Search results for: cysteine covalent binding

Authors: Choukri Lekbir, Mira Mokhtari

Abstract:

Hydrogen-Materials interaction and mechanisms can be modeled at nano scale by quantum methods. In this work, the effect of hydrogen on the electronic properties of a cluster material model «nickel» has been studied by using of density functional theoretical (DFT) method. Two types of clusters are optimized: Nickel and hydrogen-nickel system. In the case of nickel clusters (n = 1-6) without presence of hydrogen, three types of electronic structures (neutral, cationic and anionic), have been optimized according to three basis sets calculations (B3LYP/LANL2DZ, PW91PW91/DGDZVP2, PBE/DGDZVP2). The comparison of binding energies and bond lengths of the three structures of nickel clusters (neutral, cationic and anionic) obtained by those basis sets, shows that the results of neutral and anionic nickel clusters are in good agreement with the experimental results. In the case of neutral and anionic nickel clusters, comparing energies and bond lengths obtained by the three bases, shows that the basis set PBE/DGDZVP2 is most suitable to experimental results. In the case of anionic nickel clusters (n = 1-6) with presence of hydrogen, the optimization of the hydrogen-nickel (anionic) structures by using of the basis set PBE/DGDZVP2, shows that the binding energies and bond lengths increase compared to those obtained in the case of anionic nickel clusters without the presence of hydrogen, that reveals the armor effect exerted by hydrogen on the electronic structure of nickel, which due to the storing of hydrogen energy within nickel clusters structures. The comparison between the bond lengths for both clusters shows the expansion effect of clusters geometry which due to hydrogen presence.

Keywords: binding energies, bond lengths, density functional theoretical, geometry optimization, hydrogen energy, nickel cluster

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Authors: Iman Farasat, Howard M. Salis

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Engineered genetic circuits reprogram cellular behavior to act as living computers with applications in detecting cancer, creating self-controlling artificial tissues, and dynamically regulating metabolic pathways. Phenemenological models are often used to simulate and design genetic circuit behavior towards a desired behavior. While such models assume that each circuit component’s function is modular and independent, even small changes in a circuit (e.g. a new promoter, a change in transcription factor expression level, or even a new media) can have significant effects on the circuit’s function. Here, we use statistical thermodynamics to account for the several factors that control transcriptional regulation in bacteria, and experimentally demonstrate the model’s accuracy across 825 measurements in several genetic contexts and hosts. We then employ our first principles model to design, experimentally construct, and characterize a family of signal amplifying genetic circuits (genetic OpAmps) that expand the dynamic range of cell sensors. To develop these models, we needed a new approach to measuring the in vivo binding free energies of transcription factors (TFs), a key ingredient of statistical thermodynamic models of gene regulation. We developed a new high-throughput assay to measure RNA polymerase and TF binding free energies, requiring the construction and characterization of only a few constructs and data analysis (Figure 1A). We experimentally verified the assay on 6 TetR-homolog repressors and a CRISPR/dCas9 guide RNA. We found that our binding free energy measurements quantitatively explains why changing TF expression levels alters circuit function. Altogether, by combining these measurements with our biophysical model of translation (the RBS Calculator) as well as other measurements (Figure 1B), our model can account for changes in TF binding sites, TF expression levels, circuit copy number, host genome size, and host growth rate (Figure 1C). Model predictions correctly accounted for how these 8 factors control a promoter’s transcription rate (Figure 1D). Using the model, we developed a design framework for engineering multi-promoter genetic circuits that greatly reduces the number of degrees of freedom (8 factors per promoter) to a single dimensionless unit. We propose the Ptashne (Pt) number to encapsulate the 8 co-dependent factors that control transcriptional regulation into a single number. Therefore, a single number controls a promoter’s output rather than these 8 co-dependent factors, and designing a genetic circuit with N promoters requires specification of only N Pt numbers. We demonstrate how to design genetic circuits in Pt number space by constructing and characterizing 15 2-repressor OpAmp circuits that act as signal amplifiers when within an optimal Pt region. We experimentally show that OpAmp circuits using different TFs and TF expression levels will only amplify the dynamic range of input signals when their corresponding Pt numbers are within the optimal region. Thus, the use of the Pt number greatly simplifies the genetic circuit design, particularly important as circuits employ more TFs to perform increasingly complex functions.

Keywords: transcription factor, synthetic biology, genetic circuit, biophysical model, binding energy measurement

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Authors: Harriet Jane R. Caleja, Joel I. Ballesteros, Florian R. Del Mundo

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The dengue fever belongs to the world’s major cause of death, especially in the tropical areas. In the Philippines, the number of dengue cases during the first half of 2015 amounted to more than 50,000. In 2012, the total number of cases of dengue infection reached 132,046 of which 701 patients died. Dengue Nonstructural 1 virus (Dengue NS1 virus) is a recently discovered biomarker for the early detection of dengue virus. It is present in the serum of the dengue virus infected patients even during the earliest stages prior to the formation of dengue virus antibodies. A biosensor for the dengue detection using NS1 virus was developed for faster and accurate diagnostic tool. Biotinylated anti-dengue virus NS1 was used as the receptor for dengue virus NS1. Using the Diffractive Optics Technology (dotTM) technique, real time binding of the NS1 virus to the biotinylated anti-NS1 antibody is observed. The dot®-Avidin sensor recognizes the biotinylated anti-NS1 and this served as the capture molecule to the analyte, NS1 virus. The increase in the signal of the diffractive intensity signifies the binding of the capture and the analyte. The LOD was found to be 3.87 ng/mL while the LOQ is 12.9 ng/mL. The developed biosensor was also found to be specific for the NS1 virus.

