Scatchard Plot Analysis Research Articles

Entropically driven binding of Camptothecin in the minor groove of salmon testes DNA

The present study focuses on binding association of Camptothecin (CMT) towards natural deoxy-ribonucleic acid (salmon testes, ST) under physiological conditions of pH 7.4. Extensive spectroscopic and computational techniques have been employed to elucidate thermodynamics of the said interaction. UV and fluorescence analyses portrays significant intensity changes (hyper-chromic and hypsochromic) in the spectra, which confirms effective CMT binding to ST DNA. The McGhee-von Hipple method and Scatchard plot analyses estimated the binding affinities in 105 M−1 range. Associated thermodynamic data revealed spontaneous and exothermic nature of binding. Temperature-dependent fluorescence showed negative change in enthalpy and positive change in entropy, leading to the formation of a 1:1 adduct. Non-polyelectrolytic forces appeared to be the driving force of the ligand-DNA interaction, according to salt-dependent fluorescence. Dye displacement assay, viscosity study, DNA melting, iodide quenching, urea denaturation assay examined the minor groove nature of CMT. In silico docking study examined precise molecular representations of the minor groove binding mechanism that formed between the complex, and the study's findings were consistent with the experimental results. Simulation studies also validated the experimental analysis and docking data. These findings could expedite the process of creating new and improved CMT molecular derivatives and help in the creation of DNA-targeted medicines, which may be beneficial from a pharmaceutical point of view.

Synthesis, recognition properties and drug release behavior of diltiazem‐imprinted chitosan‐based biomaterials

AbstractIn this study, we prepared diltiazem (DTZ)‐imprinted biomaterials for TDDS using chitosan, PVA, plasticizers, and sulfosuccinic acid. DTZ and the prepared biomaterials were characterized using field emission scanning electron microscopy, Fourier transform infrared, and 1H nuclear magnetic resonance. DTZ recognition properties were confirmed by the binding isotherm, Scatchard plot analysis, the adsorption of materials with structures similar to DTZ, selectivity factor (α), and the imprinting‐induced promotion of binding (IPB). Results revealed that adsorbed amount (Q) of DTZ‐imprinted biomaterials was 1.63–2.53 times higher than that of non‐imprinted biomaterials. In addition, it could be verified that DTZ‐imprinted biomaterials have a binding site for DTZ according to Scatchard plot analysis. Furthermore, the results of α and IPB indicated that the recognition capacity of the prepared DTZ‐imprinted biomaterials is superior to that non‐imprinted biomaterials. DTZ release properties were evaluated under various pH buffers and artificial skin. Results indicated that the DTZ release in buffers at low pH was faster than that in buffers at high pH. The DTZ release using artificial skin was continuous over 20 days. Furthermore, the DTZ release profile in the buffer followed the pseudo‐Fickian diffusion mechanism, whereas the profile in the artificial skin test followed a non‐Fickian diffusion mechanism.

Mechanistic investigation into the binding property of Yohimbe towards natural polymeric DNAs

DNA interactions with multivalent ligand(s) have increasingly become the subject of substantial research. For several small molecules with therapeutic potential, nucleic acids serve as their primary molecular target. Such interaction has been shown to affect transcription or replication, ultimately leading to apoptotic cell death. As a result, researchers are becoming increasingly interested in understanding how small molecules interact with DNA making it possible to develop new, DNA-specific drugs. The bioactive indole alkaloid, Yohimbe (Yohimbine; Yh) has been broadly studied in pharmacological properties while its binding mode to DNA has not been explicated so far. This study adopted molecular modelling and multi-spectroscopic methods to investigate the interaction between Yohimbine and herring testes (HT DNA) in physiological conditions. Minor hypochromic and bathochromic shifts of fluorescence intensity were observed, suggesting the binding of Yh to HT DNA. The Scatchard plot analyses using the McGhee-von Hipple method revealed non-cooperative binding and affinities in the range of 105 M−1. The thermodynamic parameters suggested exothermic binding, which was favoured by negative enthalpy and positive entropy changes from temperature-dependent fluorescence experiments. Salt-dependent fluorescence suggested that the interaction between the ligand and DNA was governed by non-polyelectrolytic forces. The results of iodide quenching, urea denaturation assay, dye displacement, and in silico molecular docking, suggested groove binding of Yh to HT DNA. Thus, the groove binding mechanism of interaction was validated by both biophysical and computational techniques. The structural elucidation and energetic profiling of Yh's interaction with naturally occurring polymeric DNA can be useful to the development of DNA-targeted therapeutics.

