Affinity Assay Research Articles

Nonylphenol and Cetyl Alcohol Polyethoxylates Disrupt Thyroid Hormone Receptor Signaling to Disrupt Metabolic Health.

Surfactants are molecules with both hydrophobic and hydrophilic structural groups that adsorb at the air-water or oil-water interface and serve to decrease the surface tension. Surfactants combine to form micelles that surround and break down or remove oils, making them ideal for detergents and cleaners. Two of the most important classes of nonionic surfactants are alkylphenol ethoxylates (APEOs) and alcohol ethoxylates (AEOs). APEOs and AEOs are high production-volume chemicals that are used for many industrial and residential purposes, including laundry detergents, hard-surface cleaners, paints, and pesticide adjuvants. Commensurate with better appreciation of the toxicity of APEOs and the base alkylphenols, use of AEOs has increased, and both sets of compounds are now ubiquitous environmental contaminants. We recently demonstrated that diverse APEOs and AEOs induce triglyceride accumulation and/or pre-adipocyte proliferation in vitro. Both sets of contaminants have also been demonstrated as obesogenic and metabolism disrupting in a developmental exposure zebrafish model. While these metabolic health effects are consistent across models and species, the mechanisms underlying these effects are less clear. This study sought to evaluate causal mechanisms through reporter gene assay assessments, relative binding affinity assays, co-exposure experiments, and use of both human cell and zebrafish models. We report that antagonism of thyroid hormone receptor signaling appears to mediate at least a portion of the polyethoxylate-induced metabolic health effects. These results suggest further evaluation is needed, given the ubiquitous environmental presence of these thyroid disrupting contaminants and reproducible effects in human cell models and vertebrate animals.

Development and Optimization of a Redox Enzyme-Based Fluorescence Biosensor for the Identification of MsrB1 Inhibitors

MsrB1 is a thiol-dependent enzyme that reduces protein methionine-R-sulfoxide and regulates inflammatory response in macrophages. Therefore, MsrB1 could be a promising therapeutic target for the control of inflammation. To identify MsrB1 inhibitors, we construct a redox protein-based fluorescence biosensor composed of MsrB1, a circularly permutated fluorescent protein, and the thioredoxin1 in a single polypeptide chain. This protein-based biosensor, named RIYsense, efficiently measures protein methionine sulfoxide reduction by ratiometric fluorescence increase. We used it for high-throughput screening of potential MsrB1 inhibitors among 6868 compounds. A total of 192 compounds were selected based on their ability to reduce relative fluorescence intensity by more than 50% compared to the control. Then, we used molecular docking simulations of the compound on MsrB1, affinity assays, and MsrB1 activity measurement to identify compounds with reliable and strong inhibitory effects. Two compounds were selected as MsrB1 inhibitors: 4-[5-(4-ethylphenyl)-3-(4-hydroxyphenyl)-3,4-dihydropyrazol-2-yl]benzenesulfonamide and 6-chloro-10-(4-ethylphenyl)pyrimido[4,5-b]quinoline-2,4-dione. They are heterocyclic, polyaromatic compounds with a substituted phenyl moiety interacting with the MsrB1 active site, as revealed by docking simulation. These compounds were found to decrease the expression of anti-inflammatory cytokines such as IL-10 and IL-1rn, leading to auricular skin swelling and increased thickness in an ear edema model, effectively mimicking the effects observed in MsrB1 knockout mice. In summary, using a novel redox protein-based fluorescence biosensor, we identified potential MsrB1 inhibitors that can regulate the inflammatory response, particularly by influencing the expression of anti-inflammatory cytokines. These compounds are promising tools for understanding MsrB1’s role during inflammation and eventually controlling inflammation in therapeutic approaches.

Molecular Imaging of Melanoma VEGF-expressing Tumors through [99mTc]Tc-HYNIC-Fab(Bevacizumab).