Keywords: avidin-biotin, diffractive optics technology, immunosensor, NS1

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Authors: Syed Asif Hassan, Tabrej Khan

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Multiple sclerosis (MS) is a chronic demyelinating autoimmune disorder, of the central nervous system (CNS). In the present scenario, the current therapies either do not halt the progression of the disease or have side effects which limit the usage of current Disease Modifying Therapies (DMTs) for a longer period of time. Therefore, keeping the current treatment failure schema, we are focusing on screening novel analogues of the available DMTs that specifically bind and inhibit the Sphingosine1-phosphate receptor1 (S1PR1) thereby hindering the lymphocyte propagation toward CNS. The novel drug-like analogs molecule will decrease the frequency of relapses (recurrence of the symptoms associated with MS) with higher efficacy and lower toxicity to human system. In this study, an integrated approach involving ligand-based virtual screening protocol (Ultrafast Shape Recognition with CREDO Atom Types (USRCAT)) to identify the non-toxic drug like analogs of the approved DMTs were employed. The potency of the drug-like analog molecules to cross the Blood Brain Barrier (BBB) was estimated. Besides, molecular docking and simulation using Auto Dock Vina 1.1.2 and GOLD 3.01 were performed using the X-ray crystal structure of Mtb LprG protein to calculate the affinity and specificity of the analogs with the given LprG protein. The docking results were further confirmed by DSX (DrugScore eXtented), a robust program to evaluate the binding energy of ligands bound to the ligand binding domain of the Mtb LprG lipoprotein. The ligand, which has a higher hypothetical affinity, also has greater negative value. Further, the non-specific ligands were screened out using the structural filter proposed by Baell and Holloway. Based on the USRCAT, Lipinski’s values, toxicity and BBB analysis, the drug-like analogs of fingolimod and BG-12 showed that RTL and CHEMBL1771640, respectively are non-toxic and permeable to BBB. The successful docking and DSX analysis showed that RTL and CHEMBL1771640 could bind to the binding pocket of S1PR1 receptor protein of human with greater affinity than as compared to their parent compound (Fingolimod). In this study, we also found that all the drug-like analogs of the standard MS drugs passed the Bell and Holloway filter.

Keywords: antagonist, binding affinity, chemotherapeutics, drug-like, multiple sclerosis, S1PR1 receptor protein

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Authors: Mohammed Hakim Jafferali, Balaje Vijayaraghavan, Ricardo A. Figueroa, Ellinor Crafoord, Veronica J. Larsson, Einar Hallberg, Santhosh Gudise

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The nuclear envelope which surrounds the chromatin of eukaryotic cells contains more than a hundred transmembrane proteins. Mutations in some genes encoding nuclear envelope proteins give rise to human diseases including neurological disorders. The function of many nuclear envelope proteins is not well established. This is partly because nuclear envelope proteins and their interactions are difficult to study due to the inherent resistance to extraction of nuclear envelope proteins. We have developed a novel method called MCLIP, to identify interacting partners of nuclear envelope proteins in live cells. Using MCLIP, we found three new binding partners of the inner nuclear membrane protein Samp1: the intermediate filament protein Lamin B1, the LINC complex protein Sun1 and the G-protein Ran. Furthermore, using in vitro studies, we show that Samp1 binds both Emerin and Ran directly. We have also studied the interaction between Samp1 and Ran in detail. The results show that the Samp1 binds stronger to RanGTP than RanGDP. Samp1 is the first transmembrane protein known to bind Ran and it is tempting to speculate that Samp1 may provide local binding sites for RanGTP at membranes.

Keywords: MCLIP, nuclear envelope, ran, Samp1

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Authors: Ishan Tank, Ashmita Rupal, Sanjay Kumar Sharma

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Concrete, a widely used building material, has cement as its main constituent. An excessive amount of emissions are released into the atmosphere during the manufacture of cement, which is detrimental to the environment. To minimize this problem, innovative materials like geopolymer mortar (GPM) seem to be a better alternative. By using fly ash-based geopolymer instead of standard cement mortar as a binding ingredient, this concept has been successfully applied to the building sector. The advancement of this technology significantly reduces greenhouse gas emissions and helps in source reduction, thereby minimizing pollution of the environment. In order to produce mortar and use this geopolymer mortar in the development of building materials, the current investigation is properly introducing this geopolymeric material, namely fly ash, as a binder in place of standard cement. In the domain of the building material industry, fly ash based geopolymer is a new and optimistic replacement for traditional binding materials because it is both environmentally sustainable and has good durability. The setting behaviour and strength characteristics of fly ash, when mixed with alkaline activator solution with varied concentration of sodium hydroxide solution, alkaline liquids mix ratio, and curing temperature, must be investigated, though, in order to determine its suitability and application in comparison with the traditional binding material, by activating the raw materials, which include various elements of silica and alumina, finer material known as geopolymer mortar is created. The concentration of the activator solution has an impact on the compressive strength of the geopolymer concrete formed. An experimental examination of compressive strength after 7, 14, and 28 days of fly ash-based geopolymer concrete is presented in this paper. Furthermore, the process of geopolymerization largely relies on the curing temperature. So, the setting time of Geopolymer mortar due to different curing temperatures has been studied and discussed in this paper.

Keywords: geopolymer mortar, setting time, flyash, compressive strength, binder material

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Authors: Gülşah Çelik Gül, Figen Kurtuluş

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The first laboratory synthesis of hard materials such as diamond proceeded to attack of developing materials with high hardness to compete diamond. Boron rich solids are good candidates owing to their short interatomic bond lengths and strong covalent character. Boron containing hard material was synthesized by modified-microwave method under nitrogen atmosphere by using a fuel (glycine or urea), amorphous boron and/or boric acid in appropriate molar ratio. Characterizations were done by x-ray diffraction (XRD), fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy/energy dispersive analyze (SEM/EDS), thermo gravimetric/differantial thermal analysis (TG/DTA).

Keywords: boron containing materials, hard materials, microwave synthesis, powder X-ray diffraction

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Authors: M. Harmel, H. Khachai

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The first principles within the full potential linearized augmented plane wave (FP-LAPW) method were applied to study the structural and electronic properties of cubic perovskite-type compounds BiAlO3 and BiGaO3. The lattice constant, bulk modulus, its pressure derivative, band structure and density of states were obtained. The results show that BiGaO3 should exhibit higher hardness and stiffness than BiAlO3. The Al–O or Ga–O bonds are typically covalent with a strong hybridization as well as Bi–O ones that have a significant ionic character. Both materials are weakly ionic and exhibit wide and indirect band gaps, which are typical of insulators.

