Competition For Binding Sites Research Articles

Overcoming nucleotide bias in the nonenzymatic copying of RNA templates.

The RNA World hypothesis posits that RNA was the molecule of both heredity and function during the emergence of life. This hypothesis implies that RNA templates can be copied, and ultimately replicated, without the catalytic aid of evolved enzymes. A major problem with nonenzymatic template-directed polymerization has been the very poor copying of sequences containing rA and rU. Here, we overcome that problem by using a prebiotically plausible mixture of RNA mononucleotides and random-sequence oligonucleotides, all activated by methyl isocyanide chemistry, that direct the uniform copying of arbitrary-sequence templates, including those harboring rA and rU. We further show that the use of this mixture in copying reactions suppresses copying errors while also generating a more uniform distribution of mismatches than observed for simpler systems. We find that oligonucleotide competition for template binding sites, oligonucleotide ligation and the template binding properties of reactant intermediates work together to reduce product sequence bias and errors. Finally, we show that iterative cycling of templated polymerization and activation chemistry improves the yields of random-sequence products. These results for random-sequence template copying are a significant advance in the pursuit of nonenzymatic RNA replication.

Functional role of myosin-binding protein H in thick filaments of developing vertebrate fast-twitch skeletal muscle.

Myosin-binding protein H (MyBP-H) is a component of the vertebrate skeletal muscle sarcomere with sequence and domain homology to myosin-binding protein C (MyBP-C). Whereas skeletal muscle isoforms of MyBP-C (fMyBP-C, sMyBP-C) modulate muscle contractility via interactions with actin thin filaments and myosin motors within the muscle sarcomere "C-zone," MyBP-H has no known function. This is in part due to MyBP-H having limited expression in adult fast-twitch muscle and no known involvement in muscle disease. Quantitative proteomics reported here reveal that MyBP-H is highly expressed in prenatal rat fast-twitch muscles and larval zebrafish, suggesting a conserved role in muscle development and prompting studies to define its function. We take advantage of the genetic control of the zebrafish model and a combination of structural, functional, and biophysical techniques to interrogate the role of MyBP-H. Transgenic, FLAG-tagged MyBP-H or fMyBP-C both localize to the C-zones in larval myofibers, whereas genetic depletion of endogenous MyBP-H or fMyBP-C leads to increased accumulation of the other, suggesting competition for C-zone binding sites. Does MyBP-H modulate contractility in the C-zone? Globular domains critical to MyBP-C's modulatory functions are absent from MyBP-H, suggesting that MyBP-H may be functionally silent. However, our results suggest an active role. In vitro motility experiments indicate MyBP-H shares MyBP-C's capacity as a molecular "brake." These results provide new insights and raise questions about the role of the C-zone during muscle development.

Cotransport of Thallium(I) and kaolinite colloids in quartz sand media containing sodium humate: Ionic strength, pH and kaolinite colloid concentration

In real soil environments, humus, colloids and other components significantly affect pollutant migration behavior. Investigating Tl(I) and kaolinite colloids’ cotransport in quartz sand media containing sodium humate (HA-Na) is vital for comprehending Tl(I) migration underground. This study examined the migration of Tl(I) and kaolinite colloids across varying pH levels (5, 7), ionic strengths (ISs) (1, 5, 50 mmol/L), and kaolinite colloid concentrations. Results indicate that lower IS and pH promote Tl(I) migration when transported alone. In cotransport system, kaolinite promotes Tl(I) migration under acidic conditions but inhibits it under neutral conditions, except at high kaolinite concentrations, where the effect shifts from inhibition to promotion. This is primarily due to changes in the zeta potential of quartz sand, HA-Na, and kaolinite, as well as Tl(I) adsorption after HA-Na and kaolinite occupy binding sites. Competitive adsorption between cations and Tl(I) also plays a significant role. Conversely, in individual system, higher IS and pH inhibit kaolinite migration, while increased kaolinite concentration promotes it. In cotransport system, Tl(I) promotes kaolinite migration under acidic conditions but inhibits it under neutral conditions, except at low kaolinite concentrations. This relates to changes in the zeta potential between kaolinite and the medium, as well as the retention of HA-Na in the column and its adsorption onto kaolinite. Competitive adsorption and binding site saturation also have an impact. This study enhances understanding of Tl(I) migration by revealing the dual effect of kaolinite colloids under different environmental conditions, contributing to better knowledge of Tl(I) fate and transport in natural environments.

