ABSTRACT
Rapid, convenient and accurate detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is urgently needed to timely diagnosis of coronavirus pandemic (COVID-19) and control of the epidemic. In this study, a signal-off photoelectrochemical (PEC) immunosensor was constructed for SARS-CoV-2 nucleocapsid (N) protein detection based on a magnetic all-solid-state Z-scheme heterojunction (Fe3O4@SiO2@TiO2@CdS/Au, FSTCA). Integrating the advantages of magnetic materials and all-solid-state Z-scheme heterostructures, FSTCA was implemented to ligate the capture antibody to form magnetic capture probe (FSTCA/Ab1). It can simplify the separation and washing process to improve reproducibility and stability, while allowing immune recognition to be performed in the liquid phase instead of the traditional solid-liquid interface to improve anti-interference. Besides, the heterojunction inhibited the recombination of photogenerated electron/hole (e-/h+) and promoted the light absorption to provide superior photoelectric substrate signal. The mechanism of photogenerated e-/h+ transfer of FSTCA were investigated by the electron spin resonance (ESR) spectroscopy. SiO2 spheres loaded with Au NPs utilized as an efficient signal quencher. The steric hindrance effect of SiO2@Au labeled detection antibodies (SiO2@Au-Ab2) conjugates significantly diminished light absorption and hindered the transfer of photogenerated electrons, further amplifying the signal change value. Based on the above merits, the elaborated immunosensor had a wide linear range of 10 pg mL-1-100 ng mL-1 and a low detection limit down to 2.9 pg mL-1 (S/N = 3). The fabricated PEC immunosensor demonstrated strong anti-interference, easy operation, and high sensitivity, showing enormous potential in clinical diagnosis of SARS-CoV-2.
ABSTRACT
A sandwich "signal-off" type photoelectrochemical (PEC) immunosensor was fabricated based on a composite heterojunction of tungsten oxide/titanium oxide microspheres (WO3/TiO2) acting as signal amplification platform and carbon microspheres loaded by gold nanoparticles (Cs@Au NPs) utilized as the label for detecting antibody. WO3/TiO2 had excellent photoelectric performance, and the results of Mott-Schottky plots, open-circuit voltage, and electron spin resonance spectroscopy indicated that it belonged to the Z-scheme heterojunction transfer mechanism of photogenerated carriers. To achieve the sensitization of PEC immunosensor, Cs@Au NP-labeled immunocomplex can effectively reduce the photocurrent signal. The PEC immunosensors were fabricated under the optimal conditions of 1:1 WO3/TiO2 (molar ratio), 2.0 mg mL-1 WO3/TiO2, and 1.5 mg mL-1 Cs@Au NPs. Through comparison of the detection results of label-free and sandwich-type PEC immunosensors for nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), we found that the sensitivity of the sandwich type was 2.53 times the label-free type, and the limit of detection was 0.006 ng mL-1, i.e., 3.17 times lower than the label-free type. This demonstrates that the developed sandwich-type PEC immunosensor will have a brighter application prospect.
Subject(s)
Biosensing Techniques , COVID-19 , Metal Nanoparticles , Humans , Gold , Immunoassay , SARS-CoV-2ABSTRACT
Flavivirus, such as Dengue Virus (DENV) and Zika virus (ZIKV), infects millions of people and cause the death of thousands of people every year. Despite many efforts, there is no approved anti-flaviviral treatment available. In particular, some antiflavivirus compounds were investigated the cellular activities of DENV and ZIKV, but lacking the exploration of specific target enzyme, thereby resulting in the hindrance of structure-based drug design. One example is Montlukast, which was found to inhibit the replicon replication in DENV and ZIKV infected cells, with EC50 values as 1.03 µM (DENV) and 1.14 µM (ZIKV), while the underlying mechanism remains unclear. In our study, the inhibitory mechanisms of Montelukast against the replicon replication of DENV and ZIKV infected cells were studied by using in silico approaches including inverse virtual screening (IVS), molecular dynamics (MD) simulations and binding free energy calculation, and validated through in vitro protease assay, confirming Montelukast could bind to NS2B-NS3 proteases of DENV and ZIKV as a competitive inhibitor (IC50 for DENV: 25.65 µM, for ZIKV: 15.57 µM). Moreover, Montelukast has no potential off-target effect on NS2B-NS3 protease from thrombin and trypsin inhibitory assay. Overall, Montelukast may be used as a potential candidate to block NS2B-NS3 protease as well as lead for structural modification.
