Development of a carbon quantum dot-based sensor for the detection of acetylcholinesterase and the organophosphate pesticide.

In this study, a proper and reliable fluorometric method is introduced for screening acetylcholinesterase (AChE) and its inhibitors, using carbon quantum dots (CQDs) as the signal reporter. Pure, S-doped, and P-doped CQDs, were synthesized and their recoverable fluorescence quenching properties were observed, when exposed to Hg2+, Cu2+, and Fe3+ quenching ions, respectively. The study on the recovery of their emission showed that after the introduction of another guest substance with a stronger affinity to the quenching ions, their fluorescence is restored. The Design Expert software was employed to compare the performance of the three CQDs, as fluorescent probes, based on their quenching efficiency and the percentage of their emission recovery in the presence of AChE and acetylthiocholine (ATCh). Based on the statistical analysis, among the studied CQDs, S-doped CQD was the most suitable candidate for sensor designing. The detection mechanism for the proposed S-doped CQD-based sensor is as follows: The strong binding of Cu2+ ions to carboxyl groups of S-doped CQD quenches the fluorescence signal. Then, hydrolysis of ATCh into thiocholine (TCh) in the presence of AChE causes fluorescence recovery, due to the stronger affinity of Cu2+ to the TCh, rather than the CQD. Finally, in the presence of malathion and chlorpyrifos inhibitors, AChE loses its ability to hydrolyze ATCh to TCh, so the fluorescence emission remains quenched. Based on the proposed detection technique, the designed sensor showed detection limits of 1.70 ppb and 1.50 ppb for malathion and chlorpyrifos, respectively.

Enhancing neutralizing antibodies against receptor binding domain of SARS-CoV-2 by a safe natural adjuvant system.

The receptor binding domain (RBD) plays a pivotal role in the viral entry as it enables the engagement of severe acute respiratory syndrome 2 (SARS-CoV-2) with the human angiotensin-converting enzyme 2 (ACE2) receptor for host cell entry. RBD is the major target for developing viral inhibitors and vaccines. Expression of recombinant RBD in E.coli is highly scalable with a low-cost procedure despite its high expression level compared to expression in mammalian and yeast cells. Using an alternative natural adjuvant system instead of alum adjuvant, increased immunogenicity of RBD antigen in serological assay including direct ELISA and surrogate Virus Neutralization Test (sVNT) was demonstrated with high levels of IgGs and neutralizing antibodies in mice sera immunized with RBD:AlSa (Alum and Sodium alginate) formulation. The sVNT is a simple and fast test that can be used instead of the conventional virus neutralization test requiring live virus and BSL3 laboratory to detect total neutralizing antibodies against RBD. Additionally, results showed a safety profile for sodium alginate which supported using it as an alternative natural adjuvant.

Expression of functional eGFP-fused antigen-binding fragment of ranibizumab in Pichia pastoris.

Introduction: Ranibizumab is a mouse monoclonal antibody fragment antigen-binding (Fab) against human vascular endothelial growth factor-A (VEGF-A), inhibiting angiogenesis. This antibody is commercially produced in Escherichia coli host and used to treat wet age-related macular degeneration (AMD). Methods: In this study, the heavy and light chains of ranibizumab were expressed in Pichia pastoris. The expressed chains were incubated overnight at 4°C for interaction. The formation of an active structure was evaluated based on the interaction with substrate VEGF-A using an indirect ELISA, and an electrochemical setup. Furthermore, reconstruction of split enhanced green fluorescent protein (eGFP) reporter, chimerized at the C-terminus of the heavy and light chains, was used to characterize chains' interaction. Results: P. pastoris efficiently expressed designed constructs and secreted them into the culture medium. The anti-Fab antibody detected the constructed Fab structure in western blot analysis. Reconstruction of the split reporter confirmed the interaction between heavy and light chains. The designed ELISA and electrochemical setup results verified the binding activity of the recombinant Fab structure against VEGF-A. Conclusion: In this work, we indicated that the heavy and light chains of ranibizumab Fab fragments (with or without linkage to split parts of eGFP protein) were produced in P. pastoris. The fluorescence of reconstructed eGFP was detected after incubating the equal ratio of chimeric-heavy and light chains. Immunoassay and electrochemical tests verified the bioactivity of constructed Fab. The data suggested that P. pastoris could be considered a potential efficient eukaryotic host for ranibizumab production.

An outlook on coronavirus disease 2019 detection methods.