Identification of acetylshikonin as a novel tubulin polymerization inhibitor with antitumor activity in human hepatocellular carcinoma cells.

Microtubules are attractive targets for anticancer drugs. However, the microtubule-targeting agents (MTAs) currently in clinical use exhibit inevitable drug resistance. Therefore, there is an urgent need to discover novel MTAs for the clinical treatment of cancer. Bioactive compounds extracted from Lithospermum erythrorhizon were assessed for in vitro anti-proliferative activities against a panel of human cancer cell lines using cell counting kit-8 (CCK-8) assay. Tubulin polymerization inhibition assay, colchicine competitive binding site assay, and immunofluorescence were used to validate the tubulin inhibition effect of acetylshikonin. Flow cytometry, Hoechst staining, and caspase-3 activity evaluation were performed to assess cell cycle arrest and cell apoptosis. 5,5',6,6'-tetrachloro-1,1',3,3'-tetramethylbenzimidazolylcarbocyanine iodide (JC-1) staining and dichloro-dihydro-fluorescein diacetate (DCFH-DA) staining were used to evaluate mitochondrial membrane potential (MMP) and reactive oxygen species (ROS), respectively. Acetylshikonin exhibited potent anti-proliferative activities against a panel of human cancer cell lines (IC50 values: 1.09-7.26 µM) and displayed comparable cytotoxicity against several drug-resistant cell lines. Further mechanism studies revealed that acetylshikonin induced cell cycle arrest of MHCC-97H cells at G2/M phase, and significantly promoted apoptosis marked by a collapse of MMP and abnormal ROS accumulation. In this study, acetylshikonin was identified as MTA against hepatocellular carcinoma and can serve as a promising lead compound for further development of anti-cancer drug, underscoring its potential clinical significance.

High-solid-content paste for stereolithography self-supporting printing of Luneburg lenses with a high degree of geometric-design freedom

Luneburg lenses prepared by alumina stereolithography, have the advantages of low dielectric loss, corrosion resistance, and flexible structure. However, the supporting structure limits the degree of geometric design freedom. This paper analyzes the effects of modifiers and dispersants on paste properties from the molecular structure, which is different from the traditional process that doesn’t analyze the influence of similar mechanisms and binding site competition while ignoring the correlation. Based on the dispersant’s better flexibility bringing more obvious change in viscosity, we first determine the amount of dispersant and then adapt the amount of modifier to obtain self-supporting pastes of 82 wt % and 24.9 Pa.swithout gradation. The gain and beam deflection angle of Luneburg lenses with high geometric complexity and working bandwidth of 38° formed by stereolithography self-supporting process is matched to the simulation results without manufacturing defects, which provides a theoretical basis for more flexible design and manufacture of Luneburg lenses.

Non-Steroidal Anti-Inflammatory Drug Effect on the Binding of Plasma Protein with Antibiotic Drug Ceftazidime: Spectroscopic and In Silico Investigation.

The coexistence of ceftazidime, which is a popular third-generation of cephalosporin antibiotic, with ubiquitous paracetamol or acetaminophen, is very likely because the latter is given to the patients to reduce fever due to bacterial infection along with an antibiotic such as the former. Therefore, in this study, we investigated the detailed binding of ceftazidime with plasma protein, human serum albumin (HSA), in the absence and presence of paracetamol using spectroscopic techniques such as fluorescence, UV-visible, and circular dichroism, along with in silico methods such as molecular docking, molecular dynamics simulations, and MM/PBSA-based binding free energy analysis. The basic idea of the interaction was attained by using UV-visible spectroscopy. Further, fluorescence spectroscopy revealed that there was a fair interaction between ceftazidime and HSA, and the mechanism of the quenching was a dynamic one, i.e., the quenching constant increased with increasing temperature. The interaction was, primarily, reinforced by hydrophobic forces, which resulted in the partial unfolding of the protein. Low concentrations of paracetamol were ineffective in affecting the binding of ceftazidime with has; although, a decrease in the quenching and binding constants was observed in the presence of high concentrations of the former. Competitive binding site experiments using warfarin and ibuprofen as site markers revealed that ceftazidime neither binds at drug site 1 or at drug site 2, articulating another binding site, which was confirmed by molecular docking simulations.

