. There is an urgent need to find specific probes that may result in green and safe alternatives to the commonly used radiative technologies, to deepen the knowledge of the CB2R pathways impacting the onset of the above-mentioned pathologies. Therefore, based on one of the CB2R pharmacophores, we developed a class of fluorescent N-adamantyl-1-alkyl-4-oxo-1,4-dihydroquinoline-3-carboxamide derivatives spanning from the green to the near-infrared (NIR) regions of the light spectrum. Among the synthesized fluorescent ligands, the green-emitting compound 55 exhibited a favorable binding profile (strong CB2R affinity and high selectivity). Notably, this ligand demonstrated versatility as its use was validated in different experimental settings such as flow cytometry saturation, competitive fluorescence assays, and in vitro microglia cells mimicking inflammation states where CB2R are overexpressed.

A novel competitive color-tone change fluorescence immunochromatographic assay for the ultrasensitive detection of pesticide and veterinary drug residues

One important challenge faced by competitive immunoassay techniques is the inability to visually distinguish weak color changes in the T-line during the detection of trace analyte concentrations. In this study, we introduced multi-color fluorescence into the immunoassay system and developed a novel competitive dual-channel color-tone change fluorescent immunochromatographic assay (CFICA) for the visual and quantitative detection of kanamycin (KAN) and carbendazim (CBZ). At the same time, we have developed matching, portable fluorescence-reading equipment, which can analyse the spectral data of the RGB channel individually and can effectively solve the problems of the traditional single-fluorescence reader – large reading error and poor detection sensitivity. After optimization, our CFICA, combined with the corresponding instrument, achieved low limits of detection (LOD) for KAN and CBZ, as low as 1.98 and 12.82 pg/mL, respectively. We believe that this CFICA detection system, with its high accuracy, low error, and multi-color detection capabilities, holds tremendous potential for visual and multi-channel competitive point-of-care testing.

Multifunctional Self-Signaling nanoMIP and Its Application for a Washing-Free Assay of Human Angiotensin-Converting Enzyme 2.

Molecular imprinting techniques have attracted a lot of attention as a potential biomimetic technology, but there are still challenges in protein imprinting. Herein, multifunctional nanosized molecularly imprinted polymers (nanoMIPs) for human angiotensin-converting enzyme 2 (ACE2) were prepared by epitope imprinting of magnetic nanoparticles-anchored peptide (magNP-P) templates, which were further applied to construct a competitive displacement fluorescence assay toward ACE2. A cysteine-flanked dodecapeptide sequence was elaborately selected as an epitope for ACE2, which was immobilized onto the surface of magnetic nanoparticles and served as a magNP-P template for imprinting. During polymerization, fluorescent monomers were introduced to endow fluorescence responsiveness to the prepared self-signaling nanoMIPs. A competitive displacement fluorescence assay based on the nanoMIPs was established and operated in a washing-free manner, yielding a wide range for ACE2 (0.1-6.0 pg/mL) and a low detection limit (0.081 pg/mL). This approach offers a promising avenue in the preparation of nanoMIPs for macromolecule recognition and expands potential application of an MIP in the detection of proteins as well as peptides.

Molecular Characterization of Plant Volatile Compound Interactions with Cnaphalocrocis medinalis Odorant-Binding Proteins.

Odorant-binding proteins (OBPs) play important roles in the insect olfactory system since they bind external odor molecules to trigger insect olfactory responses. Previous studies have identified some plant-derived volatiles that attract the pervasive insect pest Cnaphalocrocis medinalis (Lepidoptera: Pyralidae), such as phenylacetaldehyde, benzyl acetate, 1-heptanol, and hexanal. To characterize the roles of CmedOBPs in the recognition of these four volatiles, we analyzed the binding abilities of selected CmedOBPs to each of the four compounds, as well as the expression patterns of CmedOBPs in different developmental stages of C. medinalis adult. Antennaes of C. medinalis adults were sensitive to the studied plant volatile combinations. Expression levels of multiple CmedOBPs were significantly increased in the antennae of 2-day-old adults after exposure to volatiles. CmedOBP1, CmedOBP6, CmedPBP1, CmedPBP2, and CmedGOBP2 were significantly up-regulated in the antennae of volatile-stimulated female and male adults when compared to untreated controls. Fluorescence competition assays confirmed that CmedOBP1 could strongly bind 1-heptanol, hexanal, and phenylacetaldehyde; CmedOBP15 strongly bound benzyl acetate and phenylacetaldehyde; and CmedOBP26 could weakly bind 1-heptanol. This study lays a theoretical foundation for further analysis of the mechanisms by which plant volatiles can attract C. medinalis. It also provides a technical basis for the future development of efficient plant volatile attractants of C. medinalis.

