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This study describes the applicability of the fluorescence polarization assay (FPA) based on the use of FITC-labeled oligosaccharide tracers of defined structure for the measurement of active lysozyme in hen egg white. Depending on the oligosaccharide chain length of the tracer, this method detects both the formation of the enzyme-to-tracer complex (because of lectin-like, i.e., carbohydrate-binding action of lysozyme) and tracer splitting (because of chitinase activity of lysozyme). Evaluation of the fluorescence polarization dynamics enables simultaneous measurement of the chitinase and lectin activities of lysozyme, which is crucial for its detection in complex biological systems. Hen egg white lysozyme (HEWL), unlike human lysozyme (HL), formed a stable complex with the chitotriose tracer that underwent no further transformations. This fact allows for easy measurement of the carbohydrate-binding activity of the HEWL. The results of the lysozyme activity measurement for hen egg samples obtained through the FPA correlated with the results obtained using the traditional turbidimetry method. The FPA does not have the drawbacks of turbidimetry, which are associated with the need to use bacterial cells that cannot be precisely standardized. Additionally, FPA offers advantages such as rapid analysis, the use of compact equipment, and standardized reagents. Therefore, the new express technique for measuring the lysozyme activity is applicable for evaluating the complex biomaterial, including for the purposes of food product quality control.

Since Chagas disease, melioidosis, and Legionnaires' disease are all potentially life-threatening infections, there is an urgent need for new treatment strategies. All causative agents, Trypanosoma cruzi , Burkholderia pseudomallei , and Legionella pneumophila , express a virulence factor, the macrophage infectivity potentiator (MIP) protein, emerging as a promising new therapeutic target. Inhibition of MIP proteins having a peptidyl-prolyl isomerase activity leads to reduced viability, proliferation, and cell invasion. The affinity of a series of pipecolic acid-type MIP inhibitors was evaluated against all MIPs using a fluorescence polarization assay. The analysis of structure-activity relationships led to highly active inhibitors of MIPs of all pathogens, characterized by a one-digit nanomolar affinity for the MIPs and a very effective inhibition of their peptidyl-prolyl isomerase activity. Docking studies, molecular dynamics simulations, and quantum mechanical calculations suggest an extended σ-hole of the meta -halogenated phenyl sulfonamide to be responsible for the high affinity.

Rapid and specific diagnosis is necessary for both the treatment and prevention of infectious diseases. Bacteria and viruses that enter the bloodstream can trigger a strong immune response in infected animals and humans. The fluorescence polarization assay (FPA) is a rapid and accurate method for detecting specific antibodies in the blood that are produced in response to infection. One of the first examples of FPA is the non-competitive test for detecting brucellosis in animals, which was followed by the development of other protocols for detecting various infections. Fluorescently labeled polysaccharides (in the case of brucellosis and salmonellosis) or specific peptides (in the case of tuberculosis and salmonellosis, etc.) can be used as biorecognition elements for detecting infections. The availability of new laboratory equipment and mobile devices for fluorescence polarization measurements outside the laboratory has stimulated the development of new fluorescence polarization assays (FPAs) and the emergence of commercial kits on the market for the detection of brucellosis, tuberculosis, and equine infectious anemia viruses. It has been shown that, in addition to antibodies, the FPA method can detect both viruses and nucleic acids. The development of more specific and sensitive biomarkers is essential for the diagnosis of infections and therapy monitoring. This review summarizes studies published between 2003 and 2023 that focus on the detection of infections using FPA. Furthermore, it demonstrates the potential for using new biorecognition elements (e.g., aptamers, proteins, peptides) and the combined use of FPA with new technologies, such as PCR and CRISPR/Cas12a systems, for detecting various infectious agents.

Brucellosis in animals is an infectious disease caused by bacteria of the genus Brucella . Known methods for diagnosing brucellosis face some challenges, due to the difficulties in isolating and standardizing the natural brucellosis antigen. In this work, we investigated the possibility of using the fluorescence polarization assay (FPA) with synthetic glycoconjugate biosensing tracers to detect antibodies against Brucella as a new methodology for diagnosing brucellosis. Based on the received results, the synthetic fluorescein-labeled trisaccharide tracer is most effective for Brucellosis detection. This tracer is structurally related to the immune determinant fragment of the Brucella LPS buildup of N-formyl-d-perosamine units, connected via α-(1→3)-linkage at the non-reducing end and α-(1→2)-linkage at the reducing end. The sensitivity and specificity in the case of the use of trisaccharide tracer 3b were 71% and 100% (Yuden's method) and 87% and 88% (Euclidean method), respectively, which is comparable with the diagnostic efficiency of traditionally used serological methods, such as the agglutination test (AT), complement fixation test (CFT), and Rose Bengal test (RBT). Given the known advantages of FPA (e.g., speed, compactness of the equipment, and standard reagents) and the increased specificity of the developed test system, it would be appropriate to consider its widespread use for the diagnosis of brucellosis in animals, including rapid testing in the field.