Deposition behaviors of carboxyl-modified polystyrene nanoplastics with goethite in aquatic environment: Effects of solution chemistry and organic macromolecules

The ubiquitous nanoplastics (NPs) in the environment are emerging contaminants due to their risks to human health and ecosystems. The interaction between NPs and minerals determines the environmental and ecological risks of NPs. In this study, the deposition behaviors of carboxyl modified polystyrene nanoplastics (COOH-PSNPs) with goethite (α-FeOOH) were systematically investigated under various solution chemistry and organic macromolecules (OMs) conditions (i.e., pH, ionic type, humic acid (HA), sodium alginate (SA), and bovine serum albumin (BSA)). The study found that electrostatic interactions dominated the interaction between COOH-PSNPs and goethite. The deposition rates of COOH-PSNPs decreased with an increase in solution pH, due to the enhanced electrostatic repulsion by higher pH. Introducing cations or anions could compress the electrostatic double layers and compete for interaction sites on COOH-PSNPs and goethite, thereby reducing the deposition rates of COOH-PSNPs. The stabilization effects, which were positive with ions valence, followed the orders of NaCl ≈ KCl < CaCl2, NaNO3 ≈ NaCl < Na2SO4 < Na3PO4. Specific adsorption of SO42− or H2PO4− caused a potential reversal of goethite from positive to negative, leading to the electrostatic forces between COOH-PSNPs and goethite changed from attraction to repulsion, and thus significantly decreasing deposition of COOH-PSNPs. Organic macromolecules could markedly inhibit the deposition of COOH-PSNPs with goethite because of enhanced electrostatic repulsion, steric hindrance, and competition of surface binding sites. The ability for inhibiting the deposition of COOH-PSNPs followed the sequence of SA > HA > BSA, which was related to their structure (SA: linear, semi-flexible, HA: globular, semi-rigid, BSA: globular, with protein tertiary structure) and surface charge density (SA > HA > BSA). The results of this study highlight the complexity of the interactions between NPs and minerals under different environments and provide valuable insights in understanding transport mechanisms and environmental fate of nanoplastics in aquatic environments.

Switch of phosphorylation to O-GlcNAcylation of AhR contributes to vascular oxidative stress induced by benzo[a]pyrene

Benzo[a]pyrene (B[a]P) is a food contaminant toxic for cardiovascular diseases. The nuclear translocation of Arylhydrocarbon receptor (AhR) plays an important role in B[a]P-induced oxidative stress and vascular diseases. We confirmed that B[a]P promoted ROS production in vascular smooth muscle cells (VSMCs) in vitro and in vivo, associated with the nuclear translocation of AhR. It is known that phosphorylation inhibits while dephosphorylation of AhR promotes nuclear translocation of AhR. However, from the posttranslational modification level, the mechanism by which B[a]P activates and regulates the nuclear translocation of AhR is unclear. Co-immunoprecipitation results showed that cytoplasmic AhR was phosphorylated before B[a]P stimulation, and switched to O-GlcNAcylation upon B[a]P 1-h stimulation in VSMCs, suggesting there may be a competitively inhibitory relationship between O-GlcNAcylation and phosphorylation of AhR. Next, siRNAs of O-linked N-acetylglucosamine transferase (OGT), O-GlcNAcase (OGA) and OGA inhibitor PUGNAc were used. SiOGT blocks but siOGA and PUGNAc promote B[a]P -dependent AhR nuclear translocation and oxidative stress. Ser11 may be the competitive binding site for phosphorylation and O-GlcNAcylation of AhR. Phosphorylation-mimic variant inhibits but O-GlcNAcylation of AhR promotes AhR nuclear translocation and oxidative stress. Our findings highlight a new perspective for AhR nuclear translocation regulated by the competitive modification between phosphorylation and O-GlcNAcylation.

Regulome analysis in B-acute lymphoblastic leukemia exposes Core Binding Factor addiction as a therapeutic vulnerability

The ETV6-RUNX1 onco-fusion arises in utero, initiating a clinically silent pre-leukemic state associated with the development of pediatric B-acute lymphoblastic leukemia (B-ALL). We characterize the ETV6-RUNX1 regulome by integrating chromatin immunoprecipitation- and RNA-sequencing and show that ETV6-RUNX1 functions primarily through competition for RUNX1 binding sites and transcriptional repression. In pre-leukemia, this results in ETV6-RUNX1 antagonization of cell cycle regulation by RUNX1 as evidenced by mass cytometry analysis of B-lineage cells derived from ETV6-RUNX1 knock-in human pluripotent stem cells. In frank leukemia, knockdown of RUNX1 or its co-factor CBFβ results in cell death suggesting sustained requirement for RUNX1 activity which is recapitulated by chemical perturbation using an allosteric CBFβ-inhibitor. Strikingly, we show that RUNX1 addiction extends to other genetic subtypes of pediatric B-ALL and also adult disease. Importantly, inhibition of RUNX1 activity spares normal hematopoiesis. Our results suggest that chemical intervention in the RUNX1 program may provide a therapeutic opportunity in ALL.