Diagnostic testing plays a fundamental role in the mitigation and containment of coronavirus disease 2019 (COVID-19), as it enables immediate quarantine of those who are infected and contagious and is essential for the epidemiological characterization of the virus and estimating the number of infected cases worldwide. Confirmation of viral infections, such as COVID-19, can be achieved through two general approaches: nucleic acid amplification tests (NAATs) or molecular tests, and serological or antibody-based tests. The genetic material of the pathogen is detected in NAAT, and in serological tests, host antibodies produced in response to the pathogen are identified. Other methods of diagnosing COVID-19 include radiological imaging of the lungs and in vitro detection of viral antigens. This review covers different approaches available to diagnosing COVID-19 by outlining their advantages and shortcomings, as well as appropriate indications for more accurate testing.

An outlook on coronavirus disease 2019 detection methods / 药物分析学报

Diagnostic testing plays a fundamental role in the mitigation and containment of coronavirus disease 2019(COVID-19),as it enables immediate quarantine of those who are infected and contagious and is essential for the epidemiological characterization of the virus and estimating the number of infected cases worldwide.Confirmation of viral infections,such as COVID-19,can be achieved through two general approaches:nucleic acid amplification tests(NAATs)or molecular tests,and serological or antibody-based tests.The genetic material of the pathogen is detected in NAAT,and in serological tests,host antibodies produced in response to the pathogen are identified.Other methods of diagnosing COVID-19 include radiological imaging of the lungs and in vitro detection of viral antigens.This review covers different approaches available to diagnosing COVID-19 by outlining their advantages and short-comings,as well as appropriate indications for more accurate testing.

Allosteric modulators enhance agonist efficacy by increasing the residence time of a GPCR in the active state.

Much hope in drug development comes from the discovery of positive allosteric modulators (PAM) that display target subtype selectivity and act by increasing agonist potency and efficacy. How such compounds can allosterically influence agonist action remains unclear. Metabotropic glutamate receptors (mGlu) are G protein-coupled receptors that represent promising targets for brain diseases, and for which PAMs acting in the transmembrane domain have been developed. Here, we explore the effect of a PAM on the structural dynamics of mGlu2 in optimized detergent micelles using single molecule FRET at submillisecond timescales. We show that glutamate only partially stabilizes the extracellular domains in the active state. Full activation is only observed in the presence of a PAM or the Gi protein. Our results provide important insights on the role of allosteric modulators in mGlu activation, by stabilizing the active state of a receptor that is otherwise rapidly oscillating between active and inactive states.

Development and characterization of a thermostable GH11/GH10 xylan degrading chimeric enzyme.

Xylanases are categorized into different family groups, two of which are glycoside hydrolases 10 (GH10) and 11 (GH11) families. These well-characterized xylanases demonstrate different modes of action in hydrolysis of xylans. Imitating certain types of microorganisms to produce bifunctional enzymes such as engineered xylanases has gained considerable attention among researchers. In this study, a recombinant chimeric enzyme (X11-10) was designed by fusing two thermostable xylanases through a peptide linker. The recombinant parental enzymes, xylanase 10 from fungus Bispora sp. MEY-1 (X10) and xylanase 11 from bacterium Thermobacillus xylanilyticus (X11), and their chimera were successfully expressed in Pichia pastoris (P. pastoris), purified, and characterized. Being active over a wide pH range, X11-10 chimera showed higher thermal stability, possessed a lower Km, and a higher catalytic efficiency (kcat/Km) in comparison to the parental enzymes. Also, molecular dynamics simulation (MDS) of X11-10 revealed that its active site residues were free to interact with substrate. This novel chimeric xylanase may have potential applications in different industrial processes since it can substitute two separate enzymes and therefore minimize the production costs.

Design and fabrication of a label-free aptasensor for rapid and sensitive detection of endoglucanase.

Endoglucanase (endocellulase, EC 3.2.1.4) is one of the most widely used enzymes in industry. Despite its importance, improved methods for the rapid, selective, quantitative assay of this enzyme have been slow to emerge. In this work, we have designed an aptasensor platform for ultrasensitive endoglucanase II detection based on DNA aptamer and reduced graphene oxide/Au nanoparticles (RGO/AuNPs). The surface morphology of RGO/AuNPs was characterized by various techniques such as transmission electron microscopy (TEM), X-ray diffraction (XRD), energy-dispersive X-ray spectroscopy (EDX) methods. The aptasensor characterization was monitored with electrochemical techniques. Using RGO/AuNPs as a nanomaterial can effectively increase the conductivity of biosensor electrode. Moreover, using an RGO/AuNPs/aptamer platform in the presence of endoglucanase, the sensor system is able to generate a signal, which significantly improves the selectivity of the aptasensor. The fabricated aptasensor exhibits high sensitivity and selectivity to endoglucanase II with a low limit of detection (LOD) ˂0.1 nM.

