Competitive Binding Assay Research Articles

An Antennae-Enriched Odorant-Binding Protein EonuOBP43 Mediate the Behavioral Response of the Tea Green Leafhopper, Empoasca onukii Matsuda to the Host and Nonhost Volatiles.

Olfaction is crucial for Empoasca onukii Matsuda to recognize odors from the host and nonhost plants, and it has been proposed that odorant binding proteins are directly required for odorant discrimination and represent potential targets of interest for pest control. Here, we cloned EonuOBP43 and expressed the recombinant EonuOBP43 protein. Furthermore, competitive fluorescence binding assays with 19 ligands indicated that terpenoids and alkanes showed a relatively higher than for other classes of chemicals. Additionally, ligand docking and site-directed mutagenesis results revealed that seven hydrophobic residues, including Val-86, Met-89, Phe-90, Ile-104, Ile-105, Leu-130, and Val-134, played a key role in the binding of EonuOBP43 to plant volatiles. In olfactometer tests, E. onukii were significantly attracted to α-farnesene and repelled to β-caryophyllene, and dsOBP43 treated adult lost response to α-farnesene and β-caryophyllene. In summary, our results demonstrated that EonuOBP43 may function as a carrier in the process of sensing plant compounds of E. onukii.

Cevipabulin induced abnormal tubulin protofilaments polymerization by binding to Vinblastine site and The Seventh site.

Microtubules, composed of αβ-tubulin heterodimers, are crucial targets for chemotherapeutic agents and possess eight binding sites. Our previous study identified cevipabulin as the only one agent capable of simultaneously binding to two different sites (Vinblastine site and The Seventh site). Binding to The Seventh site by cevipabulin induces tubulin degradation. This study aimed to investigate whether it is binding to the Vinblastine site and The Seventh site exhibited an interactive cellular effect. Surprisingly, we discovered that cevipabulin induced abnormal tubulin protofilaments polymerization, a previously undefined tubulin morphology, and we proved it was an interactive effect of Cevipabulin's binding to both Vinblastine site and The Seventh site. Immunofluorescence and transmission electron microscopy confirmed cevipabulin induced the formation of linear tubulin protofilaments and their subsequent aggregation into irregular tubulin aggregates. Competition binding assays and the αY224G mutation revealed that binding of cevipabulin to both sites was necessary for the tubulin protofilaments polymerization effect. Moreover, we found that co-treatment with a microtubule stabilization agent binding the Vinblastine site and a microtubule destabilization agent binding at the intra-dimer interface of tubulin could also induce similar tubulin protofilaments polymerization. We proposed a mechanism where a microtubule stabilization agent on the Vinblastine site enhances longitudinal interactions between tubulin dimers, while, a microtubule destabilization agent binding at the intra-dimer interface prevents the adoption of a straight conformation of the tubulin dimer and disrupts lateral interactions between tubulins, consequently leading to tubulin protofilaments polymerization. This study reported a new inhibitor-induced-tubulin-morphology-change and would provide insight into tubulin dynamic instability and also guide further study of cevipabulin.

Novel Anti-Mesothelin Nanobodies and Recombinant Immunotoxins with Pseudomonas Exotoxin Catalytic Domain for Cancer Therapeutics.

Recombinant immunotoxins (RITs) are fusion proteins consisting of a targeting domain linked to a toxin, offering a highly specific therapeutic strategy for cancer treatment. In this study, we engineered and characterized RITs aimed at mesothelin, a cell surface glycoprotein overexpressed in various malignancies. Through an extensive screening of a large nanobody library, four mesothelin-specific nanobodies were selected and genetically fused to a truncated Pseudomonas exotoxin (PE24B). Various optimizations, including the incorporation of furin cleavage sites, maltose-binding protein tags, and tobacco etch virus protease cleavage sites, were implemented to improve protein expression, solubility, and purification. The RITs were successfully overexpressed in Escherichia coli, achieving high solubility and purity post-purification. In vitro cytotoxicity assays on gastric carcinoma cell lines NCI-N87 and AGS revealed that Meso(Nb2)-PE24B demonstrated the highest cytotoxic efficacy, warranting further characterization. This RIT also displayed selective binding to human and monkey mesothelins but not to mouse mesothelin. The competitive binding assays between different RIT constructs revealed significant alterations in IC50 values, emphasizing the importance of nanobody specificity. Finally, a modification in the endoplasmic reticulum retention signal at the C-terminus further augmented its cytotoxic activity. Our findings offer valuable insights into the design and optimization of RITs, showcasing the potential of Meso(Nb2)-PE24B as a promising therapeutic candidate for targeted cancer treatment.