Cationic proteins from eosinophils bind bone morphogenetic protein receptors promoting vascular calcification and atherogenesis.

Blood eosinophil count and eosinophil cationic protein (ECP) concentration are risk factors of cardiovascular diseases. This study tested whether and how eosinophils and ECP contribute to vascular calcification and atherogenesis. Immunostaining revealed eosinophil accumulation in human and mouse atherosclerotic lesions. Eosinophil deficiency in ΔdblGATA mice slowed atherogenesis with increased lesion smooth muscle cell (SMC) content and reduced calcification. This protection in ΔdblGATA mice was muted when mice received donor eosinophils from wild-type (WT), Il4-/-, and Il13-/- mice or mouse eosinophil-associated-ribonuclease-1 (mEar1), a murine homologue of ECP. Eosinophils or mEar1 but not interleukin (IL) 4 or IL13 increased the calcification of SMC from WT mice but not those from Runt-related transcription factor-2 (Runx2) knockout mice. Immunoblot analyses showed that eosinophils and mEar1 activated Smad-1/5/8 but did not affect Smad-2/3 activation or expression of bone morphogenetic protein receptors (BMPR-1A/1B/2) or transforming growth factor (TGF)-β receptors (TGFBR1/2) in SMC from WT and Runx2 knockout mice. Immunoprecipitation showed that mEar1 formed immune complexes with BMPR-1A/1B but not TGFBR1/2. Immunofluorescence double-staining, ligand binding, and Scatchard plot analysis demonstrated that mEar1 bound to BMPR-1A and BMPR-1B with similar affinity. Likewise, human ECP and eosinophil-derived neurotoxin (EDN) also bound to BMPR-1A/1B on human vascular SMC and promoted SMC osteogenic differentiation. In a cohort of 5864 men from the Danish Cardiovascular Screening trial and its subpopulation of 394 participants, blood eosinophil counts and ECP levels correlated with the calcification scores of different arterial segments from coronary arteries to iliac arteries. Eosinophils release cationic proteins that can promote SMC calcification and atherogenesis using the BMPR-1A/1B-Smad-1/5/8-Runx2 signalling pathway.

Structural Insight into Groove Binding of Yohimbine with Calf Thymus DNA: A Spectroscopic, Calorimetric, and Computational Approach.

A variety of anticancer and antibacterial drugs target DNA as one of their primary intracellular targets. Understanding ligand-DNA interactions and developing new, promising bioactive molecules for clinical use are greatly aided by elucidating the interaction between small molecules and natural polymeric DNAs. Small molecules' ability to attach to and inhibit DNA replication and transcription provides more information on how drugs impact the expression of genes. Yohimbine has been broadly studied in pharmacological properties, while its binding mode to DNA has not been explicated so far. In this study, an attempt was made to explore the interaction between Yohimbine (YH) and calf thymus (CT-DNA) by using varying thermodynamics and in silico approaches. Minor hypochromic and bathochromic shifts of fluorescence intensity were observed, suggesting the binding of YH to CT-DNA. The Scatchard plot analysis using the McGhee-von Hipple method revealed noncooperative binding and affinities in the range of 105 M-1. The binding stoichiometry value is 2:1 (2 molecules of YH were span by 1 base pair) and was determined by Job's plot. The thermodynamic parameters suggested exothermic binding, which was favored by negative enthalpy and positive entropy changes from both isothermal titration calorimetry and temperature-dependent fluorescence experiment. Salt-dependent fluorescence suggested that the interaction between the ligand and DNA was governed by nonpolyelectrolytic forces. Kinetics experiment confirmed the static type of quenching. The results of iodide quenching, urea denaturation assay, dye displacement, DNA melting, and in silico molecular docking (MD) suggested groove binding of YH to CT-DNA. Circular dichroism spectra confirmed minimal perturbation of CT-DNA with YH binding via groove region. Therefore, the groove binding mechanism of interaction was validated by biophysical techniques and in silico, MD approaches. The findings supported here may contribute to the development of new YH therapeutics possessing better efficacy and lesser side effects.