One‐droplet saliva detection on photonic crystal‐based competitive immunoassay for precise diagnosis of migraine

AbstractMigraine exhibits a substantial prevalence worldwide. The current diagnostic criteria rests exclusively on clinical characteristics without any objective and reliable means. The calcitonin gene‐related peptide (CGRP), as a biomarker for distinguishing migraine, undergoes swift degradation, featuring a half‐life of under 10 min, which poses a significant challenge to the point‐of‐care testing of CGRP in clinical application. Here, a photonic crystal (PC)‐based biochip has been developed to detect CGRP via the fluorescence competition assay. The chip integrates the functionalities of fluorescence enhancement and hydrophilic–hydrophobic patterning enrichment, enabling rapid and sensitive detection of CGRP. After investigating the optimal enhancement distance of fluorescence near PCs, the chip allows CGRP detection using <30 μL of saliva at room temperature within 10 min. A minimum detection limit of 0.05 pg/mL is achieved. Furthermore, CGRP concentrations in the saliva of 70 subjects have been tested by PC biochips. The results exhibit strong concordance with the enzyme‐linked immunosorbent assay (ELISA), demonstrating a linear correlation coefficient of R2 of 0.97. This sensitive detection of markers within such a short duration surpasses the capacities of ELISA, which paves the way for establishing a precise diagnostic framework integrating clinical phenotypes and biomarkers for migraine.

Development of Nitroreductase‐Activatable Fluoroquinolone Prodrugs Exhibiting Attenuated Magnesium Ion Binding

AbstractFluoroquinolones such as levofloxacin and ciprofloxacin are potent antibiotics prescribed to treat various bacterial infections. Recent studies have also identified prominent examples as promising anti‐cancer agents. However, significant off‐target effects observed in human patients have precluded their wide‐spread clinical use. For instance, upon systemic administration, overt toxicity toward eukaryotic organelles (i. e., mitochondria) can result to the destruction of healthy tissue. Moreover, fluoroquinolones feature a β‐ketoacid motif which binds to and strips magnesium ions from cartilage, leading to rupture of the Achilles tendon. In this study, we have masked the β‐ketoacid of levofloxacin and ciprofloxacin (as well as two quinolones) with a nitroreductase (NTR)‐responsive warhead appended through a new self‐immolative prodrug linker. The resulting β‐ketoester functional group was found to be remarkably resistant toward spontaneous hydrolysis, esterase activity, as well as other ester‐cleaving enzymatic systems. Further testing also reveal no cross‐reactivity against commonly encountered biologically relevant analytes (e. g., ROS and thiols). Importantly, we demonstrate that upon esterification, the binding toward magnesium ions was attenuated based on an in vitro fluorescent competition assay with the unmodified parent drug.

Competitive fluorescence assay for Cd2+ based on aptamer structure-switching

Cadmium ion (Cd2+) is a heavy metal pollutant that causes serious effects on human health. Development of a simple and specific Cd2+ detection method is of great significance in food safety and environmental monitoring. In this work, we demonstrated a fluorescence assay for Cd2+ based on aptamer structure-switching. A short 17-mer aptamer against Cd2+ with fluorescein (FAM) labeled at the 5′ end was used as an affinity ligand, and a partially complementary DNA (cDNA) with black hole quencher 1 (BHQ1) labeled at the 3′ end was used as quencher. In the absence of Cd2+, the aptamer hybridized with the cDNA, forming a duplex helix structure that showed low fluorescence intensity as FAM was quenched by adjacent BHQ1. In the presence of Cd2+, the aptamer specially bound to Cd2+, and the FAM-labeled aptamer dissociated from the aptamer-cDNA duplex, showing high fluorescence intensity. We examined the effects of different lengths of cDNA, the composition of binding buffer, incubation time, and incubation temperature on the performance of the sensing system. Under the optimal experimental conditions, we achieved sensitive and selective detection of Cd2+ with a detection limit of 2.4 nM by using the 17-mer aptamer and 13-mer cDNA. The FAM-labeled aptamer exhibited a large fluorescence increase upon Cd2+, with a maximum change about 51.6-fold. The developed assay also showed good responses to Cd2+ in actual water samples, showing the potential for practical application.

