Functional characterization of a human odorant receptor

Olfaction appears to be mediated by a large family of odorant receptors (ORs) that accounts for ~ 1% of the human genome [1–4]. The functional characterization of these receptors has so far been hindered by their poor functional expression in the plasma membrane of heterologous cells. In order to investigate the problem of inefficient OR membrane targeting, sequential stages in the life cycle of the human OR17-40 were monitored. The modification of the receptor by different tags enabled the direct visualization of its biogenesis and trafficking in HEK293 cells. A multi-color labelling approach, which involved fluorescent subcellular markers as well as the spectral separation of membrane-inserted and intracellularly located receptors, indicated that high overexpression led to the accumulation of the receptor in the ER, whereas low expression levels improved its membrane targeting. To increase the efficiency of OR-mediated signalling, fluorescence-activated cell sorting of a functional OR17-40 – GFP fusion protein was used to separate low from high expressing cells, thus increasing the fraction of odorant-responsive cells up to 80% of the total cell population. Selectively labelled cell surface receptors accumulated over time in intracellular compartments, indicating constitutive receptor internalization. This process, which was independent of receptor activation, occurred along the clathrin-mediated pathway. Moreover, the imaging of single receptors in the plasma membrane indicated that, in absence of ligand, part of the receptors are already confined within small domains of 195 ± 10 nm. This fraction, which increased upon agonist or antagonist binding, probably corresponds to receptors located in clathrin-coated prepits. Odorant molecules structurally related to the cognate agonist helional were tested for their ability to activate OR17-40. It was found that an aldehyde group connected to an aromatic ring via a carbon chain of defined length and containing a methyl group in alpha or beta position is necessary for activating the receptor. In addition, first evidence for an OR17-40-specific antagonism was provided. Finally, OR17-40 activation was monitored in cell-derived native vesicles, opening new ways for assay miniaturization and the development of odorant screenings in a micro-array format

Structure-Function Analysis of Insect Olfactory Receptors

Organisms use their senses to transform external stimuli into an internal representation of the world. Insects employ their keen sense of smell for a variety of tasks including location of food sources, which can vary from yeast growing on ripe fruits for the vinegar fly Drosophila melanogaster to mammals for blood-feeding insects such as the mosquito Anopheles gambiae. The first informational relay between the external environment and the organism is the olfactory sensory neuron (OSN), whose activation translates the intensity, quality, and temporal features of volatile chemicals into spike trains. This dissertation focuses on understanding how the insect olfactory system functions at the periphery, shedding light on the molecular players involved and the interactions between environmental chemicals and OSNs. In Drosophila, most of the ~1,200 OSNs express members of the olfactory receptor (OR) protein family (Stocker, 1994; Vosshall et al., 1999). The functional OR complex comprises at least one variable odorant-binding subunit and one constant subunit named OR83b (Benton et al., 2006). Insect ORs have historically been grouped with mammalian and nematode ORs, both of which are G protein coupled receptors (GPCRs), whose activation leads to increased concentrations of intracellular second messengers and opening of cyclic nucleotide-gated channels (CNG; Buck and Axel, 1991; Colbert et al., 1997; Firestein et al., 1991; Nakamura and Gold, 1987; Troemel et ii al., 1995). Insect ORs lack similarity to GPCRs (Benton et al., 2006; Vosshall et al., 1999), and we hypothesized that they function as odorant-gated ion channels. We showed that expression of insect ORs in heterologous cells generates odorant-evoked currents that are resistant to G protein inhibitors, independent of cyclic nucleotides, and whose properties change based on OR subunit composition (Sato et al., 2008). This surprising discovery supports our hypothesis that insect ORs are indeed odorant-gated ion channels. Concurrently with these findings, we investigated the mode of action of DEET, the most widely used topical insect repellent, and showed that ORs are among its molecular targets. We demonstrated that DEET suppresses Drosophila food-seeking behavior, modulates OSN activity, and decreases OR-mediated currents in heterologous cells (Ditzen et al., 2008). Moreover, we showed that a missense polymorphism in a ligand-binding OR subunit leads to pharmacological resistance to the repellent in vivo. This is the first finding that identifies a molecular target of DEET. Within the OR complex, OR83b plays an essential role. Ligand-binding subunits fail to localize properly at the OSN dendrite in the absence of OR83b, resulting in almost complete loss of sense of smell (Benton et al., 2006; Larsson et al., 2004). We identified a putative localization motif in the OR83b protein, and showed that mutations in conserved residues abolish proper OR trafficking and impair odorant-evoked responses. This discovery defines critical amino acids that might be used as possible targets of future repellents to modulate the activity of insect OSNs. The discoveries described in this thesis will have an impact on the design of better and safer insect repellents and the control of insect-borne diseases

Formulation and In-Vitro Evaluation of Metoprolol Tartrate Microspheres by using Natural Semisynthetic and Synthetic Polymers as Controlled Release Dosage Form