Angiogenesis is a process that many tumors depend on for growth, development, and metastasis. Vascular endothelial growth factor (VEGF) is one of the major players in tumor angiogenesis in several tumor types, including melanoma. VEGF inhibition is achieved by bevacizumab, a humanized monoclonal antibody that binds with high affinity to VEGF and prevents its function. In order to successfully enable in vivo VEGF expression imaging in a murine melanoma model, we previously labeled bevacizumab with [99mTc]Tc. We observed that this was feasible, but it had prolonged blood circulation and delayed tumor uptake. The aim of this study was to develop a radiolabeled Fab bevacizumab fragment, [99mTc]Tc-HYNICFab( bevacizumab), for non-invasive in vivo VEGF expression molecular imaging. Flow cytometry was used to examine VEGF presence in the murine melanoma cell line (B16-F10). Bevacizumab was digested with papain for six hours at 37°C to produce Fab(bevacizumab), which was then conjugated to NHS-HYNIC-Tfa for radiolabeling with [99mTc]Tc. Stability and binding affinity assays were also evaluated. Biodistribution and single photon emission computed tomography/computed tomography (SPECT/CT) were performed at 1, 3, and 6 h (n = 4) after injection of [99mTc]Tc-HYNIC-Fab(Bevacizumab) in normal and B16-F10 tumor-bearing C57Bl/6J mice. Using flow cytometry, it was shown that the B16-F10 murine melanoma cell line has intracellular VEGF expression. Papain incubation resulted in the complete digestion of bevacizumab with good purity and homogeneity. The radiolabeling yield of [99mTc]Tc-HYNIC-Fab(bevacizumab) was 85.00 ± 6.06%, with a specific activity of 291.87 ± 18.84 MBq/mg (n=3), showing in vitro stability. Binding assays demonstrated significant intracellular in vitro VEGF expression. Fast blood clearance and high kidney and tumor uptake were observed in biodistribution and SPECT/CT studies. We present the development and evaluation of [99mTc]Tc-HYNIC-Fab(bevacizumab), a novel molecular VEGF expression imaging agent that may be used for precision medicine in melanoma and potentially in other VEGF-expressing tumors.

The Antifungal Effects of Berberine and Its Proposed Mechanism of Action Through CYP51 Inhibition, as Predicted by Molecular Docking and Binding Analysis.

Fungal infections present a significant health risk, particularly in immunocompromised individuals. Berberine, a natural isoquinoline alkaloid, has demonstrated broad-spectrum antimicrobial activity, though its antifungal potential and underlying mechanisms against both yeast-like and filamentous fungi are not fully understood. This study investigates the antifungal efficacy of berberine against Candida albicans, Cryptococcus neoformans, Trichophyton rubrum, and Trichophyton mentagrophytes in vitro, as well as its therapeutic potential in a murine model of cryptococcal infection. Berberine showed strong antifungal activity, with MIC values ranging from 64 to 128 µg/mL. SEM and TEM analyses revealed that berberine induced notable disruptions to the cell wall and membrane in C. neoformans. No signs of cell necrosis or apoptosis were observed in fungal cells treated with 2 × MIC berberine, and it did not increase intracellular ROS levels or affect mitochondrial membrane potential. Molecular docking and binding affinity assays demonstrated a strong interaction between berberine and the fungal enzyme CYP51, with a dissociation constant (KD) of less than 1 × 10-12 M, suggesting potent inhibition of ergosterol biosynthesis. In vivo studies further showed that berberine promoted healing in guinea pigs infected with T. mentagrophytes, and in a murine cryptococcal infection model, it prolonged survival and reduced lung inflammation, showing comparable efficacy to fluconazole. These findings indicate that berberine exerts broad-spectrum antifungal effects through membrane disruption and CYP51 inhibition, highlighting its potential as a promising therapeutic option for fungal infections.

Free Electron Density Gradients Enhanced Biosensor for Ultrasensitive and Accurate Affinity Assessment of the Immunotherapy Drugs.

Accurate affinity assessments play an important role in drug discovery, screening, and efficacy evaluation. Label-free affinity biosensors are recognized as a dependable and standard technology for addressing this challenge. This study constructs a free electron density gradient-enhanced meta-surface plasmon resonance (FED-MSPR) biosensor through a finite-difference time-domain simulation model, the biosensor demonstrates superior detection performance in accurately determining affinity and kinetic rate constants. By controlling the dielectric properties of the metal on the surface of the nanocup arrays, the plasmon resonance effects are easily tuned without changing the nanostructure design. Compared with the single-layer gold chip, the triple-layer FED-MSPR chip demonstrated a four-fold improvement in resolution at the optimal resonance peak. Additionally, the sensitivity and figure of merit (FOM) of the multi-layer chip increased by 3.5 and 7.99 times, respectively. Following modification with high- and low-staggered carboxylation, the noise-signal ratio and baseline stability of the real-time kinetic curves based on these chips are significantly enhanced. The developed carboxylation FED-MSPR platform is successfully used to perform affinity assays for Adalimumab and TNF-α protein, resulting in favorable dynamic curves. These findings validate the proposed FED-MSPR biosensor platform as cost-effective, rapid, sensitive, and label-free, facilitating real-time quality control in drug development.

