Competitive Binding Analysis Research Articles

Unraveling the enigma: Probing the unconventional binding site and binding mechanism of entrectinib with human serum albumin through spectroscopic, molecular docking, and dynamic simulation analyses

Entrectinib (ENB), a novel potent tyrosine kinase inhibitor, has demonstrated promising activity against specific genetic alterations in various solid tumors. Investigating the interaction of ENB with human serum albumin (HSA) provides valuable insights into its binding characteristics and pharmacological behavior. The interaction between ENB and HSA was investigated using various spectroscopic techniques, molecular docking, and simulations approaches. Fluorescence spectroscopy revealed a consistent decrease in the intrinsic fluorescence of HSA upon binding to ENB, indicating a static binding mechanism. The binding constant calculated from Stern Volmer and double-log equations (KSV) and (K) were determined to be 4.11 x104 M−1 and 4.38 x104 M−1, respectively at 298 K, indicating moderate binding affinity. Thermodynamic analysis demonstrated a spontaneous interaction (ΔG < 0) with an enthalpy change (ΔH) of −5.59 kJ mol−1 and an entropy change (ΔS) of + 70.36 J mol−1 K−1, indicating that the interaction is primarily driven by electrostatic forces with potential involvement of hydrogen bonding. Synchronous and 3D fluorescence analysis indicated no significant changes in the microenvironment surrounding Tyr and Trp residues upon ENB binding. Near and far UV spectrophotometric analyses confirmed the formation of the ENB-HSA complex and suggested no conformational changes in the protein structure. Fluorescence measurements and binding competition analysis using site markers revealed that ENB selectively binds to the HSA Sudlow sites I and III, displacing phenylbutazone at both sites. Molecular docking studies confirmed the binding of ENB to Sudlow sites I and III and identified critical residues involved in the interaction. Molecular dynamics simulations further validated the stability of the ENB-HSA complex at both sites, with more favorable binding at site III, and minimal fluctuations observed in the root mean square deviation, root mean square fluctuation, and radius of gyration. Hydrogen bond analysis and solvent accessible surface area calculations supported the strong binding and accessibility of ENB within the HSA structure. Overall, the experimental and computational results provided valuable insights into the ENB-HSA interaction, elucidating the binding mechanism and key residues involved.

Synthesis of bimetallic palladacycles via C[sbnd]H bond activation: Crystal structure, DNA binding and in vitro cytotoxicity study

Herein, we report the synthesis, characterization and in vitro anticancer activity of two μ-Cl and μ-O bridged dimeric cyclometalated palladium(II) complexes having general formula [Pd2(L1)Cl2] (1) and [Pd2(L2)Cl2] (2). The complexes are synthesized via palladium assisted aromatic CH bond activation of coordinating ligands (HL1/HL2) and thoroughly characterized by spectroscopic techniques. X-ray crystal structure analysis has confirmed the square planar geometry of the complexes. DFT and TDDFT results have interpreted the electronic structure and solution spectra of the complexes. Cytotoxicity of the complexes was studied by in vitro model with human gastric cancer cell lines (AGS). Cytotoxicity investigation reveals that the complexes have promising anti-cancer activity, with IC50 values of 13.03 µM and 17.86 µM for 1 and 2, respectively. UV–vis and fluorescence techniques are used to examine how the complexes interact with CT DNA. Fluorescence-based competitive binding analysis with ethidium bromide (EB) indicates that the complexes effectively displace EB from the EB-DNA complex.

Characterization of CrufCSP1 and Its Potential Involvement in Host Location by Cotesia ruficrus (Hymenoptera: Braconidae), an Indigenous Parasitoid of Spodoptera frugiperda (Lepidoptera: Noctuidae) in China.

