Search results for: molecular genetics

Authors: Abdullah Alaklabi, Ibrahim A. Arif, Sameera O. Bafeel, Ahmad H. Alfarhan, Anis Ahamed, Jacob Thomas, Mohammad A. Bakir

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Diospyros mespiliformis (Hochst. ex A.DC.; Ebenaceae) is a large deciduous medicinal plant. This plant species is currently listed as endangered in Saudi Arabia. Molecular identification of this plant species based on short sequence regions (571 and 664 bp) of plastid rbcL (ribulose-1, 5-biphosphate carboxylase) gene was investigated in this study. The endangered plant specimens were collected from Al-Baha, Saudi Arabia (GPS coordinate: 19.8543987, 41.3059349). Phylogenetic tree inferred from the rbcL gene sequences showed that this species is very closely related with D. brandisiana. The close relationship was also observed among D. bejaudii, D. Philippinensis and D. releyi (≥99.7% sequence homology). The partial rbcL gene sequence region (571 bp) that was amplified by rbcL primer-pair rbcLaF-rbcLaR failed to discriminate D. mespiliformis from the closely related plant species, D. brandisiana. In contrast, primer-pair rbcL1F-rbcL724R yielded longer amplicon, discriminated the species from D. brandisiana and demonstrated nucleotide variations in 3 different sites (645G>T; 663A>C; 710C>G). Although D. mespiliformis (EU980712) and D. brandisiana (EU980656) are very closely related species (99.4%); however, studied specimen showed 100% sequence homology with D. mespiliformis and 99.6% with D. brandisiana. The present findings showed that rbcL short sequence region (664 bp) of plastid rbcL gene, amplified by primer-pair rbcL1F-rbcL724R, can be used for authenticating samples of D. mespiliforformis and may provide help in authentic identification and management process of this medicinally valuable endangered plant species.

Keywords: Diospyros mespiliformis, endangered plant, identification partial rbcL

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Authors: Emily Rabe, Stephanie Candelaria, Rachel Malone, Olivia Lenz, Greg Newbloom

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Electrodialysis (ED) is a well-developed technology for ion removal in a variety of applications. However, many industries generate harsh wastewater streams that are incompatible with traditional ion exchange membranes. Membrion® has developed novel ceramic-based ion exchange membranes (IEMs) offering several advantages over traditional polymer membranes: high performance in low pH, chemical resistance to oxidizers, and a rigid structure that minimizes swelling. These membranes are synthesized with our patented silane-based sol-gel techniques. The pore size, shape, and network structure are engineered through a molecular self-assembly process where thermodynamic driving forces are used to direct where and how pores form. Either cationic or anionic groups can be added within the membrane nanopore structure to create cation- and anion-exchange membranes. The ceramic IEMs are produced on a roll-to-roll manufacturing line with low-temperature processing. Membrane performance testing is conducted using in-house permselectivity, area-specific resistance, and ED stack testing setups. Ceramic-based IEMs show comparable performance to traditional IEMs and offer some unique advantages. Long exposure to highly acidic solutions has a negligible impact on ED performance. Additionally, we have observed stable performance in the presence of strong oxidizing agents such as hydrogen peroxide. This stability is expected, as the ceramic backbone of these materials is already in a fully oxidized state. This data suggests ceramic membranes, made using sol-gel chemistry, could be an ideal solution for acidic and/or oxidizing wastewater streams from processes such as semiconductor manufacturing and mining.

Keywords: ion exchange, membrane, silane chemistry, nanostructure, wastewater

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Authors: Waqas Shafqat Chattha, Muhammad Iqbal, Amir Shakeel

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Transcriptional factors are proteins that play a vital role in regulating the transcription of target genes in different biological processes and are being widely studied in different plant species. In the current era of genomics, plant genomes sequencing has directed to the genome-wide identification, analyses and categorization of diverse transcription factor families and hence provide key insights into their structural as well as functional diversity. The DNA-binding with One Finger (DOF) proteins belongs to C2-C2-type zinc finger protein family. DOF proteins are plant-specific transcription factors implicated in diverse functions including seed maturation and germination, phytohormone signalling, light-mediated gene regulation, cotton-fiber elongation and responses of the plant to biotic as well as abiotic stresses. In this context, a genome-wide in-silico analysis of DOF TF family in diploid cotton species i.e. Gossypium raimondii has enabled us to identify 55 non-redundant genes encoding DOF proteins renamed as GrDofs (Gossypium raimondii Dof). Gene distribution studies have shown that all of the GrDof genes are unevenly distributed across 12 out of 13 G. raimondii chromosomes. The gene structure analysis illustrated that 34 out of 55 GrDof genes are intron-less while remaining 21 genes have a single intron. Protein sequence-based phylogenetic analysis of putative 55 GrDOFs has divided these proteins into 5 major groups with various paralogous gene pairs. Molecular evolutionary studies aided with the conserved domain as well as gene structure analysis suggested that segmental duplications were the principal contributors for the expansion of Dof genes in G. raimondii.

Keywords: diploid cotton , G. raimondii, phylogenetic analysis, transcription factor

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Authors: Sameh A. S. Thabit Alariqi

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Food is widely packaged with plastic materials to prevent microbial contamination and spoilage. Ionizing radiation is widely used to sterilize the food-packaging materials. Sterilization by γ-radiation causes degradation for the plastic packaging materials such as embrittlement, stiffening, softening, discoloration, odour generation, and decrease in molecular weight. Many antioxidants can prevent γ-degradation but most of them are toxic. The migration of antioxidants to its environment gives rise to major concerns in case of food packaging plastics. In this attempt, we have aimed to utilize synergistic mixtures of stabilizers which are approved for food-contact applications. Ethylene-propylene-diene terpolymer (EPDM) have been melt-mixed with hindered amine stabilizers (HAS), phenolic antioxidants and organo-phosphites (hydroperoxide decomposer). Results were discussed by comparing the stabilizing efficiency of mixtures with and without phenol system. Among phenol containing systems where we mostly observed discoloration due to the oxidation of hindered phenol, the combination of secondary HAS, tertiary HAS, organo-phosphite and hindered phenol exhibited improved stabilization efficiency than single or binary additive systems. The mixture of secondary HAS and tertiary HAS, has shown antagonistic effect of stabilization. However, the combination of organo-phosphite with secondary HAS, tertiary HAS and phenol antioxidants have been found to give synergistic even at higher doses of -sterilization. The effects have been explained through the interaction between the stabilizers. After γ-irradiation, the consumption of oligomeric stabilizer significantly depends on the components of stabilization mixture. The effect of the organo-phosphite antioxidant on the overall stability has been discussed.

