Search results for: nucleic acid binding

Authors: Zelal Polat, Şebnem Harsa, Semra Ülkü

Abstract:

Lactic acid exists in nature optically in two forms, L(+), D(-)-lactic acid, and has been used in food, leather, textile, pharmaceutical and cosmetic industries. Moreover, L(+)-lactic acid constitutes the raw material for the production of poly-L-lactic acid which is used in biomedical applications. Microbially produced lactic acid was aimed to be recovered from the fermentation media efficiently and economically. Among the various downstream operations, ion exchange chromatography is highly selective and yields a low cost product recovery within a short period of time. In this project, Lactobacillus casei NRRL B-441 was used for the production of L(+)-lactic acid from whey by fermentation at pH 5.5 and 37°C that took 12 hours. The product concentration was 50 g/l with 100% L(+)-lactic acid content. Next, the suitable resin was selected due to its high sorption capacity with rapid equilibrium behavior. Dowex marathon WBA, weakly basic anion exchanger in OH form reached the equilibrium in 15 minutes. The batch adsorption experiments were done approximately at pH 7.0 and 30°C and sampling was continued for 20 hours. Furthermore, the effect of temperature and pH was investigated and their influence was found to be unimportant. All the adsorption/desorption experiments were applied to both model lactic acid and biomass free fermentation broth. The ion exchange equilibria of lactic acid and L(+)-lactic acid in fermentation broth on Dowex marathon WBA was explained by Langmuir isotherm. The maximum exchange capacity (qm) for model lactic acid was 0.25 g La/g wet resin and for fermentation broth 0.04 g La/g wet resin. The equilibrium loading and exchange efficiency of L(+)-lactic acid in fermentation broth were reduced as a result of competition by other ionic species. The competing ions inhibit the binding of L(+)-lactic acid to the free sites of ion exchanger. Moreover, column operations were applied to recover adsorbed lactic acid from the ion exchanger. 2.0 M HCl was the suitable eluting agent to recover the bound L(+)-lactic acid with a flowrate of 1 ml/min at ambient temperature. About 95% of bound L(+)-lactic acid was recovered from Dowex marathon WBA. The equilibrium was reached within 15 minutes. The aim of this project was to investigate the purification of L(+)-lactic acid with ion exchange method from fermentation broth. The additional goals were to investigate the end product purity, to obtain new data on the adsorption/desorption behaviours of lactic acid and applicability of the system in industrial usage.

Keywords: fermentation, ion exchange, lactic acid, purification, whey

Procedia PDF Downloads 481

Authors: Fatih Ozaydin

Abstract:

Quantum Fisher information (QFI) is a multipartite entanglement witness and recently it has been studied extensively with separability and entanglement in the focus. On the other hand, bound entanglement is a special phenomena observed in mixed entangled states. In this work, we study the QFI of W states under a four-dimensional entanglement binding channel. Starting with initally pure W states of several qubits, we find how the QFI decreases as two qubits of the W state is subject to entanglement binding. We also show that as the size of the W state increases, the effect of entanglement binding is decreased.

Keywords: Quantum Fisher information, W states, bound entanglement, entanglement binding

Procedia PDF Downloads 450

Authors: Wei-Ting Kuo, Feng-Huei Lin

Abstract:

Epidermal growth factor receptor (EGFR) is often constitutively stimulated in cancer owing to the binding of ligands such as epidermal growth factor (EGF), so it is necessary to investigate the interaction between EGFR and its targeting biomolecules which were over ligands binding. This study would focus on the binding affinity and adhesion force of two targeting products anti-EGFR monoclonal antibody (mAb) and peptide A to EGFR comparing with EGF. Surface plasmon resonance (SPR) was used to obtain the equilibrium dissociation constant to evaluate the binding affinity. Atomic force microscopy (AFM) was performed to detect adhesion force. The result showed that binding affinity of mAb to EGFR was higher than that of EGF to EGFR, and peptide A to EGFR was lowest. The adhesion force between EGFR and mAb that was higher than EGF and peptide A to EGFR was lowest. From the studies, we could conclude that mAb had better adhesion force and binding affinity to EGFR than that of EGF and peptide A. SPR and AFM could confirm the interaction between receptor and targeting ligand easily and carefully. It provide a platform to screen ligands for receptor targeting and drug delivery.

Keywords: adhesion force, binding affinity, epidermal growth factor receptor, target molecule

Procedia PDF Downloads 408

Authors: Semuel Unwakoly, Reinner Puppela, Maresthy Rumalean, Healthy Kainama

Abstract:

Fish is a kind of food that contains many nutritions, one of those is the long chain of unsaturated fatty acids as omega-3 and omega-6 fatty acids and essential amino acid in enough amount for the necessity of our body. Like pelagic fish that found in the sea of Maluku. This research was done to identify fatty acids and amino acids composition in Moonfish (M. maculata) using transesterification reaction steps and Gas Chromatograph-Mass Spectrophotometer (GC-MS) and High-Performance Liquid Chromatography (HPLC). The result showed that fatty acids composition in Moonfish (M. maculata) contained tridecanoic acid (2.84%); palmitoleic acid (2.65%); palmitic acid (35.24%); oleic acid (6.2%); stearic acid (14.20%); and 5,8,11,14-eicosatetraenoic acid (1.29%) and 12 amino acids composition that consist of 7 essential amino acids, were leucine, isoleucine, valine, phenylalanine, methionine, lysine, and histidine, and also 5 non-essential amino acid, were tyrosine, glycine, alanine, glutamic acid, and arginine.Thus, these fishes can be used by the people to complete the necessity of essential fatty acid and amino acid.

