Search results for: enzymatic polymerization

Authors: S. Thangapandiyan, S. Miltonprabu

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Fl (Fl) exposure engenders neurodegeneration and induces oxidative stress in the brain. The Neuroprotective role of EGCG on oxidative stress-mediated neurotoxicity in Fl intoxicated rat hippocampus has not yet been explored so far. Hence, the present study is focused on witnessing whether EGCG (40mg/kg) supplementation prevents Fl induced oxidative stress in the brain of rats with special emphasis on the hippocampus. Fl (25mg/kg) intoxication for four weeks in rats showed an increase in Fl concentration along with the decrease the AChE, NP, DA, and 5-HT activity in the brain. The oxidative stress markers (ROS, TBARS, NO, and PC) were significantly increased with decreased enzymatic (SOD, CAT, GPx, GR, GST, and G6PD) and non-enzymatic antioxidants (GSH, TSH, and Vit.C) in Fl intoxicated rat hippocampus. Moreover, Fl intoxicated rats exhibited an intrinsic and extrinsic pathway mediated apoptosis in the hippocampus of rats. Fl intoxication significantly increased the DNA damage as evidenced by increased DNA fragmentation. Furthermore, the toxic impact of Fl on hippocampus was also proved by the immunohistochemical, histological, and ultrastructural studies. Pre-administration of EGCG has significantly protected the Fl induced oxidative stress, biochemical changes, cellular apoptotic, and histological alternations in the hippocampus of rats. In conclusion, EGCG supplementation significantly attenuated the Fl induced oxidative stress mediated neurotoxicity via its free radical scavenging and antioxidant activity.

Keywords: brain, hippocampal, NaF, ROS, EGCG

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Authors: Friday A. Ogori, Ojotu M. Eke, Oneh J. Abu, Abraham T. Girgih

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B.aeqygtiaca del (Balanites aegyptiaca del) seed protein concentrate (APC) was hydrolyzed using different enzymes such as pepsin+pancreatin (PP), Alcalase (Alca), and Flavourzyme (Flav). The Alca has higher yield (100%) when compared to PP (83.23%) and Flav (62.90%). The hydrophobic amino acid and Sulphur containing amino acid (40.19%, 7.04%) in PP hydrolysate were higher compared to Alcalase (38.92%, 6.69%), Flavourenzyme (37.43%,6.35%), and APC (39.97%, 6.95%) samples. The PP has stronger DPPH, Hydroxyl radical quenching, Ferric reducing activity, and linoleic acid peroxidation activity, followed by the protein concentrate (APC) and Alcalase (Alca), while Flavourenzyme (Flav) derived hydrolysate was least in scavenging and inhibiting radical peroxidation properties. Flavourenzyme derived hydrolysate had stronger Ferric reducing antioxidant potential and metal chelating property. The result showed that the Alcalase hydrolysate has promising peptide yield, and PP hydrolysate had excellent amino acid residues and good in-vitro antioxidant potentials and could be a preferred ingredients in the nutraceutical and functional food emerging industries.

Keywords: balanites aegyptiaca del, protein concentrate, protein hydrolysates, enzymatic hydrolysis, antioxidants

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Authors: M. Naveed Yasin, Robert Brooke, Andrew Chan, Geoffrey I. N. Waterhouse, Drew Evans, Darren Svirskis, Ilva D. Rupenthal

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Poly(3,4-ethylenedioxythiophene) (PEDOT) is a robust conducting polymer (CP) exhibiting high conductivity and environmental stability. It can be synthesized by either chemical, electrochemical or vapour phase polymerization (VPP). Dexamethasone sodium phosphate (dexP) is an anionic drug molecule which has previously been loaded onto PEDOT as a dopant via electrochemical polymerisation; however this technique requires conductive surfaces from which polymerization is initiated. On the other hand, VPP produces highly organized biocompatible CP structures while polymerization can be achieved onto a range of surfaces with a relatively straight forward scale-up process. Following VPP of PEDOT, dexP can be loaded and subsequently released via ion-exchange. This study aimed at preparing and characterising both non-porous and porous VPP PEDOT structures including examining drug loading and release via ion-exchange. Porous PEDOT structures were prepared by first depositing a sacrificial polystyrene (PS) colloidal template on a substrate, heat curing this deposition and then spin coating it with the oxidant solution (iron tosylate) at 1500 rpm for 20 sec. VPP of both porous and non-porous PEDOT was achieved by exposing to monomer vapours in a vacuum oven at 40 mbar and 40 °C for 3 hrs. Non-porous structures were prepared similarly on the same substrate but without any sacrificial template. Surface morphology, compositions and behaviour were then characterized by atomic force microscopy (AFM), scanning electron microscopy (SEM), x-ray photoelectron spectroscopy (XPS) and cyclic voltammetry (CV) respectively. Drug loading was achieved by 50 CV cycles in a 0.1 M dexP aqueous solution. For drug release, each sample was exposed to 20 mL of phosphate buffer saline (PBS) placed in a water bath operating at 37 °C and 100 rpm. Film was stimulated (continuous pulse of ± 1 V at 0.5 Hz for 17 mins) while immersed into PBS. Samples were collected at 1, 2, 6, 23, 24, 26 and 27 hrs and were analysed for dexP by high performance liquid chromatography (HPLC Agilent 1200 series). AFM and SEM revealed the honey comb nature of prepared porous structures. XPS data showed the elemental composition of the dexP loaded film surface, which related well with that of PEDOT and also showed that one dexP molecule was present per almost three EDOT monomer units. The reproducible electroactive nature was shown by several cycles of reduction and oxidation via CV. Drug release revealed success in drug loading via ion-exchange, with stimulated porous and non-porous structures exhibiting a proof of concept burst release upon application of an electrical stimulus. A similar drug release pattern was observed for porous and non-porous structures without any significant statistical difference, possibly due to the thin nature of these structures. To our knowledge, this is the first report to explore the potential of VPP prepared PEDOT for stimuli-responsive drug delivery via ion-exchange. The produced porous structures were ordered and highly porous as indicated by AFM and SEM. These porous structures exhibited good electroactivity as shown by CV. Future work will investigate porous structures as nano-reservoirs to increase drug loading while sealing these structures to minimize spontaneous drug leakage.