Chemosensory proteins (CSPs) are a class of soluble proteins that facilitate the recognition of chemical signals in insects. While CSP genes have been identified in many insect species, studies investigating their function remain limited. Cotesia ruficrus (Hymenoptera: Braconidae) holds promise as an indigenous biological control agent for managing the invasive pest Spodoptera frugiperda (Lepidoptera: Noctuidae) in China. This study aimed to shed light on the gene expression, ligand binding, and molecular docking of CrufCSP1 in C. ruficrus. A RT-qPCR analysis revealed that the expression of CrufCSP1 was higher in the wings, with male adults exhibiting significantly higher relative expression levels than other developmental stages. A fluorescence competitive binding analysis further demonstrated that CrufCSP1 has a high binding ability with several host-related volatiles, with trans-2-hexenal, octanal, and benzaldehyde showing the strongest affinity to CrufCSP1. A molecular docking analysis indicated that specific amino acid residues (Phe24, Asp25, Thr53, and Lys81) of CrufCSP1 can bind to these specific ligands. Together, these findings suggest that CrufCSP1 may play a crucial role in the process of C. ruficrus locating hosts. This knowledge can contribute to the development of more efficient and eco-friendly strategies for protecting crops and managing pests.

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

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

Enzyme-Catalyzed Hydrogen-Deuterium Exchange between Environmental Pollutants and Enzyme-Regulated Endogenous Metabolites.

Environmental pollutants can disrupt the homeostasis of endogenous metabolites in organisms, leading to metabolic disorders and syndromes. However, it remains highly challenging to efficiently screen for critical biological molecules affected by environmental pollutants. Herein, we found that enzyme could catalyze hydrogen-deuterium (H-D) exchange between a deuterium-labeled environmental pollutant [D38-bis(2-ethylhexyl) phthalate (D38-DEHP)] and several groups of enzyme-regulated metabolites [cardiolipins (CLs), monolysocardiolipins (MLCLs), phospholipids (PLs), and lysophospholipids (LPLs)]. A high-throughput scanning identified the D-labeled endogenous metabolites in a simple enzyme [phospholipase A2 (PLA2)], enzyme mixtures (liver microsomes), and living organisms (zebrafish embryos) exposed to D38-DEHP. Mass fragmentation and structural analyses showed that similar positions were D-labeled in the CLs, MLCLs, PLs, and LPLs, and this labeling was not attributable to natural metabolic transformations of D38-DEHP or incorporation of its D-labeled side chains. Molecular docking and competitive binding analyses revealed that DEHP competed with D-labeled lipids for binding to the active site of PLA2, and this process mediated H-D exchange. Moreover, competitive binding of DEHP against biotransformation enzymes could interfere with catabolic or anabolic lipid metabolism and thereby affect the concentrations of endogenous metabolites. Our findings provide a tool for discovering more molecular targets that complement the known toxic endpoints of metabolic disruptors.

Chemosensory Protein 2 of Male Athetis lepigone Is Involved in the Perception of Sex Pheromones and Maize Volatiles.

In moths, the interactions between chemosensory proteins (CSPs) and sex pheromones have yet to be comprehensively investigated. Here, we examined the function of AlepCSP2 in male Athetis lepigone based on protein expression, molecular docking, site-directed mutagenesis, fluorescence competitive binding analyses, and RNA interference (RNAi) experiments. We found that AlepCSP2 showed strong binding affinity for two sex pheromones and five maize volatiles and that binding was optimal under neutral conditions. Furthermore, we identified six amino acids as being key residues involved in the interaction between AlepCSP2 and multiple ligands. Further RNAi showed that siCSP2 males displayed consistently lower electroantennography responses to two sex pheromones and three maize volatiles at different dosages tested, and the mating rate also decreased significantly by 37.50%. These findings will contribute to characterizing the binding mechanisms of moth CSPs to sex pheromones and host volatiles and also identify unique targets for developing novel pest behavior disruptors.

Mutational escape prevention by combination of four neutralizing antibodies that target RBD conserved regions and stem helix.

New variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) appear rapidly every few months. They have showed powerful adaptive ability to circumvent the immune system. To further understand SARS-CoV-2's adaptability so as to seek for strategies to mitigate the emergence of new variants, herein we investigated the viral adaptation in the presence of broadly neutralizing antibodies and their combinations. First, we selected four broadly neutralizing antibodies, including pan-sarbecovirus and pan-betacoronavirus neutralizing antibodies that recognize distinct conserved regions on receptor-binding domain (RBD) or conserved stem-helix region on S2 subunit. Through binding competition analysis, we demonstrated that they were capable of simultaneously binding. Thereafter, a replication-competent vesicular stomatitis virus pseudotyped with SARS-CoV-2 spike protein was employed to study the viral adaptation. Twenty consecutive passages of the virus under the selective pressure of individual antibodies or their combinations were performed. It was found that it was not hard for the virus to adapt to broadly neutralizing antibodies, even for pan-sarbecovirus and pan-betacoronavirus antibodies. The virus was more and more difficult to escape the combinations of two/three/four antibodies. In addition, mutations in the viral population revealed by high-throughput sequencing showed that under the selective pressure of three/four combinational antibodies, viral mutations were not prone to present in the highly conserved region across betacoronaviruses (stem-helix region), while this was not true under the selective pressure of single/two antibodies. Importantly, combining neutralizing antibodies targeting RBD conserved regions and stem helix synergistically prevented the emergence of escape mutations. These studies will guide future vaccine and therapeutic development efforts and provide a rationale for the design of RBD-stem helix tandem vaccine, which may help to impede the generation of novel variants.