The goal of a controlled release dosage form is to maintain therapeutic blood or tissue levels of the drug for an extended and specified period of time. This is generally accomplished by attempting to obtain "zero-order" release from the dosage form. Zero-order release constitutes drug release from the dosage form which is independent of the amount of drug in the delivery system (i.e. a constant release rate). Sustained-release systems generally do not attain this type of release and usually try to mimic zero-order release by providing drug in a slow first-order fashion (i.e., concentration release dependent). Systems that are designated as prolonged release can also be considered as attempts at achieving sustained-release delivery. Based on all the above evaluation parameters it was concluded that the formulation F3 was found to be best formulation among the formulations from F1 to F9. The in-vitro drug released data was applied to various kinetic models such as zero order kinetics, Higuchi plot, first order kinetics and Peppas plot by predict the drug release kinetics mechanism. The formulation F3 was best fitted with Matrix kinetics and it undergoes Quasi-fickian diffusion mechanism (n<0.5). According to stability study it was found that there was no variation in Percentage yield, Entrapment efficiency, and In-vitro drug released profile of optimized formulation F3 for 3 months period

Theoretical study of the interaction of agonists with the 5-HT2A receptor

The 5-HT2A receptor (5-HT2AR) is a biogenic amine receptor that belongs to the class A of G protein coupled receptors. It is characterized by a low affinity for serotonin (5-HT) and for other primary amines. Introduction of an ortho-methoxybenzyl substituent at the amine nitrogen increases the partial agonistic activity by a factor of 40 to 1400 compared with 5-HT. The present study was to analyse the QSAR of a series of 51 5-HT2AR partial agonistic arylethylamines, tested in vascular in-vitro assays on rats, at a structure-based level and to suggest ligand binding sites. The compounds belong to three different structural classes, (1) indoles, (2) methoxybenzenes and (3) quinazolinediones. Following a hierarchical strategy, different methods have been applied which all contribute to the investigation of ligand-receptor interactions: fragment regression analysis (FRA), receptor modeling, docking studies and 3D QSAR approaches (comparative molecular field analysis, CoMFA, and comparative molecular similarity index analysis, CoMSIA). An initial FRA indicated that methoxy substituents at indole and phenyl derivatives increase the activity and may be involved in polar interactions with the 5-HT2AR. The large contribution of lipophilic substituents in p position of phenethylamines suggests fit to a specific hydrophobic pocket. Secondary benzylamines are more than one order of magnitude more active than their NH2 analogs. An ortho-OH or -OMe substituent at the benzyl moiety further increases activity. Homology models of the human and rat 5-HT2AR were generated using the crystal structure of bovine rhodopsin and of the beta2-adrenoceptor as templates. The derivation of the putative binding sites for the arylethylamines was based on the results from FRA and on mutagenesis data. Both templates led to 5-HT2AR models with similar topology of the binding pocket within the transmembrane domains TM3, TM5, TM6 and TM7. Docking studies with representative members of the three structural classes suggested that the aryl moieties and particularly para-substituents in phenyl derivatives fit into a hydrophobic pocket formed by Phe2435.47, Phe2445.48 and Phe3406.52. The 5-methoxy substituents in indole and phenyl compounds form H bonds with Ser2395.43. In each case, an additional H bond with Ser1593.36 may be assumed. The cationic amine interacts with the conserved Asp1553.32. The benzyl group of secondary arylethylamines is inserted into another hydrophobic pocket formed by Phe3396.51, Trp3677.40 and Tyr3707.43. In this region, the docking poses depend on the template used for model generation, leading to different interactions especially of ortho- substituents. The docking studies with the beta2-adrenoceptor based rat 5-HT2AR model provided templates for a structure-based alignment of the whole series which was used in 3D QSAR analyses of the partial agonistic activity. Both approaches, CoMFA and CoMSIA, led to highly predictive models with low complexity (cross-validated q2 of 0.72 and 0.81 at 4 and 3 components, respectively). The results were largely compatible with the binding site and confirm the docking studies and the suggested ligand-receptor interactions. Steric and hydrophobic field effects on the potency indicate a hydrophobic pocket around the aryl moiety and near the para position of phenyl derivatives and account for the increased activity of secondary benzylamines. The effects of electrostatic and H-bond acceptor fields suggest a favourable influence of negative charges around the aryl moiety, corresponding to the increase in potency caused by methoxy substituents in 2-, 4-, 5- and 6-position of phenethylamines and by the quinazolinedione oxygens. This is in accord with the role of Ser1593.36 and Ser2395.43 as H bond donors. At the benzyl moiety, the negative charge and the acceptor potential of 2-hydroxy and -methoxy substituents is of advantage. Agonists stabilize or induce active receptor states not reflected by the existing crystal structures. Based on models of different rhodopsin states, a homology modeling and ligand docking study on corresponding 5-HT2AR states suggested to be specific to agonist and partial agonist binding, respectively, was performed. The models indicate collective conformational changes of TM domains during activation. The different 5-HT2AR states are similar with respect to the amino acids interacting with the arylethylamines, but show individual topologies of the binding sites. The interconversion of states by TM movements may be accompanied by co-translations and rotations of the ligands. In the case of the secondary amines considered, the tight fit of the benzyl substituent into a hydrophobic pocket containing key residues in TM6 probably impedes the complete receptor activation due to inhibiting the rotation of this helix. High affinity of a partial agonist is therefore often at the expense of its ability to fully activate a receptor