Keywords: ethylene-propylene-diene terpolymer, synergistic mixtures, gamma sterilization, gamma stabilization

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Authors: Hiroyuki Aoki, Toru Asada, Tomomi Tanii

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The properties of a spin-cast film of a polymer material are different from those in the bulk material because the polymer chains are frozen in an un-equilibrium state due to the rapid evaporation of the solvent. However, there has been little information on the un-equilibrated conformation and dynamics in a spin-cast film at the single chain level. The real-space observation of individual chains would provide direct information to discuss the morphology and dynamics of single polymer chains. The recent development of super-resolution fluorescence microscopy methods allows the conformational analysis of single polymer chain. In the current study, the conformation of a polymer chain in a spin-cast film by the super-resolution microscopy. Poly(methyl methacrylate) (PMMA) with the molecular weight of 2.2 x 10^6 was spin-cast onto a glass substrate from toluene and chloroform. For the super-resolution fluorescence imaging, a small amount of the PMMA labeled by rhodamine spiroamide dye was added. The radius of gyration (Rg) was evaluated from the super-resolution fluorescence image of each PMMA chain. The mean-square-root of Rg was 48.7 and 54.0 nm in the spin-cast films prepared from the toluene and chloroform solutions, respectively. On the other hand, the chain dimension in a bulk state (a thermally annealed 10- μm-thick sample) was observed to be 43.1 nm. This indicates that the PMMA chain in the spin-cast film takes an expanded conformation compared to the unperturbed chain and that the chain dimension is dependent on the solvent quality. In a good solvent, the PMMA chain has an expanded conformation by the excluded volume effect. The polymer chain is frozen before the relaxation from an un-equilibrated expanded conformation to an unperturbed one by the rapid solvent evaporation.

Keywords: chain conformation, polymer thin film, spin-coating, super-resolution optical microscopy

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Authors: Salma E. Ahmed

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Since the mid 50’s, enormous attention has been paid towards nanocarriers and their applications in drug and gene delivery. Among these vesicles, liposomes and micelles have been heavily investigated due to their many advantages over other types. Liposomes, for instance, are mostly distinguished by their ability to encapsulate hydrophobic, hydrophilic and amphiphilic drugs. Micelles, on the other hand, are self-assembled shells of lipids, amphiphilic or oppositely charged block copolymers that, once exposed to aqueous media, can entrap hydrophobic agents, and possess prolonged circulation in the bloodstream. Both carriers are considered compatible and biodegradable. Nevertheless, they have limited stabilities, chemical versatilities, and drug encapsulation efficiencies. In order to overcome these downsides, strategies for optimizing a novel drug delivery system that has the architecture of liposomes and polymeric characteristics of micelles have been evolved. Polymersomes are vehicles with fluidic cores and hydrophobic shells that are protected and isolated from the aqueous media by the hydrated hydrophilic brushes which give the carrier its distinctive polymeric bilayer shape. Similar to liposomes, this merit enables the carrier to encapsulate a wide range of agents, despite their affinities and solubilities in water. Adding to this, the high molecular weight of the amphiphiles that build the body of the polymersomes increases their colloidal and chemical stabilities and reduces the permeability of the polymeric membranes, which makes the vesicles more protective to the encapsulated drug. These carriers can also be modified in ways that make them responsive when targeted or triggered, by manipulating their composition and attaching moieties and conjugates to the body of the carriers. These appealing characteristics, in addition to the ease of synthesis, gave the polymersomes greater potentials in the area of drug delivery. Thus, their design and characterization, in comparison with liposomes and micelles, are briefly reviewed in this work.

Keywords: controlled release, liposomes, micelles, polymersomes, targeting

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Authors: T. S. Soliman, A. G. Galyas, E. V. Rusinova, S. A. Vshivkov

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The liquid crystals combining properties of a liquid and an anisotropic crystal substance play an important role in a science and engineering. Molecules of cellulose and its derivatives have rigid helical conformation, stabilized by intramolecular hydrogen bonds. Therefore the macromolecules of these polymers are capable to be ordered at dissolution and form liquid crystals of cholesteric type. Phase diagrams of solutions of some cellulose derivatives are known. However, little is known about the effect of a magnetic field on the viscosity of polymer solutions. The systems hydroxypropyl cellulose (HPC) – ethanol, HPC – ethylene glycol, HPC–DМАA, HPC–DMF, ethyl cellulose (EC)–ethanol, EC–DMF, were studied in the presence and absence of magnetic field. The solution viscosity was determined on a Rheotest RN 4.1 rheometer. The effect of a magnetic field on the solution properties was studied with the use of two magnets, which induces a magnetic-field-lines directed perpendicularly and parallel to the rotational axis of a rotor. Application of the magnetic field is shown to be accompanied by an increase in the additional assembly of macromolecules, as is evident from a gain in the radii of light scattering particles. In the presence of a magnetic field, the long chains of macromolecules are oriented in parallel with field lines. Such an orientation is associated with the molecular diamagnetic anisotropy of macromolecules. As a result, supramolecular particles are formed, especially in the vicinity of the region of liquid crystalline phase transition. The magnetic field leads to the increase in viscosity of solutions. The results were used to plot the concentration dependence of η/η0, where η and η0 are the viscosities of solutions in the presence and absence of a magnetic field, respectively. In this case, the values of viscosity corresponding to low shear rates were chosen because the concentration dependence of viscosity at low shear rates is typical for anisotropic systems. In the investigated composition range, the values of η/η0 are described by a curve with a maximum.

Keywords: rheology, liquid crystals, magnetic field, cellulose ethers

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Authors: Jin Young Kim, Kwon Kyoo Kang

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The SGR1 (STAYGREEN1) protein is a critical regulator of plant leaves in chlorophyll degradation and senescence. The functions and mechanisms of tomato SGR1 action are poorly understood and worthy of further investigation. To investigate the function of the SGR1 gene, we generated a SGR1-knockout (KO) null line via clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated gene editing and conducted RNA sequencing and gas chromatography tandem mass spectrometry (GC-MS/MS) analysis to identify the differentially expressed genes. The SlSGR1 (Solanum lycopersicum SGR1) knockout null line clearly showed a turbid brown color with significantly higher chlorophyll and carotenoid content compared to wild-type (WT) fruit. Differential gene expression analysis revealed 728 differentially expressed genes (DEGs) between WT and sgr1 #1-6 line, including 263 and 465 downregulated and upregulated genes, respectively, for which fold change was >2, and the adjusted p-value was <0.05. Most of the DEGs were related to photosynthesis and chloroplast function. In addition, the pigment, carotenoid changes in sgr1 #1-6 line was accumulated of key primary metabolites such as sucrose and its derivatives (fructose, galactinol, raffinose), glycolytic intermediates (glucose, G6P, Fru6P) and tricarboxylic acid cycle (TCA) intermediates (malate and fumarate). Taken together, the transcriptome and metabolite profiles of SGR1-KO lines presented here provide evidence for the mechanisms underlying the effects of SGR1 and molecular pathways involved in chlorophyll degradation and carotenoid biosynthesis.