Label-free LC-MS based assay to characterize small molecule compound binding to cells.

Study of small molecule binding to live cells provides important information on the characterization of ligands pharmacologically. Here we developed and validated a label-free, liquid chromatography-mass spectrometry (LC-MS) based cell binding assay, using centrifugation to separate binders from non-binders. This assay was applied to various target classes, with particular emphasis on those for which protein-based binding assay can be difficult to achieve. In one example, to study a G protein coupled receptor (GPCR), we used one antagonist as probe and multiple other antagonists as competitor ligands. Binding of the probe was confirmed to be specific and saturable, reaching a fast equilibrium. Competition binding analysis by titration of five known ligands suggested a good correlation with their inhibition potency. In another example, this assay was applied to an ion channel target with its agonists, of which the determined binding affinity was consistent with functional assays. This versatile method allows quantitative characterization of ligand binding to cell surface expressed targets in a physiologically relevant environment.

Gram-Scale Preparation of Cannflavin A from Hemp (Cannabis sativa L.) and Its Inhibitory Effect on Tryptophan Catabolism Enzyme Kynurenine-3-Monooxygenase.

Simple SummaryKynurenine-3-monooxygenase (KMO) is an enzyme in the neurotoxic branch of the kynurenine pathway (KP) and is a target of inhibitors with therapeutic potential against neuroinflammatory and neurodegenerative diseases. Phytochemicals from hemp (Cannabis sativa L.) including phytocannabinoids and flavonoids, can modulate enzymes involved in the KP metabolism, but their direct inhibition on KMO is unknown. In the current study, we purified a unique C. sativa flavonoid, namely, cannflavin A (CFA), from the hemp aerial material at a gram-scale. We evaluated the anti-KMO activity of CFA and a panel of phytocannabinoids. CFA was the most active compound with an IC50 of 29.4 μM, which was comparable to the positive control Ro 61-8048 (IC50 = 5.1 μM). We used a modeling method to demonstrate the interactions between CFA and the KMO protein and measured the binding affinity by a biophysical tool (surface plasmon resonance; SPR). We also conducted a competitive SPR assay to show that CFA and Ro 61-8048 may bind to the same location of the KMO protein. Our findings help the understanding of C. sativa compounds’ effects on KMO.Inhibitors targeting kynurenine-3-monooxygenase (KMO), an enzyme in the neurotoxic kynurenine pathway (KP), are potential therapeutics for KP metabolites-mediated neuroinflammatory and neurodegenerative disorders. Although phytochemicals from Cannabis (C. sativa L.) have been reported to show modulating effects on enzymes involved in the KP metabolism, the inhibitory effects of C. sativa compounds, including phytocannabinoids and non-phytocannabinoids (i.e., cannflavin A; CFA), on KMO remain unknown. Herein, CFA (purified from hemp aerial material at a gram-scale) and a series of phytocannabinoids were evaluated for their anti-KMO activity. CFA showed the most active inhibitory effect on KMO, which was comparable to the positive control Ro 61-8048 (IC50 = 29.4 vs. 5.1 μM, respectively). Furthermore, a molecular docking study depicted the molecular interactions between CFA and the KMO protein and a biophysical binding assay with surface plasmon resonance (SPR) technique revealed that CFA bound to the protein with a binding affinity of . A competitive SPR binding analysis suggested that CFA and Ro 61-8048 bind to the KMO protein in a competitive manner. Our findings show that C. sativa derived phytochemicals, including CFA, are potential KMO inhibitors, which provides insight into the development of therapeutics targeting the KP and its related pathological conditions.