Keywords: PEDOT for ion-exchange drug delivery, stimuli-responsive drug delivery, template based porous PEDOT structures, vapour phase polymerization of PEDOT

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Authors: Parastoo Motallebi, Vahid Niknam, Hassan Ebrahimzadeh, Majid Hashemi

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Wheat, the most strategically important worldwide crop, is widely grown in various countries. Based on international wheat production statistics (FAOSTAT database), the total production of wheat in 2012 was 13.8 in Iran. Fusarium culmorum is one of the principal causative agents of Fusarium crown rot (FCR), an overwhelming disease of wheat and barley which is in the early stages causing yield losses, stand reductions and rotting of root and lower stem tissues. In this study inoculation of two wheat seedlings of the susceptible cultivar Falat and the partially field-resistant cultivar Pishtaz were carried out in greenhouse conditions and root samples were taken for 6 days. The activity of peroxidase (POX) and polyphenoloxidase (PPO) enzymes were analyzed to identify possible relations between resistance and enzymatic activities. Although the POX and PPO activities in both geno types increased, this significant increase was more dominant in Pishtaz. The results showed an earlier elevation in the activity of these defense related enzymes in semi-resistant cv. Pishtaz after inoculation, suggested that the activities of POX and PPO in wheat geno types play an important role in the induction of resistance to this disease.

Keywords: Defense responses, Fusarium culmorum, Wheat

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Authors: Patsarawadee Paojinda, Waranya Imprasittichai, Sudaratana R. Krungkrai, Nirianne Marie Q. Palacpac, Toshihiro Horii, Jerapan Krungkrai

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Fusion of the last two enzymes in the pyrimidine biosynthetic pathway in the inversed order by having COOH-terminal orotate phosphoribosyltransferase (OPRT) and NH2-terminal orotidine 5'-monophosphate decarboxylase (OMPDC), as OMPDC-OPRT, are described in many organisms. Here, we produced gene fusions of Plasmodium falciparum OMPDC-OPRT and expressed the bifunctional protein in Escherichia coli. The enzyme was purified to homogeneity using affinity and anion-exchange chromatography, exhibited enzymatic activities and functioned as a dimer. The activities, although unstable, can be stabilized by its substrate and product during purification and long-term storage. Furthermore, the enzyme expressed a perfect catalytic efficiency (kcat/Km). The kcat was selectively enhanced up to 3 orders of magnitude, while the Km was not much affected and remained at low µM levels when compared to the monofunctional enzymes. The fusion of the two enzymes, creating a “super-enzyme” with perfect catalytic power and more flexibility, reflects cryptic relationship of enzymatic reactivaties and metabolic functions on molecular evolution.

Keywords: bifunctional enzyme, orotate phosphoribosyltransferase, orotidine 5'-monophosphate decarboxylase, plasmodium falciparum

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Authors: Masni Mat Yusoff, Michael H. Gordon, Keshavan Niranjan

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The study reports on the effect of aqueous enzymatic extraction (AEE) parameters on the Moringa oleifera (MO) oil yield and the formation of emulsion at the end of the process. A mixture of protease and cellulase enzymes was used at 3:1 (w/w) ratio. The highest oil yield of 19% (g oil/g sample) was recovered with the use of a mixture of pH 6, 1:4 material/moisture ratio, and incubation temperature, time, and shaking speed of 50 ⁰C, 12.5 hr, and 300 stroke/min, respectively. The use of pH 6 and 8 resulted in grain emulsions, while solid-intact emulsion was observed at pH 4. Upon fixing certain parameters, higher oil yield was extracted with the use of lower material/moisture ratio and higher shaking speed. Longer incubation time of 24 hr resulted in significantly (p < 0.05) similar oil yield with that of 12.5 hr, and an incubation temperature of 50 ⁰C resulted in significantly (p < 0.05) higher oil yield than that of 60 ⁰C. In overall, each AEE parameter showed significant effects on both the MO oil yields and the emulsions formed. One of the major disadvantages of an AEE process is the formation of emulsions which require further de-emulsification step for higher oil recovery. Therefore, critical studies on the effect of each AEE parameter may assist in minimizing the amount of emulsions formed whilst extracting highest total MO oil yield possible.

Keywords: enzyme, emulsion, Moringa oleifera, oil yield

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Authors: R. Saint-Loup, H. Amedro, N. Jacquel, S. Legrand, F. Fenouillot, J. P. Pascault, A. Rousseau

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Isosorbide or 1,4-3,6 dianhydrohexitol has been developped for several years as a new biobased monomer. It is commercially available as a starch derivative, more precisely obtained derivated from starch and more precisely from sorbitol. Isosorbide can find several applications, directly as a monomer or after chemical modification, in different polymer fields like thermoplastics (obtained from polycondensation or from radical polymerization of unsaturated monomers) or like Thermosetting resins (like cross linked PU, or after modification like acrylates or epoxy coatings) Concerning aliphatic or semi-aromatic polyesters, the addition of isosorbide improves thermal stability an,d optical properties, allowing a large range of applications as semi-crystalline or amorphous polymers. The preparation of poly (ethylene-co-isosorbide) terephthalate with different ratios of isosorbide will be particularly detailed. The structure – properties relationship will permit a focus on the obtention of polyesters with semi-crystalline or amorphous structures. The influence of isosorbide on the polymerization, on the processing of the resulting polyester as well as the modification of the final properties will be enlightened. The properties of Poly (ethylene-co-isosorbide) terephthlate will be emphasized and related to their applications. The evolutions related to Isosorbide with the replacement of ethylene glycol by Cyclohexanedimethanol allowed to drastically change the properties of the resulting polyester, with a large gap on the properties and new potential applications.