Subject(s)
Flavivirus , Zika Virus Infection , Zika Virus , Acetates , Antiviral Agents/chemistry , Cyclopropanes , Enzyme Inhibitors/pharmacology , Humans , Peptide Hydrolases , Protease Inhibitors/pharmacology , Quinolines , Sulfides , Viral Nonstructural Proteins/metabolismABSTRACT
Dengue virus (DENV) has developed rapidly in the past few decades and has been becoming the most widespread arbovirus in the world. The vital role of NS2B-NS3 in virus replication and maturation of relevant proteins makes it the most promising target for anti-DENV drug discovery, although none of NS2B-NS3 inhibitors have been approved for the market so far. In this study, potent NS2B-NS3 covalent inhibitors were discovered via chemical modification of a published covalent inhibitor WSL-01 (IC50 = 129 nM), yielding promising analogs WSL-75 and WSL-84 (IC50 = 24.8 nM and IC50 = 32.89 nM, respectively) with more than 10-fold increased enzymatic activities compared to the lead compound, and no evident cellular toxicity was observed. Further comprehensive structure-activity relationship analysis through covalent docking and molecular dynamics simulation provides informative understanding of the binding modes of covalent inhibitors targeting NS2B-NS3, which would be beneficial for novel NS2B-NS3 inhibitory development.
Subject(s)
Dengue Virus , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Dengue Virus/metabolism , Molecular Docking Simulation , Protease Inhibitors/pharmacology , Serine Endopeptidases/metabolism , Viral Nonstructural Proteins/chemistryABSTRACT
Lipid self-organization and lipid-water interfaces have been an increasingly important topic positioned at the crossroads of physical chemistry and biology. Some neutral lipids can partition into the biomembrane and play an important biological role. In this study, we have used all-atom molecular dynamics simulations to dissect the partition, aggregation, flip-flop, and modulation of neutral lipids including (i) menaquinone/menaquinol, (ii) ubiquinone/ubiquinol, and (iii) triacylglycerol. The partitioning of these molecules is driven by the balancing force between headgroup hydrophilicity and acyl chain hydrophobicity as well as the lipid shapes. We then discuss the emerging questions in this area, share our own perspectives, and mention the development of the CHARMM-GUI membrane modeling platform, which enables further computational investigations into those questions.
Subject(s)
Lipid Bilayers , Molecular Dynamics Simulation , Cell Membrane/chemistry , Hydrophobic and Hydrophilic Interactions , Lipid Bilayers/chemistry , MembranesABSTRACT
Airborne transmission of pathogens is the most probable cause for the spread of respiratory diseases, which can be intercepted by personal protective equipment such as masks. In this study, an efficient antiviral personal protective filter was fabricated by coupling the biocompatible curcumin (CCM) with nanofibrous polytetrafluoroethylene (PTFE) membrane. The CCM extracted from plants was first dissolved in acidified ethanol at a certain pH and temperature to optimize its loading concentration, antiviral activation, and binding forces on the polyethylene terephthalate (PET) support to form a pre-filtration layer at the front section of the filter. Ultrathin PTFE membrane was then fabricated on the antibacterial-antiviral PET support (A-A PET) by controllable heating lamination. This functional layer of the filter exhibits good gas permeance (3423.6 m3/(m2·h·kPa)) and ultrafine particles rejection rate (>98.79%). Moreover, the obtained A-A filter exhibit a high antibacterial rate against a variety of bacteria (E. coli, B. subtilis, A. niger, and Penicillium were 99.84%, 99.02%, 93.60%, 95.23%, respectively). Forthwith virucidal (SARS-CoV-2) efficiency of the A-A filter can reach 99.90% for 5 min. The filter shows good stability after 10 heating cycles, demonstrating its reusability.