Keywords: tomato, CRISPR/Cas9, null line, RNA-sequencing, metabolite profiling

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Authors: Sarah Gaßmeyer, Nadine Hülsemann, Raphael Stoll, Kenji Miyamoto, Robert Kourist

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Enzymatic racemization allows the smooth interconversion of stereocenters under very mild reaction conditions. Racemases find frequent applications in deracemization and dynamic kinetic resolutions. Arylmalonate decarboxylase (AMDase) from Bordetella Bronchiseptica has high structural similarity to amino acid racemases. These cofactor-free racemases are able to break chemically strong CH-bonds under mild conditions. The racemase-like catalytic machinery of mutant G74C conveys it a unique activity in the racemisation of pharmacologically relevant derivates of 2-phenylpropionic acid (profenes), which makes AMDase G74C an interesting object for the mechanistic investigation of cofactor-independent racemases. Structure-guided protein engineering achieved a variant of this unique racemase with 40-fold increased activity in the racemisation of several arylaliphatic carboxylic acids. By saturation–transfer–difference NMR spectroscopy (STD-NMR), substrate binding during catalysis was investigated. All atoms of the substrate showed interactions with the enzyme. STD-NMR measurements revealed distinct nuclear Overhauser effects in experiments with and without molecular conversion. The spectroscopic analysis led to the identification of several amino acid residues whose variation increased the activity of G74C. While single-amino acid exchanges increased the activity moderately, structure-guided saturation mutagenesis yielded a quadruple mutant with a 40 times higher reaction rate. This study presents STD-NMR as versatile tool for the analysis of enzyme-substrate interactions in catalytically competent systems and for the guidance of protein engineering.

Keywords: racemase, rational protein design, STD-NMR, structure guided saturation mutagenesis

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Authors: Ivan Zupan, Tomislav Saric, Suzana Tkalcic

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IMUNO-2865® is a commercially available, β–glucan based, natural hemicellulose compound with proven immunostimulative properties in people, domestic and some aquatic animals. During the experimental feeding trial with IMUNO-2865® in juvenile wild-caught chub under laboratory conditions, supplementation resulted in overall higher growth performance for all experimental groups regardless of the concentration of the added compound. The maximum, 5% concentration of the supplement, resulted in highest weight gain and calculated specific growth rate. In sea bream, as economically most important species in the Mediterranean aquaculture, significant increases in numbers of monocytes and heterophils were observed in the group supplemented with 2.5 % of IMUNO-2865® in the feed. An overall increase of erythrocytes was noted by the end of the experiment, although with variable distribution among groups. Blood Ca++ levels, total proteins, and total NH₃ were significantly higher after 60 days of feeding in all treatment groups compared to the control and remained elevated in the treated group following the secession of supplementation. Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and serum paraoxonase PON1 (U/L) showed similar trends. All these parameters are playing a significant role in either oxygen supplementation of tissues, or anabolic and catabolic processes that on molecular levels contribute to the overall health and immune-building capacity of cells and tissues. The complete lack of mortality in sea bream and presented increases in cellular, biochemical and oxidative stress parameters in the blood suggest that the IMUNO-2865® represents a safe dietary supplement for in aquaculture, with an overall positive and potentially immunostimulative effect on farmed fish.

Keywords: IMUNO-2865®, β–glucans, Mediterranean aquaculture, fish imunnostimulans

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Authors: Sruthi Ramagiri, Rajeev Taliyan

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Background and purpose: Recent investigations on ischemia and reperfusion injury postulate that transient ischemia of remote organs after a prolonged ischemic insult confers neuroprotection. However, the molecular mechanisms of the remote limb ischemic post-conditioning (RIPOC) are yet to be elucidated. The current study was designed to investigate the protective mechanism of RIPOC against cerebral ischemic injury using global model of stroke. Materials and methods: Global ischemic reperfusion injury (IR) was achieved by 30 minutes ischemia of cerebral artery, followed by reperfusion for 24 hours. Induction of global ischemia was followed by 4 brief episodes (30 seconds each) of ischemia and reperfusion of femoral artery to accomplish RIPOC. 5-Hydroxy Decanoic acid (5-HD), a KATP channel blocker (20 mg/kg) was administered after induction of global ischemia and RIPOC intervention. Results: IR injury ensue significant behavioural deficits as manifested by rotarod performance and spontaneous locomotor activity when compared to sham control. Furthermore, IR injury significantly increased oxidonitrative stress and infarct volume as evidenced by biochemical parameters (MDA, GSH, Nitrite, SOD) and 2,3,5-triphenyltetrazolium chloride (TTC) staining respectively. Moreover, RIPOC intervention ameliorated the behavioural performance, attenuated the oxidative stress and infarct volume when compared to IR injury group. However, administration of 5-HD increased the oxidative stress and infarct size while deteriorating the behavioural parameters when compared to RIPOC group. Conclusions: In a nutshell, cerebral IR injury has significantly induced the neuronal damage, whereas RIPOC intervention decreased the neuronal injury. Moreover, 5-HD abolished the neuroprotection offered by RIPOC indicating the putative role of KATP channel opening in RIPOC against cerebral ischemic injury.

Keywords: RIPOC, cerebral injury, KATP channel, neuroprotection

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Authors: Mohammad Aladwan, Adelia Skripova

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Bioremediation aspects of crude oil-polluted fields can be achieved by isolation and identification of bacterial species from oil-contaminated soil in order to choose the most active isolates and increase the strength of others. In this study, oil-degrading bacteria were isolated and identified from oil-contaminated soil samples in northeastern Jordan. The bacterial growth count (CFU/g) was between 1.06×10⁵ and 0.75×10⁹. Eighty-two bacterial isolates were characterized by their morphology and biochemical tests. The identified bacterial genera included: Klebsiella, Staphylococcus, Citrobacter, Lactobacillus, Alcaligenes, Pseudomonas, Hafnia, Micrococcus, Rhodococcus, Serratia, Enterobacter, Bacillus, Salmonella, Mycobacterium, Corynebacterium, and Acetobacter. Molecular identification of a universal primer 16S rDNA gene was used to identify four bacterial isolates: Microbacterium esteraromaticum strain L20, Pseudomonas stutzeri strain 13636M, Klebsilla pneumoniae, and uncultured Klebsilla sp., known as new strains. Our results indicate that their specific oil-degrading bacteria isolates might have a high strength of oil degradation from oil-contaminated sites. Staphylococcus intermedius (75%), Corynebacterium xerosis (75%), and Pseudomonas fluorescens (50%) showed a high growth rate on different types of hydrocarbons, such as crude oil, toluene, naphthalene, and hexane. In addition, monooxygenase and catechol 2,3-dioxygenase were detected in 17 bacterial isolates, indicating their superior hydrocarbon degradation potential. Total petroleum hydrocarbons were analyzed using gas chromatography for soil samples. Soil samples M5, M7, and M8 showed the highest levels (43,645, 47,805, and 45,991 ppm, respectively), and M4 had the lowest level (7,514 ppm). All soil samples were analyzed for heavy metal contamination (Cu, Cd, Mn, Zn, and Pb). Site M7 contains the highest levels of Cu, Mn, and Pb, while Site M8 contains the highest levels of Mn and Zn. In the future, these isolates of bacteria can be used for the cleanup of oil-contaminated soil.