Keywords: DFT, Ab initio, electronic structure, Perovskite structure, ferroelectrics

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Authors: B. Gueridi, T. Chihi, M. Fatmi, A. Faci

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Some fundamental physical properties of BiGaO₃ were investigated under pressure and temperature effect using generalized gradient approximation and local density approximation approaches. The effect of orientation on Debye temperature and sound waves velocities were estimated from elastic constants. The value of the bulk modulus of BiGaO₃ is a sign of its high hardness because it is linked to an isotropic deformation. BiGaO₃ is a semiconductor and ductile material with covalent bonding (Ga–O), and the Bi-O bonding is ionic. The optical transitions were observed when electrons pass from the top of the valence band (O-2p) to the bottom of the conduction band (Ga-4p or Bi-6p). The thermodynamic parameters are determined in temperature and pressure ranging from 0 to 1800 K and 0 to 50 GPa.

Keywords: BiGaO₃ perovskite, optical absorption, first principle, band structure

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Authors: S. D. N. K. Bathige, G. I. Godahewa, Navaneethaiyer Umasuthan, Jehee Lee

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Kunitz-type serine protease inhibitors (KTIs) are identified in various organisms including animals, plants and microbes. These proteins shared single or multiple Kunitz inhibitory domains link together or associated with other types of domains. Characteristic Kunitz type domain composed of around 60 amino acid residues with six conserved cysteine residues to stabilize by three disulfide bridges. KTIs are involved in various physiological processes, such as ion channel blocking, blood coagulation, fibrinolysis and inflammation. In this study, two Kunitz-type domain containing protein was identified from rock bream database and designated as RbKunitz. The coding sequence of RbKunitz encoded for 507 amino acids with 56.2 kDa theoretical molecular mass and 5.7 isoelectric point (pI). There are several functional domains including MANEC superfamily domain, PKD superfamily domain, and LDLa domain were predicted in addition to the two characteristic Kunitz domain. Moreover, trypsin interaction sites were also identified in Kunitz domain. Homology analysis revealed that RbKunitz shared highest identity (77.6%) with Takifugu rubripes. Completely conserved 28 cysteine residues were recognized, when comparison of RbKunitz with other orthologs from different taxonomical groups. These structural evidences indicate the rigidity of RbKunitz folding structure to achieve the proper function. The phylogenetic tree was constructed using neighbor-joining method and exhibited that the KTIs from fish and non-fish has been evolved in separately. Rock bream was clustered with Takifugu rubripes. The SYBR Green qPCR was performed to quantify the RbKunitz transcripts in different tissues and challenged tissues. The mRNA transcripts of RbKunitz were detected in all tissues (muscle, spleen, head kidney, blood, heart, skin, liver, intestine, kidney and gills) analyzed and highest transcripts level was detected in gill tissues. Temporal transcription profile of RbKunitz in rock bream blood tissues was analyzed upon LPS (lipopolysaccharide), Poly I:C (Polyinosinic:polycytidylic acid) and Edwardsiella tarda challenge to understand the immune responses of this gene. Compare to the unchallenged control RbKunitz exhibited strong up-regulation at 24 h post injection (p.i.) after LPS and E. tarda injection. Comparatively robust expression of RbKunits was observed at 3 h p.i. upon Poly I:C challenge. Taken together all these data indicate that RbKunitz may involve into to immune responses upon pathogenic stress, in order to protect the rock bream.

Keywords: Kunitz-type, rock bream, immune response, serine protease inhibitor

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Authors: Virendra Nath, Vipin Kumar

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Background: TypeII Diabetes mellitus is a foremost health problem worldwide, predisposing to increased mortality and morbidity. Undesirable effects of the current medications have prompted the researcher to develop more potential drug(s) against the disease. The peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptors family and take part in a vital role in the regulation of metabolic equilibrium. They can induce or repress genes associated with adipogenesis, lipid, and glucose metabolism. Aims: Investigation of PPARα/γ agonistic hits were screened by hierarchical virtual screening followed by molecular dynamics simulation and knowledge-based structure-activity relation (SAR) analysis using approved PPAR α/γ dual agonist. Methods: The PPARα/γ agonistic activity of compounds was searched by using Maestro through structure-based virtual screening and molecular dynamics (MD) simulation application. Virtual screening of nuclear-receptor ligands was done, and the binding modes with protein-ligand interactions of newer entity(s) were investigated. Further, binding energy prediction, Stability studies using molecular dynamics (MD) simulation of PPARα and γ complex was performed with the most promising hit along with the structural comparative analysis of approved PPARα/γ agonists with screened hit was done for knowledge-based SAR. Results and Discussion: The silicone chip-based approach recognized the most capable nine hits and had better predictive binding energy as compared to the reference drug compound (Tesaglitazar). In this study, the key amino acid residues of binding pockets of both targets PPARα/γ were acknowledged as essential and were found to be associated in the key interactions with the most potential dual hit (ChemDiv-3269-0443). Stability studies using molecular dynamics (MD) simulation of PPARα and γ complex was performed with the most promising hit and found root mean square deviation (RMSD) stabile around 2Å and 2.1Å, respectively. Frequency distribution data also revealed that the key residues of both proteins showed maximum contacts with a potent hit during the MD simulation of 20 nanoseconds (ns). The knowledge-based SAR studies of PPARα/γ agonists were studied using 2D structures of approved drugs like aleglitazar, tesaglitazar, etc. for successful designing and synthesis of compounds PPARγ agonistic candidates with anti-hyperlipidimic potential.