Keywords: modified PET, poly(ethylene-co-isosorbide)terephthalate, specialy polyester, poly(isosorbide_co_cyclohexanediol)terephthalate

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Authors: Rajeev Ravindran, Amit K. Jaiswal

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Lignocellulose is the largest reservoir of inexpensive, renewable source of carbon. It is composed of lignin, cellulose and hemicellulose. Cellulose and hemicellulose is composed of reducing sugars glucose, xylose and several other monosaccharides which can be metabolised by microorganisms to produce several value added products such as biofuels, enzymes, aminoacids etc. Enzymatic treatment of lignocellulose leads to the release of monosaccharides such as glucose and xylose. However, factors such as the presence of lignin, crystalline cellulose, acetyl groups, pectin etc. contributes to recalcitrance restricting the effective enzymatic hydrolysis of cellulose and hemicellulose. In order to overcome these problems, pre-treatment of lignocellulose is generally carried out which essentially facilitate better degradation of lignocellulose. A range of pre-treatment strategy is commonly employed based on its mode of action viz. physical, chemical, biological and physico-chemical. However, existing pretreatment strategies result in lower sugar yield and formation of inhibitory compounds. In order to overcome these problems, we proposes a novel pre-treatment, which utilises the superior oxidising capacity of alkaline potassium permanganate assisted by ultra-sonication to break the covalent bonds in spent coffee waste to remove recalcitrant compounds such as lignin. The pre-treatment was conducted for 30 minutes using 2% (w/v) potassium permanganate at room temperature with solid to liquid ratio of 1:10. The pre-treated spent coffee waste (SCW) was subjected to enzymatic hydrolysis using enzymes cellulase and hemicellulase. Shake flask experiments were conducted with a working volume of 50mL buffer containing 1% substrate. The results showed that the novel pre-treatment strategy yielded 7 g/L of reducing sugar as compared to 3.71 g/L obtained from biomass that had undergone dilute acid hydrolysis after 24 hours. From the results obtained it is fairly certain that ultrasonication assists the oxidation of recalcitrant components in lignocellulose by potassium permanganate. Enzyme hydrolysis studies suggest that ultrasound assisted alkaline potassium permanganate pre-treatment is far superior over treatment by dilute acid. Furthermore, SEM, XRD and FTIR were carried out to analyse the effect of the new pre-treatment strategy on structure and crystallinity of pre-treated spent coffee wastes. This novel one-step pre-treatment strategy was implemented under mild conditions and exhibited high efficiency in the enzymatic hydrolysis of spent coffee waste. Further study and scale up is in progress in order to realise future industrial applications.

Keywords: spent coffee waste, alkaline potassium permanganate, ultra-sonication, physical characterisation

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Authors: Ritu Chaturvedi, Manoj Paul

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A pot experiment was conducted to investigate the effect of Arbuscular mycorrhizal fungus (AMF) on metal(loid) uptake and accumulation efficiency of Pisum sativum along with physiological and biochemical response. Plants were grown in soil spiked with 50 and 100 mg kg-1 Pb, 25 and 50 mg kg-1 Cd, 50 and 100 mg kg-1 As and a combination of all three metal(loid)s. A parallel set was maintained and inoculated with arbuscular mycorrhizal fungus for comparison. After 60 days, plants were harvested and analysed for metal(loid) content. A steady increase in metal(loid) accumulation was observed on increment of metal(loid) dose and also on AMF inoculation. Plant height, biomass, chlorophyll, carotenoid and carbohydrate content reduced upon metal(loid) exposure. Increase in enzymatic (CAT, SOD and APX) and nonenzymatic (Proline) defence proteins was observed on metal(loid) exposure. AMF inoculation leads to an increase in plant height, biomass, chlorophyll, carotenoids, carbohydrate and enzymatic defence proteins (p≤0.001) under study; whereas proline content was reduced. Considering the accumulation efficiency and adaptive response of plants and alleviation of stress by AMF, this symbiosis can be applied for on-site remediation of Pb and Cd contaminated soil.

Keywords: heavy metal, mycorrhiza, pea, phyroremediation

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Authors: Shu-Chuan Liao, Chia-Ti Chang, Ko-Shao Chen

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Titanium and its alloy are widely used in many fields such as dentistry or orthopaedics. Due to their high strength low elastic modulus that chemical inertness and bio inert. The micro-arc oxidation used to formation a micro porous ceramic oxide layer film on Titanium surface and also to improve the resistance corrosion. For improving the biocompatibility, micro-arc oxidation surfaces bio-inert need to introduce reactive group. We introduced boundary layer by used plasma enhanced chemical vapor deposition of hexamethyldisilazane (HMDS) and organic active layer by UV light graft reactive monomer acrylic acid (AAc) therefore we can immobilize Chondroitin sulphate on surface easily by crosslinking EDC/NHS. The surface properties and composition of the modified layer were measured by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS) and X-ray diffraction (XRD) and water contact angle. Water contact angle of the plasma-treated Ti surface decreases from 60° to 38°, which is an indication of hydrophilicity. The results of electrochemical polarization analysis showed that the sample plasma treated at micro-arc oxidation after plasma treatment has the best corrosion resistance. The result showed that we can immobilize chondroitin sulfate successful by a series of modification and MTT assay indicated the biocompatibility has been improved in this study.

Keywords: MAO, plasma, graft polymerization, biomedical application

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Authors: H. Merhni, A. Sbiti, I. Ratbi, A. Sefiani

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The proximal spinal muscular atrophy (SMA) is a group of neuromuscular disorders characterized by progressive muscle weakness due to the degeneration and loss of anterior motor neurons of the spinal cord. Depending on the age of onset of symptoms and their evolution, four types of SMA, varying in severity, result in a mutations of the SMN gene (survival of Motor neuron). We have analyzed the DNA of 295 patients referred to our genetic counseling; since January 1996 until October 2014; for suspected SMA. The homozygous deletion of exon 7 of the SMN gene was found in 133 patients; of which, 40.6% were born to consanguineous parents. In countries like Morocco, where the frequency of heterozygotes for SMA is high, genetic testing should be offered as first-line and, after careful clinical assessment, especially in newborns and infants with congenital hypotonia unexplained and prognosis compromise. The molecular diagnosis of SMA allows a quick and certainly diagnosis, provide adequate genetic counseling for families at risk and suggest, for couples who want prenatal diagnosis. The analysis of the SMN gene is a perfect example of genetic testing with an excellent cost/benefit ratio that can be of great interest in public health, especially in low-income countries. We emphasize in this work for the benefit of the generalization of molecular diagnosis of SMA by the technique of PCR-enzymatic digestion in other centers in Morocco.