Keywords: bioremediation, 16S rDNA gene, oil-degrading bacteria, hydrocarbons

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Authors: Yasir Arafat, Asma Shah, Hua Shao

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Background and Aims: Legume/cereal intercropping is used worldwide for enhancement in biomass and yield of cereal crops. However, because of intercropping, the belowground biological and chemical interactions and their effect on physiological parameters and yield of crops are limited. Methods: Wheat faba bean (WF) intercropping was designed to understand the underlying changes in the soil's chemical environment, soil microbial communities, and effect on growth and yield parameters. Experimental plots were established as having no root partition (NRP), semi-root partition (SRP), complete root partition (CRP), and their sole cropping (CK). Low molecular weight organic acids (LMWOAs) were determined by GC-MS, and high throughput sequencing of the 16S rRNA gene was carried out to screen microbial structure and composition in different root partitions of the WF intercropping system. Results: We show that intercropping induced a shift in the relative abundance of some genera of plant growth promoting rhizobacteria (PGPR) such as Allorhizobium, Neorhizobium, Pararhizobium, and Rhizobium species and resulted in better growth and yield performance of wheat. Moreover, as the plant's distance of wheat from faba beans decreased, the diversity of microbes increased, and a positive effect was observed on physiological traits and crop yield. Furthermore, an abundance and positive correlations of palmitic acid, arachidic acid, stearic acid, and 9-Octadecenoic with PGPR were recorded in the root zone of WF intercropping, which can play an important role in this facilitative mechanism of enhancing growth and yield of cereals. Conclusion: The two treatments clearly affected soil microbial and chemical composition, which can be reflected in growth and yield enhancement.

Keywords: intercropping, microbial community, LMWOAs, PGPR, soil chemical environment

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Authors: Amin Abbasi, Fariborz Shekari, Seyed Bahman Mousavi

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Seed aging during storage is a complex biochemical and physiological processes that can lead to reduce seed germination. This phenomenon associated with increasing of total antioxidant activity during aging. To study the effects of hormones on seed aging, aged wheat seeds (control, 90 and 80% viabilities) were treated with GA3, Salicylic Acid, and paclobutrazol and antioxidant system were investigated as molecular biomarkers for seed vigor. The results showed that, seed priming treatment significantly affected germination percentage, normality seedling percentage, H2O2, MDA, CAT, APX, and GPX activates. Maximum germination percentage achieve in GA3 priming in control treatment. Germination percentage and normal seedling percentage increased in other GA3 priming treatment compared with other hormones. Also aging increased MDA, H2O2 content. MDA is considered sensitive marker commonly used for assessing membrane lipid peroxidation and H2O2result in toxicity to cellular membrane system and damages to plant cells. Amount of H2O2 and MDA declined in GA3 treatment. CAT, GPX and APX activities were reduced by increasing the aging time and at different levels of priming. The highest APX activity was observed in Salicylic Acid control treatment and the highest GPX and CAT activity was obtained in GA3 control treatment. The lowest MDA and H2O2 showed in GA3 control treatment, too. Hormone priming increased Antioxidant enzyme activity and decreased amount of reactive oxygen space and malondialdehyde (MDA) under aging treatment. Also, GA3 priming treatments have a significant effect on germination percentage and number of normal seedling. Generally aging seed, increase ROS and lipid peroxidation. Antioxidant enzymes activity of aged seeds increased after hormone priming.

Keywords: hormones priming, wheat, aging seed, antioxidant, lipid peroxidation

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Authors: Mohammad K. Parvez, Mohammed S. Al-Dosari

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Hepatitis E virus (HEV) is an emerging RNA virus that causes acute and chronic liver disease with a global mortality rate of about 2%. Despite milestone developments in understanding of HEV biology, there is still lack of a robust culture system or animal model. Therefore, in a novel approach, two recombinant-baculoviruses (vBac-ORF2 and vBac-ORF3) that could overexpress HEV ORF2 (structural/capsid) and ORF3 (nonstructural/regulatory) proteins, respectively were constructed. The established HEV-SAR55 (genotype 1) replicon that contained GFP gene, in place of ORF2/ORF3 sequences was in vitro transcribed, and GFP production in RNA transfected S10-3 cells was scored by FACS. Enhanced infectivity, if any, of nascent virions produced by exogenously-supplied ORF2 and viral RNA by co-expression of ORF3 was tested on naïve HepG2 cells. Co-transduction with vBac-ORF2/vBac-ORF3 (108 pfu/microL) produced high amounts of native ORF2/ORF3 in approximately 60% of S10-3 cells, determined by immunofluorescence microscopy and Western analysis. FACS analysis showed about 9% GFP positivity of S10-3 cells on day6 post-transfection (i.e, day5 post-transduction). Further, FACS scoring indicated that lysates from S10-3 cultures receiving the RNA plus vBac-ORF2 were capable of producing HEV particles with about 4% infectivity in HepG2 cells. However, lysates of cultures co-transduced with vBac-ORF3, were found to further enhance virion infectivity by approximately 17%. This supported a previously proposed role of ORF3 as a minor-structural protein in HEV virion assembly and infectivity. In conclusion, the present model for efficient genomic RNA packaging and production of infectious virions could be a valuable tool to study various aspects of HEV molecular biology, in vitro.

Keywords: chronic liver disease, hepatitis E virus, ORF2, ORF3, replicon

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Authors: Tettey Afi Pamela, Sotaro Fujii, Hidetoshi Saito, Kawaii Koichiro

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Introduction: Organisms employ adaptive mechanisms when faced with any stressor or risk of being wiped out. This has made it possible for them to survive in harsh environmental conditions such as increasing temperature, low pH, and anoxia. Some of the mechanisms they utilize include the expression of heat shock proteins, synthesis of cryoprotectants, and anhydrobiosis. Heat shock proteins (HSPs) have been widely studied to determine their involvement in stress tolerance among various organism, of which chironomid species have been no exception. We examined the survival and expression of genes encoding five (5) heat shock proteins (HSP70, HSP67, HSP60, HSP27, and HSP23) from Chironomus sulfurosus larvae reared from 1st instar at 25°C, 30°C, 35°C, and 40°C. Results: The highest survival rate was recorded at 30°C, followed by 25°C, then 35°C. Only a small percentage of C. sulfurosus survived at 40°C (14.5%). With regards to HSPs expression, some HSPs responded to an increase in high temperature. The relative expression levels were lowest at 30°C for HSP70, HSP60, HSP27, and HSP23. At 25°C and 40°C, HSP70, HSP67, HSP60, HSP27, and HSP23 had the highest expression. At 35°C, all had the lowest expression. Discussion: The expression of heat shock proteins varies from one species to another. We designated the genes HSP 70, HSP 67, HSP 60, HSP 27, and HSP 23 genes based on transcriptome analysis of C. sulfurosus. Our study can be termed as a long-heat shock study as C. sulfurosus was reared from the first instar to the fourth instar, and this might have led to a continuous induction of HSPs at 25°C. 40°C had the lowest survival but highest HSPs expression as C. sulfurosus larvae had to utilize HSPs for sustenance. These results and future high-throughput studies at both the transcriptome and proteome level will improve the information needed to predict the future geographic distribution of these species within the context of global warming.