Keywords: computational, diabetes, PPAR, simulation

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Authors: Hiral D. Trivedi, Dinesh S. Patel, Sachin P. Shukla

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We have immobilized alcohol dehydrogenase on k-carrageenan, which is a natural polysaccharide obtained from seaweeds by entrapment and on copolymer of acrylamide and 2-hydroxy ethylmethaacrylate by covalent coupling. We have optimized all the immobilization parameters and also carried the comparison studies of both. In case of copolymer of acrylamide and 2-hydroxy ethylmethaacrylate, we have activated both the amino and hydroxyl group individually and simultaneously using different activating agents and obtained some interesting results. We have found that covalently bound enzyme was found to be better under all tested conditions. The reaction on ethanol was carried out using these immobilized systems.

Keywords: alcohol dehydrogenase, acrylamide-co-2-hydroxy ethylmethaacrylate, ethanol, k-carrageenan

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Authors: H.Yari, M. Mohseni, Z. Ranjbar

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In this work a typical automotive clearcoat is modified with a combination of hyperbranched polymer (HBP) and polyhedral oligomeric silsesquioxane (POSS) nanostructures to simultaneously enhance the scratch resistance and healing ability of the resulting films. Micro-scratch and healing data revealed that these goals were achieved at high loadings of modifiers. Enhanced scratch resistance was attributed to the improved elastic recovery of the clearcoats in presence of modifiers. In addition, improved healing performance due to the partial replacement of covalent cross-links with physical ones resulted from the unique globular highly branched structure of HBP and POSS macromolecules.

Keywords: automotive clearcoat, POSS building blocks scratch resistance, self-healing

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Authors: Ketaki D. Belsare, Nicholas F. Polizzi, Lior Shtayer, William F. DeGrado

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Nature utilizes metalloproteins to perform chemical transformations with activities and selectivities that have long been the inspiration for design principles in synthetic and biological systems. The chemical reactivities of metalloproteins are directly linked to local environment effects produced by the protein matrix around the metal cofactor. A complete understanding of how the protein matrix provides these interactions would allow for the design of functional metalloproteins. The de novo computational design of proteins have been successfully used in design of active sites that bind metals like di-iron, zinc, copper containing cofactors; however, precisely designing active sites that can bind small molecule ligands (e.g., substrates) along with metal cofactors is still a challenge in the field. The de novo computational design of a functional metalloprotein that contains a purposefully designed substrate binding site would allow for precise control of chemical function and reactivity. Our research strategy seeks to elucidate the design features necessary to bind the cofactor protoporphyrin IX (hemin) in close proximity to a substrate binding pocket in a four helix bundle. First- and second-shell interactions are computationally designed to control orientation, electronic structure, and reaction pathway of the cofactor and substrate. The design began with a parameterized helical backbone that positioned a single histidine residue (as an axial ligand) to receive a second-shell H-bond from a Threonine on the neighboring helix. The metallo-cofactor, hemin was then manually placed in the binding site. A structural feature, pi-bulge was introduced to give substrate access to the protoporphyrin IX. These de novo metalloproteins are currently being tested for their activity towards hydroxylation and epoxidation. The de novo designed protein shows hydroxylation of aniline to 4-aminophenol. This study will help provide structural information of utmost importance in understanding de novo computational design variables impacting the functional activities of a protein.

Keywords: metalloproteins, protein design, de novo protein, biocatalysis

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Authors: Pei-Ying Lo, Jing-Hao Ciou, Kai-Cheng Yang, Jia-Huei Zheng, Shih-Hsiang Huang, Kuen-Chan Lee, Er-Chieh Cho

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As-produced CNTs are insoluble in all organic solvents and aqueous solutions have imposed limitations to the use of CNTs. Therefore, how to debundle carbon nanotubes and to modify them for further uses is an important issue. There are several methods for the dispersion of CNTs in water using covalent attachment of hydrophilic groups to the surface of tubes. These methods, however, alter the electronic structure of the nanotubes by disrupting the network of sp2 hybridized carbons. In order to keep the nanotubes’ intrinsic mechanical and electrical properties intact, non-covalent interactions are increasingly being explored as an alternative route for dispersion. Apart from conventional surfactants such as sodium dodecylsulfate (SDS) or sodium dodecylbenzenesulfonate (SDBS) which are highly effective in dispersing CNTs, biopolymers have received much attention as dispersing agents due to the anticipated biocompatibility of the dispersed CNTs. Also, The pyrenyl group is known to interact strongly with the basal plane of graphene via π-stacking. In this study, a highly re-dispersible biopolymer is reported for the synthesis of pyrene-modified poly-L-lysine (PBPL) and poly(D-Glu, D-Lys) (PGLP). To provide the evidence of the safety of the PBPL/CNT & PGLP/CNT materials we use in this study, H1299 and HCT116 cells were incubated with PBPL/CNT & PGLP/CNT materials for toxicity analysis, MTS assays. The results from MTS assays indicated that no significant cellular toxicity was shown in H1299 and HCT116 cells. Furthermore, the fluorescence marker fluorescein isothiocyanate (FITC) was added to PBPL & PGLP dispersions. From the fluorescent measurements showed that the chemical functionalisation of the PBPL/CNT & PGLP/CNT conjugates with the fluorescence marker were successful. The fluorescent PBPL/CNT & PGLP/CNT conjugates could find application in medical imaging. In the next step, the GFP gene is immobilized onto PBPL/CNT conjugates by introducing electrostatic interaction. GFP-transfected cells that emitted fluorescence were imaged and counted under a fluorescence microscope. Due to the unique biocompatibility of PBPL modified CNTs, the GFP gene could be transported into H1299 cells without using antibodies. The applicability of such soluble and chemically functionalised polypeptide/CNT conjugates in biomedicine is currently investigated. We expect that this polypeptide/CNT system will be a safe and multi-functional nanomedical delivery platform and contribute to future medical therapy.