ABSTRACT
The coronavirus disease 2019 (COVID-19) pandemic has led to a great demand on the personal protection products such as reusable masks. As a key raw material for masks, meltblown fabrics play an important role in rejection of aerosols. However, the electrostatic dominated aerosol rejection mechanism of meltblown fabrics prevents the mask from maintaining the desired protective effect after the static charge degradation. Herein, novel reusable masks with high aerosols rejection efficiency were fabricated by the introduction of spider-web bionic nanofiber membrane (nano cobweb-biomimetic membrane). The reuse stability of meltblown and nanofiber membrane mask was separately evaluated by infiltrating water, 75% alcohol solution, and exposing under ultraviolet (UV) light. After the water immersion test, the filtration efficiency of meltblown mask was decreased to about 79%, while the nanofiber membrane was maintained at 99%. The same phenomenon could be observed after the 75% alcohol treatment, a high filtration efficiency of 99% was maintained in nanofiber membrane, but obvious negative effect was observed in meltblown mask, which decreased to about 50%. In addition, after long-term expose under UV light, no filtration efficiency decrease was observed in nanofiber membrane, which provide a suitable way to disinfect the potential carried virus. This work successfully achieved the daily disinfection and reuse of masks, which effectively alleviate the shortage of masks during this special period.
ABSTRACT
This study aimed to determine whether hydrogen sulfide (H2S) inhibits pulmonary arterial endothelial inflammation in rats with monocrotaline (MCT)-induced pulmonary hypertension and its possible mechanisms. Twenty-four male Wistar rats were divided randomly into control, MCT, and MCT+H2S treatment groups. Human pulmonary arterial endothelial cells (HPAEC) were cultured and divided into four groups: control, MCT, MCT+H2S, and H2S. Pulmonary artery pressure was determined using a right cardiac catheterization procedure 3 weeks after MCT administration. Pulmonary vascular morphological changes and inflammatory infiltration were measured. Endogenous H2S levels, cystathionine-γ-lyase (CSE) expression, and inflammatory cytokines were determined both in vivo and in vitro. In addition, phosphorylation of NF-κB p65 and IκBα was detected by western blotting, and NF-κB p65 nuclear translocation, as well as its DNA-binding activity, was determined. Pulmonary hypertension and vascular remolding developed 3 wks after MCT administration, with elevated lung tissue inflammatory infiltration and cytokine level associated with activation of the NF-κB pathway, both in vivo and in vitro. However, the endogenous H2S/CSE pathway was downregulated in MCT rats. By contrast, an H2S donor markedly reduced pulmonary artery pressure, pulmonary vascular structural remolding, and increased lung inflammatory infiltration and cytokine levels of MCT-treated rats. Meanwhile, H2S reversed the activation of the NF-κB pathway successfully. The downregulated pulmonary arterial endothelial H2S/CSE pathway is involved in the pulmonary inflammatory response in MCT-treated pulmonary hypertensive rats. H2S attenuated endothelial inflammation by inhibiting the NF-κB pathway.
Subject(s)
Endothelial Cells/drug effects , Hydrogen Sulfide/pharmacology , Hypertension, Pulmonary/metabolism , Inflammation/metabolism , Pulmonary Artery/metabolism , Active Transport, Cell Nucleus/drug effects , Animals , Blotting, Western , Cell Nucleus/metabolism , Cells, Cultured , Cystathionine gamma-Lyase/metabolism , Cytokines/metabolism , Endothelial Cells/metabolism , Humans , Hydrogen Sulfide/metabolism , Hypertension, Pulmonary/chemically induced , Hypertension, Pulmonary/physiopathology , Inflammation/physiopathology , Inflammation Mediators/metabolism , Male , Monocrotaline , Phosphorylation/drug effects , Pulmonary Artery/cytology , Pulmonary Artery/physiopathology , Rats, Wistar , Transcription Factor RelA/metabolismABSTRACT
Evolutionary game theory provides an appropriate tool for investigating the competition and diffusion of behavioral traits in biological or social populations. A core challenge in evolutionary game theory is the strategy selection problem: Given two strategies, which one is favored by the population? Recent studies suggest that the answer depends not only on the payoff functions of strategies but also on the interaction structure of the population. Group interactions are one of the fundamental interactive modes within populations. This work aims to investigate the strategy selection problem in evolutionary game dynamics on group interaction networks. In detail, the strategy selection conditions are obtained for some typical networks with group interactions. Furthermore, the obtained conditions are applied to investigate selection between cooperation and defection in populations. The conditions for evolution of cooperation are derived for both the public goods game and volunteer's dilemma game. Numerical experiments validate the above analytical results.