Molecular analysis of NlugCSP3 with behaviorally active ligands of the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae)

The insect olfactory system is sensitive to the complicated chemical environment. Insect’s chemosensory proteins (CSPs) supposedly act as transport of plant volatiles across the sensillar lymph and competitive fluorescent binding assay was commonly used to test the binding affinities with various plant volatiles. However, extensive research to determine the physiological role of CSPs is necessary and helpful through binding interaction of a protein with the plant volatiles. In this comparative study, we employed phylogenetic analysis, fluorescence spectra, quenching mode, and thermodynamic force to characterize Nilaparvata lugens CSP3 (NlugCSP3). The phylogenetic tree revealed that amino acid sequence of NlugCSP3 showed extremely close similarities with Laodelphax striatella (LstrCSP10 & LstrCSP11) and Sogatella furcifera (SurCSP3). The Stern-Volmer (SV) curve of NlugCSP3 fluorescence quenching indicated that nonadecane and 2-tridecanone clearly quenched NlugCSP3 fluorescence as a stable static quenching mode. Meanwhile, α-terpinene and farnesene collided with NlugCSP3, instead of forming stable complexes. The thermodynamic analysis of NlugCSP3 revealed that spontaneous binding interaction occurred in nonadecane and 2-tridecanone and is driven primarily by hydrophobic interactions. This study not only provides the information about the binding interaction of protein with the volatiles, but also improves the efficient recognition of behaviorally elicited volatiles that could be used in the management of brown planthopper.

Influence of pH on indole-dependent heterodimeric interactions between Anopheles gambiae odorant-binding proteins OBP1 and OBP4

Insect Odorant Binding Proteins (OBPs) constitute important components of their olfactory apparatus, as they are essential for odor recognition. OBPs undergo conformational changes upon pH change, altering their interactions with odorants. Moreover, they can form heterodimers with novel binding characteristics. Anopheles gambiae OBP1 and OBP4 were found capable of forming heterodimers possibly involved in the specific perception of the attractant indole.In order to understand how these OBPs interact in the presence of indole and to investigate the likelihood of a pH-dependent heterodimerization mechanism, the crystal structures of OBP4 at pH 4.6 and 8.5 were determined. Structural comparison to each other and with the OBP4-indole complex (3Q8I, pH 6.85) revealed a flexible N-terminus and conformational changes in the α4-loop-α5 region at acidic pH. Fluorescence competition assays showed a weak binding of indole to OBP4 that becomes further impaired at acidic pH. Additional Molecular Dynamic and Differential Scanning Calorimetry studies displayed that the influence of pH on OBP4 stability is significant compared to the modest effect of indole. Furthermore, OBP1-OBP4 heterodimeric models were generated at pH 4.5, 6.5, and 8.5, and compared concerning their interface energy and cross-correlated motions in the absence and presence of indole. The results indicate that the increase in pH may induce the stabilization of OBP4 by increasing its helicity, thereby enabling indole binding at neutral pH that further stabilizes the protein and possibly promotes the creation of a binding site for OBP1. A decrease in interface stability and loss of correlated motions upon transition to acidic pH may provoke the heterodimeric dissociation allowing indole release.Finally, we propose a potential OBP1-OBP4 heterodimer formation/disruption mechanism induced by pH change and indole binding.