Keywords: Exon7, PCR-digestion, SMA, SMN gene

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Authors: Shivam Yadav, Neelam Atri

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Cyanobacteria, the photosynthetic prokaryotes are indispensable components of paddy soil contribute substantially to the nitrogen economy however often appended with metal load. They are well known to play crucial roles in maintenance of soil fertility and rice productivity. Nickel is one such metal that plays a vital role in the cellular physiology, however at higher concentrations it exerts adverse effects. Arsenic is another toxic metalloid that negatively affects the cyanobacterial proliferation. However species-specific comparative responses under As and Ni is largely unknown. The present study focuses on the comparative effects of nickel (Ni2+) and arsenite (As(III)) on two diazotrophic cyanobacterial species (Anabaena doliolum and Anabaena sp. PCC7120) in terms of antioxidative aspects. Oxidative damage measured in terms of lipid peroxidation and peroxide content was significantly higher after As(III) than Ni treatment as compared to control. Similarly, all the studied enzymatic and non-enzymatic parameters of antioxidative defense system except glutathione reductase (GR) showed greater induction against As(III) than Ni. Moreover, integrating comparative analysis of all studied parameters also demonstrated interspecies variation in terms of stress adaptive strategies reflected through higher sensitivity of Anabaena doliolum over Anabaena PCC7120.

Keywords: antioxidative system, arsenic, cyanobacteria, nickel

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Authors: Najwa Othman, Norhidayah Suleiman, Gun Hean Chong

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Palm fatty acid distillate (PFAD) is a by-product of palm oil refineries which contains valuable compounds such as phytosterols, squalene, polycosanol, co-enzyme Q10 and vitamin E (tocopherols and tocotrienols). Approximately 0.7-1.0% of vitamin E accumulates in PFAD, and it functions as antioxidants and anti-inflammatory. The objective of this research is to evaluate the effect of manipulated variables in supercritical carbon dioxide towards the recovery of tocotrienols in PFAD. The vitamin E concentrate isolated varies depending on the pre-treatment of sample and extraction techniques. In this research, tocotrienols in PFAD was concentrated by removing the extraneous matters, especially free fatty acid (FFA) and acylglycerols. Pre-treatment method such as enzymatic hydrolysis by using lipase from Candida rugosa as an enzyme was used to remove FFA and improve recovery of vitamin E. After that, treated PFAD was extracted by using supercritical fluid extraction in co-current glass beads packed column (22 cm x 75 cm i.d) at different temperatures (40-60°C) and pressures (100-300 bar) for 5 hours. After the extraction, the sample was analyzed by using high-pressure liquid chromatography (HPLC) system to quantify the tocotrienols. The results indicated that a combined pressure (200 bar) and temperature (60°C) was predicted to provide highest tocotrienols yield and the extraction yield obtained was 106.45%.

Keywords: enzymatic hydrolysis, palm fatty acid distillate, supercritical fluid extraction, tocotrienols

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Authors: A. Bouriche, D. Merah, L.Alachaher-Bedjaoui, U. Maschke

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Intensive research continues in the field of liquid crystals (LCs) for their potential use in modern display applications. Nematic LCs has been most commonly used due to the large birefringence and their sensitivity to even weak perturbation forces induced by electric, magnetic and optical fields. Polymer dispersed liquid crystals (PDLCs), composed of micron-sized nematic LC droplets dispersed in a polymer matrix is an important class of materials for applications in different domains of technology involving large area display devices, optical switches, phase modulators, variable attenuators, polarisers, flexible displays and smart windows. In this study the composites are prepared from mixtures of monofunctional acrylic monomers, (Butylacrylate (ABu), 2-Ethylhexylacrylate (2-EHA), 2-Hydroxyethyl methacrylate (HEMA) and hydroxybutylmethacrylate (HBMA)) and two liquid crystals: (4-cyano-4'-n-pentyl-biphenyl) (5CB) and E7 which is an eutectic mixtures of four cyanoparaphenylenes. These mixtures are prepared adding the Darocur 1173 as photoinitiateor, the 1.6-hexanediol diacrylate (HDDA) as cross-linker agent, and finally they are exposed to UV irradiation. The kinetic polymerization of monomer/LC mixture were investigated with the Fourier Transform Infra Red spectroscopy (FTIR). The electro-optical properties of the PDLC films were determined by measuring the voltage dependence on the transmitted light.

Keywords: acrylic monomers, films PDLC, liquid crystal, polymerisation

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Authors: Kyle Phillips, Ndiko Ludidi

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Global climate change has resulted in altered rainfall patterns, which has resulted in annual losses in maize crop yields due to drought. Therefore it is important to produce maize cultivars that are more drought-tolerant, which is not an easily accomplished task as plants have a plethora of physical and biochemical adaptation methods. One such mechanism is the drought-induced expression of enzymatic and non-enzymatic proteins which assist plants to resist the effects of drought on their growth and development. One of these proteins is AtRD22 which has been identified in Arabidopsis thaliana. Using an in silico approach, a maize protein with 48% sequence homology to AtRD22 has been identified. This protein appears to be localized in the extracellular matrix, similarly to AtRD22. Promoter analysis of the encoding gene reveals cis-acting elements suggestive of induction of the gene’s expression by abscisic acid (ABA). Semi-quantitative transcriptomic analysis of the putative maize RD22 has revealed an increase in transcript levels after the exposure to drought. Current work elucidates the effect of up-regulation and silencing of the maize RD22 gene on the tolerance of maize to drought. The potential role of the maize RD22 gene in maize drought tolerance can be used as a tool to improve food security.

Keywords: abscisic acid, drought-responsive cis-acting elements, maize drought tolerance, RD22

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Authors: L. L. Tundisi, A. Pessoa Jr., E. B. Tambourgi, E. Silveira, P. G. Mazzola