Harnessing Light for G-Quadruplex Modulation: Dual Isomeric Effects of an Ortho-Fluoroazobenzene Derivative.

G-quadruplexes (G4s) are important therapeutic and photopharmacological targets in cancer research. Small-molecule ligands targeting G4s offer a promising strategy to block DNA transactions and induce genetic instability in cancer cells. While numerous G4-ligands have been reported, relatively few examples exist of compounds whose G4-interactive binding properties can be modulated using light. Herein, we report the photophysical characterization of a novel ortho-fluoroazobenzene derivative, Py-Azo4F-3N, that undergoes reversible two-way isomerization upon visible light exposure. Using a combination of biophysical techniques, including affinity and selectivity assays, structural and computational analysis, and cytotoxicity experiments in cancer cell lines, we carefully characterized the G4-interactive binding properties of both isomers. We identify the trans isomer as the most promising form of interacting and stabilizing G4s, enhancing their ablation capability in cancer cells. Our research highlights the importance of light-responsive molecules in achieving precise control over G4 structures, demonstrating their potential in innovative anticancer strategies.

Determining the Degree of Sulfo-tag Conjugation to AAV5 Vectors by LC-HRMS and Evaluating the Effects on Antibody Binding Affinity and Bridging Assay Sensitivity.

Pre-existing anti-AAV antibodies can be detected using ligand binding-based assay formats. One such format is the MSD-based bridging assay, which uses sulfo-tag-labeled AAV vectors as detection reagents. However, no method has been developed to accurately measure the degree of sulfo-tag labeling on AAV vectors. To fill this gap, we developed a liquid chromatography-high resolution mass spectrometry (LC-HRMS) method to assess the degree of labeling (DoL) of sulfo-tag on AAV5 vectors, enabling the measurement of the DoL on AAV5 at six increasing levels of sulfo-tag challenge ratio. In addition, a Biacore-based assay was used to evaluate the binding affinity between an anti-AAV5 monoclonal antibody and various sulfo-tag labeled AAV5 vectors. The results indicated that increased DoL of sulfo-tag labeling on AAV5 did not compromise the binding affinity.Our study further employed the MSD-bridging assay to detect the binding Signal/Noise (S/N) ratios of four anti-AAV5 monoclonal antibodies (mAbs) to various sulfo-tag-labeled AAV5 vectors. The findings revealed a strong correlation between the degree of sulfo-tag labeling and both the S/N ratios and the sensitivity of MSD bridging assays. This result underscores the importance of optimizing the labeling of detection reagents to enhance assay sensitivity for detecting anti-AAV5 antibodies.

Online ligand screening for cytochrome C from herbal medicines through “four-in-one” measurement

Affinity-oriented online ligand screening with LC coupled to different detectors is widely popular to capture active compounds from herbal medicines (HMs). However, false-positive extensively occurs because insufficient information is recorded for the existence and stability of ligand-protein complex. Here, efforts were made to advance the hit confidences via configuring post-column infusion-LC–energy-resolved-affinity MS (PCI-LC–ER-AMS) to achieve “four-in-one” monitoring of: 1) response decrement of potential ligands; 2) response decrement of protein; 3) ions relating to ligand-protein complexes; and 4) ligand-protein binding strength. Ligand fishing for Cyt C from HMs was conducted as a proof-of-concept. For utility justification, a mimic sample containing twelve well-defined ligands and two negative controls underwent LC separation and met Cyt C prior to Qtof-MS measurements. Compared to Cyt C- or ligand-free assay, twelve ligands instead of negative controls showed response decrements that were consistent with twelve negative peaks observed at retention times corresponding to the ligands in Cyt C ion current chromatogram. Serial ions correlating to each ligand-Cyt C complex were observed. After recording breakdown graphs, optimal collision energy (OCE) corresponding to the non-covalent bond dissociation was positively correlated with binding strength. Two HMs including Scutellariae Radix (SR) and Aconiti Lateralis Radix Preparata were investigated. Consequently, 24 compounds were merely fished from SR, and particularly, flavonoid glycosides exhibited greater OCEs and also binding strengths over aglycones. Affinity assays and cellular evaluations consolidated the significant interactions between each captured compound and Cyt C. Overall, PCI-LC–ER-AMS is eligible for confidence-enhanced online ligand screening for Cyt C from HMs through “four-in-one” measurement.