Accurate Determination of Human CPR Conformational Equilibrium by smFRET Using Dual Orthogonal Noncanonical Amino Acid Labeling.

Conjugation of fluorescent dyes to proteins-a prerequisite for the study of conformational dynamics by single-molecule (sm) FRET-can lead to substantial changes in a dye's photophysical properties, ultimately biasing the determination of inter-dye distances. In particular, cyanine dyes and their derivatives, the most commonly used dyes in smFRET experiments, exhibit such behavior. To overcome this, we developed a general strategy to equip proteins site-specifically with FRET pairs through chemoselective reactions with two distinct noncanonical amino acids simultaneously incorporated through genetic code expansion in Escherichia coli. Application of this technique to human NADPH-cytochrome P450 reductase (CPR) demonstrated the importance of homogenously labeled samples for accurate determination of FRET efficiencies and unveiled the effect of NADP+ on the ionic-strength-dependent modulation of the conformational equilibrium of CPR. Thanks to its generality and accuracy, the presented methodology establishes a new benchmark for deciphering of complex molecular dynamics in single molecules.

A well-balanced preexisting equilibrium governs electron flux efficiency of a multidomain diflavin reductase.

Diflavin reductases are bidomain electron transfer proteins in which structural reorientation is necessary to account for the various intramolecular and intermolecular electron transfer steps. Using small-angle x-ray scattering and nuclear magnetic resonance data, we describe the conformational free-energy landscape of the NADPH-cytochrome P450 reductase (CPR), a typical bidomain redox enzyme composed of two covalently-bound flavin domains, under various experimental conditions. The CPR enzyme exists in a salt- and pH-dependent rapid equilibrium between a previously described rigid, locked state and a newly characterized, highly flexible, unlocked state. We further establish that maximal electron flux through CPR is conditioned by adjustable stability of the locked-state domain interface under resting conditions. This is rationalized by a kinetic scheme coupling rapid conformational sampling and slow chemical reaction rates. Regulated domain interface stability associated with fast stochastic domain contacts during the catalytic cycle thus provides, to our knowledge, a new paradigm for improving our understanding of multidomain enzyme function.

Dynamic control of electron transfers in diflavin reductases.

Diflavin reductases are essential proteins capable of splitting the two-electron flux from reduced pyridine nucleotides to a variety of one electron acceptors. The primary sequence of diflavin reductases shows a conserved domain organization harboring two catalytic domains bound to the FAD and FMN flavins sandwiched by one or several non-catalytic domains. The catalytic domains are analogous to existing globular proteins: the FMN domain is analogous to flavodoxins while the FAD domain resembles ferredoxin reductases. The first structural determination of one member of the diflavin reductases family raised some questions about the architecture of the enzyme during catalysis: both FMN and FAD were in perfect position for interflavin transfers but the steric hindrance of the FAD domain rapidly prompted more complex hypotheses on the possible mechanisms for the electron transfer from FMN to external acceptors. Hypotheses of domain reorganization during catalysis in the context of the different members of this family were given by many groups during the past twenty years. This review will address the recent advances in various structural approaches that have highlighted specific dynamic features of diflavin reductases.

The closed and compact domain organization of the 70-kDa human cytochrome P450 reductase in its oxidized state as revealed by NMR.

The NADPH cytochrome P450 reductase (CPR), a diflavin enzyme, catalyzes the electron transfer (ET) from NADPH to the substrate P450. The crystal structures of mammalian and yeast CPRs show a compact organization for the two domains containing FMN (flavin mononucleotide) and FAD (flavin adenine dinucleotide), with a short interflavin distance consistent with fast ET from the NADPH-reduced FAD to the second flavin FMN. This conformation, referred as "closed", contrasts with the alternative opened or extended domain arrangements recently described for partially reduced or mutant CPR. Internal domain flexibility in this enzyme is indeed necessary to account for the apparently conflicting requirements of having FMN flavin accessible to both the FAD and the substrate P450 at the same interface. However, how interdomain dynamics influence internal and external ETs in CPR is still largely unknown. Here, we used NMR techniques to explore the global, domain-specific and residue-specific structural and dynamic properties of the nucleotide-free human CPR in solution in its oxidized state. Based on the backbone resonance assignment of this 70-kDa protein, we collected residue-specific (15)N relaxation and (1)H-(15)N residual dipolar couplings. Surprisingly and in contrast with previous studies, the analysis of these NMR data revealed that the CPR exists in a unique and predominant conformation that highly resembles the closed conformation observed in the crystalline state. Based on our findings and the previous observations of conformational equilibria of the CPR in partially reduced states, we propose that the large-scale conformational transitions of the CPR during the catalytic cycle are tightly controlled to ensure optimal electron delivery.