Abstract 4999: Preclinical evaluation of MCLA-129, a bispecific antibody targeting EGFR and c-MET on solid tumor cells, in comparison with amivantamab

Abstract The HGF/c-MET pathway is frequently upregulated in tumors that are resistant to EGFR tyrosine kinase inhibitors. MCLA-129 is an antibody-dependent cellular cytotoxicity (ADCC) enhanced Biclonics® that targets epidermal growth factor receptor (EGFR) and c-MET in non-small cell lung cancer (NSCLC) and other solid tumors. MCLA-129 is a potent inhibitor of tumor growth applying various mechanisms of action (MOA), including inhibition of c-MET and EGFR signaling, antibody-dependent cellular phagocytosis (ADCP) and ADCC. Here we compared the MOA of MCLA-129 with the MOA of the EGFR-c-MET bispecific antibody amivantamab. The binding of MCLA-129 to tumor cells expressing different levels of EGFR and c-MET was quantified using radiolabeled antibodies. Scatchard plot analysis showed that the affinity of MCLA-129 for both c-MET and EGFR is in the low nanomolar range. In FACS, MCLA-129 and amivantamab show similar binding to tumor cells harboring EGFR or c-MET exon14-skipping mutations. Crossblock experiments with the c-MET binding antibody onartuzumab analog showed that MCLA-129 competes for the same HGF-binding region of c-MET, which is distinct from the binding site of amivantamab according to Neijssen et al, 2021 (PMC8113745). MCLA-129 and amivantamab did not downmodulate EGFR or c-MET. ADCP was also comparable for MCLA-129 and amivantamab, as measured with monocyte-derived macrophages incubated with pHrodo-labeled target cells with readout of tumor cell phagocytosis on the Incucyte® system. In contrast, MCLA-129 displayed ADCC activity similar to or more potently than amivantamab in an ADCC reporter assay with either high affinity FcγRIII 158V-variant or low affinity 158F-variant effector cells in absence or presence of soluble EGFR and soluble c-MET. MCLA-129-induced ADCC appears to be less affected than amivantimab by the presence of soluble EGFR and soluble c-MET. In conclusion, MCLA-129 is a Biclonics® common light chain bispecific antibody with multiple MOA including inhibition of c-MET and EGFR ligand binding, ADCP and ADCC comparable or more potent than amivantamab. A phase 1/2 clinical trial of MCLA-129 in solid tumors is ongoing. These data support the further clinical development of MCLA-129 in patients with NSCLC and other solid tumors. Citation Format: David J.J. de Gorter, Alexandre Deshiere, Arjen Kramer, Martijn van Rosmalen, Franziska Mortensen, Cecile Geuijen. Preclinical evaluation of MCLA-129, a bispecific antibody targeting EGFR and c-MET on solid tumor cells, in comparison with amivantamab. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4999.

Chitosan-based magnetic molecularly imprinted polymer: synthesis and application in selective recognition of tricyclazole from rice and water samples

A tricyclazole selective chitosan/Fe3O4 magnetic molecularly imprinted polymer (MMIP) was synthesized using non-covalent binding polymerization involving methacrylic acid (MAA) as functional monomer, divinylbenzene (DVB-80) as crosslinker, 2,2'-azobisisobutyronitrile as initiator, acetonitrile/toluene (75:25, v/v) as porogenic solvent and tricyclazole as template. Surface morphology and magnetic characterization of the prepared imprinted and non-imprinted polymers were done using scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectrometry and vibrating sample magnetometry, respectively. The adsorption kinetic data fitted best in pseudo-second-order model. The adsorption equilibrium was achieved in 30 min and the maximum binding capacity was 4579.9 µg/g. The Freundlich isotherm model was found suitable for explaining the binding isotherm data (R2 > 0.99). Negative values of thermodynamic parameters ∆G (Gibb’s free energy), ∆H (enthalpy), and ∆S (entropy) revealed exothermic and spontaneous nature of adsorption processes. It also revealed decreased randomness at the solid–liquid interface during sorption. The scatchard plot analysis suggested heterogeneity of binding sites on MMIPs. The molecular recognition selectivity of MMIPs towards tricyclazole was much higher, as compared to its structural analogues, tebuconazole (α = 28.58) and hexaconazole (α = 37.16). The MMIPs were successfully applied to separate and enrich tricyclazole from fortified samples of rice and water, with a recovery percentage of 89.4% and 90.9%, respectively. These reusable imprinted polymers possessing high selectivity and specificity can be utilized as an adsorbent for solid-phase extraction in sample preparation for tricyclazole residue analysis in complex environmental matrices.