Keywords: carbon nanotube, nanotoxicology, GFP transfection, polypeptide/CNT hybrids

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Authors: Hassan Youssef Mohamed

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In this paper, we show new wave equations, and by using the equations, we concluded that the strong force and the weak force are not fundamental, but they are quantum effects for electromagnetism. This result is different from the current scientific understanding about strong and weak interactions at all. So, we introduce three evidences for our theory. First, we prove the asymptotic freedom phenomenon in the strong force by using our model. Second, we derive the nuclear shell model as an approximation of our model. Third, we prove that the leptons do not participate in the strong interactions, and we prove the short ranges of weak and strong interactions. So, our model is consistent with the current understanding of physics. Finally, we introduce the electron-positron model as the basic ingredients for protons, neutrons, and all matters, so we can study all particles interactions and nuclear interaction as many-body problems of electrons and positrons. Also, we prove the violation of parity conservation in weak interaction as evidence of our theory in the weak interaction. Also, we calculate the average of the binding energy per nucleon.

Keywords: new wave equations, the strong force, the grand unification theory, hydrogen atom, weak force, the nuclear shell model, the asymptotic freedom, electron-positron model, the violation of parity conservation, the binding energy

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Authors: Kinga Mylkie, Pawel Nowak, Marta Z. Borowska

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The most important proteins in human serum responsible for drug binding are human serum albumin (HSA) and α1-acid glycoprotein (AGP). The AGP molecule is a glycoconjugate containing a single polypeptide chain composed of 183 amino acids (the core of the protein), and five glycan branched chains (sugar part) covalently linked by an N-glycosidic bond with aspartyl residues (Asp(N) -15, -38, -54, -75, - 85) of polypeptide chain. This protein plays an important role in binding alkaline drugs, a large group of drugs used in psychiatry, some acid drugs (e.g., coumarin anticoagulants), and neutral drugs (steroid hormones). The main goal of the research was to obtain magnetic nanoparticles coated with biopolymers in a chemically modified form, which will have highly reactive functional groups able to effectively immobilize the glycoprotein (acid α1-glycoprotein) without losing the ability to bind active substances. The first phase of the project involved the chemical modification of biopolymer starch. Modification of starch was carried out by methods of organic synthesis, leading to the preparation of a polymer enriched on its surface with aldehyde groups, which in the next step was coupled with 3-aminophenylboronic acid. Magnetite nanoparticles coated with starch were prepared by in situ co-precipitation and then oxidized with a 1 M sodium periodate solution to form a dialdehyde starch coating. Afterward, the reaction between the magnetite nanoparticles coated with dialdehyde starch and 3-aminophenylboronic acid was carried out. The obtained materials consist of a magnetite core surrounded by a layer of modified polymer, which contains on its surface dihydroxyboryl groups of boronic acids which are capable of binding glycoproteins. Magnetic nanoparticles obtained as carriers for plasma protein immobilization were fully characterized by ATR-FTIR, TEM, SEM, and DLS. The glycoprotein was immobilized on the obtained nanoparticles. The amount of mobilized protein was determined by the Bradford method.

Keywords: glycoproteins, immobilization, magnetic nanoparticles, polysaccharides

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Authors: Raja Chinnappan, Huda Alanazi, Shanmugam Easwaramoorthi, Tanveer Mir, Balamurugan Kanagasabai, Ahmed Yaqinuddin, Sandhanasamy Devanesan, Mohamad S. AlSalhi

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Serum albumin (SA) is a highly rich water-soluble protein in plasma. It is known to maintain the living organisms' health and help to maintain the proper liver function, kidney function, and plasma osmolality in the body. Low levels of serum albumin are an indication of liver failure and chronic hepatitis. Therefore, it is important to have a low-cost, accurate and rapid method. In this study, we designed a fluorescent probe, triphenylamine rhodanine-3-acetic acid (mRA), which triggers the fluorescence signal upon binding with serum albumin (SA). mRA is a bifunctional molecule with twisted intramolecular charge transfer (TICT)-induced emission characteristics. An aqueous solution of mRA has an insignificant fluorescence signal; however, when mRA binds to SA, it undergoes TICT and turns on the fluorescence emission. A SA dose-dependent fluorescence signal was performed, and the limit of detection was found to be less than ng/mL. The specific binding of SA was tested from the cross-reactivity study using similar structural or functional proteins.

Keywords: serum albumin, fluorescent sensing probe, liver diseases, twisted intramolecular charge transfer

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Authors: Lavinia Ruta, Ileana Farcasanu

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The yeast surface display (YSD) technique enables the expression of proteins on yeast cell surfaces, facilitating the identification and isolation of proteins with targeted binding properties, such as nanobodies. Nanobodies, derived from camelid species, are single-domain antibody fragments renowned for their high affinity and specificity towards target proteins, making them valuable in research and potentially in therapeutics. Their advantages include a compact size (~15 kDa), robust stability, and the ability to target challenging epitopes. The project endeavors to establish and validate a platform for producing Green Fluorescent Protein (GFP) and mCherry nanobodies using the yeast surface display method. mCherry, a prevalent red fluorescent protein sourced from coral species, is commonly utilized as a genetic marker in biological studies due to its vibrant red fluorescence. The GFP-nanobody, a single variable domain of heavy-chain antibodies (VHH), exhibits specific binding to GFP, offering a potent means for isolating and engineering fluorescent protein fusions across various biological research domains. Both GFP and mCherry nanobodies find specific utility in cellular imaging and protein analysis applications.