Biosynthesis of Leucaena Leucocephala leaf mediated ZnO, CuO, MnO2, and MgO based nano-adsorbents for Reactive Golden Yellow-145 (RY-145) and Direct Red-31 (DR-31) dye removal from textile wastewater to reuse in agricultural purpose

Environmental pollution by textile dye-based wastewater raises a vital call for consideration to industrialists and scientists due to its influence on the ecosystem. In this study, zinc oxide (ZnO), copper oxide (CuO), Manganese dioxide (MnO2), and Magnesium Oxide (MgO) nanoparticles were successfully synthesized from Leucaena leucocephala leaves for the removal of Reactive Golden Yellow-145 (RY-145) and Direct Red-31 (DR-31) dyes. The nanoparticles were characterized by scanning electron microscope (SEM) and Fourier Transform Infrared Spectroscopy (FTIR). Optimization of various operating parameters like initial dye concentration, pH, temperature, contact time, and dose of nanoparticles was investigated. The results showed that Leucaena leucocephala leaf-mediated ZnO, CuO, MnO2, and MgO nanoparticles presented the highest adsorption capacities for RY-145 of 47.50 (mg/g), 65.53(mg/g), 53.62 (mg/g), and 58.72 (mg/g) respectively, with optimized parameter values of pH solution, contact time, adsorbent dose, initial dye concentration, and temperature were 3, 75 min, 0.05 g/50 mL, 100 mg/L and 25°C on 124 rpm, correspondingly. DR-31 presented the highest adsorption capacities of 57.08 (mg/g), 83.47(mg/g), 67.36 (mg/g), and 72.53 (mg/g) respectively at optimized parameter values of pH solution, contact time, adsorbent dose, initial dye concentration and temperature were 2 and 3, 60min, 0.05 g/50 mL, 100 mg/L and 25°C on 124 rpm. The surfactants and electrolytes effects were also scrutinized on dyes' adsorption. The surfactants and electrolytes' existence in the aqueous media reduced the adsorption of dye because of the competition for limited binding sites. The RY-145 and DR-31 adsorbents adsorption efficiencies were found in following order; MgO (65.52% and 83.47%) > MnO2 (58.72% and 72.53%) > CuO (53.62% and 67.36%) > ZnO (47.50% and 57.08%). The adsorption-based dye data were scrutinized using various isotherms, kinetics, and thermodynamics models. The studies presented that the adsorption processes were well fitted to pseudo-second-order kinetics since correlation coefficient (R2) values ranged from 0.994 to 0.999 and Intraparticle diffusion models correlation coefficient values ranged from 0.88 to 0.97, their values were relatively near the experimental values signify the adsorption kinetics in both dyes. Both dyes adsorption on green nano-adsorbents followed a Langmuir isotherm due to decent correlation coefficient values of 0.999 with value of 59.17 (mg/g), 66.22 (mg/g), 72.99 (mg/g), and 81.30 (mg/g) respectively for DR-31 and 47.62 (mg/g), 53.763 (mg/g), 58.48 (mg/g), and 64.93 (mg/g) for RY-145 with monolayer coverage. RY-145 also followed Temkin and Doubinin Radushkevich's isotherm. The thermodynamics investigations revealed spontaneous dyes adsorption onto MgO and MnO2, ZnO is non-spontaneous in both dyes while CuO is spontaneous in DR-31 and non-spontaneous in RY-145. The maximum desorption of 78–86% was attained using the 0.2 N and 0.4 N NaOH for adsorbed RY-145 and DR-31 dye respectively. The desorption data was favorable as the Leucaena leucocephala leaf-mediated ZnO, CuO, MnO2, and MgO nanoparticles adsorbents were utilized several cycles and have stability This experimental study also determines the potential efficacy of green nano-adsorbents for the removal of dye from the real textile effluent. In addition, a phytotoxicity investigation of used green nano-adsorbents is carried out on the pea seeds, to demonstrate their sustainability from a real environmental point of view. Thus, Because of their promising efficiency, green ZnO, CuO, MnO2, and MgO nanoparticles have the potential to apply for dye adsorption from textile wastewater.