Theoretical Design and Adsorption Properties of Molecularly Imprinted Polymers Obtained from Chloramphenicol and Acrylamide

Molecular simulations are widely used to model molecularly imprinted polymers(MIPs) in order to enhance their adsorption and selectivity. In this study, chloramphenicol(CAP) and acrylamide(AM) were used as the template and functional monomer, respectively, and pentaerythritol triacrylate(PETA), ethylene glycol dimethacrylate (EGDMA), and trimethylolpropane trimethylacrylate(TRIM) were used as cross-linking agents. The ωB97XD/6-31G(d,p) density functional theory method was employed to simulate binding sites, binding energy, the number of hydrogen bonds, the imprinted molar ratio, which produced the most stable complex, and the interaction mechanism. The cross-linking agent was optimized based on the binding energy. The atoms in molecules theory were used to study the nature of the imprinting effects. The theoretical calculations revealed that CAP and AM formed ordered complexes via hydrogen bonding interactions when the molar ratio between CAP and AM was 1:7 using TRIM as the cross-linking agent. The CAP-AM complex(molar ratio 1:7) had the most stable structure, the largest number of hydrogen bonds, and the smallest ΔE. The experimental results indicate that the CAP-MIPs formed perfect microspheres with an average particle size of 314 nm. Scatchard plot analysis showed that the CAP-MIPs had only one type of binding site over the studied concentration ranges. The dissociation equilibrium constant and maximum apparent adsorption capacities were 1887.35 mg/L(5.84 mmol/L) and 155.56 mg/g(0.482 mmol/g), respectively.

Calcium alginate entrapped Eupatorium adenophorum Sprengel stems powder for chromium(VI) biosorption in aqueous mediums.

A novel biosorbent, Eupatorium adenophorum Sprengel-alginate beads was used for chromium(VI) biosorption from aqueous solutions. Biosorption process was optimized at pH 2.0, biomass concentration 1.0 g/L, contact time 60 min, and temperature 30 oC respectively. Maximum uptake capacity of Cr(VI) was calculated at 28.011 mg/g. It was found that the overall biosorption process was best described by pseudo second-order kinetics with high correlation coefficient values. Intraparticle diffusion model suggested that Cr(VI) biosorption may proceed within multiple steps. Data obtained from the batch studies confirmed well to the Langmuir, Temkin, and Hill-der Boer isotherm models. Scatchard plot analysis further supported the mono-layer biosorption of Cr(VI) ions on Eupatorium adenophorum Sprengel-alginate beads as described by Langmuir isotherm model. Numerical values of E obtained from Dubinin-Radushkevich isotherm model identified the physisorption as predominant mechanism for Cr(VI) biosorption. The negative values of ΔGo confirmed the spontaneous and feasibility nature, whereas positive value of ΔHo showed the endothermic nature of biosorption process. Positive value of ΔSo indicated an increase in the randomness at the solid/solution interface during the biosorption process. The endothermic nature of Cr(VI) biosorption was also described by Temkin isotherm model. The results indicated that Cr(VI) biosorption was not significantly affected by the presence of co-ions at lower concentrations. Desorption of Cr(VI) ions from metal-loaded Eupatorium adenophorum-alginate beads was observed at 92.091% with 0.5 M HNO3 solution in solid to liquid ratio of 1.0 g/L.

Preparation and application of magnetic molecular imprinted polymers for extraction of cephalexin from pork and milk samples

A highly selective and effective method was successfully developed using magnetic molecular imprinted polymers (MMIPs) as solid-phase extraction (SPE) coupled with high performance liquid chromatography-ultraviolet detector (HPLC-UV) to rapidly determine cephalexin (CFX) in complex animal-derived food. MMIPs were creatively synthesized via suspension polymerization using Fe3O4 magnetic nanoparticles as supporter, CFX as template, acrylamide (AM) as functional monomer, and ethylene glycol dimethacrylate (EGDMA) as cross-linker. The MMIPs were characterized using X-ray diffraction (XRD), Fourier transform infrared spectrometry (FT-IR), scanning electron microscopy (SEM), and vibrating sample magnetometry (VSM). The binding process fitted well with pseudo-second-order model with good selectivity. Scatchard plot analysis suggested that MMIPs have two types of binding sites with the Qmax of 24.18 mg g−1 and 40.25 mg g−1, respectively. And Langmuir model proved that the recognition sites were uniformly distributed in a monolayer on the surface of MMIPs. The methodological assessment showed good applicability of MMIPs with excellent recovery (85.5%–94.0%), precision (1.2%–2.4%), and stability (intra-day 1.3%–3.6%; inter-day 2.6%–4.3%) in determining CFX content. In addition, the linearity of the calibration curve was good in the range of 0.02–5.00 mg L−1, with a sensitive detection limit of 5.00 μg kg−1. The results above suggest that the obtained MMIPs exert good performance for separation of CFX in animal-derived food, and the proposed method is suitable for the reliable determination of CFX in complex samples.

