High Specific Interaction Research Articles

Nonspecific interactions in transcription regulation and organization of transcriptional condensates

Eukaryotic cells are characterized by a high degree of compartmentalization of their internal contents, which ensures precise and controlled regulation of intracellular processes. During many processes, including different stages of transcription, dynamic membrane-free compartments called biomolecular condensates are formed. Transcription condensates contain various transcription factors and RNA polymerase and are formed by high and low specificity interactions between protein factors, DNA and nearby RNA. This review discusses recent data demonstrating the important role of nonspecific multivalent protein-protein and RNA-protein interactions in the organization and regulation of transcription.

Hematopoietic cell kinase as a nexus for drug repurposing: implications for cancer and HIV therapy

Hematopoietic cell kinase (HCK) has emerged as a potential target for therapeutic intervention in cancer and HIV infection because of its critical role in critical signaling pathways. Repurposing FDA-approved drugs offers an efficient strategy to identify new treatment options. Here, we address the need for novel therapies in cancer and HIV by investigating the potential of repurposed drugs against HCK. Our goal was to identify promising drug candidates with high binding affinities and specific interactions within the HCK binding pocket. We employed an integrated computational approach combining molecular docking and extensive molecular dynamics (MD) simulations. Initially, we analyzed the binding affinities and interaction patterns of a library of FDA-approved drugs sourced from DrugBank. After careful analysis, we focused on two compounds, Nilotinib and Radotinib, which exhibit exceptional binding affinities and specificity to the HCK binding pocket, including the active site. Additionally, we assessed the pharmacological properties of Nilotinib and Radotinib, making them attractive candidates for further drug development. Extensive all-atom MD simulations spanning 200 nanoseconds (ns) elucidated the conformational dynamics and stability of the HCK-Nilotinib and HCK-Radotinib complexes. These simulations demonstrate the robustness of these complexes over extended timescales. Our findings highlighted the potential of Nilotinib and Radotinib as promising candidates against HCK that offer valuable insights into their binding mechanisms. This computational approach provides a comprehensive understanding of drug interactions with HCK and sets the stage for future experimental validation and drug development endeavors. Communicated by Ramaswamy H. Sarma

High-throughput quantitation of protein-RNA UV-crosslinking efficiencies as a predictive tool for high-confidence identification of RNA-binding proteins.

UV-crosslinking has proven to be an invaluable tool for the identification of RNA-protein interactomes. The paucity of methods for distinguishing background from bona fide RNA-protein interactions, however, makes attribution of RNA-binding function on UV-crosslinking alone challenging. To address this need, we previously reported an RNA-binding protein (RBP) confidence scoring metric (RCS), incorporating both signal-to-noise (S:N) and protein abundance determinations to distinguish high- and low-confidence candidate RBPs. Although RCS has utility, we sought a direct metric for quantification and comparative evaluation of protein-RNA interactions. Here we propose the use of protein-specific UV-crosslinking efficiency (%CL), representing the molar fraction of a protein that is crosslinked to RNA, for functional evaluation of candidate RBPs. Application to the HeLa RNA interactome yielded %CL values for 1097 proteins. Remarkably, %CL values span over five orders of magnitude. For the HeLa RNA interactome, %CL values comprise a range from high efficiency, high specificity interactions, e.g., the Elav protein HuR and the Pumilio homolog Pum2, with %CL values of 45.9 and 24.2, respectively, to very low efficiency and specificity interactions, for example, the metabolic enzymes glyceraldehyde-3-phosphate dehydrogenase, fructose-bisphosphate aldolase, and alpha-enolase, with %CL values of 0.0016, 0.006, and 0.008, respectively. We further extend the utility of %CL through prediction of protein domains and classes with known RNA-binding functions, thus establishing it as a useful metric for RNA interactome analysis. We anticipate that this approach will benefit efforts to establish functional RNA interactomes and support the development of more predictive computational approaches for RBP identification.