A recognition motif is vital in determining the specificity and sensitivity of the fluorescence polarization assay (FPA) for detecting chemical contaminants in food. Four candidates (Gyrase, GyrBA, TopIV, and QepA) were prepared for this study. The applicability of QepA was confirmed through DNA cleavage assay, inhibition effects, and mechanism investigations using molecular docking, compared to other counterparts. Finally, a novel FPA based on QepA and a CIP-FITC tracer for the detection of fluoroquinolones (FQs) in eggs was developed. The limits of detection (LODs) for eight fluoroquinolones ranged from 2.2 to 5.1 ng g -1 , with enrofloxacin, danofloxacin, and difloxacin meeting the maximum residue limits (MRLs). The spiked recoveries ranged from 65.8 to 103.6% with coefficients of variation (CVs) of 5.4-12.8%. Therefore, a new recognition motif for FQs that did not belong to conventional antibodies was identified, and QepA-based FPA could be a potential tool for rapid, homogeneous, and sensitive monitoring of the residue of FQs in eggs.

Introduction: Therapeutic peptides are a significant class of drugs in the treatment of a wide range of diseases. To enhance their properties, such as stability or binding affinity, they are usually chemically modified. This includes, among other techniques, cyclization of the peptide chain by bridging, modifications to the backbone, and incorporation of unnatural amino acids. One approach previously established, is the use of halogenated aromatic amino acids. In principle, they are thereby enabled to form halogen bonds (XB). In this study, we focus on the -R-CF 2 X moiety (R = O, NHCO; X = Cl, Br) as an uncommon halogen bond donor. These groups enable more spatial variability in protein-protein interactions. The chosen approach via Fmoc-protected building blocks allows for the incorporation of these modified amino acids in peptides using solid-phase peptide synthesis., Results and Discussion: Using a competitive fluorescence polarization assay to monitor binding to Mdm4, we demonstrate that a p53-derived peptide with Lys24Nle(εNHCOCF 2 X) exhibits an improved inhibition constant K i compared to the unmodified peptide. Decreasing K i values observed with the increasing XB capacity of the halogen atoms (F ≪ Cl < Br) indicates the formation of a halogen bond. By reducing the side chain length of Nle(εNHCOCF 2 X) to Abu(γNHCOCF 2 X) as control experiments and through quantum mechanical calculations, we suggest that the observed affinity enhancement is related to halogen bond-induced intramolecular stabilization of the α-helical binding mode of the peptide or a direct interaction with His54 in human Mdm4., Competing Interests: The authors report no conflicts of interest in this work., (© 2023 Vaas et al.)

In this study, the identity of our recently produced natural TetR protein was identified by using the LC-ESI-MS/MS technique, and its recognition mechanisms, including the binding pocket, contact amino acids, intermolecular forces, binding sites, binding energies, and affinities for 10 tetracycline drugs were studied. Then, it was evolved by site-mutagenesis of an amino acid to produce a mutant, and a fluorescence polarization assay was developed to detect the 10 drugs in milk. The sensitivities for the 10 drugs were improved with IC 50 values decreasing from 30.8-80.1 ng/mL to 15.5-55.2 ng/mL, and the limits of detection were in the range of 0.4-1.5 ng/mL. Furthermore, it was found that the binding affinity for a drug was the critical factor determining its sensitivity, and the binding energy showed little influence. This is the first study reporting the recognition mechanisms of a natural TetR protein for tetracyclines and the development of a fluorescence polarization assay for the detection of tetracyclines residues in food samples., Competing Interests: All of the authors declare that they have no conflicts of interest. This article does not contain any studies with animals performed by any of the authors. This article does not contain any studies with humans. All the authors listed in the manuscript are entitled to the authorship and have approved the final version of the submitted manuscript.