Subject(s)
Biological Evolution , Game Theory , Social Behavior , Animals , Behavior, Animal , Competitive Behavior , Computer Simulation , Cooperative Behavior , Humans , Mathematical Concepts , Models, BiologicalABSTRACT
Cancer targeted therapy is essential to minimize damage to normal cells and improve treatment outcomes. The elevated activity of Cystathionine beta-synthase (CBS), an enzyme responsible for producing endogenous hydrogen sulfide (H2S), plays a significant role in promoting tumor growth, invasiveness, and metastatic potential. Consequently, the selective inhibition of CBS could represent a promising therapeutic strategy for cancer. Currently, there is much interest in combining paclitaxel with other drugs for cancer treatment. This study aimed to investigate the efficacy of combining benserazide, a CBS inhibitor, with paclitaxel in treating tumors. Firstly, we demonstrated CBS is indeed involved in the progression of multiple cancers. Then it was observed that the total binding free energy between the protein and the small molecule is -98.241 kJ/mol. The release of H2S in the group treated with 100 µM benserazide was reduced by approximately 90% compared to the negative control, and the thermal denaturation curve of the complex protein shifted to the right, suggesting that benserazide binds to and blocks the CBS protein. Next, it was found that compared to paclitaxel monotherapy, the combination of benserazide with paclitaxel demonstrated stronger antitumor activity in KYSE450, A549, and HCT8 cells, accompanied by reduced cell viability, cell migration and invasion, as well as diminished angiogenic and lymphangiogenic capabilities. In vivo studies showed that the combined administration of benserazide and paclitaxel significantly reduced the volume and weight of axillary lymph nodes in comparison to the control group and single administration group. Further mechanistic studies revealed that the combination of benserazide and paclitaxel significantly suppressed the S-sulfhydration of SIRT1 protein, thereby inhibiting the expression of SIRT1 protein and activating SIRT1 downstream Notch1/Hes1 signaling pathway in KYSE450, A549, and HCT8 cells. Meanwhile, we observed that benserazide combined with paclitaxel induced a more significant downregulation of HIF-1α, VEGF-A, VEGF-C, and VEGF-D proteins expression levels in KYSE450, A549, and HCT8 cells compared to paclitaxel alone. These findings indicated that benserazide enhances the anticancer effects of paclitaxel via inhibiting the S-sulfhydration of SIRT1 and down-regulating HIF-1α/VEGF signaling pathway. This study suggests that benserazide may have potential as a chemosensitizer in cancer treatment.
ABSTRACT
Our sensory adaptation to cold and chemically induced coolness is mediated by the intrinsic property of TRPM8 channels to desensitize. TRPM8 is also implicated in cold-evoked pain disorders and migraine, highlighting its inhibitors as an avenue for pain relief. Despite the importance, the mechanisms of TRPM8 desensitization and inhibition remained unclear. We found, using cryo-electron microscopy, electrophysiology, and molecular dynamics simulations, that TRPM8 inhibitors bind selectively to the desensitized state of the channel. These inhibitors were used to reveal the overlapping mechanisms of desensitization and inhibition and that cold and cooling agonists share a common desensitization pathway. Furthermore, we identified the structural determinants crucial for the conformational change in TRPM8 desensitization. Our study illustrates how receptor-level conformational changes alter cold sensation, providing insights into therapeutic development.
Subject(s)
Cold Temperature , Menthol , TRPM Cation Channels , TRPM Cation Channels/metabolism , TRPM Cation Channels/antagonists & inhibitors , Humans , Menthol/pharmacology , Molecular Dynamics Simulation , Adaptation, Physiological , Cryoelectron Microscopy , HEK293 Cells , Protein Conformation , AnimalsABSTRACT
Molecular dynamics simulations of membranes and membrane proteins serve as computational microscopes, revealing coordinated events at the membrane interface. As G protein-coupled receptors, ion channels, transporters, and membrane-bound enzymes are important drug targets, understanding their drug binding and action mechanisms in a realistic membrane becomes critical. Advances in materials science and physical chemistry further demand an atomistic understanding of lipid domains and interactions between materials and membranes. Despite a wide range of membrane simulation studies, generating a complex membrane assembly remains challenging. Here, we review the capability of CHARMM-GUI Membrane Builder in the context of emerging research demands, as well as the application examples from the CHARMM-GUI user community, including membrane biophysics, membrane protein drug-binding and dynamics, protein-lipid interactions, and nano-bio interface. We also provide our perspective on future Membrane Builder development.