Infrared Spectroscopy Elucidates the Inhibitor Binding Sites in a Metal-Dependent Formate Dehydrogenase.

Biological carbon dioxide (CO2 ) reduction is an important step by which organisms form valuable energy-richer molecules required for further metabolic processes. The Mo-dependent formate dehydrogenase (FDH) from Rhodobacter capsulatus catalyzes reversible formate oxidation to CO2 at a bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor. To elucidate potential substrate binding sites relevant for the mechanism, we studied herein the interaction with the inhibitory molecules azide and cyanate, which are isoelectronic to CO2 and charged as formate. We employed infrared (IR) spectroscopy in combination with density functional theory (DFT) and inhibition kinetics. One distinct inhibitory molecule was found to bind to either a non-competitive or a competitive binding site in the secondary coordination sphere of the active site. Site-directed mutagenesis of key amino acid residues in the vicinity of the bis-MGD cofactor revealed changes in both non-competitive and competitive binding, whereby the inhibitor is in case of the latter interaction presumably bound between the cofactor and the adjacent Arg587.

Simultaneous removal of atrazine and heavy metal ions using sulfonated polymeric microspheres through an adsorptive filtration process: Insights into the synergistic and competitive adsorption

The combined pollution of surface water caused by organic and inorganic contaminants has become an environmental issue worthy of attention. Adsorption has been acknowledged as a practical and efficient water treatment process. However, the combined interactions between organic and inorganic pollutants on the adsorbents remain ambiguous. In this work, a novel multi-function sulfonated polyacrylate-divinylbenzene (PADVB) microspheres were rapidly and successfully prepared for removing organic pollutants and heavy metals ions from water. The theoretical maximum adsorption capacity of ATZ and Pb(II) by sulfonated PADVB was calculated as 280 mg g −1 and 163 mg g −1 , respectively. Additionally, dynamic adsorption based on an adsorptive-filtration reactor combined with in-situ regeneration was equipped, which proved to be effective and practical for the removal of combined pollution of trace contaminants from natural water. The simultaneous adsorption of ATZ and heavy metal ions could be attributed to the combined effects of multiple adsorption mechanisms, such as electrostatic attraction , complexation, Bronsted acid-base interaction, hydrogen bonds, and π-π interactions. Besides, the complexation between ATZ and heavy metal ions was confirmed by batch adsorption experiments and multiple characterization techniques, which play an important role in the synergistic interactions. In the binary adsorption systems, the heavy metal ions acted as a connector between ATZ and the surface of the microspheres through complexation, which could partly offset the competition of adsorptive binding sites. This work provided a promising adsorptive-filtration reactor to recycle and reuse powder adsorbent for solving the trace-level combined pollution in the environmental water and expanded the insights of adsorptive behaviours between organic pollutants and heavy metal ions.

Abstract 5049: Prediction of pharmacokinetics and receptor occupancy of a bispecific antibody against CD40 and PD-L1