Keywords: chironomid, heat shock proteins, high temperature, heat shock protein expression

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Authors: Michael Josef Schwerer

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Background: The identification of unknown victims from mass disasters, violent crimes, or terrorist attacks is frequently facilitated through information from missing persons lists, portrait photos, old or recent pictures showing unique characteristics of a person such as scars or tattoos, or simply reference samples from blood relatives for DNA analysis. In contrast, the identification or at least the characterization of an unknown perpetrator from criminal or terrorist actions remains challenging, particularly in the absence of material or data for comparison, such as fingerprints, which had been previously stored in criminal records. In scenarios that result in high levels of destruction of the perpetrator’s corpse, for instance, blast or fire events, the chance for a positive identification using standard techniques is further impaired. Objectives: This study shows the forensic genetic procedures in the Legal Medicine Service of the German Air Force for the identification of unknown individuals, including such cases in which reference samples are not available. Scenarios requiring such efforts predominantly involve aircraft crash investigations, which are routinely carried out by the German Air Force Centre of Aerospace Medicine as one of the Institution’s essential missions. Further, casework by military police or military intelligence is supported based on administrative cooperation. In the talk, data from study projects, as well as examples from real casework, will be demonstrated and discussed with the audience. Methods: Forensic genetic identification in our laboratories involves the analysis of Short Tandem Repeats and Single Nucleotide Polymorphisms in nuclear DNA along with mitochondrial DNA haplotyping. Extended DNA analysis involves phenotypic markers for skin, hair, and eye color together with the investigation of a person’s biogeographic ancestry. Assessment of the biological age of an individual employs CpG-island methylation analysis using bisulfite-converted DNA. Forensic Investigative Genealogy assessment allows the detection of an unknown person’s blood relatives in reference databases. Technically, end-point-PCR, real-time PCR, capillary electrophoresis, pyrosequencing as well as next generation sequencing using flow-cell-based and chip-based systems are used. Results and Discussion: Optimization of DNA extraction from various sources, including difficult matrixes like formalin-fixed, paraffin-embedded tissues, degraded specimens from decomposed bodies or from decedents exposed to blast or fire events, provides soil for successful PCR amplification and subsequent genetic profiling. For cases with extremely low yields of extracted DNA, whole genome preamplification protocols are successfully used, particularly regarding genetic phenotyping. Improved primer design for CpG-methylation analysis, together with validated sampling strategies for the analyzed substrates from, e.g., lymphocyte-rich organs, allows successful biological age estimation even in bodies with highly degraded tissue material. Conclusions: Successful identification of unknown individuals or at least their phenotypic characterization using pigmentation markers together with age-informative methylation profiles, possibly supplemented by family tree search employing Forensic Investigative Genealogy, can be provided in specialized laboratories. However, standard laboratory procedures must be adapted to work with difficult and highly degraded sample materials.

Keywords: identification, forensic genetics, phenotypic markers, CPG methylation, biological age estimation, forensic investigative genealogy

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Authors: K. Nikovics, D. Riccobono, M. Oger, H. Morin, L. Barbier, T. Poyot, X. Holy, A. Bendahmane, M. Drouet, A. L. Favier

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Muscle regeneration after injury (as irradiation) is of great importance. However, the molecular and cellular mechanisms are still unclear. Cytokines are believed to play fundamental role in the different stages of muscle regeneration. They are secreted by many cell populations, but the predominant producers are macrophages and helper T cells. On the other hand, it has been shown that adipose tissue derived stromal/stem cell (ASC) injection could improve muscle regeneration. Stem cells probably induce the coordinated modulations of gene expression in different macrophage cells. Therefore, we investigated the patterns and timing of changes in gene expression of different cytokines occurring upon stem cells loading. Muscle regeneration was studied in an irradiated muscle of minipig animal model in presence or absence of ASC treatment (irradiated and treated with ASCs, IRR+ASC; irradiated not-treated with ASCs, IRR; and non-irradiated no-IRR). We characterized macrophage populations by immunolabeling in the different conditions. In our study, we found mostly M2 and a few M1 macrophages in the IRR+ASC samples. However, only few M2b macrophages were noticed in the IRR muscles. In addition, we found intensive fibrosis in the IRR samples. With in situ hybridization and immunolabeling, we analyzed the cytokine expression of the different macrophages and we showed that M2d macrophage are the most abundant in the IRR+ASC samples. By in situ hybridization, strong expression of the transforming growth factor β (TGF-β) was observed in the IRR+ASC but very week in the IRR samples. But when we analyzed TGF-β level with immunolabeling the expression was very different: many M2 macrophages showed week expression in IRR+ASC and few cells expressing stronger level in IRR muscles. Therefore, we investigated the MMP expressions in the different muscles. Our data showed that the M2 macrophages of the IRR+ASC muscle expressed MMP2 proteins. Our working hypothesis is that MMP2 expression of the M2 macrophages can decrease fibrosis in the IRR+ASC muscle by capturing TGF-β.

Keywords: adipose tissue derived stromal/stem cell, cytokine, macrophage, muscle regeneration

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Authors: Hao Yang, Lei Chen, Ting Mei, Jianbang Zheng

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Currently, the uncooled PbSe photodetectors in the mid-infrared range (2-5μm) with sensitization technology extract more photoelectric response than traditional ones, and enable the room temperature (300K) photo-detection with high detectivity, which have attracted wide attentions in many fields. This technology generally contains the film fabrication with vapor phase deposition (VPD) and a sensitizing process with doping of oxygen and iodine. Many works presented in the recent years almost provide and high temperature activation method with oxygen/iodine vapor diffusion, which reveals that oxygen or iodine plays an important role in the sensitization of PbSe material. In this paper, we provide our latest experimental results and discussions in the stoichiometry of oxygen and iodine and its influence on the polycrystalline structure and photo-response. The experimental results revealed that crystal orientation was transformed from (200) to (420) by sensitization, and the responsivity of 5.42 A/W was gained by the optimal stoichiometry of oxygen and iodine with molecular density of I2 of ~1.51×1012 mm-3 and oxygen pressure of ~1Mpa. We verified that I2 plays a role in transporting oxygen into the lattice of crystal, which is actually not its major role. It is revealed that samples sensitized with iodine transform atomic proportion of Pb from 34.5% to 25.0% compared with samples without iodine from XPS data, which result in the proportion of about 1:1 between Pb and Se atoms by sublimation of PbI2 during sensitization process, and Pb/Se atomic proportion is controlled by I/O atomic proportion in the polycrystalline grains, which is very an important factor for improving responsivity of uncooled PbSe photodetector. Moreover, a novel sensitization and dopant activation method is proposed using oxygen ion implantation with low ion energy of < 500eV and beam current of ~120μA/cm2. These results may be helpful to understanding the sensitization mechanism of polycrystalline lead salt materials.