Rational design, molecular docking, dynamic simulation, synthesis, PPAR-γ competitive binding and transcription analysis of novel glitazones

Over the last decade, peroxisome proliferator-activated receptor (PPAR-γ) has emerged as one of the important therapeutic targets in several metabolic and neurodegenerative disorders. The remarkable progress in drug discovery has resulted in designing novel PPAR-γ activators. Thiazolidinediones, also known as glitazones, have been demonstrated to play a significant role in treating several neurodegenerative diseases, diabetes, and cancer. Despite its wide range of adverse effects, glitazones orchestrate significant pharmacological activities. In this backdrop, we designed and synthesised novel glitazones for potential PPAR-γ binding activity. The synthesised novel compounds were structurally analysed using 1H NMR, 13C NMR and FT-IR. The interaction binding modes, binding free energy, and crucial amino acids involved in interactions of designed compounds with the target protein were studied using molecular docking. Further, molecular dynamic modeling was used to evaluate the stability of the best-docked complexes. TR-FRET, an in vitro PPAR-γ competitive binding assay, was performed to confirm the affinity of the well-docked compounds for the target protein. It demonstrated that the compounds explicitly bind to the PPAR-γ ligand-binding domain to exhibit pharmacological activity. The cytotoxicity of synthesised compounds was performed and confirmed the PPAR-γ transcription activity on SH-SY5Y cell lines

Functional and Structural Analysis of the Toxin-Binding Site of the Cadherin G-Protein-Coupled Receptor, BT-R1, for Cry1A Toxins of Bacillus thuringiensis.

The G-protein-coupled receptor BT-R1 in the moth Manduca sexta represents a class of single-membrane-spanning α-helical proteins within the cadherin family that regulate intercellular adhesion and contribute to important signaling activities that control cellular homeostasis. The Cry1A toxins, Cry1Aa, Cry1Ab, and Cry1Ac, produced by Bacillus thuringiensis bind BT-R1 very tightly (Kd = 1.1 nM) and trigger a Mg2+-dependent signaling pathway that involves the stimulation of G-protein α-subunit, which subsequently launches a coordinated signaling cascade, resulting in insect death. The three Cry1A toxins compete for the same binding site on BT-R1, and the pattern of inhibition of insecticidal activity against M. sexta is strikingly similar for all three toxins. The binding domain is localized in the 12th cadherin repeat (EC12: Asp1349 to Arg1460, 1349DR1460) in BT-R1 and to various truncation fragments derived therefrom. Fine mapping of EC12 revealed that the smallest fragment capable of binding is a highly conserved 94-amino acid polypeptide bounded by Ile1363 and Ser1456 (1363IS1456), designated as the toxin-binding site (TBS). Logistical regression analysis revealed that binding of an EC12 truncation fragment containing the TBS is antagonistic to each of the Cry1A toxins and completely inhibits the insecticidal activity of all three. Elucidation of the EC12 motif of the TBS by X-ray crystallography at a 1.9 Å resolution combined with results of competitive binding analyses, live cell experiments, and whole insect bioassays substantiate the exclusive involvement of BT-R1 in initiating insect cell death and demonstrate that the natural receptor BT-R1 contains a single TBS.

Assessment of Chilling Injury in Boar Spermatozoa by Kinematic Patterns and Competitive Sperm-Oviduct Binding In Vitro.