Possibility for detecting 14 typical odorants occurring in drinking water by employing human odor-binding protein OBP2a

Odor issues occurring in drinking water have been a big challenge to face for water suppliers globally, which highly commend to develop quick or on-site odor detection tools for the management of odor problems. Olfactory sensors based on odor-binding proteins (OBPs) have been utilized to analyze pollutants in food and air samples, while their application for the detection of typical odor-causing compounds in drinking water is rarely reported, partly due to the lack of knowledge about the binding properties of odorants. In this study, the binding affinity and mechanism of human odor-binding protein OBP2a to 14 typical odorants in water were first assessed using fluorescent competitive binding assays and molecular docking techniques. The 14 odorants include 7 aldehydes, 2 terpenes, 2 thioethers, bis(2-chloro-1-methylethyl) ether (DCIP), 2-ethyl-4-methyl-1,3-dioxolane (2E4MDL), and 2-isobutyl-3-methoxypyrazine (IBMP). The results showed that OBP2a could bind to 9 odorants (Ki = 29.91 μmol/L–48.36 μmol/L), including IBMP, 2-MIB, and six aldehydes (hexanal, heptanal, benzaldehyde, 2-octenal, decanal, and β-cyclocitral), among which stronger binding affinity for aldehydes is observed (Ki = 29.91 μmol/L–43.87 μmol/L). Molecular docking confirmed that Lys112 and Phe97 are major amino acid residues involved in the binding of the most target odorants. To be specific, IBMP and aldehydes can form hydrogen bonds with Lys112; aromatic ring-containing odorants such as IBMP and benzaldehyde can also form pi–pi stacking with Phe97. The binding affinity of OBP2a to fatty aldehydes including hexanal, heptanal, 2-octenal, decanal, and β-cyclocitral increased with the increase of hydrophobicity of aldehydes. The valuable information to the binding of OBP2a to typical odorants in this study would provide a theoretical foundation for the development of OBP-based odor detection biosensors to achieve quick detection in drinking water, further helping the improvement of water treatment processes in the water industry.Graphical

Fluorescent tamoxifen derivatives as biophotonic probes for the study of human breast cancer and estrogen-receptor directed photosensitizers

Currently, breast cancer is the most common type of cancer worldwide, being the so-called estrogen receptor (ER) positive the subtype with highest prevalence. For decades, the most successful strategy to prevent recurrence of ER + breast cancers is tamoxifen coadjuvant therapy. However, the involvement of the different estrogen receptors (ERα, ERβ and GPER) and the interaction in their signaling pathways, the evidence for side-effects in chronic tamoxifen treatments, as well as the appearance of tamoxifen-resistant breast cancers, have encouraged the need for novel receptor-targeted fluorescent probes. In line with this, multifunctional antiestrogen conjugates, exhibiting fluorescent properties while retaining the ability to antagonize estrogen actions, have been synthesized and proven to be particularly useful in the study of the molecular biology of ER + breast cancers. These novel fluorescent tamoxifen derivatives (FTDs) exhibit pharmacological features of pure antiestrogens, with similar or even greater affinity for ERα than tamoxifen, inhibit ER-dependent gene transcription and cell proliferation, and are devoid of uterotrophic effects. In the present study we have aim at providing a detailed view of their biophotonic potential, including their spectroscopic properties, their usefulness for fluorescent labelling of cellular compartments and intracellular targets, their application in the identification of non-ER antiestrogen binding sites thought fluorescence competition assays, and finally, in their ability to function as efficient ER-targeted photosensitizers.

Solid-Phase Screening and Synthesis of Molecularly Imprinted Nanoparticles for Selective Recognition and Detection of Brain Natriuretic Peptide.

A new recognition method is explored for the rapid detection of B-type natriuretic peptide (BNP) based on the rational design and solid-phase synthesis of molecularly imprinted nanoparticles (nanoMIP) encapsulated with carbon dots. The nanosized magnetic template is first prepared by attaching the epitope of BNP on amino-functionalized magnetic carriers. High-dilution polymerization of monomers in the presence of magnetic template generates lightly crosslinked imprinted nanoparticles. To obtain the optimal MIP formulation, a new combinatorial screening approach is developed by a competitive fluorescence assay using the magnetic template. The resultant nanoMIP exhibits high affinity and selectivity toward BNP with an equilibrium dissociation constant (KD ) of ≈10-11 m. The proposed assay allows fast BNP detection within ≈7min with a linear range of its concentration from 0.25 to 5000 pg mL-1 and a limit of detection of 0.208 pg mL-1 (S/N = 3). To demonstrate its practicability in clinical diagnosis, unknown real serum samples from 160 individuals are analyzed and the relative standard deviation is less than 4.43%. Compared with the routine electrochemiluminescence detection method that is widely used in hospital, the relative error is less than 4.98% and the correlation coefficient is 0.994.