The residues of aminoglycosides in foods of animal origin are a potential risk to consumers. There have been some immunoassays reported for the screening of aminoglycoside residues, but the method showing the broadest detection spectrum can only be used to detect two drugs. This is because a broad specific recognition reagent is not available. In the present study, the receptor of aminoglycosides (ribosomal protein S12 of Lysinibacillus ) was expressed, and its affinities and recognition mechanisms for 10 aminoglycosides were studied by using surface plasmon resonance and molecular docking, respectively. Then the receptor was used as a recognition reagent to develop a fluorescence polarization assay on a 96-well microplate for the detection of the 10 drugs in pork muscle samples. The limits of detection for the 10 drugs ranged from 5.25 to 30.25 ng/g. The sensitivities for the 10 drugs were generally consistent with their respective receptor affinities and binding energies. After comprehensive comparison, the method performances were better than all the previously reported immunoassays for aminoglycosides. This is the first study reporting the recognition mechanisms of ribosomal protein S12 of sphaericus ) was expressed, and its affinities and recognition mechanisms for 10 aminoglycosides were studied by using surface plasmon resonance and molecular docking, respectively. Then the receptor was used as a recognition reagent to develop a fluorescence polarization assay on a 96-well microplate for the detection of the 10 drugs in pork muscle samples. The limits of detection for the 10 drugs ranged from 5.25 to 30.25 ng/g. The sensitivities for the 10 drugs were generally consistent with their respective receptor affinities and binding energies. After comprehensive comparison, the method performances were better than all the previously reported immunoassays for aminoglycosides. This is the first study reporting the recognition mechanisms of ribosomal protein S12 of Lysinibacillus sphaericus for 10 aminoglycosides and the use of it as a recognition reagent to develop a pseudo-immunoassay for the multi-determination of aminoglycosides in food samples.

The milk ring test is a detection assay for antibodies against Brucella in bovine milk. It has good sensitivity but tends to give false positive results. In this study, we standardized the application of the fluorescence polarization assay (FPA) for the detection of antibodies against B.melitensis in goat milk. We obtained negative serum and milk samples from healthy goat flocks in the northern zone of Nuevo León. Positive milk and negative, weak, and strong controls were obtained by mixing volumes of positive control serum with negative control milk. Milk samples were treated with citric acid, after which an FPA was performed. Results were then compared with the Rose Bengal test and the FPA in serum. Milk treatment allowed the quantification of antibodies in samples. Significant differences were found between the 2%, 4%, and 6% groups, compared with the control group (F3, 67 = 17.45, p < 0.0001) but not between the 2% and 4% groups (p = 0.0718). The cut-off value was 74.1 mP, with a sensitivity (Se) of 95% and a specificity (Sp) of 100%. Se and Sp values in field milk samples were 84% and 74.55%, respectively. Despite the FPA test on milk samples showed lower Se and Sp than the FPA test on serum samples, its cutoff may be adjusted. It may be recommended as a screening test in goat milk and become useful for the control and eradication of the disease.

Background: Bovine tuberculosis (bTB) is a major bacterial disease that causes significant economic disruption across the globe., Aims: Our study was based on using a fluorescence polarization assay (FPA) that used fluorescein-labeled ESAT-6 protein to detect Mycobacterium bovis antibodies in bovine serum., Methods: The ESAT-6 protein was used in a FPA. Positive TB reactors were determined by the comparative intradermal test (CID) and interferon gamma test (IFN-γ). Antibodies against M. bovis were detected using a fluorescein isothiocyanate (FITC) labeled tracer and a whole culture FITC labeled tracer in the positive cattle., Results: Of the 192 animals tested for bTB, 37 were found to be positive by either the CID or IFN-γ assays. Using the mP values from five culture-positive serum samples, a cutoff value of more than >127 mp provided the best discrimination between positive reactors and negative bTB animals. The ESAT-6 results of FPA in comparison with CID results revealed sensitivity of 92.9% and specificity of 64.6%, and in comparison with results IFN-γ, showed sensitivity of 95.7% and specificity of 49%. FPA using FITC labelled ESAT-6 as a tracer has better sensitivity (95.7%) and specificity (49.1%) than IFN-γ test in humoral immune response in animals., Conclusion: This work revealed that the ESAT-6 protein as an antigen can be used in diagnosing bTB using a practical and sensitive humoral test., Competing Interests: There is no conflict of interest.