Keywords: YSD, nanobodies, GFP, Saccharomyces cerevisiae

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Authors: Mona Alharbi

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Melioidosis has emerged as a lethal disease. Unfortunately, the molecular mechanisms of virulence and pathogenicity of Burkholderia pseudomallei remain unknown. However, proteomics research has selected putative targets in B. pseudomallei that might play roles in the B. pseudomallei virulence. BPSL 2418 putative protein has been predicted as a free methionine sulfoxide reductase and interestingly there is a link between the level of the methionine sulfoxide in pathogen tissues and its virulence. Therefore in this work, we describe the cloning expression, purification, and crystallization of BPSL 2418 and the solution of its 3D structure using X-ray crystallography. Also, we aimed to identify the substrate binding and reduced forms of the enzyme to understand the role of BPSL 2418. The gene encoding BPSL2418 from B. pseudomallei was amplified by PCR and reclone in pETBlue-1 vector and transformed into E. coli Tuner DE3 pLacI. BPSL2418 was overexpressed using E. coli Tuner DE3 pLacI and induced by 300μM IPTG for 4h at 37°C. Then BPS2418 purified to better than 95% purity. The pure BPSL2418 was crystallized with PEG 4000 and PEG 6000 as precipitants in several conditions. Diffraction data were collected to 1.2Å resolution. The crystals belonged to space group P2 21 21 with unit-cell parameters a = 42.24Å, b = 53.48Å, c = 60.54Å, α=γ=β= 90Å. The BPSL2418 binding MES was solved by molecular replacement with the known structure 3ksf using PHASER program. The structure is composed of six antiparallel β-strands and four α-helices and two loops. BPSL2418 shows high homology with the GAF domain fRMsrs enzymes which suggest that BPSL2418 might act as methionine sulfoxide reductase. The amino acids alignment between the fRmsrs including BPSL 2418 shows that the three cysteines that thought to catalyze the reduction are fully conserved. BPSL 2418 contains the three conserved cysteines (Cys⁷⁵, Cys⁸⁵ and Cys¹⁰⁹). The active site contains the six antiparallel β-strands and two loops where the disulfide bond formed between Cys⁷⁵ and Cys¹⁰⁹. X-ray structure of free methionine sulfoxide binding and native forms of BPSL2418 were solved to increase the understanding of the BPSL2418 catalytic mechanism.

Keywords: X-Ray Crystallography, BPSL2418, Burkholderia pseudomallei, Melioidosis

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Authors: Saurabh B. Ganorkar, Atul A. Shirkhedkar

Abstract:

The need for investigations protecting against toxic impurities though seems to be a primary requirement; the impurities which may prove non - toxic can be explored for their therapeutic potential if any to assist advanced drug discovery. The essential role of pharmaceutical analysis can thus be extended effectively to achieve it. The present study successfully achieved these objectives with characterization of major degradation products as impurities for Zileuton which has been used for to treat asthma since years. The forced degradation studies were performed to identify the potential degradation products using Ultra-fine Liquid-chromatography. Liquid-chromatography-Mass spectrometry (Time of Flight) and Proton Nuclear Magnetic Resonance Studies were utilized effectively to characterize the drug along with five major oxidative and hydrolytic degradation products (DP’s). The mass fragments were identified for Zileuton and path for the degradation was investigated. The characterized DP’s were subjected to In-Silico studies as XP Molecular Docking to compare the gain or loss in binding affinity with 5-Lipooxygenase enzyme. One of the impurity of was found to have the binding affinity more than the drug itself indicating for its potential to be more bioactive as better Antiasthmatic. The close structural resemblance has the ability to potentiate or reduce bioactivity and or toxicity. The chances of being active biologically at other sites cannot be denied and the same is achieved to some extent by predictions for probability of being active with Prediction of Activity Spectrum for Substances (PASS) The impurities found to be bio-active as Antineoplastic, Antiallergic, and inhibitors of Complement Factor D. The toxicological abilities as Ames-Mutagenicity, Carcinogenicity, Developmental Toxicity and Skin Irritancy were evaluated using Toxicity Prediction by Komputer Assisted Technology (TOPKAT). Two of the impurities were found to be non-toxic as compared to original drug Zileuton. As the drugs are purposed and repurposed effectively the impurities can also be; as they can have more binding affinity; less toxicity and better ability to be bio-active at other biological targets.

Keywords: UFLC, LC-MS-TOF, NMR, Zileuton, impurities, toxicity, bio-activity

Procedia PDF Downloads 195

Authors: Tanushree Jaitly, Shailendra Gupta, Leila Taher, Gerold Schuler, Julio Vera

Abstract:

Genomics-based personalized medicine is a promising approach to fight aggressive tumors based on patient's specific tumor mutation and expression profiles. A remarkable case is, dendritic cell-based immunotherapy, in which tumor epitopes targeting patient's specific mutations are used to design a vaccine that helps in stimulating cytotoxic T cell mediated anticancer immunity. Here we present a computational pipeline for epitope-based personalized cancer vaccines using patient-specific haplotype and cancer mutation profiles. In the workflow proposed, we analyze Whole Exome Sequencing and RNA Sequencing patient data to detect patient-specific mutations and their expression level. Epitopes including the tumor mutations are computationally predicted using patient's haplotype and filtered based on their expression level, binding affinity, and immunogenicity. We calculate binding energy for each filtered major histocompatibility complex (MHC)-peptide complex using docking studies, and use this feature to select good epitope candidates further.

Keywords: cancer immunotherapy, epitope prediction, NGS data, personalized medicine

Procedia PDF Downloads 253

Authors: Hamid Hossein Khezri, Afsaneh Javdani-Mallak

Abstract:

Fast inhibitory neurotransmission in the mammalian nervous system is largely mediated by GABAA receptors, chloride-selective members of the superfamily of pentameric Cys-loop receptors. Cannabidiol (CBD) is one of the members of cannabinoid compounds found in cannabis. CBD and Cannabinol (CBN), as the other extract of plant Cannabis were able to reduce myofascial pain in rats with immunosuppressive and anti-inflammatory activities. In this study, we accomplished protein-protein BLAST, and the sequence was found to be for Gamma-aminobutyric acid receptor subunit alpha-1 (GBRA1) chain A and its 3D structure was subsequently downloaded from Protein Data Bank. The structures of the ligands, cannabinol, and cannabidiol, were obtained from PubChem. After the necessary process of the obtained files, AutoDock Vina was used to perform molecular docking. Docking between the ligands and GBRA1 chain A revealed that cannabinol has a higher affinity to GBRA1 (binding energy = -7.5 kcal/mol) compared to cannabidiol (binding energy = -6.5 kcal/mol). Furthermore, cannabinol seems to be able to interact with 10 residues of the protein, out of which 3 are in the neurotransmitter-gated ion-channel transmembrane domain of GBRA1, whereas cannabidiol interacts with two other residues. Although the results of this project do not indicate the activating /or inhibitory capability of the studied compounds, it suggests that cannabinol can act as a relatively strong ligand for GBRA1.