Development of a scintillation proximity assay for [3H]epibatidine binding sites of Tetronarce californica muscle-type nicotinic acetylcholine receptor

The therapy of intoxication with distinct organophosphorus (OP) compounds is still limited today. Especially chemical warfare agents like tabun and soman as well as novichok intoxications are difficult to address using established oxime therapeutics. These neurotoxins inhibit acetylcholinesterase (AChE), a pivotal enzyme in the synaptic cleft. The following accumulation of acetylcholine in the synaptic cleft leads to a dysfunctional, desensitized state of nicotinic acetylcholine receptors (nAChR). Without adequate treatment, the resulting cholinergic crisis leads to death by respiratory arrest. Consequently, the research approach for new therapeutic options needs to be expanded. A promising option would be substances interacting directly with nAChRs. Therefore, screening methods for new drug candidates are needed, with affinity assays playing an important role. In the present work, a saturation and competition scintillation proximity assay (SPA) for binding studies at [3H]epibatidine binding sites, conventionally classified as orthosteric binding sites of the muscle type nAChR was developed. This method offers several advantages over other assay technologies because no separation as well as washing steps are required to remove unbound ligands. Assay precision and solvent tolerance were validated according to the guidelines for validation of bioanalytical methods of the Food and Drug Administration (FDA) and European Medicines Agency (EMA). The newly developed binding assay was successfully implemented on an automated pipetting platform and is suitable for high-throughput-screening of receptor-ligand interactions at the nAChR. Furthermore, it allows to investigate/quantify competition of highly toxic agents such as nerve agents or structurally similar pesticides at the orthosteric binding site. Related to further pharmacological results, the affinity to [3H]epibatidine binding sites can provide additional information on whether potential drug candidates would be suitable for treatment of nerve agent poisoning.

Computational design and experimental confirmation of a disulfide-stapled YAP helixα1-trap derived from TEAD4 helical hairpin to selectively capture YAP α1-helix with potent antitumor activity.

Human Hippo signaling pathway is an evolutionarily conserved regulator network that controls organ development and has been implicated in various cancers. Transcriptional enhanced associate domain-4 (TEAD4) is the final nuclear effector of Hippo pathway, which is activated by Yes-associated protein (YAP) through binding to two separated YAP regions of α1-helix and Ω-loop. Previous efforts have all been addressed on deriving peptide inhibitors from the YAP to target TEAD4. Instead, we herein attempted to rationally design a so-called 'YAP helixα1-trap' based on the TEAD4 to target YAP by using dynamics simulation and energetics analysis as well as experimental assays at molecular and cellular levels. The trap represents a native double-stranded helical hairpin covering a specific YAP-binding site on TEAD4 surface, which is expected to form a three-helix bundle with the α1-helical region of YAP, thus competitively disrupting TEAD4-YAP interaction. The hairpin was further stapled by a disulfide bridge across its two helical arms. Circular dichroism characterized that the stapling can effectively constrain the trap into a native-like structured conformation in free state, thus largely minimizing the entropy penalty upon its binding to YAP. Affinity assays revealed that the stapling can considerably improve the trap binding potency to YAP α1-helix by up to 8.5-fold at molecular level, which also exhibited a good tumor-suppressing effect at cellular level if fused with TAT cell permeation sequence. In this respect, it is considered that the YAP helixα1-trap-mediated blockade of Hippo pathway may be a new and promising therapeutic strategy against cancers.

Luminescent photon-upconversion nanoparticles with advanced functionalization for smart sensing and imaging.