Keywords: Moonfish (M. maculata), fatty acid, amino acid, GC-MS, HPLC

Procedia PDF Downloads 221

Authors: Dileep Kumar, Janhavi Ramchandra Rao Kumar, Rao

Abstract:

COVID-19 has ravaged the globe, and it is spreading its Spectre day by day. In the absence of established drugs, this disease has created havoc. Some of the infected persons are symptomatic or asymptomatic. The respiratory system, cardiac system, digestive system, etc. in human beings are affected by this virus. In our present investigation, we have undertaken a study of the Indian Ayurvedic herb, Ocimum sanctum against SARS-CoV-2 using molecular docking and dynamics studies. The docking analysis was performed on the Glide module of Schrödinger suite on two different proteins from SARS-CoV-2 viz. NSP15 Endoribonuclease and spike receptor-binding domain. MM-GBSA based binding free energy calculations also suggest the most favorable binding affinities of carvacrol, β elemene, and β caryophyllene with binding energies of −61.61, 58.23, and −54.19 Kcal/mol respectively with spike receptor-binding domain and NSP15 Endoribonuclease. It rekindles our hope for the design and development of new drug candidates for the treatment of COVID19.

Keywords: molecular docking, COVID-19, ocimum sanctum, binding energy

Procedia PDF Downloads 154

Authors: Amit Chaudhary, B. S. Yadav, P. K. Maurya, A. M., S. Srivastava, S. Singh, A. Mani

Abstract:

Cold shock protein A (CspA) is major cold inducible protein present in Escherichia coli. The protein is involved in stabilizing secondary structure of RNA by working as chaperone during cold temperature. Two RNA binding motifs play key role in the stabilizing activity. This study aimed to investigate implications of low temperature on structure and RNA binding activity of E. coli CspA. Molecular dynamics simulations were performed to compare the stability of the protein at 37°C and 10 °C. The protein was mutated at RNA binding motifs and docked with RNA to assess the stability of both complexes. Results suggest that CspA as well as CspA-RNA complex is more stable at low temperature. It was also confirmed that RNP1 and RNP2 play key role in RNA binding.

Keywords: CspA, homology modelling, mutation, molecular dynamics simulation

Procedia PDF Downloads 348

Authors: A. Jamsheera, F. Arjmand, D. K. Mohapatra

Abstract:

Small molecules binding to specific sites along DNA molecule are considered as potential chemotherapeutic agents. Their role as mediators of key biological functions and their unique intrinsic properties make them particularly attractive therapeutic agents. Keeping in view, novel dipeptide complexes Cu(II)-Val-Pro (1), Zn(II)-Val-Pro (2), Cu(II)-Ala-Pro (3) and Zn(II)-Ala-Pro (4) were synthesized and thoroughly characterized using different spectroscopic techniques including elemental analyses, IR, NMR, ESI–MS and molar conductance measurements. The solution stability study carried out by UV–vis absorption titration over a broad range of pH proved the stability of the complexes in solution. In vitro DNA binding studies of complexes 1–4 carried out employing absorption, fluorescence, circular dichroism and viscometric studies revealed the binding of complexes to DNA via groove binding. UV–vis titrations of 1–4 with mononucleotides of interest viz., 5´-GMP and 5´-TMP were also carried out. The DNA cleavage activity of the complexes 1 and 2 were ascertained by gel electrophoresis assay which revealed that the complexes are good DNA cleavage agents and the cleavage mechanism involved a hydrolytic pathway. Furthermore, in vitro antitumor activity of complex 1 was screened against human cancer cell lines of different histological origin.

Keywords: dipeptide Cu(II) and Zn(II) complexes, DNA binding profile, pBR322 DNA cleavage, in vitro anticancer activity

Procedia PDF Downloads 321

Authors: Meiling Yu, Nadia Rouatbi, Khuloud T. Al-Jamal

Abstract:

Background: Treatment of neurological disorders with modern medical and surgical approaches remains difficult. Gene therapy, allowing the delivery of genetic materials that encodes potential therapeutic molecules, represents an attractive option. The treatment of brain diseases with gene therapy requires the gene-editing tool to be delivered efficiently to the central nervous system. In this study, we explored the efficiency of different delivery routes, namely intravenous (i.v.), intra-cranial (i.c.), and intra-nasal (i.n.), to deliver stable nucleic acid-lipid particles (SNALPs) containing gene-editing tools namely Cas9 mRNA and sgRNA encoding for GFP as a reporter protein. We hypothesise that SNALPs can reach the brain and perform gene-editing to different extents depending on the administration route. Intranasal administration (i.n.) offers an attractive and non-invasive way to access the brain circumventing the blood–brain barrier. Successful delivery of gene-editing tools to the brain offers a great opportunity for therapeutic target validation and nucleic acids therapeutics delivery to improve treatment options for a range of neurodegenerative diseases. In this study, we utilised Rosa26-Cas9 knock-in mice, expressing GFP, to study brain distribution and gene-editing efficiency of SNALPs after i.v.; i.c. and i.n. routes of administration. Methods: Single guide RNA (sgRNA) against GFP has been designed and validated by in vitro nuclease assay. SNALPs were formulated and characterised using dynamic light scattering. The encapsulation efficiency of nucleic acids (NA) was measured by RiboGreen™ assay. SNALPs were incubated in serum to assess their ability to protect NA from degradation. Rosa26-Cas9 knock-in mice were i.v., i.n., or i.c. administered with SNALPs to test in vivo gene-editing (GFP knockout) efficiency. SNALPs were given as three doses of 0.64 mg/kg sgGFP following i.v. and i.n. or a single dose of 0.25 mg/kg sgGFP following i.c.. knockout efficiency was assessed after seven days using Sanger Sequencing and Inference of CRISPR Edits (ICE) analysis. In vivo, the biodistribution of DiR labelled SNALPs (SNALPs-DiR) was assessed at 24h post-administration using IVIS Lumina Series III. Results: Serum-stable SNALPs produced were 130-140 nm in diameter with ~90% nucleic acid loading efficiency. SNALPs could reach and stay in the brain for up to 24h following i.v.; i.n. and i.c. administration. Decreasing GFP expression (around 50% after i.v. and i.c. and 20% following i.n.) was confirmed by optical imaging. Despite the small number of mice used, ICE analysis confirmed GFP knockout in mice brains. Additional studies are currently taking place to increase mice numbers. Conclusion: Results confirmed efficient gene knockout achieved by SNALPs in Rosa26-Cas9 knock-in mice expressing GFP following different routes of administrations in the following order i.v.= i.c.> i.n. Each of the administration routes has its pros and cons. The next stages of the project involve assessing gene-editing efficiency in wild-type mice and replacing GFP as a model target with therapeutic target genes implicated in Motor Neuron Disease pathology.