Ligand Profiling to Characterize Different Polymorphic Forms of α-Synuclein Aggregates.

The presence of amyloid fibrils is a characteristic feature of many diseases, most famously neurodegenerative disease. The supramolecular structure of these fibrils appears to be disease-specific. Identifying the unique morphologies of amyloid fibrils could, therefore, form the basis of a diagnostic tool. Here we report a method to characterize the morphology of α-synuclein (αSyn) fibrils based on profiling multiple different ligand binding sites that are present on the surfaces of fibrils. By employing various competition binding assays, seven different types of binding sites were identified on four different morphologies of αSyn fibrils. Similar binding sites on different fibrils were shown to bind ligands with significantly different affinities. We combined this information to construct individual profiles for different αSyn fibrils based on the distribution of binding sites and ligand interactions. These results demonstrate that ligand-based profiling can be used as an analytical method to characterize fibril morphologies with operationally simple fluorescence binding assays.

Molecular and Functional Characterization of Pheromone Binding Protein 2 from Cyrtotrachelus buqueti (Coleoptera: Curculionidae)

Pheromone-binding proteins (PBPs) play important roles in binding and transporting sex pheromones. However, the PBP genes identified in coleopteran insects and their information sensing mechanism are largely unknown. Cyrtotrachelus buqueti (Coleoptera: Curculionidae) is a major insect pest of bamboo plantations. In this study, a novel PBP gene, CbuqPBP2, from C. buqueti was functionally characterized. CbuqPBP2 was more abundantly expressed in the antennae of both sexes than other body parts, and its expression level was significantly male-biased. Fluorescence competitive binding assays showed that CbuqPBP2 exhibited the strongest binding affinity to dibutyl phthalate (Ki = 6.32 μM), followed by styrene (Ki = 11.37 μM), among twelve C. buqueti volatiles. CbuqPBP2, on the other hand, showed high binding affinity to linalool (Ki = 10.55), the main volatile of host plant Neosinocalamus affinis. Furthermore, molecular docking also demonstrated the strong binding ability of CbuqPBP2 to dibutyl phthalate, styrene, and linalool, with binding energy values of −5.7, −6.6, and −6.0 kcal/mol, respectively, and hydrophobic interactions were the prevailing forces. The knockdown of CbuqPBP2 expression via RNA interference significantly reduced the electroantennography (EAG) responses of male adults to dibutyl phthalate and styrene. In conclusion, these results will be conducive to understanding the olfactory mechanisms of C. buqueti and promoting the development of novel strategies for controlling this insect pest.

In Silico and In Vitro Study towards the Rational Design of 4,4'-Disarylbisthiazoles as a Selective α-Synucleinopathy Biomarker.

The α-synucleinopathies are a group of neurodegenerative diseases characterized by the deposition of α-synuclein aggregates (α-syn) in the brain. Currently, there is no suitable tracer to enable a definitive early diagnosis of these diseases. We reported candidates based on 4,4'-disarylbisthiazole (DABTA) scaffold with a high affinity towards α-syn and excellent selectivity over Aβ and tau fibrils. Based on prior in silico studies, a focused library of 23 halogen-containing and O-methylated DABTAs was prepared. The DABTAs were synthesized via a modified two-step Hantzsch thiazole synthesis, characterized, and used in competitive binding assays against [3H]PiB and [3H]DCVJ. The DABTAs were obtained with an overall chemical yield of 15-71%, and showed a calculated lipophilicity of 2.5-5.7. The ligands demonstrated an excellent affinity to α-syn with both [3H]PiB and [3H]DCVJ: Ki 0.1-4.9 nM and up to 20-3900-fold selectivity over Aβ and tau fibrils. It could be concluded that in silico simulation is useful for the rational design of a new generation of DABTAs. Further investigation of the leads in the next step is encouraged: radiolabeling of the ligands with radioisotopes such as fluorine-18 or carbon-11 for in vivo, ex vivo, and translational research and for further in vitro experiments on human-derived protein aggregates.

Discovery of Ligands for TRIM58, a Novel Tissue-Selective E3 Ligase.