In this study, the dihydropteroate synthase of Staphylococcus aureus was obtained, and its recognition mechanisms for 31 sulfonamide drugs were studied. Results showed that their core structure matched well with the binding pocket of para -aminobenzoic acid, and all the sulfonamide side chains were out of the binding pocket. Hydrogen bonds and hydrophobic interactions were the main intermolecular forces, and the key amino acids were Gly171 and Lys203. The binding sites in sulfonamide molecules were mainly around the para -aminobenzenesulfonamide part. This enzyme was used to develop a fluorescence polarization assay for detection of these drugs in chicken muscles. The change trends of half of inhibition concentrations and cross-reactivities for the 31 drugs were identical with the receptor-ligand affinities. The limits of detection were in the range of 2.0-38.5 ng/g, and one assay could be finished within several minutes. Therefore, this method could be used for multiscreening of sulfonamide residues in meat samples.

O-GlcNAc transferase (OGT) is involved in many cellular processes, and selective OGT inhibitors are valuable tools to investigate O-GlcNAcylation functions, and could potentially lead to therapeutics. However, high-throughput OGT assays that are suitable for large-scale HTS and can identify inhibitors targeting both acceptor, donor sites, and allosteric binding-sites are still lacking. Here, we report the development of a high-throughput "FP-Tag" OGT assay with bovine serum albumin (BSA) as a low-cost and superior "FP-Tag". With this assay, 2-methyleurotinone was identified as a low-micromolar OGT inhibitor. This type of assay with BSA as "FP-Tag" would find more applications with other glycosyltransferases., (© 2020 Wiley-VCH GmbH.)

We evaluated hemolyzed, bacterially contaminated, and Nobuto filter paper-derived serum, collected from 50 Rocky Mountain elk (Cervus elaphus nelson) in 2017 and 2019, divided into eight treatments to determine antibody retention. Serum was analyzed on Brucella abortus-specific fluorescence polarization assay utilizing plates and tubes. Reference titers and serostatus were compared to serum held at 22 C for 4, 8, 12, and 16 d; frozen clotted blood; blood with 2% and 10% elk rumen content (held for 8 d at 22 C); and serum eluted from Nobuto filter paper. Using Cohen's kappa test of agreement, plate assay serostatus agreement was substantial or outstanding in all treatments. Serostatus agreement was outstanding in all treatments utilizing tubes. The mean change in score (treatment minus reference) showed significant negative bias in serosuspect or seropositive animals in the frozen, 2% rumen, and 10% rumen treatments on the plate assay, and the day 16 and 10% rumen treatments on the tube assay, that could ultimately result in an animal being misclassified into a serosuspect or seronegative category. Serum eluted from Nobuto filter paper produced inconsistent results and is not recommended as an alternative to serum derived from blood. Although the potential for misclassification of animals with low titers exists, analyzing hemolyzed and bacterially contaminated serum from Brucella abortus nonendemic areas can increase sample size and the potential to detect seropositive animals., (© Wildlife Disease Association 2021.)

Background: Brucellosis is an infectious-allergic zoonotic disease caused by bacteria of the genus Brucella. Early diagnosis is the key to preventing, treating, and controlling brucellosis. Fluorescence polarization immunoassay (FPA) is a new immunoassay for relatively rapid and accurate detection of antibodies or antigens based on antigen-antibody interaction. However, there is no report on FPA-based detection of human brucellosis in China. Therefore, this study is to evaluate the value of FPA for the diagnosis of human brucellosis in China., Methods: We recruited 320 suspected brucellosis cases who had the clinical symptoms and epidemiological risk factors between January and December, 2019. According to China Guideline for Human Brucellosis Diagnosis, the Rose Bengal test (RBT) was used for the screening test, and the serum agglutination test (SAT) was used as the confirmatory test. Brucellosis was confirmed only if the results of both tests were positive. Additionally, FPA and enzyme linked immune sorbent assay (ELISA) were compared with SAT, and their sensitivity, specificity, coincidence rate and consistency coefficient (Kappa value) as diagnostic tests were analyzed individually and in combination. The optimal cut-off value of FPA was also determined using the receiver operator characteristic (ROC) curve., Results: The optimum cut-off value of FPA was determined to be 88.5 millipolarization (mP) units, with a sensitivity of 94.5% and specificity of 100.0%. Additionally, the coincidence rate with the SAT test was 96.6%, and the Kappa value (0.9) showed excellent consistency. The sensitivity and specificity of FPA and ELISA combined were higher at 98.0% and 100.0% respectively., Conclusions: When the cut-off value of FPA test is set at 88.5 mP, it has high value for the diagnosis of brucellosis. Additionally, when FPA and ELISA are combined, the sensitivity of diagnosis is significantly improved. Thus, FPA may have potential in the future as a diagnostic method for human brucellosis in China.