Keywords: CRISPR, nanoparticles, brain diseases, administration routes

Procedia PDF Downloads 66

Authors: Sinethemba Yakobi, Lindiwe Zuma, Ofentse Pooe

Abstract:

Gonococcal infections present a notable public health issue, and the major approach for treatment involves using β-lactam antibiotics that specifically target penicillin-binding protein 2 (PBP2) in Neisseria gonorrhoeae. This study examines the influence of flavonoids, namely rutin, on the structural changes of PBP2 in both penicillin-resistant (FA6140) and penicillin-susceptible (FA19) strains. The research clarifies the structural effects of particular mutations, such as inserting an aspartate residue at position 345 (Asp-345a) in the PBP2 protein. The strain FA6140, which is resistant to penicillin, shows specific changes that lead to a decrease in penicillin binding. These mutations, namely P551S and F504L, significantly impact the pace at which acylation occurs and the stability of the strain under high temperatures. Molecular docking analyses investigate the antibacterial activities of rutin and other phytocompounds, emphasizing its exceptional binding affinity and potential as an inhibitor of PBP2. Quercetin and protocatechuic acid have encouraging antibacterial effectiveness, with quercetin displaying characteristics similar to those of drugs. Molecular dynamics simulations offer a detailed comprehension of the interactions between flavonoids and PBP2, highlighting rutin's exceptional antioxidant effects and strong affinity for the substrate binding site. The study's wider ramifications pertain to the pressing requirement for antiviral treatments in the context of the ongoing COVID-19 epidemic. Flavonoids have a strong affinity for binding to PBP2, indicating their potential as inhibitors to impair cell wall formation in N. gonorrhoeae. Ultimately, this study provides extensive knowledge on the interactions between proteins and ligands, the dynamics of the structure, and the ability of flavonoids to combat penicillin-resistant N. gonorrhoeae bacteria. The verified simulation outcomes establish a basis for creating potent inhibitors and medicinal therapies to combat infectious illnesses.

Keywords: phytochemicals, penicillin-binding protein 2, gonococcal infection, ligand-protein interaction, binding energy, neisseria gonorrhoeae FA19, neisseria gonorrhoeae FA6140, flavonoids

Procedia PDF Downloads 29

Authors: Abayomi T. Ogunjimi, Gbenga Alebiowu

Abstract:

Mechanical properties of paracetamol tablets containing Neem (Azadirachta indica) gum were compared with standard Acacia gum BP as binder. Two dimensionless binding quantities BEN and BEC were used in assessing the influence of binder type on two mechanical properties, Tensile Strength (TS) and Brittle Fracture Index (BFI). The two quantities were also used to assess the influence of relative density and binder concentration on TS and BFI as well as compare Binding Efficiencies (BE). The result shows that TS is dependent on relative density, binder type and binder concentration while BFI is dependent on the binder type and binder concentration; and that although, the inclusion of NMG in a paracetamol tablet formulation may not enhance the TS of the tablets produced, however it will decrease the tendency of the tablets to cap or laminate. This work concludes that BEN may be useful in quantitative assessment while BEC may be appropriate for qualitative assessment.

Keywords: binding efficiency, brittle fracture index, dimensionless binding, tensile strength

Procedia PDF Downloads 225

Authors: Mehwish Shahzadi

Abstract:

Sunflower is cultivated all over the world not only as an ornament plant but also for the purpose of getting oil. It is the third most cultivated plant in the history because its oil considered best for health. The present study deals with the preparation of sunflower oil from commercial seed sample which was obtained from local market. The physicochemical properties of the oil were determined which included saponification value, acid value and ester value. Results showed that saponification value of the oil was 191.675, acid value was 0.64 and ester value to be 191.035 for the sample under observation. GC-MS analysis of sunflower oil was carried out to check its composition. Oleic acid was determined with linoleic acid and isopropyl palmitate. It represents the presence of three major components of sunflower oil. Other compounds detected were, p-toluylic acid, butylated hydroxytoluene, 1,2-benzenedicarboxylic acid, benzoic acid, 2,4,6-trimethyl-, 2,4,6-trimethylphenyl ester and 2,4-decadienal, (E,E).