Redirecting E3 ligases to neo-substrates, leading to their proteasomal disassembly, known as targeted protein degradation (TPD), has emerged as a promising alternative to traditional, occupancy-driven pharmacology. Although the field has expanded tremendously over the past years, the choice of E3 ligases remains limited, with an almost exclusive focus on CRBN and VHL. Here, we report the discovery of novel ligands to the PRY-SPRY domain of TRIM58, a RING ligase that is specifically expressed in erythroid precursor cells. A DSF screen, followed by validation using additional biophysical methods, led to the identification of TRIM58 ligand TRIM-473. A basic SAR around the chemotype was established by utilizing a competitive binding assay employing a short FP peptide probe derived from an endogenous TRIM58 substrate. The X-ray co-crystal structure of TRIM58 in complex with TRIM-473 gave insights into the binding mode and potential exit vectors for bifunctional degrader design.

Large chemokine binding spectrum of human and mouse atypical chemokine receptor GPR182 (ACKR5).

Atypical chemokine receptors (ACKRs) play pivotal roles in immune regulation by binding chemokines and regulating their spatial distribution without inducing G-protein activation. Recently, GPR182, provisionally named ACKR5, was identified as a novel ACKR expressed in microvascular and lymphatic endothelial cells, with functions in hematopoietic stem cell homeostasis. Here, we comprehensively investigated the chemokine binding profile of human and mouse GPR182. Competitive binding assays using flow cytometry revealed that besides CXCL10, CXCL12 and CXCL13, also human and mouse CXCL11, CXCL14 and CCL25, as well as human CCL1, CCL11, CCL19, CCL26, XCL1 and mouse CCL22, CCL24, CCL27 and CCL28 bind with an affinity of less than 100nM to GPR182. In line with the binding affinity observed in vitro, elevated serum levels of CCL22, CCL24, CCL25, and CCL27 were observed in GPR182-deficient mice, underscoring the role of GPR182 in chemokine scavenging. These data show a broader chemokine binding repertoire of GPR182 than previously reported and they will be important for future work exploring the physiological and pathophysiological roles of GPR182, which we propose to be renamed atypical chemokine receptor 5 (ACKR5).

Molecular Characterization of Three Chemosensory Proteins from Carposina sasakii

The peach fruit moth, Carposina sasakii, is part of the Carposinidae, and is harmful to the families Rosaceae and Rhamnaceae. C. sasakii lays eggs on the hairy surface of the fruit’s stalk cavity and calyx end. After hatching, the moth can bore into the fruits and feed on the flesh inside. Chemosensory proteins (CSPs) are a class of low-molecular-weight soluble carrier proteins that are highly evolutionarily conserved. To enhance our understanding of the recognition of host plant volatiles by CSPs of C. sasakii, the expression patterns and binding characteristics of CsasCSP7, CsasCSP9 and CsasCSP11 in C. sasakii were investigated. In our study, the results of real-time quantitative polymerase chain reaction (qPCR) assays demonstrate that CsasCSP7 and CsasCSP9 transcripts were abundantly expressed in the antennae of males, and CsasCSP11 was highly expressed in the wings of females. Fluorescence competitive binding assays with 38 candidate ligands showed that CsasCSP7 could bind to benzaldehyde and dodecanal, whereas CsasCSP9 bound to butyl octanoate, decanal and (-)-beta-pinene. CsasCSP11 could also bind to1-hexanol, beta-ocimene and 6-methyl-5-hepten-2-one. Our results suggest that CsasCSP7, CsasCSP9 and CsasCSP11 may play a crucial role in locating the host plant of C. sasakii.

A competitive ligand binding assay for detection of neutralizing antibodies against an insulin analog

Biotherapeutics have the potential to trigger undesired immune responses in the patients. For therapeutic proteins, immunogenicity is manifested as anti-drug antibodies (ADA). Because ADA could compromise pharmacokinetics, efficacy, and safety, regulatory agencies require immunogenicity assessment during clinical development. A tiered bioanalytical approach is recommended to monitor clinical immunogenicity, and neutralizing antibodies (NAb) are studied in Tier 4 if the molecule is immunogenic. Although cell-based assays, which reflect the pharmacological mechanism of action, are in some cases the preferred assay format for detecting NAbs, they are associated with operational complexity and sometimes suboptimal assay performance. Alternatively, non-cell-based assays have also been developed and implemented. In our current study, a competitive ligand binding assay (CLBA) was developed to detect NAbs for insulin efsitora alfa (efsitora, basal insulin Fc, LY3209590), a novel fusion protein being studied for the treatment of Type 1 diabetes and Type 2 diabetes. The CLBA demonstrated acceptable sensitivity, drug tolerance, precision, and robustness, and thus provides a suitable approach for detecting NAbs against efsitora.