Preparation and performance of FeCo bimetallic organic frameworks with super adsorption and excellent peroxymonosulfate activation for tetracycline removal

As bifunctional materials for adsorption and peroxymonosulfate (PMS) activation, iron − cobalt bimetallic organic frameworks (FeCo − MOF) were successfully fabricated via a facile method at room temperature, and characterized by XRD, SEM, TEM, XPS, TGA, BET and FTIR. Due to the high specific surface area and electrostatic interaction, FeCo − MOF showed excellent adsorption capacity. 30 mg of FeCo − MOF can adsorb 87.5 % of tetracycline (TC) in a 100 mL aqueous solution (70 mg/L). The adsorption data are well fitted by the Langmuir model and pseudo − second − order adsorption kinetics, indicating the TC capture probably was monolayer molecular adsorption and chemisorption. Meanwhile, FeCo − MOF/PMS had the outstanding catalytic activity for TC degradation via PMS activation. 10 mg of FeCo − MOF can remove 91 % of TC within 5 min in a 100 mL aqueous solution (20 mg/L) with PMS (0.1 mmol·L−1). Quenching experiments showed that •OH, SO4•−, O2•− and 1O2 all participate in the TC degradation. The effects of PMS concentration, pH value, reaction temperature on TC degradation were investigated. Although the coexisting inorganic anions and small molecule acids inhibited the catalytic performance of FeCo − MOF/PMS, the TC removal rates were still greater than 80 %, indicating the FeCo − MOF/PMS system has a strong anti − interference ability to complex water environments. In addition, the complete degradation of various pollutants, including TC, methylene orange (MO) and methyl blue (MB), and TC degradation in different water bodies confirmed the wide applicability of the FeCo − MOF/PMS system.

Novel vinylene-based covalent organic framework as a promising adsorbent for the rapid extraction of beta-agonists in meat samples

Beta-agonists are potent bronchodilators approved for the treatment of asthma and tocolysis. However, they have been extensively misused as feed additives in the veterinary field to improve feed efficiency. The concern over their potential hazard to health has come to the fore again. In this study, a novel vinylene-based covalent organic framework (V-COF-1) with a two-dimensional structure was developed. The structure shows good tolerance in a variety of mediums, which can be attributed to the low polarity linkage. The high specific surface area and variable interaction with analytes accelerate the extraction time. Furthermore, the swelling resulting from the formation of hydrogen bonds by the protic solvent intercalation with the triazine group also improves the adsorption efficiency. Finally, due to its great reusability, it is economical material in sample preparation application. The V-COF-1 based μ-dSPE approach was coupled with UHPLC–MS/MS to develop a highly sensitive and selective method. The linearity of the method ranged from 0.05 to 20 ng g−1 with a correlation coefficient (R2) higher than 0.9958, and the limits of detection and quantification fell in the ranges of 0.01–0.10 ng g−1 and 0.04–0.32 ng g−1. The proposed method has been successfully applied to determine beta-agonists in meat samples, and the results indicated good recovery of 82.2–116%. The intra-day and inter-day precision were less than 6.61%, indicating the potential for sustainable application in food analysis.

Persistent Degradation of HER2 Protein by Hybrid nanoPROTAC for Programmed Cell Death.

Proteolysis-targeting chimera (PROTAC) has emerged as a promising strategy for degrading proteins of interest. Peptide-based PROTACs offer several advantages over small-molecule-based PROTACs, such as high specificity, low toxicity, and large protein-protein interaction surfaces. However, peptide-based PROTACs have several intrinsic shortcomings that strongly limit their application including poor cell permeability and low stability and potency. Herein, we designed a nanosized hybrid PROTAC (GNCTACs) to target and degrade human epidermal growth factor receptor 2 (HER2) in tumor cells. Gold nanoclusters (GNCs) were utilized to connect HER2-targeting peptides and cereblon (CRBN)-targeting ligands. GNCTACs could overcome the intrinsic barriers of peptide-based PROTACs, efficiently delivering HER2-targeting peptides in the cytoplasm and protecting them from degradation. Furthermore, a fasting-mimicking diet was applied to enhance the cellular uptake and proteasome activity. Consequently, more than 95% of HER2 in SKBR3 cells was degraded by GNCTACs, and the degradation lasted for at least 72 h, showing a catalytic-like reaction.