Photon-upconversion nanoparticles (UCNP) have already been established as labels for affinity assays in analog and digital formats. Here, advanced, or smart, systems based on UCNPs coated with active shells, fluorescent dyes, and metal and semiconductor nanoparticles participating in energy transfer reactions are reviewed. In addition, switching elements can be embedded in such assemblies and provide temporal and spatial control of action, which is important for intracellular imaging and monitoring activities. Demonstration and critical comments on representative approaches demonstrating the progress in the use of such UCNPs in bioanalytical assays, imaging, and monitoring of target molecules in cells are reported, including particular examples in the field of cancer theranostics.

A ligand discovery toolbox for the WWE domain family of human E3 ligases

The WWE domain is a relatively under-researched domain found in twelve human proteins and characterized by a conserved tryptophan-tryptophan-glutamate (WWE) sequence motif. Six of these WWE domain-containing proteins also contain domains with E3 ubiquitin ligase activity. The general recognition of poly-ADP-ribosylated substrates by WWE domains suggests a potential avenue for development of Proteolysis-Targeting Chimeras (PROTACs). Here, we present novel crystal structures of the HUWE1, TRIP12, and DTX1 WWE domains in complex with PAR building blocks and their analogs, thus enabling a comprehensive analysis of the PAR binding site structural diversity. Furthermore, we introduce a versatile toolbox of biophysical and biochemical assays for the discovery and characterization of novel WWE domain binders, including fluorescence polarization-based PAR binding and displacement assays, 15N-NMR-based binding affinity assays and 19F-NMR-based competition assays. Through these assays, we have characterized the binding of monomeric iso-ADP-ribose (iso-ADPr) and its nucleotide analogs with the aforementioned WWE proteins. Finally, we have utilized the assay toolbox to screen a small molecule fragment library leading to the successful discovery of novel ligands targeting the HUWE1 WWE domain.

Structural Optimization of Azacryptands for Targeting Three-Way DNA Junctions.

Transient melting of the duplex-DNA (B-DNA) during DNA transactions allows repeated sequences to fold into non-B-DNA structures, including DNA junctions and G-quadruplexes. These noncanonical structures can act as impediments to DNA polymerase progression along the duplex, thereby triggering DNA damage and ultimately jeopardizing genomic stability. Their stabilization by ad hoc ligands is currently being explored as a putative anticancer strategy since it might represent an efficient way to inflict toxic DNA damage specifically to rapidly dividing cancer cells. The relevance of this strategy is only emerging for three-way DNA junctions (TWJs) and, to date, no molecule has been recognized as a reference TWJ ligand, featuring both high affinity and selectivity. Herein, we characterize such reference ligands through a combination of in vitro techniques comprising affinity and selectivity assays (competitive FRET-melting and TWJ Screen assays), functional tests (qPCR and Taq stop assays) and structural analyses (molecular dynamics and NMR investigations). We identify novel azacryptands TrisNP-amphi and TrisNP-ana as the most promising ligands, interacting with TWJs with high affinity and selectivity. These ligands represent new molecular tools to investigate the cellular roles of TWJs and explore how they can be exploited in innovative anticancer therapies.

Strengthened binding affinity of bispecific antibody nanoplatforms improved the anti-tumor efficacy

Dual receptor targeting (DRT) strategies use bispecific antibodies to simultaneously target two distinct disease mediators and thereby overcome major escape mechanisms evident in mono-targeted therapy. DRT enhances the binding affinity between the ligand and receptors and the specific targeted delivery of nanoparticles (NPs) into cancer cells. In this study we developed anti-EGFR cetuximab (CTX) and Herceptin (HER) decorated poly(lactic-co-glycolic acid) (PLGA) NPs for the targeted delivery of docetaxel (DTX), which we call DRT-DTX-PLGA-NPs, and we quantified their binding affinity with EGFR and HER2 positive cancer cells.The binding affinity assay indicated that dual conjugation of CTX and HER on PLGA-NPsenhanced the binding affinity between the NPs and cells additively in terms of affinity and synergistically in terms of adhesion energy, compared with PLGA-NPs conjugated with CTX and HER. Furthermore, the DRT-DTX-PLGA-NPs exhibited higher cellular uptake and higher cancer cell cytotoxicity than the single receptor-targeted NPs. In SW480 xenograft mice, the DRT-DTX-PLGA-NPs were more concentrated and retained at the tumor site due to their enhanced binding affinity, and as a result, tumor volume and tumor weight were significantly inhibited compared with tumors treated with the single-ligand targeted NPs.Therefore, DRT-NPs have the potential to improve cancer therapy by providing high affinity between NPs and cells and improving selectivity compared with single-ligand-targeted NPs.