Increased Endocrine Activity of Xenobiotic Chemicals as Mediated by Metabolic Activation.

The US Environmental Protection Agency (USEPA) is faced with long lists of chemicals that require hazard assessment. The present study is part of a larger effort to develop in vitro assays and quantitative structure-activity relationships applicable to untested chemicals on USEPA inventories through study of estrogen receptor (ER) binding and estrogen-mediated gene expression in fish. The present effort investigates metabolic activation of chemicals resulting in increased estrogenicity. Phenolphthalin (PLIN) was shown not to bind rainbow trout (Oncorhynchus mykiss) ER (rtER) in a competitive binding assay, but vitellogenin (Vtg) expression was induced in trout liver slices exposed to 10-4 and 10-3.7 M PLIN. Phenolphthalein (PLEIN), a metabolite of PLIN, was subsequently determined to be formed when slices were exposed to PLIN. It binds rtER with a relative binding affinity to 17β-estradiol of 0.020%. Slices exposed to PLEIN expressed Vtg messenger RNA (mRNA) at 10-4.3 , 10-4 , and 10-3.7 M, with no detectable PLIN present. Thus, Vtg expression noted in PLIN slice exposures was explained by metabolism to PLEIN in trout liver slices. A second model chemical, 4,4'-methylenedianiline (MDA), was not shown to bind rtER but did induce Vtg mRNA production in tissue slices at 10-4.3 , 10-4 , and 10-3.7 M in amounts nearly equal to reference estradiol induction, thus indicating metabolic activation of MDA. A series of experiments were performed to identify a potential metabolite responsible for the observed increase in activity. Potential metabolites hydroxylamine-MDA, nitroso-MDA, azo-MDA, and azoxy-MDA were not observed. However, acetylated MDA was observed and tested in both ER-binding and tissue slice Vtg induction assays. Environ Toxicol Chem 2023;42:2747-2757. © 2023 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Biomimetic System Based on Reconstituted Macrophage Membranes for Analyzing and Selection of Higher-Affinity Ligands Specific to Mannose Receptor to Develop the Macrophage-Focused Medicines.

Progress in macrophage research is crucial for numerous applications in medicine, including cancer and infectious diseases. However, the existing methods to manipulate living macrophages are labor-intense and inconvenient. Here, we show that macrophage membranes can be reconstituted after storage for months at 4 °C, with their CD206 receptor selectivity and specificity being similar to those in the living cells. Then, we have developed a mannose ligand, specific to CD206, linked with PEG as an IR spectroscopy marker to detect binding with the macrophage receptor. PEG was selected due to its unique adsorption band of the C-O-C group at IR spectra, which does not overlap with other biomolecules' spectroscopic feature. Next, competitive binding assay versus the PEG-bound ligand has enabled the selection of other higher-affinity ligands specific to CD206. Furthermore, those higher-affinity ligands were used to differentiate activated macrophages in a patient's bronchoalveolar (BAL) or nasopharyngeal (NPL) lavage. CD206- control cells (HEK293T) showed only non-specific binding. Therefore, biochips based on reconstituted macrophage membranes as well as PEG-trimannoside as an IR spectroscopic marker can be used to develop new methods facilitating macrophage research and macrophage-focused drug discovery.

Emerging organophosphate ester resorcinol bis(diphenyl phosphate) exerts estrogenic effects via estrogen receptor pathways