Subject(s)
Membrane Proteins , Molecular Dynamics Simulation , Lipids/chemistryABSTRACT
The novel CuMnS nanoflower fluorescent probe based on Mn-doped CuS was developed to achieve the fluorescence detection of oxytetracycline hydrochloride (OTC), the fluorescent sensor has good selectivity and stability. The doping of Mn significantly increased the fluorescence intensity of CuS, which was above 10 times that of CuS. When the predominant species of OTC molecule was zwitterionic OTC+/-at the solution pH of about 5.00, the fluorescence quenching efficiency of CuMnS by OTC reached the highest. Through fluorescence lifetime and UV absorption, the sensing mechanism between CuMnS and OTC was found to be static quenching. Moreover, Multiwfn wavefunction analysis program based on density function theory (DFT) calculation was applied to compare the interactions between different OTC species and CuMnS at different pH, to reveal the micromechanism of fluorescence quenching of CuMnS by OTC from the views of atoms. The molecular surface quantitative analysis and basin analysis of different OTC species demonstrated that the N atom and O atoms of tricarbonylamide moiety of zwitterionic OTC+/- can provide lone pair electrons to form a non-fluorescent ground state complex with CuMnS. Meanwhile, the electrostatic attraction of OTC+/- with negatively charged CuMnS was also beneficial to the interaction, resulting in the effective fluorescence quenching of CuMnS. This work offers a convenient method for sensitively detecting OTC and broadens the application of CuMnS in the field of fluorescence detection.
Subject(s)
Oxytetracycline , Copper , Fluorescent Dyes/chemistry , Oxytetracycline/analysis , Oxytetracycline/chemistry , Spectrometry, Fluorescence/methodsABSTRACT
Transient receptor potential vanilloid (TRPV) channels play various important roles in human physiology. As membrane proteins, these channels are modulated by their endogenous lipid environment as the recent wealth of structural studies has revealed functional and structural lipid binding sites. Additionally, it has been shown that exogenous ligands can exchange with some of these lipids to alter channel gating. Here, we used molecular dynamics simulations to examine how one member of the TRPV family, TRPV2, interacts with endogenous lipids and the pharmacological modulator cannabidiol (CBD). By computationally reconstituting TRPV2 into a typical plasma membrane environment, which includes phospholipids, cholesterol, and phosphatidylinositol (PIP) in the inner leaflet, we showed that most of the interacting surface lipids are phospholipids without strong specificity for headgroup types. Intriguingly, we observed that the C-terminal membrane proximal region of the channel binds preferentially to PIP lipids. We also modelled two structural lipids in the simulation: one in the vanilloid pocket and the other in the voltage sensor-like domain (VSLD) pocket. The simulation shows that the VSLD lipid dampens the fluctuation of the VSLD residues, while the vanilloid lipid exhibits heterogeneity both in its binding pose and in its influence on protein dynamics. Addition of CBD to our simulation system led to an open selectivity filter and a structural rearrangement that includes a clockwise rotation of the ankyrin repeat domains, TRP helix, and VSLD. Together, these results reveal the interplay between endogenous lipids and an exogenous ligand and their effect on TRPV2 stability and channel gating.