A dummy molecularly imprinted solid-phase extraction coupled with liquid chromatography-tandem mass spectrometry for selective determination of four pyridine carboxylic acid herbicides in milk

We aim to develop a solid-phase extraction (SPE) based on a dummy molecularly imprinted polymer (MIP) and liquid chromatography-tandem quadrupole mass spectrometry (LC-MS/MS) for selective determination of four pyridine carboxylic acid herbicides (aminopyralid, picloram, fluroxypyr, and clopyralid) in milk samples. Using picloram as the dummy template, MIP nanocomposites with highly selective recognition and adsorption for the target molecule and its structural analogs were successfully synthesized by precipitation polymerization. Adsorption isotherms and kinetics were used to determine the adsorption performance and specific recognition mechanism of both MIPs and non-molecularly imprinted polymers (NIP). The Scatchard plot analysis revealed that two different binding sites were formed in the MIP with maximum binding capacity (Qmax) of 1171.8 μg·g−1 and 3022.5 μg·g−1, respectively. Recovery at three spiking levels of 10, 20, and 50 μg·L−1 ranged between 75.3 and 89.8% with relative standard deviations (RSDs) <14.3%.The limit of detection (LOD) was estimated to be 0.124 μg·L−1. Finally, the feasibility of the proposed methodology was successfully applied to quantify aminopyralid and another three pyridine carboxylic acid herbicides in milk.

Impact of Nicotine Consumption on Hyper Acidic Patients Taking PPI: An &amp;lt;i&amp;gt;In-Vitro&amp;lt;/i&amp;gt; and Computational Analysis

The study aims to investigate the protein binding kinetics of nicotine and a PPI (pantoprazole) with Bovine Serum Albumin (BSA) through UV spectroscopy and computational modeling. Data was obtained by using nicotine and pantoprazole and warfarin and diazepam as the two site specific probes on Bovine serum albumin (BSA). In-vitro and in-silico modeling was carried out in creating an environment that simulates the body environment. Cellulose membrane tubes were cut into 9 cm and tied tightly not to let any mixtures leak out. To determine number of binding sites, association constants by using Scatchard plot, predominant binding site of each drug and rise in % of free fraction of one by the other were analyzed using equilibrium dialysis method. Molecular docking further verifies the observations. In Scatchard plot analysis, for nicotine, n1, n2, k1 and k2 = 2.2, 7.6, 0.18 μM−1 and 0.02 μM−1 and for pantoprazole, n1, n2, k1 and k2 = 0.42, 1.2, 0.40 μM−1 and 0.03 μM−1. Nicotine binds more to diazepam site (site-II) and pantoprazole mainly to warfarin site (site-I). In molecular docking, the binding affinity of nicotine being −5.7 kcal/mole demonstrates higher affinity for site-II than that of pantoprazole whose binding affinity is −8.0 kcal/mole. In absence and presence of warfarin, the free fraction of pantoprazole bound to BSA (1:1) was increased from 37.79% to 82.44% and 51.78% to 98.80% respectively by nicotine. On the other hand, free fraction of nicotine was raised by pantoprazole from 12.89% to 75.70% and 50.08% to 99.66% in the absence and presence of diazepam. Both the results of spectroscopic and computational molecular docking suggest that administering pantoprazole with nicotine might increase the % free fraction of pantoprazole more. Thus, nicotine consumption can be beneficial for smoker people taking PPI like pantoprazole.

Ultrasonication assisted extraction of chlorpyrifos from honey and brinjal using magnetic molecularly imprinted polymers followed by GLC-ECD analysis

Chlorpyrifos selective magnetic molecularly imprinted polymers (MMIPs) were synthesized by precipitation polymerization using acrylic acid as monomer and ethylene glycol dimethacrylate as cross linker. Properties of prepared polymers were determined using Fourier Transform Infrared Spectroscopy (FTIR), Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM) and Vibrating Sample Magnetometer (VSM) techniques. The imprinted polymers showed much higher adsorption capacity towards the analyte (Kd = 12,059.5 mL g−1) than the magnetic non-imprinted polymers (MNIPs) (Kd = 962.2 mL g−1). Adsorption data fitted well into linearized Freundlich equation (R2 = 0.981) and followed pseudo-second-order kinetic model (R2 = 0.999). Scatchard plot analysis revealed heterogeneous binding sites on MMIPs while it was homogenous on MNIPs. Polymers were highly selective in extracting chlorpyrifos even in the presence of its structural analogue quinalphos (α = 16.2) and triazophos (α = 15.6). Imprinted polymer after regeneration was successfully used three times for rebinding the template without any apparent loss in adsorption capacity. Using prepared polymers, about 86.23–92.04% and 89.42–102.36% of the chlorpyrifos were successfully recovered from fortified market samples of honey and brinjal with relative standard deviation of 1.70–3.93% and 0.17–5.50%, respectively.