Keywords: protein-ligand docking, cannabinol, cannabidiol, GBRA1

Procedia PDF Downloads 110

Authors: Mariam Mojally, Randa Abdou, Wisal Bokhari, Sultan Sab, Mohammed Dawoud, Amjad Albohy

Abstract:

Secondary metabolites produced by endophytes are an excellent source of biologically active compounds. In our current study, we evaluated terezine E and 14-hydroxyterezine D for binding to the active site of histone deacetylase (PDB ID: 4CBT) and matrix metalloproteinase 9 (PDB ID: 4H3X) by molecular docking using AutoDock Vina software after having tested their cytotoxic activities on three cell lines (human ductal breast epithelial tumor cells (T47D)-HCC1937), human hepatocarcinoma cell line (HepG2)-HB8065), and human colorectal carcinoma cells (HCT-116)-TCP1006, purchased from ATCC, USA)). Additionally, their antimicrobial activities were investigated, and their minimum inhibitory concentration (MIC) values were determined against P. notatum and S. aureus by the broth microdilution method. Higher cytotoxicity was observed for terezine E against all tested cell lines compared to 14-hydroxyterezine D. Molecular docking results supported the high cytotoxicity of terezine E and showed higher binding affinity with 4CBT with an energy score of 9 kcal/mol. Terezine E showed higher antibacterial and antifungal activities than 14-hydroxyrerezine D: MIC values were 15.45 and 21.73 mg/mL against S. aureus and 8.61 and 11.54 mg/mL against P. notatum, respectively

Keywords: Terezine E, 14-Hydroxyterezine D, cytotoxicity, antimicrobial activity, molecular docking

Procedia PDF Downloads 73

Authors: Abdul Matin, Salik Nawaz, Suk-Yul Jung

Abstract:

Balamuthia mandrillaris is well known to cause fatal Balamuthia amoebic encephalitis (BAE). Amoebic transmission into the central nervous system (CNS), haematogenous spread is thought to be the prime step, followed by blood-brain barrier (BBB) dissemination. Macrophages are considered to be the foremost line of defense and present in excessive numbers during amoebic infections. The aim of the present investigation was to evaluate the effects of macrophages alone or primed with cytokines on the biological characteristics of Balamuthia in vitro. Using human brain microvascular endothelial cells (HBMEC), which constitutes the BBB, we have shown that Balamuthia demonstrated > 90% binding and > 70% cytotoxicity to host cells. However, macrophages further increased amoebic binding and Balamuthia-mediated cell cytotoxicity. Furthermore, macrophages exhibited no amoebicidal effect against Balamuthia. Zymography assay demonstrated that macrophages exhibited no inhibitory effect on proteolytic activity of Balamuthia. Overall, to our best knowledge, we have shown for the first time macrophages has no inhibitory effects on the biological properties of Balamuthia in vitro. This also strengthened the concept that how and why Balamuthia can cause infections in both immuno-competent and immuno-compromised individuals.

Keywords: Balamuthia mandrillaris, macrophages, cytokines, human brain microvascular endothelial cells, Balamuthia amoebic encephalitis

Procedia PDF Downloads 156

Authors: Aidan Battison, Neliswa Mama

Abstract:

Environmental pollution by ionic species has been identified as one of the biggest challenges to the sustainable development of communities. The widespread use of organic and inorganic chemical products and the release of toxic chemical species from industrial waste have resulted in a need for advanced monitoring technologies for environment protection, remediation and restoration. Some of the disadvantages of conventional sensing methods include expensive instrumentation, well-controlled experimental conditions, time-consuming procedures and sometimes complicated sample preparation. On the contrary, the development of fluorescent chemosensors for biological and environmental detection of metal ions has attracted a great deal of attention due to their simplicity, high selectivity, eidetic recognition, rapid response and real-life monitoring. Coumarin derivatives S1 and S2 (Scheme 1) containing 1,2,3-triazole moieties at position -3- have been designed and synthesized from azide and alkyne derivatives by CuAAC “click” reactions for the detection of metal ions. These compounds displayed a strong preference for Fe3+ ions with complexation resulting in fluorescent quenching through photo-induced electron transfer (PET) by the “sphere of action” static quenching model. The tested metal ions included Cd2+, Pb2+, Ag+, Na+, Ca2+, Cr3+, Fe3+, Al3+, Cd2+, Ba2+, Cu2+, Co2+, Hg2+, Zn2+ and Ni2+. The detection limits of S1 and S2 were determined to be 4.1 and 5.1 uM, respectively. Compound S1 displayed the greatest selectivity towards Fe3+ in the presence of competing for metal cations. S1 could also be used for the detection of Fe3+ in a mixture of CH3CN/H¬2¬O. Binding stoichiometry between S1 and Fe3+ was determined by using both Jobs-plot and Benesi-Hildebrand analysis. The binding was shown to occur in a 1:1 ratio between the sensor and a metal cation. Reversibility studies between S1 and Fe3+ were conducted by using EDTA. The binding site of Fe3+ to S1 was determined by using 13 C NMR and Molecular Modelling studies. Complexation was suggested to occur between the lone-pair of electrons from the coumarin-carbonyl and the triazole-carbon double bond.