Abstract Background: Currently bispecific monoclonal antibodies gain traction in pharmacology as a way to simultaneously target different surface molecules on immune and tumor cells and modulate their activity. While antibodies against a single target, such as CD40 on antigen-presenting cells (APCs) and PD1 on T cells, are widely used in clinic to treat various forms of cancer, bispecific antibodies are still in the early stages of clinical development. The aim of this study was to predict pharmacokinetics (PK), receptor occupancy (RO) and optimal dose for a bispecific antibody YH008 against CD40 and PD1 using the PBPK/RO model. Methods: The PBPK/RO model describes distribution of an antibody (mAb) between physiological compartments, transport across the endothelium, binding with CD40 on dendritic cells (DCs), B cells, and macrophages (Mph) and PD1 on CD4+ and CD8+ T cells in the immunological synapses (ISs). The model takes into account number of cells expressing target molecules and their ligands, expression levels, clearance of the antibody. Two scenarios of mAb binding with CD40 were considered: a case when the mAb competes with CD40L for the binding site and a case when the mAb binds to a different site. Binding affinities of YH008 for human CD40 and PD1 were taken from [1]. The model was validated using PK data on monospecific antibodies against CD40 and PD1. ROs of PD1 on CD4+ and CD8+ T cells and CD40 for Mph, DCs, B cells and percentages of the trimer complex (CD40-YH008-PD1) were calculated. Optimal dose of YH008 was selected on the basis of maximal exposure/area under curve (AUC) of the trimers in ISs between APCs and CD8+ T cells in the tumor. Results: Model simulations show good agreement with the experimental PK and RO data for monospecific mAbs against PD1 (Pembrolizumab) and CD40 (Selicrelumab, BI 655064). For the bispecific antibody, the simulations show that competition for the CD40 binding site between CD40L and the mAb does not significantly affect PK and RO in blood or tissue. The model predicts 99% RO on antigen-presenting cells (APCs) and 98% RO on T cells in blood at dose 80 mg. In the tumor, 95% RO on APCs and 85% RO on T cells, as well as 5-to-6-fold decrease in the number of PD1-PDL1 complexes in ISs, are achieved at dose 240 mg. The dose dependence for YH008 was calculated, and the optimal dose based on the numbers of the trimers and signaling complexes in the ISs was estimated. Conclusions: The PBPK/RO model for a bispecific mAb based on physiological levels of immune cells, target molecule expression, and binding parameters for YH008 antibody against CD40 and PD1 predicts PK and RO in peripheral blood and tumor. The model shows that competition for CD40 binding site between the mAb and CD40L does not significantly affect PK and RO. The optimal dose for maximal effect of YH008 was estimated. Citation Format: Alexandra Diakonova, Oleg Demin, Dmitry Shchelokov, Oleg Demin. Prediction of pharmacokinetics and receptor occupancy of a bispecific antibody against CD40 and PD-L1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5049.

Nanoplastic stimulates metalloid leaching from historically contaminated soil via indirect displacement

It is generally believed that the ability of nanoplastics (NPs) to mobilize other contaminants is due to direct adsorption; however, this intuitive belief is questioned in this study when it comes to a historically contaminated soil where mining activities since 1958 have resulted in arsenic (As) and cadmium (Cd) enrichment. Negatively charged polystyrene (PS) NPs were used in this study, which should theoretically stimulate Cd (metal cation) instead of As (negatively charged oxyanion) leaching if direct adsorption accounted for co-transport. Surprisingly, PS NPs enhanced the leaching of As by up to over 5 times (p value < 0.05), but had almost no effect on Cd leaching (p value > 0.05). A novel indirect displacement model was therefore developed to describe the phenomenon of enhanced As leaching. It has been found that negatively charged NPs interacted with As via competition for soil binding sites. Underlying mechanistic insights were further explored via both theoretical calculations with the Extended Derjaguin–Landau–Verwey–Overbeek (XDLVO) approach, and direct characterization using Scanning Electron Microscopy (SEM) and Computed X-ray Microtomography (μCT) showing binding sites and flow channels, respectively. The overall results provide new and valuable insights into NP-metal(loid) interactions in the natural soil environment, which can be integrated in future studies regarding the transport and risk assessment of NPs, and toxic metal(loid)s.

Neuroprotective effect of the standardised extract of Bacopa monnieri (BacoMind) in valproic acid model of autism spectrum disorder in rats