A Review of the Treatment Process of Perfluorooctane Compounds in the Waters: Adsorption, Flocculation, and Advanced Oxidative Process

Perfluorinated compounds (PFCs) are recognized as a new type of refractory organic pollutants. Due to the persistent environmental pollution, bioaccumulation, and biotoxicity of PFCs, they have received extensive attention in recent years. To deal with the environmental risks caused by PFCs, the pollution and distribution of PFCs in the aquatic environment are discussed in detail, mainly for the most widely used PFCs—perfluorooctanoate (PFOA) and perfluorooctane sulfonate (PFOS). The latest progress in the current processing technology of PFOA and PFOS is comprehensively introduced. It includes a variety of physical techniques to remove PFCs such as adsorption and flocculation. It has been confirmed that various adsorbents can play a key role in the enrichment and removal of PFCs through high specific surface area and hydrophobic interaction. In addition, traditional degradation processes are often unsatisfactory for PFCs, prompting the search for more efficient and cost-effective methods, with great progress having been made in advanced oxidation processes (AOPs) based on radical decomposition of pollutants. This review also integrates multiple advanced oxidation processes (AOPs) such as photocatalysis, electrochemical processes, ozone, the Fenton process, and ultrasound. This paper provides an overview of the various PFCs removal techniques and discusses their efficacy. It also explores future possible developments for PFCs elimination technologies for water treatment.

Amino-functional metal–organic framework as a general applicable adsorbent for simultaneous enrichment of nine neonicotinoids

Four functional metal–organic frameworks (MOFs) were facilely synthesized through one-pot procedure or post-synthetic modification method and used as adsorbents for simultaneous enrichment of nine neonicotinoids (NEOs) for the first time. Of which, amino-functional MIL-101-NH2 showed general high affinity towards nine NEOs and performed the best due to extraordinarily high surface area and specific interaction with analytes. A simple, efficient and robust method was developed by dispersive solid phase extraction with MIL-101-NH2 combining with liquid chromatography mass spectrometry (dSPE-LC-MS). Key factors that affecting enrichment efficiency were investigated and the established method demonstrated high sensitivity with detection limits as low as 0.02–0.1 ng/mL and wide linearity range over 3–4 orders of magnitude. Besides, compared with commercial SPE cartridges, this adsorbent showed superior enrichment efficiency and could be used for at least five times without obvious recovery decrease. The method was also applied for determination of trace NEOs in aqueous environmental samples and NEO contamination was found. This study demonstrated the practicability of facile synthetic MOF in simultaneous enrichment of nine NEOs and offered a simple and general method for trace amount NEOs sample preparation in real samples.

Discovery of Small Molecule NSC290956 as a Therapeutic Agent for KRas Mutant Non-Small-Cell Lung Cancer.

HRas-GTP has a transient intermediate state with a “non-signaling open conformation” in GTP hydrolysis and nucleotide exchange. Due to the same hydrolysis process and the structural homology, it can be speculated that the active KRas adopts the same characteristics with the “open conformation.” This implies that agents locking this “open conformation” may theoretically block KRas-dependent signaling. Applying our specificity-affinity drug screening approach, NSC290956 was chosen by high affinity and specificity interaction with the “open conformation” structure HRasG60A-GppNp. In mutant KRas-driven non-small-cell lung cancer (NSCLC) model system, NSC290956 effectively suppresses the KRas-GTP state and gives pharmacological KRas inhibition with concomitant blockages of both the MAPK-ERK and AKT-mTOR pathways. The dual inhibitory effects lead to the metabolic phenotype switching from glycolysis to mitochondrial metabolism, which promotes the cancer cell death. In the xenograft model, NSC290956 significantly reduces H358 tumor growth in nude mice by mechanisms similar to those observed in the cells. Our work indicates that NSC290956 can be a promising agent for the mutant KRas-driven NSCLC therapy.

Babesia Bovis Ligand-Receptor Interaction: AMA-1 Contains Small Regions Governing Bovine Erythrocyte Binding.