Engineered Protein Copolymers for Heparin Neutralization and Detection.

Heparin is a widely applied anticoagulant agent. However, in clinical practice, it is of vital importance to reverse its anticoagulant effect to restore the blood-clotting cascade and circumvent side effects. Inspired by protein cages that can encapsulate and protect their cargo from surroundings, we utilize three designed protein copolymers to sequester heparin into inert nanoparticles. In our design, a silk-like sequence provides cooperativity between proteins, generating a multivalency effect that enhances the heparin-binding ability. Protein copolymers complex heparin into well-defined nanoparticles with diameters below 200 nm. We also develop a competitive fluorescent switch-on assay for heparin detection, with a detection limit of 0.01 IU mL-1 in plasma that is significantly below the therapeutic range (0.2-8 IU mL-1). Moreover, moderate cytocompatibility is demonstrated by in vitro cell studies. Therefore, such engineered protein copolymers present a promising alternative for neutralizing and sensing heparin, but further optimization is required for in vivo applications.

A competitive, bead-based assay combined with microfluidics for multiplexed toxin detection.

The requirement for rapid, in-field detection of cyanotoxins in water resources necessitates the developing of an easy-to-use and miniaturized system for their detection. We present a novel bead-based, competitive fluorescence assay for multiplexed detection of two types of toxins: microcystin-LR (MC-LR) and okadaic acid (OA). To automate the detection process, a reusable microfluidic device, termed toxin-chip, was designed and validated. The toxin-chip consists of a micromixer where the target toxins were efficiently mixed with a reagent solution, and a detection chamber for magnetic retainment of beads for downstream analysis. Quantum dots (QDs) were used as the reporter molecules to enhance the sensitivity of the assay and the emitted fluorescence signal from QDs was reversely proportional to the amount of toxins in the solution. An image analysis program was also developed to further automate the detection and analysis steps. Two toxins were simultaneously analyzed on a single microfluidic chip, and the device exhibited a low detection limit of 10-4 μg ml-1 for MC-LR and 4 × 10-5 μg ml-1 for OA detection. The bead-based, competitive assay also showed remarkable chemical specificity against potential interfering toxins. We also validated the device performance using natural lake water samples from Sunfish Lake of Waterloo. The toxin-chip holds promise as a versatile and simple quantification tool for cyanotoxin detection, with the potential of detecting more toxins.

Insertion of circularly permuted cyan fluorescent protein into the ligand-binding domain of inositol 1,4,5-trisphosphate receptor for enhanced FRET upon binding of fluorescent ligand

Binding of fluorescent ligand (FL) to the cyan fluorescent protein (CFP)-coupled ligand-binding domain of the inositol 1,4,5-trisphosphate (IP3) receptor (CFP-LBP) produces fluorescence (Förster) resonance energy transfer (FRET). A competitive fluorescent ligand assay (CFLA), using the FRET signal from competition between FLs and IP3, can measure IP3 concentration. The FRET signal should be enhanced by attaching a FRET donor to an appropriate position. Herein, we inserted five different circularly permuted CFPs in the loop between the second and third α-helices to generate membrane-targeted fluorescent ligand-binding proteins (LBPs). Two such proteins, LBP-cpC157 and LBP-cpC173, localized at the plasma membrane, displayed FRET upon binding the high-affinity ligand fluorescent adenophostin A (F-ADA), and exhibited a decreased fluorescence emission ratio (480 nm / 535 nm) by 1.6- to 1.8-fold that of CFP-LBP. In addition, binding of a fluorescent low-affinity ligand (F-LL) also reduced the fluorescence ratio in a concentration-dependent manner, with EC50 values for LBP-cpC157 and LBP-cpC173 of 34.7 nM and 27.6 nM, respectively. These values are comparable to that with CFP-LBP (29.2 nM), indicating that insertion of cpC157 and cpC173 did not disrupt LBP structure and function. The effect of 100 nM F-LL on the decrease in fluorescence ratio was reversed upon addition of IP3, indicating binding competition between F-LL and IP3. We also constructed cytoplasmic fluorescent proteins cyLBP-cpC157 and cyLBP-cpC173, and bound them to DYK beads for imaging analyses. Application of F-ADA decreased the fluorescence ratio of the beads from the periphery to the center over 3 − 5 min. Application of F-LL also decreased the fluorescence ratio of cyLBP-cpC157 and cyLBP-cpC173 by 20−25%, and subsequent addition of IP3 recovered the fluorescence ratio in a concentration-dependent manner. The EC50 value and Hill coefficient obtained by curve fitting against the IP3-dependent recovery of fluorescence ratio can be used to estimate the IP3 concentration.