Ethnopharmacological relevanceBacopa monnieri (BM) is commonly employed in the Indian traditional system of medicines, i.e. Ayurveda as a memory booster, antioxidant, anti-inflammatory, antipyretic, analgesic, sedative and anti-epileptic for decades. Aim of the studyTo evaluate the neuroprotective effect of Bacopa monnieri (BM) in experimental model of autism spectrum disorder (ASD) in Wistar rats and explore its mechanism of action. Materials and methodsBacoMind, was evaluated for its neuroprotective effect in valproic acid (VPA) model of ASD. For in-vivo study, the pregnant female Wistar rats were divided in two groups; normal control (NC) and VPA group who received single dose of normal saline (0.9%) or 600 mg/kg dose of VPA respectively on gestation day (G.D) 12.5. After the birth, all pups were segregated according to the sex. All the male pups from the dams were divided into six groups: Group 1 (NC, treated with only 0.9% normal saline, group 2 (VPA, treated 600 mg/kg on G.D12.5 and normal saline from post natal day (PND) 23 to 43), group 3 (risperidone 2.5 mg/kg, PND 23 to 43) and groups 4, 5 and 6 (BM 20, 40, 80 mg/kg, PND 23 to 43). All experimental groups were subjected to batteries of behavior parameters (three chamber sociability test, Morris Water Maze, elevated plus maze, open field and rota rod test), biochemical parameters such as oxidative stress (GSH, SOD, Catalase, MDA), inflammatory cytokines (Il-1β, IL-6, IL-10, TNF-α), histopathological examination (cresyl violet staining) of hippocampus (HC) and prefrontal cortex (PFC) regions. Further, the mRNA as well as protein expression of AMPA receptor was evaluated using RT-PCR and western blot respectively to study the mechanism of neuroprotective effect of BM. The in-silico analysis followed evaluating the binding profile of different constituents of BacoMind with AMPA receptor. ResultsThe results of the in-vivo study indicated BM at 80 mg/kg ameliorated abnormal behavioral paradigms such as social deficits, repetitive behavior, learning and memory impairments, and motor coordination exhibited by the VPA model of ASD in rats. Furthermore, BM was found to have a significant anti-oxidant (increasing GSH, SOD, and catalase and decreasing MDA levels) and anti-inflammatory properties (decreasing IL-1β, 6, TNF- α). The histopathological score was also found to be significantly improved by BM in a dose dependent manner in both HC and PFC. In addition to this, the up-regulated mRNA as well as protein expression of AMPA receptor was significantly reduced by 80 mg/kg dose of BM in both HC and PFC. Further, the in-silico analysis of different constituents of BacoMind with AMPA receptor demonstrated that luteolin and apigenin showed good binding to both the competitive antagonist binding site, non-competitive antagonist binding site and allosteric modulator site while Bacosaponin C showed good binding to the non-competitive antagonist binding site. ConclusionThe present study concluded that BM can be a potential candidate for ameliorating the ASD symptoms in rats and acts via modulating the up-regulated AMPA receptor expression.

Comparison of in vitro and in vivo binding site competition of Bacillus thuringiensis Cry1 proteins in two important maize pests.

Binding site models, derived from in vitro competition binding studies, have been widely used for predicting potential cross-resistance among insecticidal proteins from Bacillus thuringiensis. However, because discrepancies have been found between binding data and observed cross-resistance patterns in some insect species, new tools are required to study the functional relevance of the shared binding sites. Here, an in vivo approach has been applied to the competition studies to establish the functional relevance of shared binding sites as determined by in vitro competition assays. Using Cry disabled proteins as competitors in mixed protein overlay assays, we assessed the preference of Cry1Ab, Cry1Fa, and Cry1A.105 proteins for shared binding sites in vivo in two important corn pests, Ostrinia nubilalis and Spodoptera frugiperda. This study shows that in vivo and in vitro binding site competition assays can provide useful information to better ascertain whether different Cry proteins share binding sites and, consequently, whether cross-resistance due to binding site alteration can occur. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Heavy Metals and Human Health: Possible Exposure Pathways and the Competition for Protein Binding Sites.

Heavy metals enter the human body through the gastrointestinal tract, skin, or via inhalation. Toxic metals have proven to be a major threat to human health, mostly because of their ability to cause membrane and DNA damage, and to perturb protein function and enzyme activity. These metals disturb native proteins’ functions by binding to free thiols or other functional groups, catalyzing the oxidation of amino acid side chains, perturbing protein folding, and/or displacing essential metal ions in enzymes. The review shows the physiological and biochemical effects of selected toxic metals interactions with proteins and enzymes. As environmental contamination by heavy metals is one of the most significant global problems, some detoxification strategies are also mentioned.

Removal of munition constituents in stormwater runoff: Screening of native and cationized cellulosic sorbents for removal of insensitive munition constituents NTO, DNAN, and NQ, and legacy munition constituents HMX, RDX, TNT, and perchlorate.