Interference of hemoglobin variants with HbA1c measurements by six commonly used HbA1c methods.

Glycated hemoglobin, or hemoglobin A1c (HbA1c), serves as a crucial marker for diagnosing diabetes and monitoring its progression. We aimed to assess the interference posed by common Hb variants on popular HbA1c measurement systems. A total of 63 variant and nonvariant samples with target values assigned by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC)-calibrated methods were included. We assessed 6 methods for measuring HbA1c in the presence of HbS, HbC, HbD, HbE, and fetal hemoglobin (HbF): 2 cation-exchange high-performance liquid chromatography (HPLC) methods (Bio-Rad D-100 and HLC-723 G8), a capillary electrophoresis (CE) method (Sebia Capillarys 3 TERA), an immunoassay (Roche c501), an enzyme assay system (Mindray BS-600M), and a boronate affinity method (Primus Premier Hb9210). The HbA1c results for nonvariant samples from the 6 methods were in good agreement with the IFCC-calibrated method results. The Bio-Rad D-100, Capillarys 3, Mindray BS-600M, Premier Hb9210, and Roche c501 showed no interference from HbS, HbC, HbD, and HbE. Clinically significant interference was observed for the HLC-723 G8 standard mode. Elevated HbF levels caused significant negative biases for all 6 methods, which increased with increasing HbF concentration. Elevated levels of HbF can severely affect HbA1c measurements by borate affinity, immunoassays, and enzyme assays.

Fibrinogen binding to histones in circulation protects against adverse cellular and clinical outcomes

BackgroundCirculating histones are released by extensive tissue injury or cell death and play important pathogenic roles in critical illnesses. Their interaction with circulating plasma components and the potential roles in the clinical setting are not fully understood. ObjectivesWe aimed to characterize the interaction of histones with fibrinogen and explore its roles in vitro, in vivo, and in patient samples. MethodsHistone–fibrinogen binding was assessed by electrophoresis and enzyme-linked immunosorbent assay-based affinity assay. Functional significance was explored using washed platelets and endothelial cells in vitro and histone-infusion mouse models in vivo. To determine clinical translatability, a retrospective single-center cohort study was conducted on patients requiring intensive care admission (n = 199) and validated in a cohort of hospitalized patients with COVID-19 (n = 69). ResultsFibrinogen binds histones through its D-domain with high affinity (calf thymus histones, KD = 18.0 ± 5.6 nM; histone 3, KD = 2.7 ± 0.8 nM; and histone 4, KD = 2.0 ± 0.7 nM) and significantly reduces histone-induced endothelial damage and platelet aggregation in vitro and in vivo in a histone-infusion mouse model. Physiologic concentrations of fibrinogen can neutralize low levels of circulating histones and increase the cytotoxicity threshold of histones to 50 μg/mL. In a cohort of patients requiring intensive care, a histone:fibrinogen ratio of ≥6 on admission was associated with moderate–severe thrombocytopenia and independently predicted mortality. This finding was validated in a cohort of hospitalized patients with COVID-19. ConclusionFibrinogen buffers the cytotoxic properties of circulating histones. Detection and monitoring of circulating histones and histone:fibrinogen ratios will help identify critically ill patients at highest risk of adverse outcomes who might benefit from antihistone therapy.

Preparation and evaluation of decellularized epineurium as an anti-Adhesive biofilm in peripheral nerve repair

Abstract Following peripheral nerve anastomosis, the anastomotic site is prone to adhesions with surrounding tissues, consequently impacting the effectiveness of nerve repair. This study explores the development and efficacy of a decellularized epineurium as an anti-adhesive biofilm in peripheral nerve repair. Firstly, the entire epineurium was extracted from fresh porcine sciatic nerves, followed by a decellularization process. The decellularization efficiency was then thoroughly assessed. Subsequently, the decellularized epineurium underwent proteomic analysis to determine the remaining bioactive components. To ensure biosafety, the decellularized epineurium underwent cytotoxicity assays, hemolysis tests, cell affinity assays, and assessments of immune response following subcutaneous implantation. Finally, the functionality of the biofilm was determined using a sciatic nerve transection and anastomosis model in rat. The result indicated that the decellularization process effectively removed cellular components from the epineurium while preserving a number of bioactive molecules, and this decellularized epineurium was effective in preventing adhesion while promoting nerve repairment and functional recovery. In conclusion, the decellularized epineurium represents a novel and promising anti-adhesion biofilm for enhancing surgical outcomes of peripheral nerve repair.