Environmental occurrence and human exposure of emerging organophosphate esters (eOPEs) have increased significantly in recent years. Resorcinol bis(diphenyl) phosphate (RDP) is one of the major eOPEs detected in indoor dust, but the knowledge on its toxicities and health risks is rather limited. In this study, we investigated the in vitro estrogenic effects and underlying mechanism of RDP in comparison with a legacy OPE triphenyl phosphate (TPHP). Our results showed that RDP promoted MCF-7 cell proliferation with the lowest effect concentration of 2.5 μM, and the maximum enhancement of 1.6 folds is greater than that of TPHP (1.3 folds). The effect was inhibited completely by an estrogen receptor (ER) antagonist, suggesting that ER activation was responsible for the enhancement. In luciferase reporter gene assays both RDP and TPHP activated ER transcriptional activity at 2.5 μM, but RDP activity was higher than TPHP. Competitive fluorescence binding assays showed that RDP bound to ER with an IC10 of 0.26 μM, which is 20 folds lower than TPHP (5.6 μM). Molecular docking simulation revealed that both RDP and TPHP interacted with ER at the binding pocket of estradiol, although the hydrogen bonds were different. Taken together, RDP exerted stronger estrogenic effects than TPHP through ER-mediated pathways and may pose more health risks.

Molecular mechanism of sex pheromone perception in male Mythimna loreyi revealed by in vitro system.

Mythimna loreyi is an important agricultural pest with a sensitive sex pheromone communication system. To clarify the pheromone binding proteins (PBPs) and pheromone receptors (PRs) involved in sex pheromone perception is important for both understanding the molecular olfactory mechanism and developing a new pest control strategy in M. loreyi. First, the electroantennogram (EAG) assay showed that male M. loreyi displayed the highest response to the major sex pheromone component Z9-14:Ac, and higher responses to two minor components, Z7-12:Ac and Z11-16:Ac. Second, the fluorescence competition binding assay showed that PBP1 bound all three pheromones and other tested compounds with high or moderate affinity, while PBP2 and PBP3 each bound only one pheromone component and few other compounds. Third, functional study using the Xenopus oocyte system demonstrated that, of the six candidate PRs, PR2 was weakly sensitive to the major pheromone Z9-14:Ac, but was strongly sensitive to pheromone analog Z9-14:OH; PR3 was strongly and specifically sensitive to a minor component Z7-12:Ac; PR4 and OR33 were both weakly sensitive to another minor component, Z11-16:Ac. Finally, phylogenetic relationship and ligand profiles of PRs were compared among six species from two closely related genera Mythimna and Spodoptera, suggesting functional shifts of M. loreyi PRs toward Spodoptera PRs. Functional differentiations were revealed among three PBPs and six PRs in sex pheromone perception, laying an important basis for understanding the molecular mechanism of sex pheromone perception and for developing new control strategies in M. loreyi. © 2023 Society of Chemical Industry.

Native SEC and Reversed-Phase LC-MS Reveal Impact of Fab Glycosylation of Anti-SARS-COV-2 Antibodies on Binding to the Receptor Binding Domain.

The binding affinity of monoclonal antibodies (mAbs) for their intended therapeutic targets is often affected by chemical and post-translational modifications in the antigen binding (Fab) domains. A new two-dimensional analytical approach is described here utilizing native size exclusion chromatography (SEC) to separate populations of antibodies and bound antibody-antigen complexes for subsequent characterization of these modifications by reversed-phase (RP) liquid chromatography-mass spectrometry (LC-MS) at the intact antibody level. Previously, we utilized peptide mapping to measure modifications impacting binding. However, in this study, the large size of the modification (N-glycosylation) allowed assessing its impact from small amounts (∼20 ug) of intact antibody, without the need for peptide mapping. Here, we apply the native SEC-based competitive binding assay to quickly and qualitatively investigate the effects of Fab glycosylation of four antispike protein mAbs that were developed for use in the treatment of COVID-19 disease. Three of the mAbs were observed to have consensus N-glycosylation sites (N-X-T/S) in the Fab domains, a relatively rare occurrence in therapeutic mAbs. The goal of the study was to characterize the levels of Fab glycosylation present, as well as determine the impact of glycosylation on binding to the spike protein receptor binding domain (RBD) and the ability of the mAbs to inhibit RBD-ACE2 interaction at the intact antibody level, with minimal sample treatment and preparation. The three mAbs with Fab N-glycans were found to have glycosylation profiles ranging from full occupancy at each Fab (in one mAb) to partially glycosylated with mixed populations of two, one, or no glycan moieties. Competitive SEC analysis of mAb-RBD revealed that the glycosylated antibody populations outcompete their nonglycosylated counterparts for the available RBD molecules. This competitive SEC binding analysis was applied to investigate the three-body interaction of a glycosylated mAb blocking the interaction between endogenous binding partners RBD-ACE2, finding that both glycosylated and nonglycosylated mAb populations bound to RBD with high enough affinity to block RBD-ACE2 binding.