Technologies are needed to address contamination with energetic compounds at military installations. This research developed and evaluated novel and sustainable materials that can be used to remove munition constituents (MC) from stormwater runoff. Initial work focused on 3-nitro-1,2,4-triazol-5-one (NTO), as it is both highly soluble and ionized at environmentally relevant pH values. Screening cellulosic materials indicated that cationized (CAT) versions of pine shavings (pine, henceforth) and burlap (jute) demonstrated >70% removal of NTO from artificial surface runoff. CAT materials also demonstrated >90% removal of the anionic propellant perchlorate. NTO removal (~80%) by CAT pine was similar across initial pH values from 4 to 8.5 S.U. An inverse relationship was observed between NTO removal and the concentration of the major anions chloride, nitrate, and sulfate due to competition for anion binding sites. Sorption isotherms were performed using a mixture of the three primary legacy explosives (octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX), hexahydro-1,3,5-trinitro-s-triazine (RDX), 2,4,6-trinitrotoluene (TNT)), the three insensitive MC (nitroguanidine (NQ), NTO, 2,4-dinitroanisole (DNAN)), and perchlorate. Isotherm results indicated that effective removal of both legacy and insensitive MC would best be achieved using a mixture of peat moss plus one or more of the cationized cellulosic materials.

Exploring multi-target inhibitors using in silico approach targeting cell cycle dysregulator–CDK proteins

Cyclin-dependent kinases (CDKs) belong to a family of multifunctional enzymes that control cell cycle modifications, transcription, and cell proliferation. Their dysfunctions result in different diseases like cancer making them an important drug target in oncology and beyond. The present study aims at identifying the selective inhibitors for ATP binding site in CDK proteins (CDK1, CDK2, CDK4, and CDK5) following a multi-target drug designing approach. Significant challenges lie in identifying the selective inhibitor for the ATP binding site as this region is highly conserved in all protein kinases. Molecular docking coupled with molecular dynamics simulation and free energy of binding calculations (MMPBSA/MMGBSA) were used to identify the potent competitive ATP binding site inhibitors. All the four proteins were docked against the library of drug-like compounds and the outcomes of the docking study were further analyzed by Molecular dynamics (total of 6μs) and MMPB/GBSA techniques. Five different inhibitors for structurally distant protein kinases, i.e. CDK1, CDK2, CDK4, and CDK5 are identified with the binding energy (ΔG bind-PB) in the range −18.24 to −28.43Kcal/mol. Mechanistic complexities associated with the binding of the inhibitor are unraveled by carefully analyzing the MD trajectories. It is observed that certain residues (Lys33, Asp127, Asp145, Tyr15, Gly16, Asn144) and regions are critical for the retention of inhibitors in active pocket, and significant conformational changes take place in the active site region as well as its neighbor following the entry of the ligand inside active pocket as inferred by RMSD and RMSF. It is observed that LIG3 and LIG4 are the best possible inhibitors as reflected from their high binding energy, interaction pattern, and their retention inside the active pocket. This study will facilitate the process of multi-target drug designing against CDK proteins and can be used in the development of potential therapeutics against different diseases.

Anti‐β2‐glycoprotein I and anti‐prothrombin antibodies cause lupus anticoagulant through different mechanisms of action

BackgroundThe presence of lupus anticoagulant (LA) is an independent risk factor for thrombosis. This laboratory phenomenon is detected as a phospholipid‐dependent prolongation of the clotting time and is caused by autoantibodies against beta2‐glycoprotein I (β2GPI) or prothrombin. How these autoantibodies cause LA is unclear. ObjectiveTo elucidate how anti‐β2GPI and anti‐prothrombin antibodies cause the LA phenomenon. MethodsThe effects of monoclonal anti‐β2GPI and anti‐prothrombin antibodies on coagulation were analyzed in plasma and with purified coagulation factors. ResultsDetection of LA caused by anti‐β2GPI or anti‐prothrombin antibodies required the presence of the procofactor factor V (FV) in plasma. LA effect disappeared when FV was replaced by activated FV (FVa), both in a model system and in patient plasma, although differences between anti‐β2GPI and anti‐prothrombin antibodies were observed. Further exploration of the effects of the antibodies on coagulation showed that the anti‐β2GPI antibody attenuated FV activation by activated faxtor X (FXa), whereas the anti‐prothrombin antibody did not. Binding studies showed that β2GPI‐‐antibody complexes directly interacted with FV with high affinity. Anti‐prothrombin complexes caused the LA phenomenon through competition for phospholipid binding sites with coagulation factors as reduced FXa binding to lipospheres was observed with flow cytometry in the presence of these antibodies. ConclusionAnti‐β2GPI and anti‐prothrombin antibodies cause LA through different mechanisms of action: While anti‐β2GPI antibodies interfere with FV activation by FXa through a direct interaction with FV, anti‐prothrombin antibodies compete with FXa for phospholipid binding sites. These data provide leads for understanding the paradoxical association between thrombosis and a prolonged clotting time in the antiphospholipid syndrome.