Inhibitory Effect of a Novel DNA Aptamer with Artificial Nucleic Bases for Von Willebrand Factor A1 Domain on Platelet Thrombosis Formation

<Introduction>von Willebrand factor (VWF) is a blood glycoprotein that plays an important role in platelet thrombus formation through interaction with its A1 domain and platelet glycoprotein 1b. Thus, VWF A1 domain was thought to be a good therapeutic target candidate for VWF mediated thrombosis. In this study, we analyzed the inhibitory effects of a novel DNA aptamer (TAGX-0004) on platelet aggregation compared with another VWF A1 domain binding aptamer (ARC1779) which had entered to Phase II clinical trial for acquired thrombotic thrombocytopenic purpura (TTP) in 2011.<Methods>TAGX-0004 was generated by SELEX (systematic evolution of ligands by exponential enrichment) and consisted of artificial nucleic acid base, 7-(2-thienyl) imidazo [4,5-b] pyridine (Ds) as well as natural bases (Adenine, Thymine, Cytosine and Guanine). The dissociation constant (Kd) of the aptamers was analyzed by electrophoretic mobility shift assay (EMSA). 100 nM of each DNA aptamer was mixed with A1 domain of VWF protein (final concentration was 0 to 800 nM) in binding buffer (1x PBS, 0.005% NP-40) and incubated for 30 min at 37 °C. The samples were subjected to EMSA with 8% native PAGE, then stained by SYBR Gold. Kd value was determined with scatchard plot analysis. To characterize the binding sites of VWF A1 to these DNA aptamers, various mutants of VWF A1 domain were generated with an alanine substitution technique.Platelet aggregation was induced with ristocetin (RIPA), botrocetin (BIPA), collagen, epinephrine and adenosine diphosphate (ADP). The change of light transmission rate of platelet rich plasma (PRP) compared to platelet poor plasma (PPP) at 37 °C was recorded for 6 min, then the inhibition ratio of platelet aggregation was determined. Total thrombus-formation analysis system (T-TAS) (Fujimori Kogyo Co. Ltd., Tokyo, Japan), which is a novel micro-chip flow-chamber system, was employed to analyze thrombus formation visually and quantitatively in whole blood samples. The micro-chip coated with type 1 collagen was used for this analysis. Anti-VWF A1 inhibitory effects of these aptamers at high shear stress (initial rate of 2000 s-1) were calculated by continuous pressure levels within its narrow capillary. The course of thrombus formation was also optically recorded with a video-microscope located under the microchip.<Results>Biophysical interaction analysis using EMSA showed that TAGX-0004 had approximately 15-times higher binding activity to VWF A1 domain than ARC1779. Based on an alanine scan analysis, it was revealed that a couple of residues we tested were critical for binding of ARC1779 but not for TAGX-0004, which indicating that TAGX-0004 interacts with VWF A1 domain via different amino acid residue(s).Both aptamers did not inhibit the platelet aggregation induced by collagen, epinephrine or ADP. In RIPA, the 80% inhibition was observed by TAGX-0004 and ARC1779 at a final concentration of 10 nM and 750 nM, respectively. In BIPA, those were seen at those of 50 nM and 1000 nM, respectively. In T-TAS analysis, TAGX-0004 inhibited the thrombus formation completely at a final concentration of 100 nM, whereas ARC1779 exhibited partial inhibition of thrombosis formation even at 1000 nM. These results indicated that TAGX-0004 had stronger inhibitory effect on the platelet aggregation compared with ARC1779 under both static and high-shear conditions.<Conclusions>In the present study, we showed the affinity to VWF-A1 domain of TAGX-0004 was stronger than that of ARC1779. In addition, TAGX-0004 was more effective in suppressing platelet aggregation under both static and high-shear stress condition than ARC1779. In the published results of clinical study of ARC1779, there were no hemorrhagic complications in the small clinical trial of patients (7 patients of ARC1779 group) despite the almost complete suppression of VWF function in severely thrombocytopenic patients. Therefore, TAGX-0004 could be developed as a promising therapeutic agent for VWF mediated thrombotic disorders, such as acute coronary syndrome and TTP. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Adsorption of 1-naphthyl methyl carbamate in water by utilizing a surface molecularly imprinted polymer