Subject(s)
Antineoplastic Agents , TRPV Cation Channels , Humans , TRPV Cation Channels/chemistry , Ligands , Ankyrin Repeat , Binding Sites , PhospholipidsABSTRACT
L-cysteine (L-Cys) capped ZnS fluorescent probe (L-ZnS) were synthesized by binding ZnS nanoparticles in situ with L-Cys, the fluorescence intensity of L-ZnS increased more than 3.5 times than that of ZnS due to the cleavage of S-H bonds and the formation of Zn-S bonds between the thiol group of L-Cys and ZnS. The addition of copper ions (Cu2+) can effectively quench the fluorescence of L-ZnS to realize the rapid detection of trace Cu2+. The L-ZnS showed high sensitivity and selectivity to Cu2+. The LOD (limit of detection) of Cu2+ was as low as 7.28 nM and linearity in the concentration range of 3.5-25.5 µM. Meanwhile, for the first time, electron localization function (ELF), bond order density (BOD), and natural adaptive orbital (NAdO) analysis in the Multiwfn wavefunction program based on density functional theory were carried out to probe the binding sites and binding mode of L-Cys with Cu2+, it indicated that the deprotonated carboxyl oxygen atoms of L-Cys had the lowest electrostatic potential (ESP) and provided lone pair electrons to coordinate with Cu2+ to form non-luminescent ground state complexes, which led to fluorescence quenching of L-ZnS. From the microscopic point of view of atoms, the mechanism of fluorescence enhancement of L-Cys capped ZnS and the mechanism of fluorescence quenching after adding Cu2+ were revealed in depth, the theoretical analysis results were accordance with the experiments.
ABSTRACT
Aiming for precise, real-time, and on-site analysis of proteins, an innovative binary-emission fluorescence imprinted polymer was designed by sol-gel method after mixing MIL-101(Cr), green CdTe (g-CdTe) and red CdTe (r-CdTe) for detection of protein. In this proposal, MIL-101(Cr), as a favorable supporter, provided high surface area and porosity for imprinting sites, which ameliorated the transfer rate and the sensitivity of the nanosensor. And g-CdTe and r-CdTe were served as signal transduction for dual-emission response. Based on strengthened recognition reaction between high-affinity imprinting sites and protein, the fluorescence intensities of g-CdTe and r-CdTe yielded conspicuous two responses at 528 nm and 634 nm for protein under the excitation of 350 nm. The cytochrome c (Cyt c) and trypsin were served as model proteins to verify the generality of strategy. Given prominent merits of MIL-101(Cr), g-CdTe/r-CdTe@MIL-101(Cr)@MIP exhibited good linear range of 1-30 µM for Cyt c and 0.15-4 µM for trypsin, and the limit of detection were 0.13 µM and 0.014 µM, respectively. Significantly, an unsophisticated smartphone-based sensing device was developed by integrating g-CdTe/r-CdTe@MIL-101(Cr)@MIP with a 3D printing portable device to obtain precise on-site results. As expected, this portable platform was successfully applied for monitoring Cyt c and trypsin with a detection limit of 0.71 µM and 0.026 µM, respectively. These results indicated this dual-response molecularly imprinted fluorescence senor based on smartphone provided promising perspectives on futural on-site protein analysis.
Subject(s)
Cadmium Compounds , Molecular Imprinting , Quantum Dots , Smartphone , Trypsin , Tellurium , Molecular Imprinting/methods , Limit of DetectionABSTRACT
Crosstalk between ion channels and small GTPases is critical during homeostasis and disease, but little is known about the structural underpinnings of these interactions. TRPV4 is a polymodal, calcium-permeable cation channel that has emerged as a potential therapeutic target in multiple conditions. Gain-of-function mutations also cause hereditary neuromuscular disease. Here, we present cryo-EM structures of human TRPV4 in complex with RhoA in the ligand-free, antagonist-bound closed, and agonist-bound open states. These structures reveal the mechanism of ligand-dependent TRPV4 gating. Channel activation is associated with rigid-body rotation of the intracellular ankyrin repeat domain, but state-dependent interaction with membrane-anchored RhoA constrains this movement. Notably, many residues at the TRPV4-RhoA interface are mutated in disease and perturbing this interface by introducing mutations into either TRPV4 or RhoA increases TRPV4 channel activity. Together, these results suggest that RhoA serves as an auxiliary subunit for TRPV4, regulating TRPV4-mediated calcium homeostasis and disruption of TRPV4-RhoA interactions can lead to TRPV4-related neuromuscular disease. These insights will help facilitate TRPV4 therapeutics development.