Simple SummaryArtificial insemination is widely used for pig reproduction. Boar spermatozoa for use in artificial insemination are usually preserved at 17 °C for several days. Storage at lower temperature would be beneficial to reduce the risk of bacterial growth but is hindered by the high chilling sensitivity of boar spermatozoa. Currently, new preservation concepts are evolving which aim to store boar sperm at 5 °C. Before introducing these for animal breeding, sensitive tests are necessary to detect chilling-induced sperm damage. The aim of the study was to examine the potential of two tests systems for detecting sperm injury after chilling in two different semen extenders. It was shown that the analysis of sperm movement patterns and the binding of sperm to oviductal tissue sensitively indicate subtle differences of semen extenders to preserve the quality of chilled spermatozoa. The application of these tests is recommended for testing new hypothermic preservation strategies for boar semen.Sensitive detection of chilling injury in boar spermatozoa is required to evaluate novel hypothermic preservation concepts. The study’s aim was to examine whether analyses of motility patterns and sperm binding in a competitive oviduct explant assay (cOEA) sensitively detect chilling-induced alterations in sperm function. Semen samples (n = seven boars) were split into four subsamples by dilution either in Beltsville Thawing Solution (BTS) or Androstar® Plus and stored at 5 °C or 17 °C. Storage temperature had a significant effect on the distribution of spermatozoa in seven major kinematic clusters. The effect size of chilling at 5 °C as estimated by Cramer’s V was higher (p < 0.05) in the BTS medium (0.21) compared to AndroStar® Plus (0.11). Spermatozoa extended in Androstar® Plus had higher relative binding capacity compared to sperm in BTS (p < 0.05). Binding indices correlated with the percentage of viable, acrosome-intact (r = 0.62) and motile spermatozoa (r = 0.72, both p < 0.001). The cluster size of sperm with slow, vigorous movement was negatively correlated with sperm-oviduct binding (r = −0.43, p < 0.05). In conclusion, the cluster analysis of sperm kinematics and competitive sperm oviduct binding in vitro present meaningful biological tests to assess novel concepts for hypothermic semen preservation.

Potent neutralization of Rift Valley fever virus by human monoclonal antibodies through fusion inhibition

Rift Valley fever virus (RVFV), an emerging arboviral and zoonotic bunyavirus, causes severe disease in livestock and humans. Here, we report the isolation of a panel of monoclonal antibodies (mAbs) from the B cells of immune individuals following natural infection in Kenya or immunization with MP-12 vaccine. The B cell responses of individuals who were vaccinated or naturally infected recognized similar epitopes on both Gc and Gn proteins. The Gn-specific mAbs and two mAbs that do not recognize either monomeric Gc or Gn alone but recognized the hetero-oligomer glycoprotein complex (Gc+Gn) when Gc and Gn were coexpressed exhibited potent neutralizing activities in vitro, while Gc-specific mAbs exhibited relatively lower neutralizing capacity. The two Gc+Gn-specific mAbs and the Gn domain A-specific mAbs inhibited RVFV fusion to cells, suggesting that mAbs can inhibit the exposure of the fusion loop in Gc, a class II fusion protein, and thus prevent fusion by an indirect mechanism without direct fusion loop contact. Competition-binding analysis with coexpressed Gc/Gn and mutagenesis library screening indicated that these mAbs recognize four major antigenic sites, with two sites of vulnerability for neutralization on Gn. In experimental models of infection in mice, representative mAbs recognizing three of the antigenic sites reduced morbidity and mortality when used at a low dose in both prophylactic and therapeutic settings. This study identifies multiple candidate mAbs that may be suitable for use in humans against RVFV infection and highlights fusion inhibition against bunyaviruses as a potential contributor to potent antibody-mediated neutralization.

Crystal structure of death-associated protein kinase 1 in complex with the dietary compound resveratrol.

Death-associated protein kinase 1 (DAPK1) is a large multidomain protein with an N-terminal serine/threonine protein kinase domain. DAPK1 is considered to be a promising molecular target for the treatment of Alzheimer's disease (AD). In the present study, the inhibitory potency of resveratrol (RSV), a dietary polyphenol found in red wine, against the catalytic activity of DAPK1 was investigated. Kinetic and fluorescent probe competitive binding analyses revealed that RSV directly inhibited the catalytic activity of DAPK1 by binding to the ATP-binding site. Crystallographic analysis of DAPK1 in complex with RSV revealed that the A-ring of RSVoccupied the nucleobase-binding position. Determination of the binding mode provided a structural basis for the design of more potent DAPK1 inhibitors. In conclusion, the data here clearly show that RSV is an ATP-competitive inhibitor of DAPK1, encouraging speculation that RSV may be useful for the development of AD inhibitors.

Preparation of a Growth Hormone Receptor/Prolactin Receptor Bispecific Antibody Antagonist Which Exhibited Anti-Cancer Activity.