Synthesis and biological evaluation of 18F-labelled deuterated tropane derivatives as dopamine transporter probes

PurposeDopamine transporter (DAT) is a promising target for positron emission tomography (PET) imaging of many neuropsychiatric diseases. 18F-labelled N-alkyl tropane analogues were reported to be useful PET radioligands for DAT. However, the drawback of 18F-labelled tropane analogues is that N-alkyl on tropane is easily metabolized in vivo, which interferes with brain imaging. To develop a more in vivo stable DAT-targeted PET radioligand with high DAT affinity and specificity, in this study, we synthesized and compared a series of 18F-labelled novel deuterated N-fluoropropyl tropane derivatives [18F]4a-e for DAT tracing. ProceduresFive deuterated N-fluoropropyl-d6 tropane derivatives (4a-e) and corresponding non-deuterated compounds (6a-e) were synthesized, and their semi-inhibitory concentrations (IC50) were measured by competitive binding assay. The radioligands [18F]4a-e and [18F]6a-e were obtained by two-step one-pot radio-labelling reactions. The selectivity and specificity of these radioligands were evaluated by cellular uptake and microPET in normal rats. [18F]4e was selected for further investigation with microPET of the PD model, autoradiography and biodistribution experiments and compared with its non-deuterated [18F]6e. Finally, in vivo metabolic stability was analyzed by radio-HPLC. ResultsTen tropane compounds had high DAT affinity (IC50 = 2–21 nM), in which FP-CIT-d6 (4e) had the lowest IC50 value of 2.7 nM. Radiolabelled [18F]4a-e and [18F]6a-e were obtained with radiochemical yields ranging from 10.6 ± 2.8% to 35.1 ± 5.4% with molar activities >20 GBq/μmol and the radiochemical purities >99%. [18F]4e showed the highest cell uptake (12%) and CFT inhibition efficacy (∼72%) among [18F]4a-e. MicroPET results showed [18F]4e has the highest target to non-target ratio (striatum/cerebellum). Therefore, [18F]4e was then selected for further biological evaluation. Ex vivo autoradiography experiment confirmed high specific binding of [18F]4e towards DAT. Biodistribution results indicated that [18F]4e has a higher striatum/cerebellum value than [18F]FP-CIT ([18F]6e) at 30–120 min. Furthermore, in vivo metabolism studies in rats revealed improved stability of [18F]4e as compared with that of [18F]6e. ConclusionsThe new probe [18F]4e is a promising candidate with good DAT affinity, specificity and metabolic stability for PET imaging, and might provide reliable diagnosis, treatment and prognostic detection of DAT-related neuropsychiatric diseases.

Electrochemical sensor based on molecularly imprinted polymer for rapid quantitative detection of brain-derived neurotrophic factor

Neurodegenerative disorders necessitate early detection for timely diagnosis and treatment. Here, we present an electrochemical sensor comprising a robust biomimetic receptor, a molecularly imprinted polymer (MIP), integrated with a system of thin film metal electrodes for rapid detection of brain-derived neurotrophic factor (BDNF) as a potential neurodegenerative disorder biomarker. The MIP-based sensor utilizes the surface imprinting approach, where controlled polymer growth around the protein template creates geometrically-fit imprinted sites on the receptor surface. Following thorough characterization, the performance of the sensor was studied by differential pulse voltammetry. Under optimal conditions, the sensor exhibits an impressive limit of detection of 9 pg/mL for BDNF, showing over 5-fold selectivity against closely related neurotrophic factor proteins (CDNF and MANF) and 30-fold selectivity against proteins with similar pI values (e.g., CD48). Competitive binding assays further confirmed the sensor's selectivity, with BDNF inhibiting the binding of interfering proteins. Successful validation of BDNF detection in complex biological samples, specifically fetal bovine serum, highlighted the sensor's potential for real sample analysis. Compared to conventional biosensors, the presented strategy could unlock a new route to develop a highly desirable, robust sensing platform with strong affinity, specificity, and cost-effectiveness for diagnosing neurodegenerative disorders.

Competitive Assay for the Ultrasensitive Detection of Organophosphate Pesticides Based on a Fiber-Optic Particle Plasmon Resonance Biosensor and an Acetylcholinesterase Binding Peptide.