Keywords: GC-MS, oleic acid, saponification value, sunflower oil

Procedia PDF Downloads 278

Authors: Mina Kalantarzadeh, Claire Lockie-Williams, Caroline Howard

Abstract:

DNA barcoding is increasingly used for identification of medicinal plants worldwide. In the last decade, a large number of DNA barcodes have been generated, and their application in species identification explored. The success of DNA barcoding process relies on the accuracy of the results from polymerase chain reaction (PCR) amplification step which could be negatively affected due to a presence of inhibitors or degraded DNA in herbal samples. An established DNA reference material can be used to support molecular characterisation protocols and prove system suitability, for fast and accurate identification of plant species. The present study describes the use of a novel reference material, the trnH-psbA British Pharmacopoeia Nucleic Acid Reference Material (trnH-psbA BPNARM), which was produced to aid in the identification of Ocimum tenuiflorum L., a widely used herb. During DNA barcoding of O. tenuiflorum, PCR amplifications of isolated DNA produced inconsistent results, suggesting an issue with either the method or DNA quality of the tested samples. The trnH-psbA BPNARM was produced and tested to check for the issues caused during PCR amplification. It was added to the plant material as control DNA before extraction and was co-extracted and amplified by PCR. PCR analyses revealed that the amplification was not as successful as expected which suggested that the amplification is affected by presence of inhibitors co-extracted from plant materials. Various potential issues were assessed during DNA extraction and optimisations were made accordingly. A DNA barcoding protocol for O. tenuiflorum was published in the British Pharmacopoeia 2016, which included the reference sequence. The trnH-psbA BPNARM accelerated degradation test which investigates the stability of the reference material over time demonstrated that it has been stable when stored at 56 °C for a year. Using this protocol and trnH-psbA reference material provides a fast and accurate method for identification of O. tenuiflorum. The optimisations of the DNA extraction using the trnH-psbA BPNARM provided a signposting method which can assist in overcoming common problems encountered when using molecular methods with medicinal plants.

Keywords: degradation, DNA extraction, nucleic acid reference material, trnH-psbA

Procedia PDF Downloads 176

Authors: N. Demertzis, J. L. Bowen

Abstract:

Sepsis is a complex and rapidly evolving condition, resulting from uncontrolled prolonged activation of host immune system due to pathogenic insult. The aim of this study is the development of a multiplex electrochemical sensing platform, capable of detecting both pathogen associated and host immune markers to enable the rapid and definitive diagnosis of sepsis. A combination of aptamers and molecular imprinting approaches have been employed to generate sensing systems for lipopolysaccharide (LPS), c-reactive protein (CRP) and procalcitonin (PCT). Gold working electrodes were mechanically polished and electrochemically cleaned with 0.1 M sulphuric acid using cyclic voltammetry (CV). Following activation, a self-assembled monolayer (SAM) was generated, by incubating the electrodes with a thiolated anti-LPS aptamer / dithiodibutiric acid (DTBA) mixture (1:20). 3-aminophenylboronic acid (3-APBA) in combination with the anti-LPS aptamer was used for the development of the hybrid molecularly imprinted sensor (apta-MIP). Aptasensors, targeting PCT and CRP were also fabricated, following the same approach as in the case of LPS, with mercaptohexanol (MCH) replacing DTBA. In the case of the CRP aptasensor, the SAM was formed following incubation of a 1:1 aptamer: MCH mixture. However, in the case of PCT, the SAM was formed with the aptamer itself, with subsequent backfilling with 1 μM MCH. The binding performance of all systems has been evaluated using electrochemical impedance spectroscopy. The apta-MIP’s polymer thickness is controlled by varying the number of electropolymerisation cycles. In the ideal number of polymerisation cycles, the polymer must cover the electrode surface and create a binding pocket around LPS and its aptamer binding site. Less polymerisation cycles will create a hybrid system which resembles an aptasensor, while more cycles will be able to cover the complex and demonstrate a bulk polymer-like behaviour. Both aptasensor and apta-MIP were challenged with LPS and compared to conventional imprinted (absence of aptamer from the binding site, polymer formed in presence of LPS) and non-imprinted polymers (NIPS, absence of LPS whilst hybrid polymer is formed). A stable LPS aptasensor, capable of detecting down to 5 pg/ml of LPS was generated. The apparent Kd of the system was estimated at 17 pM, with a Bmax of approximately 50 pM. The aptasensor demonstrated high specificity to LPS. The apta-MIP demonstrated superior recognition properties with a limit of detection of 1 fg/ml and a Bmax of 100 pg/ml. The CRP and PCT aptasensors were both able to detect down to 5 pg/ml. Whilst full binding performance is currently being evaluated, there is none of the sensors demonstrate cross-reactivity towards LPS, CRP or PCT. In conclusion, stable aptasensors capable of detecting LPS, PCT and CRP at low concentrations have been generated. The realisation of a multiplex panel such as described herein, will effectively contribute to the rapid, personalised diagnosis of sepsis.

Keywords: aptamer, electrochemical impedance spectroscopy, molecularly imprinted polymers, sepsis

Procedia PDF Downloads 106

Authors: Ho-Wa Li, Sai-Wing Tsang

Abstract:

The excitonic effect in organic semiconductors plays a key role in determining the electronic devices performance. Strong exciton binding energy has been regarded as the detrimental factor limiting the further improvement in organic photovoltaic cells. To the best of our knowledge, only limited reported can be found in measuring the exciton binding energy in organic photovoltaic materials. Conventional sophisticated approach using photoemission spectroscopy (UPS and IPES) would limit the wide access of the investigation. Here, we demonstrate a facile approach to study the electrical and optical quantum efficiencies of a series of conjugated photovoltaic polymer, fullerene and non-fullerene materials. Quantitative values of the exciton binding energy in those prototypical materials were obtained with concise photovoltaic device structure. And the extracted binding energies have excellent agreement with those determined by the conventional photoemission technique. More importantly, our findings can provide valuable information on the excitonic dissociation in the first excited state. Particularly, we find that the high binding energy of some non-fullerene acceptors limits the combination of polymer acceptors for efficiency exciton dissociation. The results bring insight into the engineering of excitonic effect for the development of efficient organic photovoltaic cells.