Surface molecularly imprinting polymer (SMIP) was utilized in the removal of a residual pesticide (carbaryl (CBL)) in water and simulated fruit juice. Being the crosslinking agent, ethylene glycol dimethacrylate (EGDMA) was copolymerized with the monomer, methacrylic acid (MAA) and CBL as the template molecules on the surface of the silica gel particles to produce the SMIP adsorbents. The SMIP adsorbents showed good selectivity and good adsorption capacity for CBL in the competitive adsorptions with two structurally related carbamate pesticides. The effect of the pretreatment solvents on the adsorption capacity of the SMIP adsorbent was investigated with the results of the numerical simulations. The adsorption isotherms and the adsorption kinetics were well described by the Freundlich equilibrium model and the pseudo-second-order kinetic model, respectively. Scatchard plot analysis revealed that there were two classes of binding sites populated in the SMIP adsorbents. In addition, the good selective adsorption of CBL by the SMIP adsorbent in a simulated fruit juice containing vitamin C and fructose indicated the great potential of the SMIP adsorbents to remove residual pesticide in food industry and processing industry for agricultural products.

Application of magnetic molecularly imprinted polymers for extraction of imidacloprid from eggplant and honey

A magnetic molecularly imprinted polymer (MMIP) adsorbent for imidacloprid was prepared using non-covalent approach with functionalized nano Fe3O4 particles (magnetic cores), imidacloprid (template), acrylic acid (functional monomer), ethylene glycol dimethacrylate (cross linker) and azobisisobutyronitrile (initiator) and used for selective separation of imidacloprid from honey and vegetable samples. The polymers were characterized using FT-IR spectroscopy, SEM and TEM images. For analysis of imidacloprid LC-MS/MS equipment was used. Adsorption kinetics was best explained by pseudo-second-order kinetic model. Adsorption data fitted well into linearized Freundlich equation (R2 > 0.98). Scatchard plot analysis indicates the presence of two classes of binding sites in the MMIPs with the Cmax of 1889.6 µg g−1 and 65448.9 µg g−1, respectively. MMIPs demonstrated much higher affinity for imidacloprid over structurally similar analogues acetamiprid (α = 23.59) and thiamethoxam (α = 17.15). About 87.1 ± 5.0% and 90.6 ± 5.6% of the added imidacloprid was recovered from MMIPs in case of fortified eggplant and honey samples, respectively.

Identification of a high affinity binding site for abscisic acid on human lanthionine synthetase component C-like protein 2.

Lanthionine synthetase component C-like protein 2 (LANCL2) has been identified as the mammalian receptor mediating the functional effects of the universal stress hormone abscisic acid (ABA) in mammals. ABA stimulates insulin independent glucose uptake in myocytes and adipocytes via LANCL2 binding in vitro, improves glucose tolerance in vivo and induces brown fat activity in vitro and in vivo. The emerging role of the ABA/LANCL2 system in glucose and lipid metabolism makes it an attractive target for pharmacological interventions in diabetes mellitus and the metabolic syndrome. The aim of this study was to investigate the presence of ABA binding site(s) on LANCL2 and identify the amino acid residues involved in ABA binding. Equilibrium binding assays ([3H]-ABA saturation binding and surface plasmon resonance analysis) suggested multiple ABA-binding sites, prompting us to perform a computational study that indicated one putative high-affinity and two low-affinity binding sites. Site-directed mutagenesis (single mutant R118I, triple mutants R118I/R22I/K362I and R118I/S41A/E46I) and equilibrium binding experiments on the mutated LANCL2 proteins identified a high-affinity ABA-binding site involving R118, with a KD of 2.6 nM ± 1.2 nM, as determined by surface plasmon resonance. Scatchard plot analysis of binding curves from both types of equilibrium binding assays revealed a Hill coefficient >1, suggesting cooperativity of ABA binding to LANCL2. Identification of the high-affinity ABA-binding site is expected to allow the design of ABA agonists/antagonists, which will help to understand the role of the ABA/LANCL2 system in human physiology and disease.

Molecular and Thermodynamic Studies on DNA Triplex Formed in the Promoter Region of HMGB1 Gene as a Selective Target for Anticancer Drugs.