We evaluated serologic responses of cattle, bison, elk, and swine representing negative control, early vaccination (4-8 wk), late vaccination (21-28 wk) or booster vaccination, early after-experimental challenge (2-4 wk), and late after-experimental challenge (8-21 wk), in a brucellosis fluorescence polarization assay (FPA; n = 10 sera per species per treatment) using negative control sera from cattle, bison, elk, and swine ( n = 5 per species). Sera from cattle shedding Brucella abortus strain RB51 in milk were also evaluated against the 20 negative control sera. The species of negative control sera used in the FPA could increase ( p < 0.05) delta millipolarization (mP; delta mP = sample mP - negative control mP) results. In general, the species of negative control sera did not alter the interpretation of FPA results in control, vaccinated, or infected animals. Even after repeated RB51 vaccinations in bison, cattle, or elk, or in cattle shedding RB51 in milk, serologic results from the FPA remained negative. Species differences in FPA results were noted; elk developed robust humoral responses very quickly after infection that resulted in strong positive FPA results. In cattle and bison, humoral responses appeared to develop over a longer period of time, and greater delta mP values were detected at later times after infection. Sensitivity of the FPA for detecting infected animals was greatest for elk in early challenge samples and bison in late challenge samples. Our data suggest that species of origin of negative control sera does not influence interpretation of the FPA in natural hosts of Brucella abortus .

The mitogen-activated protein kinase kinase 4 (MKK4) plays a key role in liver regeneration and is under investigation as a target for stimulating hepatocytes to increased proliferation. Therefore, new small molecules inhibiting MKK4 may represent a promising approach for treating acute and chronic liver diseases. Fluorescently labeled compounds are useful tools for high-throughput screenings of large compound libraries. Here we utilized the azaindole-based scaffold of FDA-approved BRAF inhibitor vemurafenib 1, which displays off-target activity on MKK4, as a starting point in our fluorescent compound design. Chemical variation of the scaffold and optimization led to a selection of fluorescent 5-TAMRA derivatives which possess high binding affinities on MKK4. Compound 45 represents a suitable tool compound for Fluorescence polarization assays to identify new small-molecule inhibitors of MKK4., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: SL is stock owner <3% of Heparegenix GmbH. However this relationship has not influenced any work related to this manuscript., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

The phytohormone ethylene is widely involved in many developmental processes and is a crucial regulator of defense responses against biotic and abiotic stresses in plants. Ethylene-responsive element binding protein, a member of the APETALA2/ethylene response factor (AP2/ERF) superfamily, is a transcription factor that regulates stress-responsive genes by recognizing a specific cis-acting element of target DNA. A previous study showed only the NMR structure of the AP2/ERF domain of AtERF100 in complex with a GCC box DNA motif. In this report, we determined the crystal structure of AtERF96 in complex with a GCC box at atomic resolution. We analyzed the binding residues of the conserved AP2/ERF domain in the DNA recognition sequence. In addition to the AP2/ERF domain, an N-terminal α-helix of AtERF96 participates in DNA interaction in the flanking region. We also demonstrated the structure of AtERF96 EDLL motif, a unique conserved motif in the group IX of AP2/ERF family, might involve in the transactivation of defense-related genes. Our study establishes the structural basis of the AtERF96 transcription factor in complex with the GCC box, as well as the DNA binding mechanisms of the N-terminal α-helix and AP2/ERF domain.