An acetylcholinesterase (AChE) binding-based biosensor was developed for the ultrasensitive detection of organophosphate (OP) pesticides. The biosensor integrates the technique based on fiber-optic particle plasmon resonance detection and a synthetic AChE binding peptide conjugated with gold nanoparticles on the optical fiber surface via an AChE competitive binding assay. The OP pesticides present in the solution hinder the binding of AChE to the peptide on the biosensor by competing for the binding sites present in AChE. The limit of detection obtained for parathion using this method was observed to be 0.66 ppt (2.3 pM). This method shows a wide linear dynamic range of 6 orders. Furthermore, the use of the AChE binding peptide in the biosensor can better discriminate OPs against carbamates by using only a single biosensor. The practical application of this method was tested using spiked samples, which yielded good recovery and reproducibility. The spiked sample required minimal pretreatment before analysis; hence, this biosensor may also be used in the field.

Novel progestogenic androgens for male contraception: design, synthesis, and activity of C7 α-substituted testosterone†.

Male contraceptive development has included use of testosterone (T) with or without a progestin or the use of a single molecule such as progestogenic androgens (PA) for suppression of testicular T production. Expanding upon the vast amount of data accumulated from nortestosterone (NT), NT analogs, and their prodrugs, a new series of PA, the C7 methyl, and ethyl α-substituted T analogs 7α-Methyltestosterone (7α-MT) and 7α-Ethyltestosterone (7α-ET), respectively, were hypothesized and designed to have superior androgenic and progestogenic activities when compared with parent T. Results from androgen receptor and progesterone receptor competitive binding and transcriptional activation assays showed favorable activities for these T analogs. Additionally, 7α-MT and 7α-ET were shown to be active substrates for aromatase in vitro, mitigating a potential negative impact on bone mineral density with long-term use. In conjunction with this observation, the diminished metabolism of these T analogs by 5α-reductase may reduce potential concerns for prostatic growth. In the Hershberger in vivo rat bioassay, 7α-MT and 7α-ET showed superior androgenic and anabolic activities as compared with T. These C7 α-substituted T analogs also showed clear progestogenic activity in the McPhail bioassay which evaluated endometrial glandular arborization in a rabbit model. The discovery of aromatizable molecules with reduced metabolism by 5α-reductase that have androgenic, anabolic, and progestogenic properties indicates that the core and/or prodrugs of 7α-MT and 7α-ET are promising molecules for further development as male contraceptive PAs.

Contributions of the N-terminal flanking residues of an antigenic peptide from the Japanese cedar pollen allergen Cry j 1 to the T-cell activation by HLA-DP5.

Cry j 1 is a major allergen present in Japanese cedar (Cryptomeria japonica) pollens. Peptides with the core sequence of KVTVAFNQF from Cry j 1 ('pCj1') bind to HLA-DP5 and activate Th2 cells. In this study, we noticed that Ser and Lys at positions -2 and -3, respectively, in the N-terminal flanking (NF) region to pCj1 are conserved well in HLA-DP5-binding allergen peptides. A competitive binding assay showed that the double mutation of Ser(-2) and Lys(-3) to Glu [S(P-2)E/K(P-3)E] in a 13-residue Cry j 1 peptide (NF-pCj1) decreased its affinity for HLA-DP5 by about 2-fold. Similarly, this double mutation reduced, by about 2-fold, the amount of NF-pCj1 presented on the surface of mouse antigen-presenting dendritic cell line 1 (mDC1) cells stably expressing HLA-DP5. We established NF-pCj1-specific and HLA-DP5-restricted CD4+ T-cell clones from HLA-DP5 positive cedar pollinosis (CP) patients, and analyzed their IL-2 production due to the activation of mouse TG40 cells expressing the cloned T-cell receptor by the NF-pCj1-presenting mDC1 cells. The T-cell activation was actually decreased by the S(P-2)E/K(P-3)E mutation, corresponding to the reduction in the peptide presentation by this mutation. In contrast, the affinity of NF-pCj1·HLA-DP5 for the T-cell receptor was not affected by the S(P-2)E/K(P-3)E mutation, as analyzed by surface plasmon resonance. Considering the positional and side-chain differences of these NF residues from previously reported T-cell activating sequences, the mechanisms of enhanced T-cell activation by Ser(-2) and Lys(-3) of NF-pCj1 may be novel.