Apical membrane antigen 1 is a microneme protein which plays an indispensable role during Apicomplexa parasite invasion. The detailed mechanism of AMA-1 molecular interaction with its receptor on bovine erythrocytes has not been completely defined in Babesia bovis. This study was focused on identifying the minimum B. bovis AMA-1-derived regions governing specific and high-affinity binding to its target cells. Different approaches were used for detecting ama-1 locus genetic variability and natural selection signatures. The binding properties of twelve highly conserved 20-residue-long peptides were evaluated using a sensitive and specific binding assay based on radio-iodination. B. bovis AMA-1 ectodomain structure was modelled and refined using molecular modelling software. NetMHCIIpan software was used for calculating B- and T-cell epitopes. The B. bovis ama-1 gene had regions under functional constraint, having the highest negative selective pressure intensity in the Domain I encoding region. Interestingly, B. bovis AMA-1-DI (100YMQKFDIPRNHGSGIYVDLG119 and 120GYESVGSKSYRMPVGKCPVV139) and DII (302CPMHPVRDAIFGKWSGGSCV321)-derived peptides had high specificity interaction with erythrocytes and bound to a chymotrypsin and neuraminidase-treatment sensitive receptor. DI-derived peptides appear to be exposed on the protein’s surface and contain predicted B- and T-cell epitopes. These findings provide data (for the first-time) concerning B. bovis AMA-1 functional subunits which are important for establishing receptor-ligand interactions which could be used in synthetic vaccine development.

Production of microporous Cu-doped BTC (Cu-BTC) metal-organic framework composite materials, superior adsorbents for the removal of methylene blue (Basic Blue 9)

Abstract Cellulosic woven waste was used as a biomass material to prepare a Cu-doped BTC (Cu-BTC) adsorbent, which was then used to remove methylene blue (Basic Blue 9) from wastewater. Cellulosic woven waste was used as a biomass material to prepare a Cu-doped BTC (Cu-BTC) adsorbent, which was then used to remove methylene blue (Basic Blue 9) from wastewater. The Cu-BTC had higher adsorption capacity for methylene blue (BB9) than pure woven waste because it had high specific surface area and electrostatic interaction with cationic methylene blue molecules. The Cu-BTC removed methylene blue from wastewater rapidly and effectively and had an excellent adsorption capacity (197.90 mg/g). In batch process, the adsorption efficiency of the adsorbent for removal of BB9 was evaluated within 20 °C–60 °C, with initial BB9 concentrations of 50−200 mg/L and initial pH of 2−11. The Cu-BTC activation tailored the topological and textural properties of the obtained adsorbent, leading to a relatively large surface area of 1418.3 m2/g and pores with a volume of 0.491 cm3/g and an average size of 2.11 nm. The adsorption process fitted well with the Langmuir isotherm and the pseudo-second-order kinetic model. The possible mechanism for methylene blue removal mainly involved electrostatic attraction and micro pores. This study can serve as a guide for value-added utilization of cellulosic woven waste and as a practical method for the removal of methylene blue from wastewater. Adsorption of methylene blue onto the Cu-BTC is an effective and eco-friendly method for its removal from wastewater.

Clay/au nanoparticle composites as acetylcholinesterase carriers and modified-electrode materials: A comparative study

This work presents the first comparative study on syntheses, characterization, and applications of different clay/gold nanoparticle composites (clay/AuNPs) as acetylcholinesterase (AChE) carriers and modified-electrode materials to detect chlorpyrifos, an organophosphate pesticide. The selected based-clays were plate-like kaolinite (Kaol; 1:1 aluminum phyllosilicate), globular montmorillonite (Mt; 2:1 aluminum phyllosilicate), globular bentonite (Bent; 2:1 aluminum phyllosilicate), and fibrous sepiolite (Sep; 2:1 inverted ribbons of magnesium phyllosilicate). The clay/AuNPs were synthesized using in situ chemical reduction of the gold precursor with anchoring 3-aminopropyl triethoxysilane (APTES) on the clay surfaces. The studied materials were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), nitrogen adsorption/desorption, Fourier transformed infrared (FTIR) spectroscopy, thermogravimetric analyzer (TGA), zeta-potential analysis, and electrochemical impedance spectroscopy (EIS). Surface area, surface charges, and hydrophilic/hydrophobic nature of based-clays and the clay/AuNPs played major roles on AChE loading, residual activity, and sensor storage stability. The Mt/AuNPs was determined, among the studied clays, as the best supports for AChE loading and residual activities due to its high specific surface area and weak electrostatic interaction with the immobilized enzyme. Since apparent catalytic efficiencies kcatapp/KMappof the immobilized AChE in the four clay/AuNPs were found statistically similar, Mt/AuNPs which contained the highest enzyme loading was the favorite modified-electrode material. For chlorpyrifos detection, Kaol/AuNPs-based sensors resulted in the lowest detection limit, presumably due to the strong hydrophobic surface that helps to concentrate the nonpolar pesticide to the vicinity of the immobilized enzyme. Unfortunately, the highest electrostatic repulsion between the Kaol/AuNPs and AChE, and the dehydrated Kaol surface resulted in the lowest storage stability of the AChE pesticide sensors. Overall, Mt/AuNPs showed the highest potential as the AChE carrier and the modified electrode material for chlorpyrifos determination.