Keywords: chemosensor, "click" chemistry, coumarin, fluorescence, static quenching, triazole

Procedia PDF Downloads 163

Authors: Hamid Hossein Khezri, Afsaneh Javdani-Mallak

Abstract:

Fast inhibitory neurotransmission in the mammalian nervous system is largely mediated by GABAA receptors, chloride-selective members of the superfamily of pentameric Cys-loop receptors. Cannabidiol (CBD) is one of the members of cannabinoid compounds found in cannabis. CBD and Cannabinol (CBN), as the other extract of plant Cannabis, were able to reduce myofascial pain in rats with immunosuppressive and anti-inflammatory activities. In this study, we accomplished protein-protein BLAST and the sequence was found to be for Gamma-aminobutyric acid receptor subunit alpha-1 (GBRA1) chain A and its 3D structure was subsequently downloaded from Protein Data Bank. The structures of the ligands cannabinol and cannabidiol were obtained from PubChem. After a necessary process of the obtained files, AutoDock Vina was used to performing molecular docking. Docking between the ligands and GBRA1 chain A revealed that cannabinol has a higher affinity to GBRA1 (binding energy = -7.5 kcal/mol) compared to cannabidiol (binding energy = -6.5 kcal/mol). Furthermore, cannabinol seems to be able to interact with 10 residues of the protein, out of which 3 are in the neurotransmitter-gated ion-channel transmembrane domain of GBRA1, whereas cannabidiol interacts with two other residues. Although the results of this project do not indicate the activating /or inhibitory capability of the studied compounds, it suggests that cannabinol can act as a relatively strong ligand for GBRA1.

Keywords: protein-ligand docking, cannabinol, cannabidiol, GBRA1

Procedia PDF Downloads 118

Authors: M. Fatmi, B. Gueridi, Z. Zerrougui

Abstract:

Some physical properties of hexahalometallate K₂XBr₆(X=Se, Pt) were computed in the zinc blend structure using generalized gradient approximation. The cell constant of K₂SeBr₆ and K₂PtBr₆ is consistent with the experiment value quoted in the literature, where the error is 0.95 % and 1 %. K₂SeBr₆ and K₂PtBr₆ present covalent bonding, high anisotropy and are ductile. The elastic constants of K₂SeBr₆ and K₂PtBr₆ are significantly smaller due to their larger reticular distances and lower Colombian forces, and then they are soft and damage tolerant. The interatomic separation is greater in K₂SeBr₆ than in K₂PtBr₆; hence the Colombian interaction in K₂PtBr₆ is greater than that of K2SeBr₆. The internal coordinate of the Br atom in K₂PtBr₆ is lower than that of the same atom in K2SeBr₆, and this can be explained by the fact that it is inversely proportional to the atom radius of Se and Pt. There are two major plasmonic processes, with intensities of 3.7 and 1.35, located around 53.5 nm and 72.8 nm for K₂SeBr₆ and K₂PtBr₆.

Keywords: hexahalometallate, band structure, morphology, absorption, band gap, absorber

Procedia PDF Downloads 93

Authors: Azhar Jabareen, Mahmoud Huleihel

Abstract:

BRCA-1 is a multifunctional tumor suppressor, whose expression is activated by the estrogen (E2)-liganded ERα receptor. The activated ERα is a transcriptional factor which activates various genes either by direct binding to the DNA at E2-responsive elements (EREs) and indirectly associated with a range of alternative non-ERE elements. Interference with BRCA-1 expression and/or functions leads to high risk of breast or/and ovarian cancer. Our lab investigated the involvement of Human T-cell leukemia Virus Type 1 (HTLV-1) in breast cancer, since HTLV-1 Tax was found to strongly inhibit BRCA-1 expression. In addition, long exposure of 12-O-tetradecanoylphorbol-13-acetate (TPA), which is one of the stress-inducing agents activated the HTLV-1 promoter. So here the involvement of TPA in breast cancer had been examined by testing the effect of TPA on BRCA-1 and ERE expression. The results showed that TPA activated both BRCA-1 and ERE expression. In the 12 hours TPA activated the tow promoters more than others time, and after 24 hours the level of the tow promoters was decreased. Tax inhibited BRCA-1 expression but did not succeed to inhibit the effect of TPA. Then the activation of the two promoters was not through ERα pathway because TPA had no effect on ERα binding to the two promoters of the BRCA-1 and ERE. Also, the activation was not via nuclear factor kappa B (NF-κB) pathway because when the inhibitory of NF-κB had been added to the TPA, it still activated the tow promoters. However, it seems that 53BP1 may be involved in TPA activation of these promoters because ectopic high expression of 53BP1 significantly reduced the TPA activity. In addition, in the presence of Bisindolylmaleimide-I (BI)- the inhibitor of Protein Kinase C (PKC)- there was no activation for the two promoters, so the PKC is agonized BRCA-1 and ERE activation.

Keywords: BRCA-1, ERE, HTLV-1, TPA

Procedia PDF Downloads 248

Authors: William Thomas Worster

Abstract:

This paper will highlight the failure of international adjudication to prevent a state from evading an order of provisional measures by simply issuing a diplomatic assurance to the court. This practice changes the positions of the litigants as equals before a court, prevents the court from inquiring into the reliability of the political pledge as it would with assurances from a state to an individual, and diminishes the court’s ability to control its own proceedings in the face of concerns over sovereignty. Both the European Court of Human Rights (ECtHR) and International Court of Justice (ICJ) will entertain these kinds of unilateral pledges, but they consider them differently when the declaration is made between states or between a state and an individual, and when made directly to the court. In short, diplomatic assurances issued between states or to individuals are usually considered not to be legally binding and are essentially questions of fact, but unilateral assurances issued directly to an international court are questions of law, and usually legally binding. At the same time, orders for provisional measures are now understood also to be legally binding, yet international courts will sometimes permit a state to substitute an assurance in place of an order for provisional measures. This emerging practice has brought the nature of a state as a sovereign capable of creating legal obligations into the forum of adjudication where the parties should have equality of arms and permitted states to create legal obligations that escape inquiry into the reliability of the outcome. While most recent practice has occurred at the ICJ in state-to-state litigation, there is some practice potentially extending the practice to human rights courts. Especially where the litigants are factually unequal – a state and an individual – this practice is problematic since states could more easily overcome factual failings in their pledges and evade the control of the court. Consider, for example, the potential for evading non-refoulement obligations by extending the current diplomatic assurances practice from the state-to-state context to the state-to-court context. The dual nature of assurances, as both legal and factual instruments, should be considered as addressed to distinct questions, each with its own considerations, and that we need to be more demanding about their precise legal and factual effects.

Keywords: unilateral, diplomacy, assurances, undertakings, provisional measures, interim measures

Procedia PDF Downloads 170