Keywords: polycrystalline PbSe, sensitization, transport, stoichiometry

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Authors: Abdolreza Farhadian, Anh Phan, Zahra Taheri Rizi, Elaheh Sadeh

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The utilization of solidified gas technology based on hydrates exhibits considerable promise for carbon capture, storage, and natural gas transportation applications. The pivotal factor impeding the industrial implementation of hydrates lies in the need for efficient and non-foaming promoters. In this study, a biosurfactant with sulfonate, amide, and carboxyl groups (BS) was synthesized as a methane hydrate formation promoter, replicating the chemical characteristics of amino acids and sodium dodecyl sulfate (SDS). The synthesis of BS was achieved using an eco-friendly and three-step process. The first two steps were solvent-free, while a water-isopropanol mixture was utilized in the final step. High-pressure autoclave experiments demonstrated a significant enhancement in methane hydrate formation kinetics with low BS concentrations. 50 ppm of BS yielded a maximum water-to-hydrate conversion of 66.9%, equivalent to a storage capacity of 119.9 v/v in distilled water. With increasing BS concentration to 500 ppm, the conversion degree and storage capacity reached 97% and 162.6 v/v, respectively. Molecular dynamic simulation revealed that BS molecules acted as collectors for methane molecules, augmenting hydrate growth rate and increasing the number of hydrate cavities. Additionally, BS demonstrated a biodegradability exceeding 60% within 28 days. Toxicity assessments confirmed BS's biocompatibility, with cell viability above 70% for skin and lung cells at concentrations up to 160 and 80 µg/mL, respectively. BS showed significant potential as an environmentally friendly alternative to synthetic surfactants like SDS for methane storage. These findings suggest that the synthesis of effective, such as BS, holds promise for diverse applications, including seawater desalination, carbon capture, and gas storage.

Keywords: solidified methane, gas storage, gas hydrates, green surfactant, gas hydrate promoter, computational simulation, sustainability

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Authors: Neslihan Ozman Say, Jim Gilmour, Pratik Desai, William Zimmerman

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Landfill leachate treatment has been attracting researchers recently for various environmental and economical reasons. Leachate discharge to receiving waterbodies without treatment causes serious detrimental effects including partial oxygen depletion due to high biological oxygen demand (BOD) and chemical oxygen demand (COD) concentrations besides toxicity of heavy metals it contains and high ammonia concentrations. In this study, it is aimed to show microalgal ammonia removal performances of a wild microalgae consortia as an alternative treatment method and determine the dominant leachate tolerant species for this consortia. For the microalgae species identification experiments a microalgal consortium which has been isolated from a local pond in Sheffield inoculated in %5 diluted raw landfill leachate and acclimated to the leachate by batch feeding for a month. In order to determine the most tolerant microalgal consortium, four different untreated landfill leachate samples have been used as diluted in four different ratios as 5%, 10%, 20%, and 40%. Microalgae cell samples have been collected from all experiment sets and have been examined by using 18S rDNA sequencing and specialised gel electrophoresis which are adapted molecular biodiversity methods. The best leachate tolerant algal consortium is being used in order to determine ammonia removal performances of the culture in a microbubble assisted photobioreactor (PBR). A porous microbubble diffuser which is supported by a fluidic oscillator is being used for dosing CO₂ and air mixture in the PBR. It is known that high mass transfer performance of microbubble technology provides a better removal efficiency and a better mixing in the photobioreactor. Ammonia concentrations and microalgal growth are being monitored for PBR currently. It is aimed to present all the results of the study in final paper submission.

Keywords: ammonia removal from leachate, landfill leachate treatment, microalgae species identification, microbubble assisted photobioreactors

Procedia PDF Downloads 160

Authors: Moses Ikechukwu Benjamin

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The continued increase in methicillin-resistant Staphylococcuspseudintermedius (MRSP) among dogs and the zoonotic transmission event of MRSP from dogs to humans threaten veterinary medicine and public health. The cardinal objective of this study was to determine the antibiogram and frequency of toxingenes in MRSP obtained from shelter dogs with skin infections and dog owners in Abakaliki, Eastern Nigeria. Skinswabs from 61 shelter dogs with skin infections and 33 nasal swabs from dog owners were processed and analyzed using standard microbiological techniques. Susceptibility to antibiotics was determined by Kirby Bauer disc diffusion technique. The screening for Seccanine, lukD, siet, and exitoxin genes was carried out by PCR. A total of 23 (37.7 %) and 1 (3 %) MRSP strains were obtained from shelter dogs and dog owners, respectively. Generally, isolates exhibited high resistance to amoxicillin-clavulanic acid, ceftazidime, and cefepime (100 % - 66.7 %) but were very susceptible (100 % - 70.7 %) to chloramphenicol and doripenem. The only isolate from dog owners harbouredseccanine, lukD, and siet toxin genes while solatesfrom shelter dogs harbouredseccanine16 (69.6 %), lukD 17 (73.9 %), siet 20 (87 %), and exi1 (4.4 %) toxin genes. Isolates were generally observed to be more resistant than other reports from the literature. Interesting, there was a similarity in the resistance antibiotypes and frequency of toxin genes harboured by MRSP isolates between shelter dogs with skin infections and their owner in a sampled household, thus suggesting a likely zoonotic transmission event. This report of the occurrence of MRSP and high frequency of toxin genes (Seccanine,lukD, and siet) in shelter dogs and dog owners represent a major challenge, especially in terms of antibiotic therapy, and is a serious concern for both animal and public health.

Keywords: methicillin-resistant S. pseudintermedius, zoonotic transmission, antibiotic resistance, companion dogs, toxin genes

Procedia PDF Downloads 162

Authors: Marzia Bilkiss, Muhammad J. A. Shiddiky, Mostafa K. Masud, Prabhakaran Sambasivam, Ido Bar, Jeremy Brownlie, Rebecca Ford

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A greater achievement in the Integrated Disease Management (IDM) to prevent the loss would result from early diagnosis and quantitation of the causal pathogen species for accurate and timely disease control. This could significantly reduce costs to the growers and reduce any flow on impacts to the environment from excessive chemical spraying. Necrotrophic fungal disease botrytis grey mould, caused by Botrytis cinerea and Botrytis fabae, significantly reduce temperate legume yield and grain quality during favourable environmental condition in Australia and worldwide. Several immunogenic and molecular probe-type protocols have been developed for their diagnosis, but these have varying levels of species-specificity, sensitivity, and consequent usefulness within the paddock. To substantially improve speed, accuracy, and sensitivity, advanced nanoparticle-based biosensor approaches have been developed. For this, two sets of primers were designed for both Botrytis cinerea and Botrytis fabae which have shown the species specificity with initial sensitivity of two genomic copies/µl in pure fungal backgrounds using multiplexed quantitative PCR. During further validation, quantitative PCR detected 100 spores on artificially infected legume leaves. Simultaneously an electro-catalytic assay was developed for both target fungal DNA using functionalised magnetic nanoparticles. This was extremely sensitive, able to detect a single spore within a raw total plant nucleic acid extract background. We believe that the translation of this technology to the field will enable quantitative assessment of pathogen load for future accurate decision support of informed botrytis grey mould management.