High-performance capacitive deionization using nitrogen and phosphorus-doped three-dimensional graphene with tunable pore size

Capacitive deionization (CDI) is considered as a potential water purification technology for electrochemical removal of the salt from aqueous solutions. However, the development of an electrode material with adequate electro adsorption rate and capacity is still a major challenge. In this study, a N and P-doped three-dimensional (3D) structure of graphene with high conductivity and capacitance for high performance CDI was synthesized and possessed tunable pore size through phosphoric acid activation. It had a large specific surface area of 567.14 m2 g−1 due to the 3D hierarchical porous structure and highly crosslinked networks of graphene sheets. This novel material also had a high specific capacitance (177.19 F g−1). High specific capacitance and interaction of micropores with mesopores improved the desalination capacity up to 20.93 mg g−1. Consequently, the materials with high specific surface area, better specific capacitance and tunable pore size can have a great potential for efficient and practical desalination applications.

Acceleration of cellodextrin phosphorolysis for bioelectricity generation from cellulosic biomass by integrating a synthetic two-enzyme complex into an in vitro synthetic enzymatic biosystem

BackgroundCellulosic biomass, the earth’s most abundant renewable resource, can be used as substrates for biomanufacturing biofuels or biochemicals via in vitro synthetic enzymatic biosystems in which the first step is the enzymatic phosphorolysis of cellodextrin to glucose 1-phosphate (G1P) by cellodextrin phosphorylase (CDP). However, almost all the CDPs prefer cellodextrin synthesis to phosphorolysis, resulting in the low reaction rate of cellodextrin phosphorolysis for biomanufacturing.ResultsTo increase the reaction rate of cellodextrin phosphorolysis, synthetic enzyme complexes containing CDP and phosphoglucomutase (PGM) were constructed to convert G1P to glucose 6-phosphate (G6P) rapidly, which is an important intermediate for biomanufacturing. Four self-assembled synthetic enzyme complexes were constructed with different spatial organizations based on the high-affinity and high-specific interaction between cohesins and dockerins from natural cellulosomes. Thus, the CDP–PGM enzyme complex with the highest enhancement of initial reaction rate was integrated into an in vitro synthetic enzymatic biosystem for generating bioelectricity from cellodextrin. The in vitro biosystem containing the best CDP–PGM enzyme complex exhibited a much higher current density (3.35-fold) and power density (2.14-fold) than its counterpart biosystem containing free CDP and PGM mixture.ConclusionsHereby, we first reported bioelectricity generation from cellulosic biomass via in vitro synthetic enzymatic biosystems. This work provided a strategy of how to link non-energetically favorable reaction (cellodextrin phosphorolysis) and energetically favorable reaction (G1P to G6P) together to circumvent unfavorable reaction equilibrium and shed light on improving the reaction efficiency of in vitro synthetic enzymatic biosystems through the construction of synthetic enzyme complexes.

Excellent adsorption capacity and photocatalytic regeneration of nanoparticles-assembled mesoporous Cu2O/Bi2O3 composites for removal of methyl orange

A series of novel nanoparticles-assembled Cu2O/Bi2O3 composites were synthesized by simple wet chemical reduction method. The synthesized materials were characterized by XRD, SEM, BET and FTIR. The microstructures are considered to be the nanoparticles-assembled mesoporous structure with large specific surface. The composited Bi2O3 refine the grain of Cu2O and increase the surface area of composites. When the molar ratio of composite is 5:1, the sample shows excellent adsorption capacity of removing methyl orange and the maximum adsorption capacity is 1533.2 mg g−1. The performance of adsorption is attributed to the synergistic effects of high specific surface area and electrostatic interaction. In this work, the adsorption kinetic was investigated, which follows the pseudo-second-order kinetic model for the adsorption of methyl orange. The Cu2O/Bi2O3 composites can be reused through further photocatalytic regeneration. The results indicate that the adsorption-photocatalysis have great potential in water treatment applications.