The transcription factor ΔFosB accumulates in response to chronic insults such as drugs of abuse, L-3,4-dihydroxyphenylalanine (l-DOPA) or stress in specific regions of the brain, triggering long lasting neural and behavioral changes that underlie aspects of drug addiction, dyskinesia, and depression. Thus, small molecule chemical probes are urgently needed to investigate biological functions of ΔFosB. Herein we describe the identification of a novel phenanthridine analogue ZL0220 (27) as an active and promising ΔFosB chemical probe with micromolar inhibitory activities against ΔFosB homodimers and ΔFosB/JunD heterodimers., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Aim: The [1,2,4]triazolo[1,5- a ]pyrimidine core is highly privileged in medicinal chemistry due to its versatile pharmacological activity profile. Recently, the search for novel anticancer agents has focused on [1,2,4]triazolo[1,5- a ]pyrimidine derivatives. Results: Our hit functionalization has led to the discovery of new [1,2,4]triazolo[1,5- a ]pyrimidinium salts with potential anticancer activity. Among a small library of molecules, compound 9 significantly inhibits cancer cell growth in a panel of in vitro models. Molecular docking studies and preliminary binding assay have displayed that  9 could directly bind the Src homology 2 (SH2) domain of STAT3 protein. Conclusion: Compound 9 is a novel promising lead compound that motivates additional evaluation of [1,2,4]triazolo[1,5- a ]pyrimidinium salts as novel potential chemotherapeutics.

The eight mammalian Src-family tyrosine kinases are dynamic, multi-domain structures, which adopt distinct "open" and "closed" conformations. In the closed conformation, the regulatory SH3 and SH2 domains pack against the back of the kinase domain, providing allosteric control of kinase activity. Small molecule ligands that engage the regulatory SH3-SH2 region have the potential to modulate Src-family kinase activity for therapeutic advantage. Here we describe an HTS-compatible fluorescence polarization assay to identify small molecules that interact with the unique-SH3-SH2-linker (U32L) region of Hck, a Src-family member expressed exclusively in cells of myeloid lineage. Hck has significant potential as a drug target in acute myeloid leukemia, an aggressive form of cancer with substantial unmet clinical need. The assay combines recombinant Hck U32L protein with a fluorescent probe peptide that binds to the SH3 domain in U32L, resulting in an increased FP signal. Library compounds that interact with the U32L protein and interfere with probe binding reduce the FP signal, scoring as hits. Automated 384-well high-throughput screening of 60,000 compounds yielded Z'-factor coefficients > 0.7 across nearly 200 assay plates, and identified a series of hit compounds with a shared pyrimidine diamine substructure. Surface plasmon resonance assays confirmed direct binding of hit compounds to the Hck U32L target protein as well as near-full-length Hck. Binding was not observed with the individual SH3 and SH2 domains, demonstrating that these compounds recognize a specific three-dimensional conformation of the regulatory regions. This conclusion is supported by computational docking studies, which predict ligand contacts with a pocket formed by the juxtaposition of the SH3 domain, the SH3-SH2 domain connector, and the SH2-kinase linker. Each of the four validated hits stimulated recombinant, near-full-length Hck activity in vitro , providing evidence for allosteric effects on the kinase domain. These results provide a path to discovery and development of chemical scaffolds to target the regulatory regions of Hck and other Src family kinases as a new approach to pharmacological kinase control., (Copyright © 2019 Dorman, Close, Wingert, Camacho, Johnston and Smithgall.)

Brucellosis is a zoonosis of economic and public health concern. While most diagnostic tests for brucellosis can only be performed in the laboratory, the Fluorescence Polarization Assay (FPA) was developed as a rapid point-of-care field test. This pilot project aimed to validate the use of FPA for rapid diagnosis of ruminant brucellosis on the field, and to compare the FPA performance with that of the more commonly used Rose Bengal Test (RBT). Blood samples were first collected from ruminants in a livestock market, and later from a nearby slaughterhouse in Port Bouët, Abidjan, Côte d'Ivoire. Samples collected in the livestock market were processed and tested with the FPA in a central laboratory, while samples collected in the slaughterhouse were processed immediately and the FPA was performed on site. To assess the FPA intra-test agreement, a portion of the serum samples tested at the slaughterhouse were re-tested with the FPA in the laboratory later the same day. To assess inter-test agreement, all serum samples were retested with the RBT. A total of 232 samples were tested with the FPA, 106 and 126 from the livestock market and slaughterhouse, respectively. Of these, 26 tested positive and 39 were doubtful for brucellosis. The FPA was repeated on 28 of the samples collected at the slaughterhouse, and comparison of results indicated a moderate intra-test agreement (Kappa = 0.41). The agreement improved when the doubtful category was treated as negative (Kappa = 0.65), and when cattle were excluded (Kappa = 0.56 to 0.61). The RBT was performed on 229 samples, and of these 10 tested positive. A comparison of FPA and RBT results indicated poor agreement (Kappa = 0.00); this improved to slight when only samples taken at the livestock market and tested in the laboratory were considered (Kappa = 0.14). The FPA did not perform well in tropical field conditions, possibly due to the high ambient temperatures in the slaughterhouse. Moreover, a difference in performance was noted in relation to the species tested, whereby the FPA seemed to perform better on sheep and goat samples, compared to cattle samples. These findings highlight that further adjustments are needed before implementing the FPA on the field.