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L-asparaginase (L-ASNase) is the gold standard treatment for acute lymphoblastic leukemia that mainly affects pediatric patients; treatment increases survival from 20% to 90%. The characterization of other L-Asparaginases, apart from the most used from Escherichia coli and Erwinia chrysanthemi, has been reported, but the choice of the most appropriate is still under debate. This choice should be based on its pharmacokinetics, immune hypersensitivity, doses, prices, pharmacodynamics. The main factors influencing the antileukemic activity of ASNase are enzymatic activity, Km, glutaminase activity, clearance of the enzyme and development of resistance. However, most of the commercialized enzyme present an intrinsic glutaminase activity, which is responsible for some side effects. In this study, glutaminase free asparaginase produced from Aspergillus oryzae was precipitated in different percentages of ethanol (0–80%), until optimum ethanol concentration of 60% (w/w) was found. Following, precipitation of crude L-ASNase was performed in a single step, using 60% (w/w) ethanol, under constant agitation and temperature. It presented activity of 135.45 U/mg and after gel filtration chromatography with Sephadex G-the enzymatic activity was 322.02 U/mg. The apparent molecular mass of the purified L-ASNase fraction was estimated by 10% SDS-PAGE. Proteins were stained with Coomassie Brilliant Blue R-250. The molar mass range was from 10 kDa to 250 kDa. L-ASNase from Aspergillus oryzae was characterized aiming possible therapeutic use. Four different buffers (phosphate-citrate buffer pH 2.6 to 5.8; phosphate buffer pH 5.8 to 7.4; Tris - HCl pH 7.4 to 9.0; and carbonate buffer pH 9.8 to 10.6) were used to measure the optimum pH for L-ASNase activity. The optimum temperature for enzyme activity was measured at optimal pH conditions (Tris-HCl and phosphate buffer, pH 7.4) at different temperatures ranging from 5 to 55°C. All activities were calculated by quantifying the free ammonia, using the Nessler reagent. The kinetic parameters calculation, e.g. Michaelis-Menten constant (Km), maximum velocity (Vmax) and Hills coefficient (n), were performed by incubating the enzyme in different concentrations of the substrate at optimum conditions of pH and fitted on Hill’s equation. This glutaminase free asparaginase showed a low Km (3.39 mM and 3.81 mM) and enzymatic activity of 135.45 U/mg after precipitation with ethanol. After gel filtration chromatography it rose to 322.02 U/mg. Optimum activity was found between pH 5.8 - 9.0, best activity results with phosphate buffer pH 7.4 and Tris-HCl pH 7.4 and showed activity from 5°C to 55°C. These results indicate that L-ASNase from A. oryzae has the potential for human use.

Keywords: biopharmaceuticals, bioprocessing, bioproducts, biotechnology, enzyme activity, ethanol precipitation

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Authors: A. Aslihan Gokaltun, Ali Tuncel

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The unique properties of capillary electrochromatography (CEC) such as high performance, high selectivity, low consumption of both reagents and analytes ensure this technique an attractive one for the separation of biomolecules including nucleosides and nucleotides, peptides, proteins, carbohydrates. Monoliths have become a well-established separation media for CEC in the format that can be compared to a single large 'particle' that does not include interparticular voids. Convective flow through the pores of monolith significantly accelerates the rate of mass transfer and enables a substantial increase in the speed of the separation. In this work, we propose a new approach for the preparation of cationic monolithic stationary phase for capillary electrochromatography. Instead of utilizing a charge bearing monomer during polymerization, the desired charge-bearing group is generated on the capillary monolith after polymerization by using the reactive moiety of the monolithic support via one-pot, simple reaction. Optimized monolithic column compensates the disadvantages of frequently used reversed phases, which are difficult for separation of polar solutes. Rapid separation and high column efficiencies are achieved for the separation of neutral analytes, nucleic acid bases and nucleosides in reversed phase mode. Capillary monolith showed satisfactory hydrodynamic permeability and mechanical stability with relative standard deviation (RSD) values below 2 %. A new promising, reactive support that has a 'ligand selection flexibility' due to its reactive functionality represent a new family of separation media for CEC.

Keywords: biomolecules, capillary electrochromatography, cationic monolith, neutral analytes

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Authors: Xuan Li, Weian Huang, Jinsheng Sun, Fuhao Zhao, Zhiyuan Wang, Jintang Wang

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Natural gas hydrates (NGHs) as promising alternative energy sources have gained increasing attention. Hydrate-bearing formation in marine areas is highly unconsolidated formation and is fragile, which is composed of weakly cemented sand-clay and silty sediments. During the drilling process, the invasion of drilling fluid can easily lead to excessive water content in the formation. It will change the soil liquid plastic limit index, which significantly affects the formation quality, leading to wellbore instability due to the metastable character of hydrate-bearing sediments. Therefore, controlling the filtrate loss into the formation in the drilling process has to be highly regarded for protecting the stability of the wellbore. In this study, the temperature-sensitive nanogel of P(NIPAM-co-AMPS-co-tBA) was prepared by soap-free emulsion polymerization, and the temperature-sensitive behavior was employed to achieve self-adaptive plugging in hydrate sediments. First, the effects of additional amounts of AMPS, tBA, and cross-linker MBA on the microgel synthesis process and temperature-sensitive behaviors were investigated. Results showed that, as a reactive emulsifier, AMPS can not only participate in the polymerization reaction but also act as an emulsifier to stabilize micelles and enhance the stability of nanoparticles. The volume phase transition temperature (VPTT) of nanogels gradually decreased with the increase of the contents of hydrophobic monomer tBA. An increase in the content of the cross-linking agent MBA can lead to a rise in the coagulum content and instability of the emulsion. The plugging performance of nanogel was evaluated in a core sample with a pore size distribution range of 100-1000nm. The temperature-sensitive nanogel can effectively improve the microfiltration performance of drilling fluid. Since a combination of a series of nanogels could have a wide particle size distribution at any temperature, around 200nm to 800nm, the self-adaptive plugging capacity of nanogels for the hydrate sediments was revealed. Thermosensitive nanogel is a potential intelligent plugging material for drilling operations in natural gas hydrate-bearing sediments.

Keywords: temperature-sensitive nanogel, NIPAM, self-adaptive plugging performance, drilling operations, hydrate-bearing sediments

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Authors: Waranya Pongpaiboon, Warangkana Srichamnong, Supat Chaiyakul

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Enzymatic modification of rice flour can produce highly functional derivatives use in food industries. This study aimed to evaluate the physical properties and resistant starch content of rice flour residues hydrolyzed by α-amylase. Rice flour hydrolyzed by α-amylase (60 and 300 u/g) for 1, 24 and 48 hours were investigated. Increasing enzyme concentration and hydrolysis time resulted in decreased rice flour residue’s lightness (L*) but increased redness (a*) and yellowness (b*) of rice flour residues. The resistant starch content and peak viscosity increased when hydrolysis time increased. Pasting temperature, trough viscosity, breakdown, final viscosity, setback and peak time of the hydrolyzed flours were not significantly different (p>0.05). The morphology of native flour was smooth without observable pores and polygonal with sharp angles and edges. However, after hydrolysis, granules with a slightly rough and porous surface were observed and a rough and porous surface was increased with increasing hydrolyzed time. The X-ray diffraction patterns of native flour showed A-type configuration, which hydrolyzed flour showed almost 0% crystallinity indicated that both amorphous and crystalline structures of starch were simultaneously hydrolyzed by α-amylase.