Keywords: biosensor, botrytis grey mould, sensitive, species specific

Procedia PDF Downloads 173

Authors: Yu-Chieh Chen, Li-Yun Wang, Chi-Chih Chen, Huy Hùng Đào, Ya-Mei Chen, Ming-Chu Cheng, Wen-Bin Chung, Hso-Chi Chaung, Guan-Ming Ke

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Many recent researches have demonstrated that CD154, a protein primarily expressed on activated T cell molecules, has potentially acted as a molecular adjuvant to improve the immunogenicity of subunit vaccines against viral infections. Classical swine fever (CSF) affects the swine industry worldwide that is one of the most devastating and highly contagious pig diseases. It is listed by the World Organization for Animal Health (OIE) as an infectious animal disease that must be reported. Although pigs vaccinated with subunit vaccines can be differentially diagnosed from those infected animals, subunit vaccines usually need adjuvants to enhance and elicit immune responses. In this study, CD154 was linked with CSFV E2 sequences and then expressed in CHO cells to produce the fusion protein as E2-CD154. The porcine specific CpG adjuvant was also used in one of the formulations. The specific pathogen-free pigs (SPF) at the age of 4-week-old were randomly separated into four groups, vaccinated with E2-CpG, E2-CD154, E2-CD154-CpG or the commercial Bayovac® CSF-E2 vaccine and boosted two weeks after primary vaccination. The results showed that the percentages of CD4+ and CD4+IL2+ in peripheral blood mononuclear cells (PBMC) in E2-CD154 vaccinated piglets seven days after primary vaccination were gained by 1-5% relative to the control group. In addition, the percentages of CD4+IFNγ+ T cells had slightly edged up 0.1-0.3% compared with the control group. Also, increased E2-specific IFNγ levels had edged up CD4+CD8+ T cells found in E2-CD154 and E2-CD154-CpG groups, particularly in the E2-CD154-CpG group. These results implicate that CD154 may enhance cellular immunity and synergistically act with species-specific CpG adjuvant as a dual-phase adjuvant. Therefore, the CD154 may be beneficial as a promising adjuvant in subunit vaccines.

Keywords: CD154, CpG adjuvant, cellular immunity, subunit vaccine, pig

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Authors: Barbara Zapala, Marek Dziechciowski, Olaf Chmura, Monika Piwowar, Katarzyna Gawlik, Dorota Pawlicka-Gosiewska, Krzysztof Skotniczny, Bogdan Solnica, Kazimierz Pitynski

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BACKGROUND: Endometrial cancer is one of the most common gynecologic malignancy in developed countries.We hypothesized that amount of circulating micro-particles in blood may be connected with the development of endometrial hyperplasia and endometrial cancer. The aim of this study was to measure the micro-particles amount in uterine venous blood and in peripheral venous blood in women with atypical endometrial hyperplasia and endometrioid endometrial cancer. MATERIALS AND METHODS: By using flow cytometry (BD Canto II cytometer) we measured micro-particles amount in citrate plasma samples from peripheral and uterine venous blood of women with atypical hyperplasia of endometrium or endometrial cancer. We determined the amount of total (TF+), endothelial (CD144+) and monocytic (CD14+) micro- particles. RESULTS: Here we show statistically significant higher micro-particle levels in women with atypical hyperplasia of endometrium or endometrial cancer in comparison to healthy women. Performing measurements of the amounts of total, endothelial and monocytic microparticles allow for reliable differentiation between healthy, atypical hyperplasia and endometrial cancer groups. In blood samples from uterine veins the circulating micro-particle levels were significantly different from peripheral blood samples. The micro-particle levels in uterine blood samples were 7-fold higher than in those from peripheral blood of women with both atypical hyperplasia of endometrium and endometrial cancer when compared to the control group of healthy women. CONCLUSION: These results strongly suggested that the level of circulating micro-particles may be a sign of endometrial cancer development, however the detailed study is needed focusing on molecular processes passed through this small circulating molecules.

Keywords: endometrial cancer, endometrial hyperplasia, microvesicles, uterine blood

Procedia PDF Downloads 135

Authors: S. Redjala, N. Ait Hocine, M. Gratton, N. Poirot, R. Ferhoum, S. Azem

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Polycarbonate (PC) is a promising polymer with high transparency in the range of the visible spectrum and is used in various fields, for example medical, electronic, automotive. Its low weight, chemical inertia, high impact resistance and relatively low cost are of major importance. In recent decades, some materials such as metals and ceramics have been replaced by polymers because of their superior advantages. However, some characteristics of the polymers are highly modified under the effect of ultraviolet (UV) radiation and temperature. The changes induced in the material by such aging depend on the exposure time, the wavelength of the UV radiation and the temperature level. The UV energy is sufficient to break the chemical bonds leading to a cleavage of the molecular chains. This causes changes in the mechanical, thermal, optical and morphological properties of the material. The present work is focused on the study of the effects of aging under ultraviolet (UV) radiation and under different temperature values on the physical-chemical and mechanical properties of a PC. Thus, various investigations, such as FTIR and XRD analyses, SEM and optical microscopy observations, micro-hardness measurements and monotonic and cyclic tensile tests, were carried out on the PC in the initial state and after aging. Results have shown the impact of aging on the properties of the PC studied. In fact, the MEB highlighted changes in the superficial morphology of the material by the presence of cracks and material de-bonding in the form of debris. The FTIR spectra reveal an attenuation of the peaks like the hydroxyl (OH) groups located at 3520 cm-1. The XRD lines shift towards a larger angle, reaching a maximum of 3°. In addition, Vickers micro-hardness measurements show that aging affects the surface and the core of the material, which results in different mechanical behaviours under monotonic and cyclic tensile tests. This study pointed out effects of aging on the macroscopic properties of the PC studied, in relationship with its microstructural changes.

Keywords: mechanical properties, physical-chemical properties, polycarbonate, UV aging, temperature aging

Procedia PDF Downloads 142

Authors: Daina Jonkus, Solvita Petrovska, Dace Smiltina, Lasma Cielava

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The beta-lactoglobulin and kappa-casein are milk proteins which are important for milk composition. Cows with beta-lactoglobulin and kappa-casein gene BB genotypes have highest milk crude protein and fat content. The aim of the study was to determinate the frequencies of milk protein gene polymorphisms in local Latvian Brown (LB) cows breed and analyze the influence of beta-lactoglobulin and kappa-casein genotypes to milk productivity traits. 102 cows’ genotypes of milk protein genes were detected using Polymerase Chain Reaction and Restriction Fragment Length Polymorphism (PCR-RFLP) and electrophoresis on 3% agarose gel. For beta-lactoglobulin were observed 2 types of alleles A and B and for kappa-casein 3 types: A, B and E. Highest frequency in beta-lactoglobulin gene was observed for B allele – 0.926. Molecular analysis of beta-lactoglobulin gene shows 86.3% of individuals are homozygous by B allele and animals are with genotypes BB and 12.7% of individuals are heterozygous with genotypes AB. The highest milk yield 4711.7 kg was for 1st lactation cows with AB genotypes, whereas the highest milk protein content (3.35%) and fat content (4.46 %) was for BB genotypes. Analysis of the kappa-casein locus showed a prevalence of the A allele – 0.750. The genetic variant of B was characterized by a low frequency – 0.240. Moreover, the frequency of E occurred in the LB cows’ population with very low frequency – 0.010. 54.9 % of cows are homozygous with genotypes AA, and only 4.9 % are homozygous with genotypes BB. 32.8 % of individuals are heterozygous with genotypes AB, and 2.0 % are with AE. The highest milk productivity was for 1st lactation cows with AB genotypes: milk yield 4620.3 kg, milk protein content 3.39% and fat content 4.53 %. According to the results, in local Latvian brown there are only 2.9% of cows are with BB-BB genotypes, which is related to milk coagulation ability and affected cheese production yield. Acknowledgment: the investigation is supported by VPP 2014-2017 AgroBioRes Project No. 3 LIVESTOCK.