Combining the Benefits of Biotin-Streptavidin Aptamer Immobilization with the Versatility of Ni-NTA Regeneration Strategies for SPR.

The high affinity of the biotin-streptavidin interaction has made this non-covalent coupling an indispensable strategy for the immobilization and enrichment of biomolecular affinity reagents. However, the irreversible nature of the biotin-streptavidin bond renders surfaces functionalized using this strategy permanently modified and not amenable to regeneration strategies that could increase assay reusability and throughput. To increase the utility of biotinylated targets, we here introduce a method for reversibly immobilizing biotinylated thrombin-binding aptamers onto a Ni-nitrilotriacetic acid (Ni-NTA) sensor chip using 6xHis-tagged streptavidin as a regenerable capture ligand. This approach enabled the reproducible immobilization of aptamers and measurements of aptamer-protein interaction in a surface plasmon resonance assay. The immobilized aptamer surface was stable during five experiments over two days, despite the reversible attachment of 6xHis-streptavidin to the Ni-NTA surface. In addition, we demonstrate the reproducibility of this immobilization method and the affinity assays performed using it. Finally, we verify the specificity of the biotin tag-streptavidin interaction and assess the efficiency of a straightforward method to regenerate and reuse the surface. The method described here will allow researchers to leverage the versatility and stability of the biotin-streptavidin interaction while increasing throughput and improving assay efficiency.

Unveiling new myrsinoic acids and AChE ligands from Myrsine guianensis (Aubl.) Kuntze

Molecular dereplication and drug-like discovery are important tools for exploring the chemical profile of metabolites in a complex mixture. In order to establish a workflow for discovering novel acetylcholinesterase (AChE) ligands, we performed the chemical study of Myrsine guianensis (Aubl.) Kuntze (Primulaceae). To carry out the bioprospection, nine extracts were obtained from different parts of the plant. Through the dereplication approaches, seventeen metabolites were annotated. In order to confirm the putative inferences, a HPLC preparative method was developed to isolate three known myrsinoic acids, A(1), B(2) and C(3). Along with, we are reporting the obtention of two new congeners, G(5) and H(6), which their structures were elucidated by NMR and HRMS data. Besides that, two extracts were submitted to affinity assays to accelerate the discovery of AChE ligands. Desorbates were analyzed through LC-HRMS for calculating the affinity ratio (AR). Thus, (1) presented AR = 4.59, therefore was considered a potential ligand.

Loop PDE of viral capsid protein is involved in immune escape of the emerging novel variant infectious bursal disease virus

Infectious bursa disease (IBD) is an acute, highly contactable, lethal, immunosuppressive infectious disease caused by the Infectious bursa disease virus (IBDV). Currently, the emerged novel variant IBDV (nVarIBDV) and the sustainedly prevalent very virulent IBDV (vvIBDV) are the two most prevalent strains of IBDV in China. The antigenic properties of the two prevalent strains differed significantly, which led to the escape of nVarIBDV from the immune protection provided by the existing vvIBDV vaccine. However, the molecular basis of the nVarIBDV immune escape remains unclear. In this study, we demonstrated, for the first time, that residues 252, 254, and 256 in the PDE of VP2 are involved in the immune escape of the emerging nVarIBDV. Firstly, the IFA-mediated antigen-antibody affinity assay showed that PBC and PDE of VP2 could affect the affinity of vvIBDV antiserum to VP2, of which PDE was more significant. The key amino acids of PDE influencing the antigen-antibody affinity were also identified, with G254N being the most significant, followed by V252I and I256V. Then the mutated virus with point or combined mutations was rescued by reverse genetics. it was further demonstrated that mutations of V252I, G254N, and I256V in PDE could individually or collaboratively reduce antigen–antibody affinity and interfere with antiserum neutralization, with G254N being the most significant. This study revealed the reasons for the widespread prevalence of nVarIBDV in immunized chicken flocks and provided innovative ideas for designing novel vaccines that match the antigen of the epidemic strain.