Computational design and experimental confirmation of conformationally constrained peptides to compete with coactivators for pediatric PPAR[Formula: see text] by minimizing indirect readout effect.

The peroxisome proliferator-activated receptor-[Formula: see text] (PPAR[Formula: see text]) is a member of PPAR nuclear receptor family, and its antagonists have been widely used to treat pediatric metabolic disorders. Traditional type-1 and type-2 PPAR[Formula: see text] antagonists are all small-molecule compounds that have been developed to target the ligand-binding site (LBS) of PPAR[Formula: see text], which is not overlapped with the coactivator-interacting site (CIS) of PPAR[Formula: see text]. In this study, we described the rational design of type-3 peptidic antagonists that can directly disrupt PPAR[Formula: see text]-coactivator interaction by physically competing with coactivator proteins for the CIS site. In the procedure, seven reported PPAR[Formula: see text] coactivator proteins were collected and eight 11-mer helical peptide segments that contain the core PPAR[Formula: see text]-binding LXXLL motif were identified in these coactivators, which, however, possessed a large flexibility and intrinsic disorder when splitting from coactivator protein context, and thus would incur a considerable entropy penalty (i.e. indirect readout) upon binding to PPAR[Formula: see text] CIS site. By carefully examining the natively folded conformation of these helical peptides in their parent protein context and in their interaction mode with the CIS site, we rationally designed a hydrocarbon bridge across the solvent-exposed, ([Formula: see text], [Formula: see text]+ 4) residues to constrain their helical conformation, thus largely minimizing the unfavorable indirect readout effect but having only a moderate influence on favorable enthalpy contribution (i.e. direct readout) upon PPAR[Formula: see text]-peptide binding. The computational findings were further substantiated by fluorescence competition assays.

Cellular and Humoral Immunity against Different SARS-CoV-2 Variants Is Detectable but Reduced in Vaccinated Kidney Transplant Patients.

In kidney transplant (KTX) patients, immune responses after booster vaccination against SARS-CoV-2 are inadequately examined. We analyzed these patients a median of four months after a third/fourth vaccination and compared them to healthy controls. Cellular responses were analyzed by interferon-gamma (IFN-γ) and interleukin-2 (IL-2) ELISpot assays. Neutralizing antibody titers were assessed against SARS-CoV-2 D614G (wild type) and the variants alpha, delta, and omicron by a cell culture-based neutralization assay. Humoral immunity was also determined by a competitive fluorescence assay, using 11 different variants of SARS-CoV-2. Antibody ratios were measured by ELISA. KTX patients showed significantly lower SARS-CoV-2-specific IFN-γ responses after booster vaccination than healthy controls. However, SARS-CoV-2-specific IL-2 responses were comparable to the T cell responses of healthy controls. Cell culture-based neutralizing antibody titers were 1.3-fold higher in healthy controls for D614G, alpha, and delta, and 7.8-fold higher for omicron (p < 0.01). Healthy controls had approximately 2-fold higher concentrations of potential neutralizing antibodies against all 11 variants than KTX patients. However, more than 60% of the KTX patients displayed antibodies to variants of SARS-CoV-2. Thus, KTX patients should be partly protected, due to neutralizing antibodies to variants of SARS-CoV-2 or by cross-reactive T cells, especially those producing IL-2.