Keywords: α-Amylase, enzymatic hydrolysis, pasting properties, resistant starch

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Authors: Jessica Carolina Hernandez Galeano, Juan Carlos Moreno Pirajan, Liliana Giraldo

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Carbon gels are materials whose structure and porous texture can be designed and controlled on a nanoscale. Among their characteristics it is found their low density, large surface area and high degree of porosity. These materials are produced by a sol-gel polymerization of organic monomers using basic or acid catalysts, followed by drying and controlled carbonization. In this work, the synthesis and characterization of carbon aerogels from resorcinol, phenol and formaldehyde in ethanol is described. The aim of this study is obtaining different carbonaceous materials in the form of spheres using the Stöber method to perform a further evaluation of CO₂ adsorption of each material. In general, the synthesis consisted of a sol-gel polymerization process that generates a cluster (cross-linked organic monomers) from the precursors in the presence of NH₃ as a catalyst. This cluster was subjected to specific conditions of gelling and curing (30°C for 24 hours and 100°C for 24 hours, respectively) and CO₂ supercritical drying. Finally, the dry material was subjected to a process of carbonization or pyrolysis, in N₂ atmosphere at 350°C (1° C / min) for 2 h and 600°C (1°C / min) for 4 hours, to obtain porous solids that retain the structure initially desired. For this work, both the concentrations of the precursors and the proportion of ammonia in the medium where modify to describe the effect of the use of phenol and the amount of catalyst in the resulting material. Carbon aerogels were characterized by Scanning Electron Microscope (SEM), N₂ isotherms, infrared spectroscopy (IR) and X-ray Powder Diffraction (XRD) showing the obtention of carbon spheres in the nanometric scale with BET areas around 500 m2g-1.

Keywords: carbon aerogels, carbon spheres, CO₂ adsorption, Stöber method

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Authors: S. Hallal, Z. Chami, A. Hadji-Lehtihet, S. Sokhal-Boudella, A. Berhoune, L. Yargui

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Gaucher disease is the most common lysosomal storage in our population, it is due to a deficiency of β –glucosidase acid. The enzyme deficiency causes a pathological accumulation of undegraded substrate in lysosomes. This metabolic overload is responsible for a multisystemic disease with hepatosplenomegaly, anemia, thrombocytopenia, and bone involvement. Neurological involvement is rare. The laboratory diagnosis of Gaucher disease consists of phenotypic diagnosis by determining the enzymatic activity of β - glucosidase by fluorimetric method, a study by genotypic diagnosis in the GBA gene, limiting the search recurrent mutations (N370S, L444P, 84 GG); PCR followed by an enzymatic digestion. Abnormal profiles were verified by sequencing. Monitoring of treated patients is provided by the determination of chitotriosidase. Our experience spaning a period of 6 years (2007-2014) has enabled us to diagnose 78 patients out of a total of 328 requests from the various departments of pediatrics, internal medicine, neurology. Genotypic diagnosis focused on the entire family of 9 children treated at pediatric CHU Mustapha, which help define the clinical form; or 5 of them had type III disease, carrying the L444P mutation in the homozygous state. Three others were composite (N370/L444P) (N370S/other unintended mutation in our study), and only in one family no recurrent mutation has been found. This molecular study permits screening of heterozygous essential for genetic counseling.

Keywords: Gaucher disease, mutations, N370S, L444P

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Authors: Bilal Murtaza, Xiaoyu Li, Liming Dong, Muhammad Kashif Saleemi, Gen Li, Bowen Jin, Lili Wang, Yongping Xu

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Fusarium spp. produce numerous mycotoxins, such as zearalenone (ZEN), deoxynivalenol (DON), and its acetylated compounds, 3-acetyl-deoxynivalenol (3-ADON) and 15-acetyl-deoxynivalenol (15-ADON) (15-ADON). In a co-culture system, the soil-derived Escherichia marmotae strain degrades ZEN and DON into 3-keto-DON and DOM-1 via enzymatic deep-oxidation. When pure mycotoxins were subjected to Escherichia marmotae in culture flasks, degradation, and detoxification were also attained. DON and ZEN concentrations, ambient pH, incubation temperatures, bacterium concentrations, and the impact of acid treatment on degradation were all evaluated. The results of the ELISA and high-performance liquid chromatography-electrospray ionization-high resolution mass spectrometry (HPLC-ESI-HRMS) tests demonstrated that the concentration of mycotoxins exposed to Escherichia marmotae was significantly lower than the control. ZEN levels were reduced by 43.9%, while zearalenone sulfate ([M/z 397.1052 C18H21O8S1) was discovered as a derivative of ZEN converted by microbes to a less toxic molecule. Furthermore, Escherichia marmotae appeared to metabolize DON 35.10% into less toxic derivatives (DOM-1 at m/z 281 of [DON - O]+ and 3-keto-DON at m/z 295 of [DON - 2H]+). These results show that Escherichia marmotae can reduce Fusarium mycotoxins production, degrade pure mycotoxins, and convert them to less harmful compounds, opening up new possibilities for study and innovation in mycotoxin detoxification.

Keywords: mycotoxins, zearalenone, deoxynivalenol, bacterial degradation

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Authors: Redouane Krini, Lutz Nuhn, Hicham El Mard Cheol Woo Ha, Yoondeok Han, Kwang-Sup Lee, Dong-Yol Yang, Jinsoo Joo, Rudolf Zentel

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To use Quantum Dots (QDs) in the two photon initiated polymerization technique (TPIP) for 3D patternings, QDs were modified on the surface with photosensitive end groups which are able to undergo a photopolymerization. We were able to fabricate fluorescent 3D lattice structures using photopatternable QDs by TPIP for photonic devices such as photonic crystals and metamaterials. The QDs in different diameter have different emission colors and through mixing of RGB QDs white light fluorescent from the polymeric structures has been created. Metamaterials are capable for unique interaction with the electrical and magnetic components of the electromagnetic radiation and for manipulating light it is crucial to have a negative refractive index. In combination with QDs via TPIP technique polymeric structures can be designed with properties which cannot be found in nature. This makes these artificial materials gaining a huge importance for real-life applications in photonic and optoelectronic. Understanding of interactions between nanoparticles and biological systems is of a huge interest in the biomedical research field. We developed a synthetic strategy of polymer functionalized nanoparticles for biomedical studies to obtain hybrid systems of QDs and copolymers with a strong binding network in an inner shell and which can be modified in the end through their poly(ethylene glycol) functionalized outer shell. These hybrid systems can be used as models for investigation of cell penetration and drug delivery by using measurements combination between CryoTEM and fluorescence studies.