HMGB1 (High Mobility Group Box-1) is a very versatile highly abundant architectural protein that plays multiple roles in human health and diseases. Under physiological condition it serves as an amazing assortment of roles in different compartments of cell. The reported high expression of HMGB1 in almost all types of human cancers and inflammatory diseases makes it a critical molecular therapeutic target. In the present study, we have mobilized a proximal twenty one base pair nucleotide (21RY) which is in the promoter region (-55 to-75) of hmgb1 gene and targeted it with triplex forming oligonucleotide (TFO) in combination with two widely used chemotherapeutic drugs, actinomycin (ACT) and adriamycin (ADM). The interaction of actinomycin and adriamycin to 21R*R•Y DNA triplex was studied using UV melting profiles, CD spectroscopy, spectrofluorimetry and Isothermal titration calorimetry. The 21R*R•Y formation was confirmed from biphasic thermal melting profiles, continuous variation method, analysis of CD marker band and thermodynamic parameters. The binding of ADM and ACT to 21R*R•Y was characterized by hypochromic and bathochromic shift in their respective absorption spectrum, quenching (ADM) and enhanced fluorescence (ACT) of steady-state fluorescence intensity, perturbation in the circular dichroic spectrum and change in thermal melting temperatures. The ITC profile and Scatchard plot analysis indicate non-cooperative and higher binding affinity of these drugs to 21R*R•Y compared to their corresponding duplexes. Therefore, combining these chemotherapeutic drugs with triplex forming oligonucleotide may offer new diagnostic and therapeutic options in targeting a gene of interest more specifically with fewer side effects. This study shows that ACT and ADM effectively recognize 21R*R•Y triplex DNA formed on the hmgb1 promoter region.

Targeted therapy of osteosarcoma with radiolabeled monoclonal antibody to an insulin-like growth factor-2 receptor (IGF2R)

IntroductionOsteosarcoma overall survival has plateaued around 70%, without meaningful improvements in over 30years. Outcomes for patients with overt metastatic disease at presentation or who relapse are dismal. In this study we investigated a novel osteosarcoma therapy utilizing radioimmunotherapy (RIT) targeted to IGF2R, which is widely expressed in OS. MethodsBinding efficiency of the Rhenium-188(188Re)-labeled IGF2R-specific monoclonal antibody (mAb) to IGF2R on OS17 OS cells was assessed with Scatchard plot analysis. Biodistribution studies were performed in heterotopic murine osteosarcoma xenografts. Tumor growth was compared over a 24-day period post-treatment between mice randomized to receive 188Re-labeled IGF2R-specific murine mAb MEM-238 (188Re-MEM-238) or one of three controls: 188Re-labeled isotype control mAb, unlabeled MEM-238, or no treatment. ResultsResults demonstrate that the radioimmunoconjugate had a high binding constant to IGF2R. Both 188Re-MEM-238 and the isotype control had similar initial distribution in normal tissue. After 48h 188Re-MEM-238 exhibited a 1.8 fold selective uptake within tumor compared to the isotype control (p=0.057). Over 24days, the tumor growth ratio was suppressed in animals treated with RIT compared to unlabeled and untreated controls (p=0.005) as demonstrated by a 38% reduction of IGF2R expressing osteosarcoma cells in the RIT group (p=0.002). ConclusionsIn conclusion, given the lack of new effective therapies in osteosarcoma, additional investigation into this target is warranted. Advances in knowledgeHigh expression of IGF2R on osteosarcoma tumors, paired with the specificity and in vivo anti-cancer activity of 188Re-labeled IGF2R-specific mAb suggests that IGF2R may represent a novel therapeutic target in the treatment of osteosarcoma. Implications for patient careThis targeted approach offers the benefits of being independent of a specific pathway, a resistance mechanism, and/or an inherent biologic tumor trait and therefore is relevant to all OS tumors that express IGF2R.

Determination of malachite green in aquatic products based on magnetic molecularly imprinted polymers

Magnetic molecularly imprinted polymers (MMIPs) were synthesized through precipitation polymerization using malachite green (MG) as template, methacrylic acid as monomer, ethylene dimethacrylate as crosslinker, and Fe3O4 magnetite as magnetic component. MMIPs were characterized by scanning electron microscopy, Fourier transform infrared spectrometry, and vibrating sample magnetometry. Under the optimum condition, the MMIPs obtained exhibited quick binding kinetics and high affinity to MG in the solution. Scatchard plot analysis revealed that the MMIPs contained only one type of binding site with dissociation constant of 24.0μgmL−1. The selectivity experiment confirmed that the MMIPs exhibited higher selective binding capacity for MG than its structurally related compound (e.g., crystal violet). As a sorbent for the extraction of MG in sample preparation, MMIPs together with the absorbed analytes could easily be separated from the sample matrix with an external magnet. After elution with methanol/acetic acid (9:1, v/v), MG in the eluent was determined by high-performance liquid chromatography coupled with UV detector with recoveries of 94.0–115%. Results indicated that the as-prepared MMIPs are promising materials for MG analysis in aquatic products.