Keywords: organic photovoltaics, quantum efficiency, exciton binding energy, device physics

Procedia PDF Downloads 118

Authors: Ozcan Baris Citil, Mehmet Akoz

Abstract:

Effects of fatty acid composition and punicic acid contents of abdominal fat of Hy-line hens were investigated by the gas chromatographic method. Total 30 different fatty acids were determined in fatty acid compositions of eggs. These fatty acids were varied between C 8 to C 22. The punicic acid content of abdominal fats analysed was found to be higher percentages in the 90th day than those of 30th and 60th day. At the end of the experiment, total punicic acid contents of abdominal fats were significantly increased.

Keywords: fatty acids, gas chromatography, punicic acid, abdominal fats

Procedia PDF Downloads 316

Authors: Yaxuan Wang

Abstract:

It has long been a mystery that why the Mongolian possessive suffix, which is constrained by Condition A of binding theory, has the ability to probe a potential antecedent outside of its binding domain. This squib argues that binding theory alone is not sufficient to explain the linguistic facts and proposes an analysis adopting the Agree operation. The current analysis correctly predicts all the possible and impossible structures, with an additional hypothesis that Mongolian possessive suffixes serve as an antecedent for PROs in adjunct. The findings thus provide insights into how Agree operates in Mongolian language.

Keywords: syntax, Mongolian, agreement, possessive particles

Procedia PDF Downloads 77

Authors: Lei Wang, Yingzhou Ni, Amr M. Bakry, Hao Cheng, Li Liang

Abstract:

Food proteins have been widely used as carrier materials because of their multiple functional properties. In this study, alfa-tocopherol was encapsulated in the oil phase of an oil-in-water emulsion stabilized with whey protein isolate (WPI). The influence of WPI concentration and resveratrol or ascorbic acid on the decomposition of alfa-tocopherol in the emulsion during storage is discussed. Decomposition decreased as WPI concentrations increased. Decomposition was delayed at ascorbic acid/WPI molar ratios lower than 5 but was promoted at higher ratios. Resveratrol partitioned into the oil-water interface by binding to WPI and its cis-isomer is believed to have contributed most of the protective effect of this polyphenol. These results suggest the possibility of using the emulsifying and ligand-binging properties of WPI to produce carriers for simultaneous encapsulation of alfa-tocopherol and resveratrol in a single emulsion system.

Keywords: stability, alfa-tocopherol, resveratrol, whey protein isolate

Procedia PDF Downloads 493

Authors: Saqib Ali, Man-Qun Wang

Abstract:

The Japanese pine sawyer, Monochamus alternatus Hope (Coleoptera: Cerambycidae), is a major pest of pines and it is also the key vector of the exotic pinewood nematode in China. In the present study, we cloned, expressed, and purified a chemosensory protein (CSP) in M. alternatus. We surveyed its expression in various developmental stages of male and female adult tissues and determined its binding affinities for different pine volatiles using a competitive binding fluorescence assay. A CSP known as CSP5 in M. alternatus was obtained from an antennal cDNA library and expressed in Escherichia coli. Quantitative reverse transcription polymerase chain reaction results indicated that the CSP5 gene was mainly expressed in male and female antennae. Competitive binding assays were performed to test the binding affinity of recombinant CSP5 to 13 odour molecules of pine volatiles. The results showed that CSP5 showed very strong binding abilities to myrcene, (+)-β-pinene, and (−)-isolongifolene, whereas the volatiles 2-methoxy-4-vinylphenol, p-cymene, and (+)-limonene oxide have relatively weak binding affinity at pH 5.0. Three volatiles myrcene, (+)-β-pinene, and (−)-isolongifolene may play crucial roles in CSP5 binding with ligands, but this needs further study for confirmation. The sensitivity of insect to host plant volatiles can effectively be used to control and monitor the population through mass trapping as part of integrated pest management programs.

Keywords: olfactory-specific protein, volatiles, competitive binding assay, expression characteristics, qPCR

Procedia PDF Downloads 108

Authors: Ozcan Baris Citil

Abstract:

The aim of the present study is the determination of the effects of variations in the proximate analysis, cholesterol content and fatty acid compositions of Helix aspersa. Garden snails (Helix aspersa) were picked up by hand from the Central Anatolia Region of Turkey, in autumn (November) in 2015. Fatty acid methyl esters (FAMEs) and cholesterol analysis were analyzed by gas chromatography (GC). The protein contents of snail muscle were determined with Kjeldahl distillation units. Statistical comparisons were made by using SPSS Software (version 16.0). Thirty different fatty acids of different saturation levels were detected. As the predominant fatty acids, stearic acid (C18:0), oleic acid (C18:1ω9), linoleic acid (C18:2ω6), palmitic acid (C16:0), arachidonic acid (C20:4ω6), eicosadienoic acid (C20:2) and linolenic acid (C18:3ω3) were found in Helix aspersa. Palmitic acid (C16:0) was identified as the major SFA in autumn. Linoleic acid (C18:2ω6), eicosadienoic acid (C20:2) and arachidonic acid (C20:4ω6) have the highest levels among the PUFAs. In the present study, ω3 were found 5.48% in autumn. Linolenic acid and omega-3 fatty acid amounts in the autumn decreased significantly but cholesterol content was not affected in Helix aspersa in autumn (November) in 2015.