Prolactin receptor (PRLR) and growth hormone receptor (GHR) are closely related to the occurrence and development of breast cancer, and breast cancer cell endogenously express GHR, PRLR and GHR-PRLR heterodimer. In this case, the combined use of PRLR or GHR inhibitors may produce better anti-breast cancer potential than PRLR or GHR inhibitors alone. In this case, it is necessary to develop the dual-function GHR/PRLR antagonists with anti-breast cancer potential. For this, we used hybridoma technology to generate an anti-idiotypic antibody (termed H53). We then used various techniques, including competitive ELISA, competitive receptor binding analysis, and indirect immunofluorescence assay to identify H53, and the results show that H53 behaves as a typical internal image anti-idiotypic antibody (Ab2β). Further experiments indicate that H53 is a dual-function inhibitor, which not only inhibited PRLR-mediated intracellular signaling, but also blocked GHR-mediated intracellular signaling in a dose-dependent manner. Furthermore, H53 could inhibit PRL/GH-driven cancer cell proliferation in vivo and in vitro. This study indicates that H53 exhibits potential biological activity against breast tumors, which implies that internal image anti-idiotypic antibodies may be a useful strategy for the development of PRLR/GHR dual-function antagonists for breast cancer therapy.

Preparation and identification of an anti-idiotypic antibody antagonist (FG8) for EGFR that shows potential activity against liver cancer cells.

Currently, there are two categories of epidermal growth factor receptor (EGFR) antagonists, small molecule antagonists and anti-EGFR antibodies. In the current study, we developed a new EGFR antagonist employing the anti-idiotypic antibodies strategy. First, using EGF as an antigen, through a series of immunological protocols and hybridoma technology, we obtained an anti-idiotypic antibody against EGF receptor-binding epitopes. On this basis, we screened and characterized the anti-idiotype antibodies against EGFR through competitive ELISA, co-localization analysis, competitive receptor binding analysis, and immunofluorescence. Finally, an internal image anti-idiotype antibody called FG8 was successfully prepared. Experiment result shows that FG8 inhibits EGFR-mediated signaling pathways in vitro. Additionally, FG8 inhibits liver tumor cell proliferation as well as induces tumor cell apoptosis. The present study suggests that FG8 is a potential therapeutic agent for liver cancer. In addition, this study provides a novel method for the preparation of EGFR antagonists.

Boosting CO2 Conversion with Terminal Alkynes by Molecular Architecture of Graphene Oxide-Supported Ag Nanoparticles

Summary Carboxylation of terminal alkynes with CO2 via direct C–H bond activation is a highly appealing way to capture and convert CO2 but faces great challenges and difficulty. Here, we report an efficient catalyst that is prepared simply through introduction of p-tert-butylaniline into graphite oxide powders followed by localized growth of Ag nanoparticles (NPs). The bulky nature of p-tert-butylaniline facilitates the powder exfoliation, giving rise to mass production of uniform modified graphene oxide (tert-GO) nanosheets of 4 nm in thickness. Notably, Ag/tert-GO shows excellent catalytic activity for converting alkynes regardless of containing electron-donating or electron-withdrawing groups under mild reaction conditions. The synergy of the amide linkages on tert-GO nanosheets and the supported small-sized Ag NPs is recognized as the vital role in promoting adsorption of CO2 and subsequent conversion.

Crystal Structure of Flagellar Export Chaperone FliS in Complex With Flagellin and HP1076 of Helicobacter pylori.

Functional flagella formation is a widespread virulence factor that plays a critical role in survival and host colonization. Flagellar synthesis is a complex and highly coordinated process. The assembly of the axial structure beyond the cell membrane is mediated by export chaperone proteins that transport their cognate substrates to the export gate complex. The export chaperone FliS interacts with flagellin, the basic component used to construct the filament. Unlike enterobacteria, the gastric pathogen Helicobacter pylori produces two different flagellins, FlaA and FlaB, which exhibit distinct spatial localization patterns in the filament. Previously, we demonstrated a molecular interaction between FliS and an uncharacterized protein, HP1076, in H. pylori. Here, we present the crystal structure of FliS in complex with both the C-terminal D0 domain of FlaB and HP1076. Although this ternary complex reveals that FliS interacts with flagellin using a conserved binding mode demonstrated previously in Aquifex aeolicus, Bacillus subtilis, and Salmonella enterica serovar Typhimurium, the helical conformation of FlaB in this complex was different. Moreover, HP1076 and the D1 domain of flagellin share structural similarity and interact with the same binding interface on FliS. This observation was further validated through competitive pull-down assays and kinetic binding analyses. Interestingly, we did not observe any detrimental flagellation or motility phenotypes in an hp1076-null strain. Our localization studies suggest that HP1076 is a membrane-associated protein with a cellular localization independent of FliS. As HP1076 is uniquely expressed in H. pylori and related species, we propose that this protein may contribute to the divergence of the flagellar system, although its relationship with FliS remains incompletely elucidated.