Detection of quinocetone in animal feed by quantum dots‐based indirect competitive fluorescent immunosorbent assay

AbstractA sensitive monoclonal antibody against quinocetone (QCT) was produced. Subsequently, the quantum dots‐based indirect competitive fluorescent immunosorbent assay (ic‐FLISA) for the rapid detection of QCT in animal feed was developed. The 50% inhibitory concentration (IC50) was 4.39 ng/ml, the limit of detection was 0.252 ng/ml, and the detection ranged from 0.515 to 37.150 ng/ml. The recoveries of the spiked QCT in the animal feed ranged from 96.48 to 102.52% and the coefficient of variation ranged from 2.20 to 6.59%. The established ic‐FLISA has high sensitivity which can provide an ideal tool to detect QCT in animal feed.

Cellular and Humoral Immunity after the Third Vaccination against SARS-CoV-2 in Hematopoietic Stem-Cell Transplant Recipients.

Protecting vulnerable groups from severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection is mandatory. Immune responses after a third vaccination against SARS-CoV-2 are insufficiently studied in patients after hematopoietic stem-cell transplantation (HSCT). We analyzed immune responses before and after a third vaccination in HSCT patients and healthy controls. Cellular immunity was assessed using interferon-gamma (IFN-γ) and interleukin-2 (IL-2) ELISpots. Furthermore, this is the first report on neutralizing antibodies against 11 variants of SARS-CoV-2, analyzed by competitive fluorescence assay. Humoral immunity was also measured by neutralization tests assessing cytopathic effects and by ELISA. Neither HSCT patients nor healthy controls displayed significantly higher SARS-CoV-2-specific IFN-γ or IL-2 responses after the third vaccination. However, after the third vaccination, cellular responses were 2.6-fold higher for IFN-γ and 3.2-fold higher for IL-2 in healthy subjects compared with HSCT patients. After the third vaccination, neutralizing antibodies were significantly higher (p < 0.01) in healthy controls, but not in HSCT patients. Healthy controls vs. HSCT patients had 1.5-fold higher concentrations of neutralizing antibodies against variants and 1.2-fold higher antibody concentrations against wildtype. However, half of the HSCT patients exhibited neutralizing antibodies to variants of SARS-CoV-2, which increased only slightly after a third vaccination.

Screening of Non‐Purine Ligands Binding to Adenosine Receptors A1, A2A, A2B, and A3

Adenosine receptors (ARs) are G‐protein coupled receptors (GPCRs) which have been shown to have therapeutic potential. ARs are made up of four subtypes, A1, A2A, A2B, and A3. A3AR ligands have previously been indicated to lower inflammation, prevent or treat cancer, and produce antihyperalgesic effects in many preclinical pain models, specifically for neuropathy. We have previously published in animal models that ferulic acid dimer, a non‐purine compound with the 3‐methoxy phenyl moiety, has non‐opioid antinociceptive properties through primarily binding to A3ARs. Current literature suggests that the antinociceptive properties of a natural compound, incarvillateine, which contains the 3‐methoxy phenyl moiety is mediated through ARs, however there is a lack of sufficient evidence of in vitro binding of such compounds at ARs. We thus hypothesized that compounds with the 3‐methoxy phenyl moiety show binding to ARs, with an affinity in the submicromolar range. For this study, we performed fluorescent competitive binding assays using cells transfected with ARs: Chinese Hamster Ovary (CHO) cells for A1 or A3 receptors and Human Embryonic Kidney (HEK) cells for A2A or A2B receptors. The compounds tested included trans cinnamic acid analogs and curcumin analogs. The compounds were synthesized using cavitand‐mediated photoisomerization and cavitand‐mediated photodimerization. Our fluorescent binding protocol employed the separation method, in which the unbound ligand was removed after equilibrium was reached, via PBS wash. The binding assays performed suggested that 3‐methoxy cinnamic acid dimer was the most effective in binding to A3ARs, while the monomer did not bind. There was very little to no binding observed at the other receptors. Other analogs of trans cinnamic acid and curcumin did not show binding at any of the ARs so far. In conclusion, 3‐methoxy cinnamic acid dimer binds to G‐inhibitory coupled adenosine receptor, A3. Future studies will investigate structure‐activity‐relationship of 3‐methoxy phenyl dimers to demonstrate why dimerization leads to superior binding compared to similar monomeric compounds.