Keywords: Helix aspersa, fatty acid, SFA, PUFA, cholesterol

Procedia PDF Downloads 311

Authors: Bongmun Kang, Kagnari Yamakawa, Yoshihisa Hagihara, Yuji Ito, Michimasa Kishimoto, Yoichi Kumada

Abstract:

The PMMA-tag was genetically fused with the C-terminal region of VHH molecules. This antibody, VHH, is known as a single-chain domain, which is devoid of light chains. The PMMA-tag, which could affect the isoelectric point (pI) changeable with a charge of amino acid in VHHs were closely related to the solubility of VHH molecules during refolding. The genetic fusion of PMMA-tag to C-terminal region of VHHs significantly affects the recovery of their soluble protein during refolding by 50 mM TAPS at pH 8.5. It could be refolded with a recovery of more than 95% by dialysis at pH 8.5. A marked difference in the antigen-binding activities in the adsorption state was significantly high in VHH-PM compared to the wild type of VHH. There are approximately 8-fold differences in the antigen-binding activities in the adsorption state between VHH-PM and VHH.

Keywords: VHH, PMMA-tag, isoelectric point, pH, Solubility, refolding, immobilization, ELISA

Procedia PDF Downloads 394

Authors: William Thomas Worster

Abstract:

Diplomatic assurances issued by states declaring that they will not mistreat individuals returned to them occupy a strange middle ground between being legal and non-legal obligations. States assert that they are non-binding, yet at other times that they are binding. However, this assertion may not be the end of the discussion. The International Court of Justice and other tribunals have concluded that similar instruments were binding, states have disagreed that certain similar instruments were binding, and the Vienna Convention on the Law of Treaties and its travaux prépératoires do not appear to contemplate non-binding instruments. This paper is a case study of diplomatic assurances but, by necessity, touches on the delicate question of whether certain texts are treaties, promises, or non-binding political statements. International law, and law in general, requires a binary approach to obligation. All communications must be binding or not, even if the fit is not precise. Through this study, we will find that some of the obligations in certain assurances can be understood as legal and some not. We will attempt to state the current methodology for determining which obligations are legal under the law of treaties and law on binding unilateral promises. The paper begins with some background of the legal environment of diplomatic assurances and their use in cases of expulsion. The paper then turns to discuss the legal nature of diplomatic assurances, proceeding to address various possibilities for legal value as treaties and as binding unilateral statements. This paper will not examine the legal value of diplomatic assurances solely under customary international law other than the way in which customary international law might further refine the treaty definition. In order to identify whether any assurances are contained in legal acts, this study identifies a pool of relevant assurances and qualitatively analyzes whether any of those are contained in treaties or binding unilateral statements. To the author’s best knowledge, this study is the first large-scale, qualitative qualitative analysis of assurances as a group of instruments that accounts for their heterogenous nature. It is also the first study to identify the indicators of whether an instrument is a treaty or promise.

Keywords: diplomatic assurances, deportation, extradition, expulsion, non-refoulement, torture, persecution, death penalty, human rights, memorandum of understanding, promises, secret, monitoring, compliance, enforcement

Procedia PDF Downloads 61

Authors: Mateen A. Khan, Elizabeth J. Theil, Dixie J. Goss

Abstract:

Cellular iron homeostasis is accomplished by the coordinated regulated expression of iron uptake, storage, and export. Iron regulate the translation of ferritin and mitochondrial aconitase iron responsive element (IRE)-mRNA by interaction with an iron regulatory protein (IRPs). Iron increases protein biosynthesis encoded in iron responsive element. The noncoding structure IRE-mRNA, approximately 30-nt, folds into a stem loop to control synthesis of proteins in iron trafficking, cell cycling, and nervous system function. Fluorescence anisotropy measurements showed the presence of one binding site on IRP1 for ferritin and mitochondrial aconitase IRE-mRNA. Scatchard analysis revealed the binding affinity (Kₐ) and average binding sites (n) for ferritin and mitochondrial aconitase IRE-mRNA were 68.7 x 10⁶ M⁻¹ and 9.2 x 10⁶ M⁻¹, respectively. In order to understand the relative importance of equilibrium and stability, we further report the contribution of electrostatic interactions in the overall binding of two IRE-mRNA with IRP1. The fluorescence quenching of IRP1 protein was measured at different ionic strengths. The binding affinity of IRE-mRNA to IRP1 decreases with increasing ionic strength, but the number of binding sites was independent of ionic strength. Such results indicate a differential contribution of electrostatics to the interaction of IRE-mRNA with IRP1, possibly related to helix bending or stem interactions and an overall conformational change. Selective destabilization of ferritin and mitochondrial aconitase RNA/protein complexes as reported here explain in part the quantitative differences in signal response to iron in vivo and indicate possible new regulatory interactions.