Keywords: biomedical study models, lithography, photo induced polymerization, quantum dots

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Authors: Deepak Mohan, Sushma Sharma, Mohammad Asif

Abstract:

Diclofenac sodium being one of the most common non-steroidal anti-inflammatory drugs is normally used as painkiller and to reduce inflammation. The drug is known to alter the enzymatic activities of acid and alkaline phosphatase, glutamate oxaloacetate transaminase and glutamate pyruvate transaminases. The drug also results in change in the concentration of proteins and lipids in the body. The present study is an attempt to study different biochemical changes electrophoretically due to administration of different doses of diclofenac (4mg/kg/body weight and 14mg/kg/body weight) on liver and testes of mice from 7-28 days of investigation. Homogenization of the tissue was done, supernatant separated was loaded in the gel and native polyacrylamide gel electrophoresis was conducted. Diclofenac administration resulted in alterations of all these biochemical parameters which were observed in native polyacrylamide gel electrophoretic studies. The severe degenerative changes as observed during later stages of the experiment showed correlation with increase or decrease in the activities of all the enzymes studied in the present investigation. Image analysis of gel in liver showed a decline of 7.4 and 5.3 % in low and high dose group after 7 days whereas a decline of 9.6 and 7.5% was registered after 28 days of investigation. Similar analysis for testis also showed an appreciable decline in the activity of alkaline phosphatase after 28 days. Gel analysis of serum was also performed to find a correlation in the enzymatic activities between the tissue and blood.

Keywords: diclofenac, inflammation, polyacrylamide, phosphatase

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Authors: Joan O. Ogundele, Aladesanmi A. Oshodi, Adekunle I. Amoo

Abstract:

Abstract In-vitro multi-enzyme protein digestibility (IVMPD) and some anti-nutritional factors (ANF) of five melon (egusi) seed flours (MSF) and their protein isolates (PI) were carried out. Their PI have potentials comparable to that of soya beans. It is important to know the IVMPD and ANF of these protein sources as to ensure their safety when adapted for use as alternate protein sources to substitute for cow milk, which is relatively expensive in Nigeria. Standard methods were used to produce PI of Citrullus colocynthis, Citrullus vulgaris, African Wine Kettle gourd (Lageneria siceraria I), Basket Ball gourd (Lagenaria siceraria II) and Bushel Giant Gourd (Lageneria siceraria III) seeds and to determine the ANF and IVMPD of the MSF and PI unheated and at 37oC. Multi-enzymes used were trypsin, chymotrypsin and peptidase. IVMPD of MSF ranged from (70.67±0.70) % (C. vulgaris) to (72.07± 1.79) % (L.siceraria I) while for their PI ranged from 74.33% (C.vulgaris) to 77.55% (L.siceraria III). IVMPD of the PI were higher than those of MSF. Heating increased IVMPD of MSF with average value of 79.40% and those of PI with average of 84.14%. ANF average in MSF are tannin (0.11mg/g), phytate (0.23%). Differences in IVMPD of MSF and their PI at different temperatures may arise from processing conditions that alter the release of amino acids from proteins by enzymatic processes. ANF in MSF were relatively low, but were found to be lower in the PI, therefor making the PI safer for human consumption as an alternate source of protein.

Keywords: Anti-nutrients, Enzymatic protein digestibility, Melon (egusi)., Protein Isolates.

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Authors: Marco Soto-Arriaza, Eduardo Cena-Ahumada, Jaime Melendez-Rojel

Abstract:

Background: Liposomes have been widely used as a model of lipid bilayer to study the physicochemical properties of biological membrane, encapsulation, transport and release of different molecules. Furthermore, extensive research has focused on improving the efficiency in the transport of drugs, developing tools that improve the release of the encapsulated drug from liposomes. In this context, the enzymatic activity of PLA₂, despite having been shown to be an effective tool to promote the release of drugs from liposomes, is still an open field of research. Aim: The aim of the present study is to explore the effect of cholesterol (Cho) and amphipathic di- and tri-block copolymers, on calcein release mediated by enzymatic activity of PLA2 in Dipalmitoylphosphatidylcholine (DPPC) liposomes under physiological conditions. Methods: Different dispersions of DPPC, cholesterol, di-block POE₄₅-PCL₅₂ or tri-block PCL₁₂-POE₄₅-PCL₁₂ were prepared by the extrusion method after five freezing/thawing cycles; in Phosphate buffer 10mM pH 7.4 in presence of calcein. DPPC liposomes/Calcein were centrifuged at 15000rpm 10 min to separate free calcein. Enzymatic activity assays of PLA₂ were performed at 37°C using the TBS buffer pH 7.4. The size distribution, polydispersity, Z-potential and Calcein encapsulation of DPPC liposomes was monitored. Results: PLA₂ activity showed a slower kinetic of calcein release up to 20 mol% of cholesterol, evidencing a minimum at 10 mol% and then a maximum at 18 mol%. Regardless of the percentage of cholesterol, up to 18 mol% a one-hundred percentage release of calcein was observed. At higher cholesterol concentrations, PLA₂ showed to be inefficient or not to be involved in calcein release. In assays where copolymers were added in a concentration lower than their cmc, a similar behavior to those showed in the presence of Cho was observed, that is a slower kinetic in calcein release. In both experimental approaches, a one-hundred percentage of calcein release was observed. PLA₂ was shown to be sensitive to the 4-(4-Octadecylphenyl)-4-oxobutenoic acid inhibitor and calcium, reducing the release of calcein to 0%. Cell viability of HeLa cells decreased 7% in the presence of DPPC liposomes after 3 hours of incubation and 17% and 23% at 5 and 15 hours, respectively. Conclusion: Calcein release from DPPC liposomes, mediated by PLA₂ activity, depends on the percentage of cholesterol and the presence of copolymers. Both, cholesterol up to 20 mol% and copolymers below it cmc could be applied to the regulation of the kinetics of antitumoral drugs release without inducing cell toxicity per se.