Carbon Nanotubes as A High-Performance Platform for Target Delivery of Anticancer Quinones.

In spite of considerable efforts of researchers the cancer deseases remain to be incurable and a percentage of cancer deseases in the structure of mortality increases every year. At that, high systemic toxicity of antitumor drugs hampers their effective use. Because of this fact, the development of nanosystems for targeted delivery of antitumor drugs is one of the leading problem in nanomedicine and nanopharmacy. To critically examine the modern strategies for carbon nanotubes (CNTs)-based delivery of anticancer quinones and to summarize the mechanisms which can provide high effectiveness and multifunctionality of the CNT-based quinone delivery platform. Quinones, including anthracycline antibiotics - doxorubicin and daunorubicin, are among the most prospective group of natural and syntetic compounds which exhibit high antitumor activity against different type of tumors. In this review, we focus on the possibilities of using CNTs for targeted delivery of antitumor compounds with quinoid moiety which is ordinarily characterized by high specific interaction with DNA molecules. Quinones can be non-covalently adsorbed on CNT surface due to their aromatic structure and π-conjugated system of double bonds. The characteristic features of doxorubicine-CNT complex are high loading efficiency, pH-dependent release in acidic tumor microenviroment, enough stability in biological fluid. Different types of CNT functionalization, targeting strategies and designs for multifunctional CNT-based doxorubicine delivery platform are disscussed. Nanosystems based on functionalized CNTs are very promising platform for quinone delivery resulting in significant enhancement of cancer treatment efficiency. Functionalization of CNTs with the polymeric shell, especially DNA-based shells, can provide the greatest affinity and mimicry with biological structures.

A genetically-encoded synthetic self-assembled multienzyme complex of lipase and P450 fatty acid decarboxylase for efficient bioproduction of fatty alkenes

We develop an efficient and economic cascade multienzymes for fatty alkene bioproduction based on the lipase hydrolysis coupled to the P450 decarboxylation in the form of multiple enzyme complex. One step preparation of a multienzyme complex was based on a mixture of cell extracts including dockerin-enzyme fusions and one cohesin-cellulose binding module (CBM) fusion through high specific interaction of dockerin and cohesin. Simultaneously, the CBM was bound to cellulose carrier to form co-immobilized multienzyme. The key factors affecting overall efficiency of alkene bioproduction including substrate channeling of hydrolysis and decarboxylation, the ratio and position of two enzymes, stability were all addressed by genetically engineering of the synthetic CBM-cohesin fusions. The multienzymes exhibited more than 9.2 fold enhancement in initial reaction rate and much higher conversion yields (69%–72%) compared to mixture of free enzyme counterpart. The enzymatic cascade based multienzymes could efficiently convert renewable triglycerides to alkenes.

Purification of equine IgG3 by lectin affinity and an interaction analysis via microscale thermophoresis

The availability of purified antibodies is a prerequisite for many applications and the appropriate choice(s) for antibody-purification is crucial. Numerous methods have been developed for the purification of antibodies from different sources with affinity chromatography-based methods being the most extensively utilized. These methods are based on high specificity, easy reversibility and biological interactions between two molecules (e.g., between receptor and ligand or antibody and antigen). However, no simple techniques have yet been described to characterize and purify subclasses of immunoglobulins (Ig) from some animals of biotechnology importance such as equines, which are frequently used to produce biotherapeutic antibodies. The sera of these animals present a large number of Ig classes that have a greater complexity than other animals. The implementation of an effective protocol to purify the desired antibody class/subclasses requires meticulous planning to achieve yields at a high purity. The IgG3 subclass of equine-Ig has recently been used as antigen in a new diagnostic test for allergic responses to horse sera-based therapies. Here, we defined a simple method using Jacalin lectin immobilized on Sepharose beads to prepare highly pure equine IgG3 antibodies with a determination of the affinity constants for Jacalin lectin and horse IgG3.