Keywords: IRE-mRNA, IRP1, binding, ionic strength

Procedia PDF Downloads 108

Authors: Muhammed Saeed Rasheed

Abstract:

Knowledge of the zinc and phytic acid (PA) concentrations of staple cereal crops are essential when evaluating the nutritional health of national and regional populations. In the present study, a total of 120 farmers’ fields in Iraqi Kurdistan were surveyed for zinc status in soil and wheat grain samples; wheat is the staple carbohydrate source in the region. Soils were analysed for total concentrations of phosphorus (PT) and zinc (ZnT), available P (POlsen) and Zn (ZnDTPA) and for pH. Average values (mg kg-1) ranged between 403-3740 (PT), 42.0-203 (ZnT), 2.13-28.1 (POlsen) and 0.14-5.23 (ZnDTPA); pH was in the range 7.46-8.67. The concentrations of Zn, PA/Zn molar ratio and estimated Zn bioavailability were also determined in wheat grain. The ranges of Zn and PA concentrations (mg kg⁻¹) were 12.3-63.2 and 5400 – 9300, respectively, giving a PA/Zn molar ratio of 15.7-30.6. A trivariate model was used to estimate intake of bioaccessible Zn, employing the following parameter values: (i) maximum Zn absorption = 0.09 (AMAX), (ii) equilibrium dissociation constant of zinc-receptor binding reaction = 0.680 (KP), and (iii) equilibrium dissociation constant of Zn-PA binding reaction = 0.033 (KR). In the model, total daily absorbed Zn (TAZ) (mg d⁻¹) as a function of total daily nutritional PA (mmole d⁻¹) and total daily nutritional Zn (mmole Zn d⁻¹) was estimated assuming an average wheat flour consumption of 300 g day⁻¹ in the region. Consideration of the PA and Zn intake suggest only 21.5±2.9% of grain Zn is bioavailable so that the effective Zn intake from wheat is only 1.84-2.63 mg d-1 for the local population. Overall results suggest available dietary Zn is below recommended levels (11 mg d⁻¹), partly due to low uptake by wheat but also due to the presence of large concentrations of PA in wheat grains. A crop breeding program combined with enhanced agronomic management methods is needed to enhance both Zn uptake and bioavailability in grains of cultivated wheat types.

Keywords: phosphorus, zinc, phytic acid, phytic acid to zinc molar ratio, zinc bioavailability

Procedia PDF Downloads 97

Authors: Meltem Betül Sağlam, Halil İbrahim Güler, Aykut Sağlam

Abstract:

In this work, phthalocyanine compounds containing α-naphtholbenzeinunits have been synthesized. Mutagenicity and DNA binding properties of the compounds were investigated by Salmonella/Microsome Assay and spectrophotometer. According to the results of the preliminary range finding tests, the compounds gave no toxic effect to all tester strain S. typhimurium TA98 and TA100 at doses of 500, 1100, 350, 500 and 750 µg/plate in the presence and absence of S9, respectively. This study showed that all compounds exhibited efficient DNA-binding activity. In conclusion, these non-toxic compounds may be used as effective DNA dyes for molecular biology studies.

Keywords: dye, mutagenicity, phthalocyanine, toxicity

Procedia PDF Downloads 203

Authors: Shweta Sinha, Mukesh Doble, Manju S. L.

Abstract:

Pyrazole scaffold is an important group of compound in heterocyclic chemistry and is found to possess numerous uses in chemistry. Pyrazole derivatives are also known to possess important biological activities including antitumor, antimicrobial, antiviral, antifungal, anticancer and anti-inflammatory. Inflammation is associated with pain, allergy and asthma. Leukotrienes are mediators of various inflammatory and allergic disorders. 5-Lipoxygenase (5-LOX) is an important enzyme involved in the biosynthesis of leukotrienes and metabolism of arachidonic acid (AA) and thus targeted for anti-inflammation. In vitro inhibitory activity of pyrazol-3-yl thiazole 4-carboxylic acid derivatives is tested against enzyme 5-LOX. Most of these compounds exhibit good inhibitory activity against this enzyme. Binding mode study of these compounds is determined by computational tool. Further experiments are being done to understand the mechanism of action of these compounds in inhibiting this enzyme. To conclude, these compounds appear to be a promising target in drug design against 5-LOX.

Keywords: inflammation, inhibition, 5-lipoxygenase, pyrazole

Procedia PDF Downloads 216

Authors: Yusra Tariq

Abstract:

The Grass 37 gene is presently known in tomatoes, which are the source of healthy substances such as ascorbic acid, polyphenols, carotenoids and nutrients. It has a significant impact on the growth and development of humans. The GRASS 37 gene is a plant Transcription factor group assuming significant parts in various reactions of different Abiotic stresses such as (drought, salinity, thermal stresses, temperature, and bright waves) which could highly affect the growth. Tomatoes are very sensitive to temperature, and their growth or production occurs optimally in a temperature range from 21 C to 29.5 C during the daytime and from 18.5 C to 21 C during the night. This protein acts as a positive regulator of salt stress response and abscisic acid signaling. This study summarizes the structure characterized by molecular formula and protein-binding domains by different bioinformatics tools such as Expasy translate tool, Expasy Portparam, Swiss Prot and Inter Pro Scan, Clustal W tool regulatory procedure of GRASS gene components, also their reactions to both biotic and Abiotic stresses.

Keywords: GRAS37, gene, bioinformatics, tool

Procedia PDF Downloads 14

Authors: Rohit Singh Dangi, Ravi Kant Pal, Monica Sundd

Abstract:

Acyl-CoA binding protein (ACBP) is a housekeeping protein which functions as an intracellular carrier of acyl-CoA esters. Given the fact that the amastigote stage (blood stage) of Leishmania depends largely on fatty acids as the energy source, of which a large part is derived from its host, these proteins might have an important role in its survival. In Leishmania major, genome sequencing suggests the presence of six ACBPs, whose function remains largely unknown. For functional and structural characterization, one of the ACBP genes was cloned, and the protein was expressed and purified heterologously. Acyl-CoA ester binding and stoichiometry were analyzed by isothermal titration calorimetry and Dynamic light scattering. Our results shed light on high affinity of ACBP towards longer acyl-CoA esters, such as myristoyl-CoA to arachidonoyl-CoA with single binding site. To understand the binding mechanism & dynamics, Nuclear magnetic resonance assignments of this protein are being done. The protein's crystal structure was determined at 1.5Å resolution and revealed a classical topology for ACBP, containing four alpha-helical bundles. In the binding pocket, the loop between the first and the second helix (16 – 26AA) is four residues longer from other extensively studied ACBPs (PfACBP) and it curls upwards towards the pantothenate moiety of CoA to provide a large tunnel space for long acyl chain insertion.