Keywords: amphipathic copolymers, calcein release, cholesterol, DPPC liposome, phospholipase A₂

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Authors: Mehdi Nasr-Esfahani, Leila Mohammad Bagheri, Ava Nasr-Esfahani

Abstract:

Root and collar rot disease caused by Phytophthora capsici (Leonian) is one of the most serious diseases in pepper, Capsicum annuum L. In this study, a diverse collection of 37 commercial edible and ornamental pepper genotypes infected with P. capsici were investigated for biomass parameters and enzymatic activity of peroxidase or peroxide reductases (EC), superoxide dismutase (SOD), polyphenol oxidase (PPOs), catalase (CAT) and phenylalanine ammonia-lyase (PAL). Seven candidate DEG genes were also evaluated on resistant and susceptible pepper cultivars, through measuring product formation, using spectrophotometry and real-time polymerase chain reaction. All the five enzymes and seven defense-gene candidates were up-regulated in all inoculated pepper accessions to P. capsici. But, the enzymes and DEG genes were highly expressed in resistant cv. 19OrnP-PBI, 37ChillP-Paleo, and “23CherryP-Orsh". The expression level of enzymes were 1.5 to 5.6-fold higher in the resistant peppers, than the control non-inoculated genotypes. Also, the transcriptional levels of related candidate DEG genes were 3.16 to 5.90-fold higher in the resistant genotypes. There was a direct and high correlation coefficient between resistance, bio-mass parameters, enzymatic activity, and resistance gene expression. The related enzymes and candidate genes expressed herein will provide a basis for further gene cloning and functional verification studies, and also will aid in an understanding of the regulatory mechanism of pepper resistance to P. capsici.

Keywords: AP2/ERF, cDNA, enzymes, MIP gene, q-RTPCR, XLOC

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Authors: Popy Bora, L. C. Bora, A. Bhattacharya, Sehnaz S. Ahmed

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Microbial bioagents, viz., Pseudomonas fluorescens, Bacillus subtilis, and Trichoderma viride were assessed for their ability to suppress grey blight caused by Pestalotiopsis theae, a major disease of tea crop in Assam. The expression of defense-related phytochemicals due to the application of these bioagents was also evaluated. The individual bioagents, as well as their combinations, were screened for their bioefficacy against P. theae in vitro using nutrient agar (NA) as basal medium. The treatment comprising a combination of the three bioagents, P. fluorescens, B. subtilis, and T. viride showed significantly the highest inhibition against the pathogen. Bioformulation of effective bioagent combinations was further evaluated under field condition, where significantly highest reduction of grey blight (90.30%), as well as the highest increase in the green leaf yield (10.52q/ha), was recorded due to application of the bioformulation containing the three bioagents. The application of the three bioformulation also recorded an enhanced level of caffeine (4.15%) and polyphenols (22.87%). A significant increase in the enzymatic activity of phenylalanine ammonia-lyase, peroxidase and polyphenol oxidase were recorded in the plants treated with the microbial bioformulation of the three bioagents. The present investigation indicates the role of microbial agents in suppressing disease, inducing plant defense response, as well as improving the quality of tea.

Keywords: enzymatic activity, grey blight, microbial bioagents, Pestalotiopsis theae, phytochemicals, plant defense, tea

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Authors: Munisath Khandoker, Stephan Haefele, Andy Gregory

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Extracellular enzymes play a key role in soil organic carbon (SOC) decomposition and nutrient cycling and are known indicators for soil health; however, it is not understood how these enzymes respond to different land uses and their relationships to other soil properties have not been extensively reviewed. The relationships among the activities of three soil enzymes: β-glucosaminidase (NAG), phosphomonoesterase (PHO) and β-glucosidase (GLU), were examined. The impact of soil organic amendments, soil types and land management on soil enzyme activities were reviewed, and it was hypothesized that soils with increased SOC have increased enzyme activity. Long-term experiments at Rothamsted Research Woburn and Harpenden sites in the UK were used to evaluate how different management practices affect enzyme activity involved in carbon (C) and nitrogen (N) cycling in the soil. Samples were collected from soils with different organic treatments such as straw, farmyard manure (FYM), compost additions, cover crops and permanent grass cover to assess whether SOC can be linked with increased levels of enzymatic activity and what influence, if any, enzymatic activity has on total C and N in the soil. Investigating the interactions of important enzymes with soil characteristics and SOC can help to better understand the health of soils. Studies on long-term experiments with known histories and large datasets can better help with this. SOC tends to decrease during land use changes from natural ecosystems to agricultural systems; therefore, it is imperative that agricultural lands find ways to increase and/or maintain SOC in the soil.

Keywords: biological soil health indicators, extracellular enzymes, soil health, soil, microbiology

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Authors: Carmen M. Gonzalez-Henriquez, Mauricio A. Sarabia-Vallejos, Juan Rodriguez-Hernandez

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DMAEMA, as a monomer, has been widely studied and used in several application fields due to their pH-sensitive capacity (tertiary amine protonation), being relevant in the biomedical area as a potential carrier for drugs focused on the treatment of genetic or acquired diseases (efficient gene transfection), among others. Additionally, the inhibition of bacterial growth and, therefore, their antimicrobial activity, can be used as dual-functional antifogging/antimicrobial polymer coatings. According to their interesting physicochemical characteristics and biocompatible properties, DMAEMA was used as a monomer to synthesize a smart pH-sensitive hydrogel, namely poly(HEMA-co-PEGDA575-co-DMAEMA). Thus, different mole ratios (ranging from 5:1:0 to 0:1:5, according to the mole ratio between HEMA, PEGDA, and DEAEMA, respectively) were used in this research. The surface patterns formed via a two-step polymerization (redox- and photo-polymerization) were first chemically studied via 1H-NMR and elemental analysis. Secondly, the samples were morphologically analyzed by using Field-Emission Scanning Electron Microscopy (FE-SEM) and Atomic Force Microscopy (AFM) techniques. Then, a particular relation between HEMA, PEGDA, and DEAEMA (0:1:5) was also characterized at three different pH (5.4, 7.4 and 8.3). The hydrodynamic radius and zeta potential of the micro-hydrogel particles (emulsion) were carried out as a possible control for morphology, exploring the effect that produces hydrogel micelle dimensions in the wavelength, height, and roughness of the wrinkled patterns. Finally, contact angle and cross-hatch adhesion test was carried out for the hydrogels supported on glass using TSM-silanized surfaces in order to measure their mechanical properties.

Keywords: wrinkled patterns, smart pH-sensitive hydrogels, hydrogel micelle diameter, adhesion tests

Procedia PDF Downloads 180