Based on streptavidin coated nanospheres and T7 exonuclease assisted dual-cycle signal amplification, we developed a novel sensitive fluorescence polarization detection method for miRNA. When target miRNA was present in the system, hairpin probe hybridized with miRNA, forming a double-stranded structure. The 5' end of hairpin probe was then recognized and digested by T7 exonuclease, releasing the non-degraded single strand DNA fragments and miRNA. The released target miRNA could trigger the next cycle of hybridization and digestion, releasing more non-degraded fragments from hairpin probe. The fragments could hybridize with a signal probe (with carboxyfluorescein modification at 5'-end and biotin modification at 3'-end). The formed blunt 5'-end of signal probe was then recognized and degraded by T7 exonuclease, releasing the fragments and the fluorophore carboxyfluorescein. The next cycle of hybridization and digestion of signal probe was triggered by the released fragment at the same time. The free carboxyfluorescein cannot connect with streptavidin coated nanospheres which were used as the fluorescence polarization signal amplifier. So, there was a big change of fluorescence polarization signal after adding miRNA into the detection system, due to the different fluorescence polarization signal between nanospheres-captured intact signal probe and free carboxyfluorescein. The detection limit of this method is about 0.001 nM, and it has a good selectivity. In addition, it was also applicable to determine target miRNA in total miRNA extracts and compare the expression level of target miRNA in different cells. Consequently, the proposed method is expected to be used for the potential cancer diagnosis and the related biomedical research., (Copyright © 2019 Elsevier B.V. All rights reserved.)

This paper demonstrates the use of a smartphone-based sensor for fluorescence polarization (FP) analysis of biomolecules. The FP detection can rapidly sense ligand-analyte bindings by measuring molecule mobility, and thus, FP-based assays have been widely used for rapid diagnostics in clinics. Here, we implemented the FP detection apparatus using a 3D-printed compact holder and the built-in camera of a smartphone. The system offers accurate measurements of the degree of polarization by simultaneously detecting the fluorescence intensities parallel and perpendicular to the polarization of the excitation. The fluorescence signal of the sample is excited by a laser or light-emitting diode and separated by a polarization beam cube depending on the polarization. Parallel and perpendicular polarized emissions are projected onto two different regions of the sensor chip in the smartphone camera. A custom software app was developed to count the average intensity in the areas of interest and compute the degree of polarization. We validated the system by measuring the polarization of dye molecules dissolved in solutions with different viscosities. As an example of biomolecule sensing, a competitive FP immunoassay of Prostaglandin E 2 was demonstrated using the developed system and exhibited the limit of detection of 1.57 ng/mL. The smartphone-based FP assay platform can also be implemented for the detection of toxins, disease biomarkers, and pathogens in resource-limited settings., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Self-propagating amyloid-β (Aβ) aggregates or seeds possibly drive pathogenesis of Alzheimer's disease (AD). Small molecules targeting such structures might act therapeutically in vivo. Here, a fluorescence polarization assay was established that enables the detection of compound effects on both seeded and spontaneous Aβ42 aggregation. In a focused screen of anti-amyloid compounds, we identified Disperse Orange 1 (DO1) ([4-((4-nitrophenyl)diazenyl)-N-phenylaniline]), a small molecule that potently delays both seeded and non-seeded Aβ42 polymerization at substoichiometric concentrations. Mechanistic studies revealed that DO1 disrupts preformed fibrillar assemblies of synthetic Aβ42 peptides and decreases the seeding activity of Aβ aggregates from brain extracts of AD transgenic mice. DO1 also reduced the size and abundance of diffuse Aβ plaques and decreased neuroinflammation-related gene expression changes in brains of 5xFAD transgenic mice. Finally, improved nesting behavior was observed upon treatment with the compound. Together, our evidence supports targeting of self-propagating Aβ structures with small molecules as a valid therapeutic strategy., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Brucellosis caused by the bacteria of the genus Brucella is an important zoonosis and constitutes a serious public health hazard. Brucellosis is diagnosed mainly by the Rose Bengal plate test and indirect ELISA, both of which have poor specificity because false positive serological reactions occur when screening animals that have been vaccinated with B. abortus S19. Fluorescence polarization assay (FPA) was evaluated for screening samples from cattle and buffalo calves with persistent antibody titers induced by vaccination. The standardized FPA exhibited relative sensitivity and specificity of 0.94 and 0.95, respectively, and the area under the curve, kappa and accuracy were 0.98, 0.87 and 0.95, respectively. Comparison of competitive ELISA and FPA revealed that, FPA is more specific than competitive ELISA. The high specificity, sensitivity and 95% accuracy of FPA indicate that, it is suitable for testing vaccinated animals because it can distinguish between infected from vaccinated animals., (© 2018 The Societies and John Wiley & Sons Australia, Ltd.)