Subject(s)
TRPV Cation Channels , rhoA GTP-Binding Protein , Humans , Ankyrin Repeat , Calcium/metabolism , Mutation , TRPV Cation Channels/chemistry , rhoA GTP-Binding Protein/chemistryABSTRACT
G-protein coupled receptors (GPCRs) and ion channels serve as key molecular switches through which extracellular stimuli are transformed into intracellular effects, and it has long been postulated that ion channels are direct effector molecules of the alpha subunit of G-proteins (Gα). However, no complete structural evidence supporting the direct interaction between Gα and ion channels is available. Here, we present the cryo-electron microscopy structures of the human transient receptor potential canonical 5 (TRPC5)-Gαi3 complexes with a 4:4 stoichiometry in lipid nanodiscs. Remarkably, Gαi3 binds to the ankyrin repeat edge of TRPC5 ~ 50 Å away from the cell membrane. Electrophysiological analysis shows that Gαi3 increases the sensitivity of TRPC5 to phosphatidylinositol 4,5-bisphosphate (PIP2), thereby rendering TRPC5 more easily opened in the cell membrane, where the concentration of PIP2 is physiologically regulated. Our results demonstrate that ion channels are one of the direct effector molecules of Gα proteins triggered by GPCR activation-providing a structural framework for unraveling the crosstalk between two major classes of transmembrane proteins: GPCRs and ion channels.
Subject(s)
Transient Receptor Potential Channels , Humans , Cryoelectron Microscopy , Receptors, G-Protein-Coupled/metabolism , Cell Membrane/metabolism , TRPC Cation Channels/metabolismABSTRACT
Since the global outbreak of coronavirus disease 2019 (COVID-19), it has spread rapidly around the world. The nucleocapsid (N) protein is one of the most abundant SARS-CoV-2 proteins. Therefore, a sensitive and effective detection method for SARS-CoV-2 N protein is the focus of research. Here, we developed a surface plasmon resonance (SPR) biosensor based on the dual signal-amplification strategy of Au@Ag@Au nanoparticles (NPs) and graphene oxide (GO). Additionally, a sandwich immunoassay was utilized to sensitively and efficiently detect SARS-CoV-2 N protein. On the one hand, Au@Ag@Au NPs have a high refractive index and the capability to electromagnetically couple with the plasma waves propagating on the surface of gold film, which are harnessed for amplifying the SPR response signal. On the other hand, GO, which has the large specific surface area and the abundant oxygen-containing functional groups, could provide unique light absorption bands that can enhance plasmonic coupling to further amplify the SPR response signal. The proposed biosensor could efficiently detect SARS-CoV-2 N protein for 15 min and the detection limit for SARS-CoV-2 N protein was 0.083 ng/mL, with a linear range of 0.1 ng/mL~1000 ng/mL. This novel method can meet the analytical requirements of artificial saliva simulated samples, and the developed biosensor had a good anti-interference capability.
Subject(s)
Biosensing Techniques , COVID-19 , Metal Nanoparticles , Humans , Surface Plasmon Resonance/methods , Biosensing Techniques/methods , SARS-CoV-2 , Gold , Immunoassay/methods , COVID-19/diagnosisABSTRACT
The wide clinical application of d-penicillamine (D-PA) makes it inevitably accumulates in the environment, seriously threatening human health and the ecological environment. To better supervisory control D-PA, a highly sensitive and reliable photoelectrochemical (PEC) sensor based on gold nanoparticles (Au NPs) loaded on graphitic carbon nitride sheet and hexagonal NH2-UiO-66 composite (g-C3N4/Au/NH2-UiO-66) was synthesized. Tactfully using the strong bonding between D-PA and Au NPs and the effective carrier separation of Z-scheme heterojunction, the designed g-C3N4/Au/NH2-UiO-66 PEC sensor without an extra recognition unit exhibited a selective and sensitive photocurrent to D-PA. With the aid of UV diffuse reflectance spectra (UV-DRs), electron paramagnetic resonance (EPR) characterization, and free radical capture experiments, the electron transfer path of the PEC sensing system was deduced. The proposed g-C3N4/Au/NH2-UiO-66 PEC-based sensor achieved a low detection limit of 0.0046 µM (S/N = 3) with a wide linear response ranging from 10 nM to 400 µM. In addition, its good stability and selectivity also laid a good foundation for practical applications.