Novel superabsorbent materials obtained from plant proteins

This work reviews the potential of plant protein-based materials as superabsorbent polymers (SAP). The review also discusses important topics of relevance for the current state of petroleum-based SAP and explains the background of the high water uptake of such materials. As diapers represent one of the most significant example of SAP applications, their industrial assemblying is highlighted. The research in absorbent materials has shown that treated and functionalized proteins may play a role in daily-care applications where super absorbency is required. Consequently, a description of proteins and the interactions that can take place within these natural polymers are reviewed. Protein sidestream candidates from biorefinery processes are listed and a further description of protein extraction mechanisms is given. This review also summarizes the results that have been obtained so far in the area of protein-based SAP materials. Finally, the challenges that protein-based SAPs face, as a possible candidate to replace petroleum-based ones, are discussed

Volatile organic compounds by-products generation in photocatalytic oxidation reactor: Experimental and modelling

The presence of volatile organic compounds (VOCs) in indoor air is inevitable. Their adverse effect on human health has encouraged researchers to develop various technologies for air pollution remediation. Photocatalytic oxidation (PCO) has been regarded as a promising and emerging technique for air purification and extensively investigated in the last two decades to characterize and improve the effectiveness and performance of this technology. In addition, the development of appropriate models can enhance the understanding of reactor performance and the evaluation of intrinsic kinetic parameters that enable the scale-up or re-design of more efficient large-scale photocatalytic reactors. This research works on mathematical modeling of gas phase photocatalytic reactors and analyses different key factors that can enhance pollutants decomposition. At the first step, a one-dimensional time-dependent mathematical model for continuous flow UV-PCO reactor has been developed. In this model, transfer of pollutants by advection and dispersion in bulk phase incorporates with the reaction rate based on the extended Langmuir Hinshelwood model in the catalyst phase. CFD modeling was also used to determine the flow distribution in the reactor at various airflow rates. Moreover, the light intensity distribution on the photocatalyst surface was simulated using the linear source spherical emission model. A dimensionless form of the model was then proposed to generalize the result for any scale. The proposed model was validated first by comparing with predictions of other models (inter-model comparison) and then by experimental data from two different scales (pilot and bench) of UV-PCO reactors. Furthermore, a sensitivity analysis using dimensionless parameters was conducted to find the controlling step in the PCO process. To validate the model, acetone, MEK, and toluene were tested in the UV-PCO reactor with a commercial PCO filter (TiO2 coated on silica fiber felts) at various operating conditions, such as concentration, relative humidity, irradiance and air velocity. The main issue for applying PCO technology in the indoor environment is the generation of hazardous by-products. The effect of by-products formation was usually ignored in former modeling studies. The next effort was to improve the model and build a comprehensive one to consider by-products generation in the UV-PCO reactor. To achieve this goal, a possible reaction pathway for degradation of each challenge compound was proposed based on identified by-products in analytical methods (GC-MS and HPLC). Different possible reaction rate scenarios were evaluated to find the best expression fitted to experimental data at the steady-state condition. The obtained reaction coefficients were then used to validate the model under various operating conditions. Finally, the Health Risk Index was used to investigate the implications of generated by-products on human health under varying operating conditions. The results indicated that the proposed model has a great potential to simulate the behavior of UV-PCO reactor in a real application

ESSE 2017. Proceedings of the International Conference on Environmental Science and Sustainable Energy

Environmental science is an interdisciplinary academic field that integrates physical-, biological-, and information sciences to study and solve environmental problems. ESSE - The International Conference on Environmental Science and Sustainable Energy provides a platform for experts, professionals, and researchers to share updated information and stimulate the communication with each other. In 2017 it was held in Suzhou, China June 23-25, 2017

Current Air Quality Issues

Air pollution is thus far one of the key environmental issues in urban areas. Comprehensive air quality plans are required to manage air pollution for a particular area. Consequently, air should be continuously sampled, monitored, and modeled to examine different action plans. Reviews and research papers describe air pollution in five main contexts: Monitoring, Modeling, Risk Assessment, Health, and Indoor Air Pollution. The book is recommended to experts interested in health and air pollution issues

Effect of curing conditions and harvesting stage of maturity on Ethiopian onion bulb drying properties

The study was conducted to investigate the impact of curing conditions and harvesting stageson the drying quality of onion bulbs. The onion bulbs (Bombay Red cultivar) were harvested at three harvesting stages (early, optimum, and late maturity) and cured at three different temperatures (30, 40 and 50 oC) and relative humidity (30, 50 and 70%). The results revealed that curing temperature, RH, and maturity stage had significant effects on all measuredattributesexcept total soluble solids