Nucleic acid binding by the Argonaute protein is an important trigger step in the Argonaute-dependent gene silencing system. We established an in vitro method to detect the nucleic acid binding activity of the Argonaute protein by fluorescence polarization. In this chapter, we will describe the expression and purification of the prokaryotic (Rhodobacter sphaeroides) Argonaute protein, and the nucleic acid-binding analysis using a Fluorescence Polarization System (Beacon 2000).

The 2014 Ebola outbreak in West Africa, the largest outbreak on record, highlighted the need for novel approaches to therapeutics targeting Ebola virus (EBOV). Within the EBOV replication complex, the interaction between polymerase cofactor, viral protein 35 (VP35), and nucleoprotein (NP) is critical for viral RNA synthesis. We recently identified a peptide at the N-terminus of VP35 (termed NPBP) that is sufficient for interaction with NP and suppresses EBOV replication, suggesting that the NPBP binding pocket can serve as a potential drug target. Here we describe the development and validation of a sensitive high-throughput screen (HTS) using a fluorescence polarization assay. Initial hits from this HTS include the FDA-approved compound tolcapone, whose potency against EBOV infection was validated in a nonfluorescent secondary assay. High conservation of the NP-VP35 interface among filoviruses suggests that this assay has the capacity to identify pan-filoviral inhibitors for development as antivirals.

Prolyl hydroxylase domain 2 (PHD2) enzyme, a Fe(II) and 2-oxoglutarate (2-OG) dependent oxygenase, mediates key physiological responses to hypoxia by modulating the levels of hypoxia inducible factor 1-α (HIF1α). PHD2 has been shown to have the therapeutic potentials for conditions including anemia and ischemic disease. Currently, many activity-based assays have been developed for identifying PHD2 inhibitors. Here we report an affinity-based fluorescence polarization method using FITC-labeled HIF1α (556-574) peptide as a probe for quantitative and site-specific screening of small molecule PHD2 inhibitors.

Myt1 kinase is a member of the Wee-kinase family involved in G2/M checkpoint regulation of the cell cycle. So far, no peptide substrate suitable for activity-based screening has been reported, hampering systematic development of Myt1 kinase inhibitors. Myt1 inhibitors had to be identified by using either binding assays or activity assays with expensive proteinous substrates. Here, a peptide microarray approach was used to identify peptidic Myt1 substrates. Wee1 kinase was profiled for comparison using the same technology. Myt1 hits from peptide microarray experiments were verified in solution by a fluorescence polarization assay and several peptide substrates derived from cellular proteins were identified. Subsequently, phosphorylation site determination was carried out by MS fragmentation studies and identified substrates were validated by kinase inhibitor profiling., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

There are strong indications for the involvement of cyclophilin 40 in diseases caused by misregulation of steroid hormone receptors, like prostate or breast cancer. To identify novel inhibitors for this immunophilin, we developed a simplified fluorescence polarization assay based on the synthesis of a fluorescein-labeled tracer. This tracer was produced by a facile four-step synthesis involving Grubbs metathesis and standard amide bond coupling, to label cyclosporin A with fluorescein. We show the binding of this tracer to Cyp40 and Cyp18 with K D values of 106 ± 13 or 12 ± 1 nM, respectively, by analyzing the anisotropy change and demonstrate its competition with cyclosporin A. Binding data obtained by fluorescence polarization were corroborated by an enzymatic activity assay. The described tracer allows for a robust assay in a high-throughput format to support the development of novel Cyp40 ligands.