Keywords: acyl-coa binding protein (ACBP), acyl-coa esters, crystal structure, isothermal titration, calorimetry, Leishmania

Procedia PDF Downloads 420

Authors: Nusrat Masood, Vijaya Dubey, Suaib Luqman

Abstract:

Caspase 3, a member of cysteine-aspartic acid protease family, is an imperative indicator for cell death particularly when substantiating apoptosis. Thus, caspase 3 is an interesting target for the discovery and development of anticancer agent. We adopted a four level assessment of both terpenoids and flavonoids and thus experimentally performed the enzymatic assay in cell free system as well as in cancer cell line which was validated through real time expression and molecular interaction studies. A significant difference was observed with both the class of natural products indicating terpenoids as better activators of caspase 3 compared to flavonoids both in the cell free system as well as in cell lines. The expression analysis, activation constant and binding energy also correlate well with the enzyme activity. Overall, terpenoids had an unswerving effect on caspase 3 in all the tested system while flavonoids indirectly affect enzyme activity.

Keywords: Caspase 3, terpenoids, flavonoids, activation constant, binding energy

Procedia PDF Downloads 208

Authors: Sameer N. Goyal, Chandragauda R. Patil

Abstract:

Oleanolic acid (3/3-hydroxy-olea-12-en-28-oic acid) and its isomer, Ursolic acid (38-hydroxy-urs-12-en-28-oic acid) are triterpenoids compounds which exist widely in plant kingdom in the free acid form or as glycosidic triterpenoids saponins. The aim of the study is to evaluate intravenously administered oleanolic acid and ursolic acid in doxorubicin induced cardiotoxicity. Cardiotoxicity was induced in albino wistar rat with single intravenous injection of doxorubicin at dose of 67.75mg/kg i.v for 48 hrs at 12 hrs interval following doxorubicin administration in the same model cardioprotective effect of amifostine (90 mg/kg i.v, single dose prior 30 min before doxorubicin administration) was evaluated as standard treatment. Induction of cardiotoxicity was confirmed by rise in cardiac markers in serum such as CK–MB, LDH and also by electrocardiographically. The doxorubicin treated group significantly increased in QT interval, serum CK-MB, serum LDH, SGOT, SGPT and antioxidant parameter. Both the treatment group showed significant protective effect on Hemodynamic, electrocardiographic, biochemical, and antioxidant parameters. The oleanolic acid showed slight protective effect in histological lesions in doxorubicin induced cardiotoxicity. Hence, the results indicate that Oleanolic acid has more cardioprotective potential than ursolic acid against doxorubicin induced cardiotoxicity in rats.

Keywords: cardioprotection, doxorubicin, oleanolic acid, ursolic acid

Procedia PDF Downloads 498

Authors: Junie B. Billones, Maria Constancia O. Carrillo, Voltaire G. Organo, Stephani Joy Y. Macalino, Inno A. Emnacen, Jamie Bernadette A. Sy

Abstract:

The drug discovery and development process is generally known to be a very lengthy and labor-intensive process. Therefore, in order to be able to deliver prompt and effective responses to cure certain diseases, there is an urgent need to reduce the time and resources needed to design, develop, and optimize potential drugs. Computer-aided drug design (CADD) is able to alleviate this issue by applying computational power in order to streamline the whole drug discovery process, starting from target identification to lead optimization. This drug design approach can be predominantly applied to diseases that cause major public health concerns, such as tuberculosis. Hitherto, there has been no concrete cure for this disease, especially with the continuing emergence of drug resistant strains. In this study, CADD is employed for tuberculosis by first identifying a key enzyme in the mycobacterium’s metabolic pathway that would make a good drug target. One such potential target is the lipoate protein ligase B enzyme (LipB), which is a key enzyme in the M. tuberculosis metabolic pathway involved in the biosynthesis of the lipoic acid cofactor. Its expression is considerably up-regulated in patients with multi-drug resistant tuberculosis (MDR-TB) and it has no known back-up mechanism that can take over its function when inhibited, making it an extremely attractive target. Using cutting-edge computational methods, compounds from AnalytiCon Discovery Natural Derivatives database were screened and docked against the LipB enzyme in order to rank them based on their binding affinities. Compounds which have better binding affinities than LipB’s known inhibitor, decanoic acid, were subjected to in silico toxicity evaluation using the ADMET and TOPKAT protocols. Out of the 31,692 compounds in the database, 112 of these showed better binding energies than decanoic acid. Furthermore, 12 out of the 112 compounds showed highly promising ADMET and TOPKAT properties. Future studies involving in vitro or in vivo bioassays may be done to further confirm the therapeutic efficacy of these 12 compounds, which eventually may then lead to a novel class of anti-tuberculosis drugs.

Keywords: pharmacophore, molecular docking, lipoate protein ligase B (LipB), ADMET, TOPKAT

Procedia PDF Downloads 395