Synthesis of g-C3N4 nanosheets decorated flower-like tin oxide composites and their improved ethanol gas sensing properties

The g-C3N4 nanosheets decorated three-dimensional (3D) hierarchical flower-like SnO2 nanocomposites were synthesized via a facile hydrothermal method by using the g-C3N4 and SnCl2·2H2O as the precursors. The structure and morphology of the as-synthesized samples were characterized by the X-ray powder diffraction (XRD), electron microscopy (FESEM and TEM), N2 sorption, X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectrometer (FT-IR) techniques. It is supposed that g-C3N4 nanosheets act as a template in the hydrothermal process, accelerating the preferential growth of SnO2 nanocrystals and preventing the agglomeration of the SnO2 nanoparticles. The gas sensing performances of the as-synthesized samples to ethanol vapor were tested, and the response of the sensor based on 3 wt.% g-C3N4 decorated SnO2 composite to 500 ppm ethanol vapor was 360 at 300 °C, which was much higher than that of pure flower-like SnO2 based sensor. The improved ethanol gas sensing property is proposed to relate to the good conductivity of g-C3N4, high specific surface area and interactions between g-C3N4 and SnO2.

Detection of Parathyroid Hormone Receptor 1 (PTH1R): A Novel Approach Using Enzymatic Ligands Applicable to Class B GPCRs

BACKGROUNDClass B GPCRs (receptors of the secretin family) are characterised by long C‐ and N‐terminal domains as well as by their binding of large peptide hormones. One of those, parathyroid hormone (PTH), has been reported to support C‐terminal extension without significant affinity loss for PTH1R. Indeed, the N‐terminal domain of PTH that interacts with the transmembrane domains of PTH1R leading to the activation of this receptor whereas the C‐terminal domain of PTH is involved in affinity interactions. Accordingly, we previously described a fusion protein based on the enhanced green fluorescent protein (EGFP), PTH‐EGFP. This fusion protein supported the detection of recombinant PTH1Rs, but failed to detect endogenous populations of this receptor. The objective of this study is to develop new fusion protein ligands with enzymatic activity which would allow the detection of endogenously expressed PTH1R.METHODSTo detect endogenous populations of PTH1R, we generated two new fusion proteins by linking PTH to the N‐terminal of a peroxidase. These peroxidases were either the horseradish peroxidase (HRP; PTH‐HRP) or the genetically modified soybean peroxidase APEX2 (PTH‐APEX2). Reaction of receptor‐bound enzymatic PTH‐fusion protein with widely available substrates allowed a significant signal amplification resulting in the detection of PTH1R. Alternatively, we also used the previously reported ligand PTH‐myc. The myc epitope (EQKLISEEDL) reacts with a number of monoclonal antibodies, some of them conjugated to a peroxidase. The three PTH‐fusion proteins were produced as conditioned medium (CM) following transfection of producer HEK 293a cells with the appropriate vector.RESULTSThe stimulation of recipient HEK 293a expressing PTH1R with a trimolecular complex composed of PTH‐myc, a monoclonal mouse anti‐myc antibody and an anti‐mouse HRP‐conjugated antibody led to the specific detection of this receptor either using a colorimetric peroxidase substrate (TrueBlue™) or a luminol‐based luminescent peroxidase substrate. These schemes also supported the detection of endogenously expressed receptors in the osteoblastic cell line HOS. The PTH‐APEX2 CM also supported the detection of recombinant populations of PTH1R using the colorimetric or luminescent approach but failed to detect endogenous populations of PTH1R. However, the PTH‐HRP construction was more sensitive and allowed the detection of endogenously expressed PTH1R in HOS and evidenced recombinant PTH1R as well. The specificity of the PTH1R labeling was confirmed by competing the binding of the PTH fusion proteins by an excess of PTH1R ligand (PTH1‐34 1 μM).CONCLUSIONSGiven that anti‐GPCRs antibodies are rarely valid and adapted to detect cell surface receptors, we proposed to detect intact receptors using the high affinity and specificity interaction between the receptor and its ligand. We illustrated this by generating PTH analogs prolonged at their C‐terminal by peroxidases or by an epitope recognized by widely available monoclonal antibodies that support enzymatic detection of PTH1R. Since all class B GPCRs ligands bind their receptor according to the two domain model, it could be possible to generalize the enzymatic ligand strategy to all the class B GPCRs.Support or Funding InformationCHU de Québec ‐ Université Laval, Quebec City, QC, Canada G1V 4G2