Response of Midgut Trypsin- and Chymotrypsin-Like Proteases of Helicoverpa armigera Larvae Upon Feeding With Peanut BBI: Biochemical and Biophysical Characterization of PnBBI.

Proteinase/Protease inhibitors (PIs) from higher plants play an important role in defense and confer resistance against various insect pests and pathogens. In the present study, Bowman-Birk Inhibitor (BBI) was purified from mature seeds of an interspecific advanced hybrid peanut variety (4368-1) using chromatographic techniques. The biochemical and biophysical characteristics such as low molecular mass, presence of several isoinhibitors and higher-ordered dimer/tetramer, predominance of antiparallel β-sheets and random coils in secondary structure, reactive sites against trypsin and chymotrypsin, broad spectrum of stability toward extreme pH and temperature along with MALDI TOF-TOF analysis (ProteomeXchange identifier PXD016933) ascertained the purified biomolecule from peanut as BBI (PnBBI). Surface plasmon resonance competitive binding analysis revealed the bifunctional PnBBI is a trypsin specific inhibitor with 1:2 stoichiometry as compared to chymotrypsin. A concentration-dependent self-association tendency of PnBBI was further confirmed by ‘red shift’ in the far-UV CD spectra. Furthermore, the insecticidal potential of PnBBI against Helicoverpa armigera was assessed by in vitro assays and in vivo feeding experiments. A significant reduction in larval body weight was observed with concomitant attenuation in the activity of midgut trypsin-like proteases of H. armigera (HaTPs) fed on PnBBI supplemented diet. The one and two-dimensional zymography studies revealed the disappearance of several isoforms of HaTP upon feeding with PnBBI. qRT-PCR analysis further suggests the role of PnBBI in not only inhibiting the activity of midgut trypsin and chymotrypsin-like proteases but also in modulating their expression. Taken together, the results provide a biochemical and molecular basis for introgressed resistance in peanut interspecific advanced hybrid variety against H. armigera.

Separating dopamine D2 and D3 receptor sources of [11C]-(+)-PHNO binding potential: Independent component analysis of competitive binding

Development of medications selective for dopamine D2 or D3 receptors is an active area of research in numerous neuropsychiatric disorders including addiction and Parkinson’s disease. The positron emission tomography (PET) radiotracer [11C]-(+)-PHNO, an agonist that binds with high affinity to both D2 and D3 receptors, has been used to estimate relative receptor subtype occupancy by drugs based on a priori knowledge of regional variation in the expression of D2 and D3 receptors. The objective of this work was to use a data-driven independent component analysis (ICA) of receptor blocking scans to separate D2-and D3-related signal in [11C]-(+)-PHNO binding data in order to improve the precision of subtype specific measurements of binding and occupancy. Eight healthy volunteers underwent [11C]-(+)-PHNO PET scans at baseline and at two time points following administration of the D3-preferring antagonist ABT-728 (150–1000 ​mg). Parametric binding potential (BPND) images were analyzed as four-dimensional image series using ICA to extract two independent sources of variation in [11C]-(+)-PHNO BPND. Spatial source maps for each component were consistent with respective regional patterns of D2-and D3-related binding. ICA-derived occupancy estimates from each component were similar to D2-and D3-specific occupancy estimated from a region-based approach (intraclass correlation coefficients ​> ​0.95). ICA-derived estimates of D3 receptor occupancy improved quality of fit to a single site binding model. Furthermore, ICA-derived estimates of the regional fraction of [11C]-(+)-PHNO binding related to D3 receptors was generated for each subject and values showed good agreement with region-based model estimates and prior literature values. In summary, ICA successfully separated D2-and D3-related components of the [11C]-(+)-PHNO binding signal, establishing this approach as a powerful data-driven method to quantify distinct biological features from PET data composed of mixed data sources.