Keywords: beta-lactoglobulin, cows, genotype frequencies, kappa-casein

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Authors: Md. Z. Alam, Amal A. Elgharbawy, Muhammad Moniruzzaman, Nassereldeen A. Kabbashi, Parveen Jamal

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The enzymatic hydrolysis of lignocellulosic biomass is one of the obstacles in the process of sugar production, due to the presence of lignin that protects the cellulose molecules against cellulases. Although the pretreatment of lignocellulose in ionic liquid (IL) system has been receiving a lot of interest; however, it requires IL removal with an anti-solvent in order to proceed with the enzymatic hydrolysis. At this point, introducing a compatible cellulase enzyme seems more efficient in this process. A cellulase enzyme that was produced by Trichoderma reesei on palm kernel cake (PKC) exhibited a promising stability in several ILs. The enzyme called PKC-Cel was tested for its optimum pH and temperature as well as its molecular weight. One among evaluated ILs, 1,3-diethylimidazolium dimethyl phosphate [DEMIM] DMP was applied in this study. Evaluation of six factors was executed in Stat-Ease Design Expert V.9, definitive screening design, which are IL/ buffer ratio, temperature, hydrolysis retention time, biomass loading, cellulase loading and empty fruit bunches (EFB) particle size. According to the obtained data, IL-enzyme system shows the highest sugar concentration at 70 °C, 27 hours, 10% IL-buffer, 35% biomass loading, 60 Units/g cellulase and 200 μm particle size. As concluded from the obtained data, not only the PKC-Cel was stable in the presence of the IL, also it was actually stable at a higher temperature than its optimum one. The reducing sugar obtained was 53.468±4.58 g/L which was equivalent to 0.3055 g reducing sugar/g EFB. This approach opens an insight for more studies in order to understand the actual effect of ILs on cellulases and their interactions in the aqueous system. It could also benefit in an efficient production of bioethanol from lignocellulosic biomass.

Keywords: cellulase, hydrolysis, lignocellulose, pretreatment

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Authors: Fadime Eroglu

Abstract:

Acanthamoeba genus belongs to kingdom protozoa, and it is known as free-living amoebae. Acanthamoeba genus has been isolated from human bodies, swimming pools, bottled mineral water, contact lens solutions, dust, and soil. The members of the genus Acanthamoeba causes Acanthamoeba Keratitis which is a painful sight-threatening disease of the eyes. In recent years, the prevalence of Acanthamoeba keratitis has been high rate reported. The eight different Acanthamoeba species are known to be effective in Acanthamoeba keratitis. These species are Acanthamoeba castellanii, Acanthamoeba polyphaga, Acanthamoeba griffini, Acanthamoeba hatchetti, Acanthamoeba culbertsoni and Acanhtamoeba rhysodes. The conventional diagnosis of Acanthamoeba Keratitis has relied on cytological preparations and growth of Acanthamoeba in culture. However molecular methods such as real-time PCR has been found to be more sensitive. The real-time PCR has now emerged as an effective method for more rapid testing for the diagnosis of infectious disease in decade. Therefore, a real-time PCR assay for the detection of Acanthamoeba keratitis and Acanthamoeba species have been developed in this study. The 18S rRNA sequences from Acanthamoeba species were obtained from National Center for Biotechnology Information and sequences were aligned with MEGA 6 programme. Primers and probe were designed using Custom Primers-OligoPerfectTMDesigner (ThermoFisherScientific, Waltham, MA, USA). They were also assayed for hairpin formation and degree of primer-dimer formation with Multiple Primer Analyzer ( ThermoFisherScientific, Watham, MA, USA). The eight different ATCC Acanthamoeba species were obtained, and DNA was extracted using the Qiagen Mini DNA extraction kit (Qiagen, Hilden, Germany). The DNA of Acanthamoeba species were analyzed using newly designed primer and probe set in real-time PCR assay. The early definitive laboratory diagnosis of Acanthamoeba Keratitis and the rapid initiation of suitable therapy is necessary for clinical prognosis. The results of the study have been showed that new primer and probes could be used for detection and distinguish for Acanthamoeba species. These new developing methods are helpful for diagnosis of Acanthamoeba Keratitis.

Keywords: Acathamoeba Keratitis, Acanthamoeba species, fast diagnosis, Real-Time PCR

Procedia PDF Downloads 119

Authors: Gargi Ghoshal, Ashay Jain, Deepika Thakur, U. S. Shivhare, O. P. Katare

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The main objectives of this work were the rheological characterization of dispersions, emulsions at different pH used in the microcapsules preparation and the microcapsules obtain from gum arabic (A), guar gum (G), casein (C) and whey protein isolate (W) to keep β-carotene protected from degradation using the complex coacervation microencapsulation technique (CCM). The evaluation of rheological properties of dispersions, emulsions of different pH and so obtained microencapsules manifest the changes occur in the molecular structure of wall materials during the encapsulation process of β-carotene. These dispersions, emulsions of different pH and formulated microencapsules were subjected to go through various conducted experiments (flow curve test, amplitude sweep, and frequency sweep test) using controlled stress dynamic rheometer. Flow properties were evaluated as a function of apparent viscosity under steady shear rate ranging from 0.1 to 100 s-1. The frequency sweep test was conducted to determine the extent of viscosity and elasticity present in the samples at constant strain under changing angular frequency range from 0.1 to 100 rad/s at 25ºC. The dispersions and emulsion exhibited a shear thinning non-Newtonian behavior whereas microencapsules are considered as shear-thickening respectively. The apparent viscosity for dispersion, emulsions were decreased at low shear rates 20 s-1 and for microencapsules, it decreases up to ~50 s-1 besides these value, it has shown constant pattern. Oscillatory shear experiments showed a predominant viscous liquid behavior up to crossover frequencies of dispersions of C, W, A at 49.47 rad/s, 57.60 rad/s and 21.45 rad/s emulsion sample of AW at pH 5.0 it was 17.85 rad/s and GW microencapsules 61.40 rad/s respectively whereas no such crossover was found in G dispersion, emulsion with C and microencapsules still it showed more viscous behavior. Storage and loss modulus decreases with time also a shift of the crossover towards lower frequencies for A, W and C was observed respectively. However, their microencapsules showed more viscous behavior as compared to samples prior to blending.

Keywords: viscosity, gums, proteins, frequency sweep test, apparent viscosity

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