Search results for: monoamine oxidase

Authors: Uc-Cayetano E. G., Ake-Uh O. E., Villanueva-Mena I. E., Ordonez L. C.

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Carbon nanotubes (CNTs) and other graphitic nanostructures are materials with extraordinary physical, physicochemical and electrochemical properties which are being aggressively investigated for a variety of sensing applications. Thus, sensing of biological molecules such as proteins, DNA, glucose and other enzymes using either single wall or multiwall carbon nanotubes (MWCNTs) has been widely reported. Despite the current progress in this area, the electrochemical response of CNTs used in a variety of sensing arrangements still needs to be improved. An alternative towards the enhancement of this CNTs' electrochemical response is to chemically (or physically) modify its surface. The influence of the decoration with iron oxide nanoparticles in different types of MWCNTs on the amperometric sensing of glucose, urea, and cholesterol in solution is investigated. Commercial MWCNTs were oxidized in acid media and subsequently decorated with iron oxide nanoparticles; finally, the enzymes glucose oxidase, urease, and cholesterol oxidase are chemically immobilized to oxidized and decorated MWCNTs for glucose, urease, and cholesterol electrochemical sensing. The results of the electrochemical characterizations consistently show that the presence of iron oxide nanoparticles decorating the surface of MWCNTs enhance the amperometric response and the sensitivity to increments in glucose, urease, and cholesterol concentration when compared to non-decorated MWCNTs.

Keywords: WCNTs, enzymes, oxidation, decoration

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Authors: Gourav Jain, Sameer Dixit, Surjeet Kumar Arya, Praveen C. Verma

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Plant cell wall plays a major role in defence mechanism against biotic and abiotic stress as it constitutes the physical barrier between the microenvironment and internal component of the cell. It is a complex structure composed of mostly carbohydrates among which cellulose and hemicelluloses are most abundant that is embedded in a matrix of pectins and proteins. Multiple enzymes have been reported which plays a vital role in cell wall modification, Pectin Methylesterase (PME) is one of them which catalyses the demethylesterification of homogalacturonans component of pectin which releases acidic pectin and methanol. As emitted methanol is toxic to the insect pest, we use PME gene for the better methanol production. In the current study we showed overexpression of PME gene isolated from Withania somnifera under the insect inducible promoter causes enhancement of methanol production at the time of insect feeds to plants, and that provides better insect resistance property. We found that the 85-90% mortality causes by transgenic tobacco in both chewing (Spodoptera litura larvae and Helicoverpa armigera) and sap-sucking (Aphid, mealybug, and whitefly) pest. The methanol content and emission level were also enhanced by 10-15 folds at different inducible time point interval (15min, 30min, 45min, 60min) which would be analysed by Purpald/Alcohol Oxidase method.

Keywords: methanol, Pectin methylesterase, inducible promoters, Purpald/Alcohol oxidase

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Authors: R. M. Srivastava, Subbanna A.R.N.S, Md Abbas Ahmad, S. P.More, Shivashankar, B. Kalyanbabu

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The similar morphology associated with high genetic variability poses problems in phylogenetic studies of Helicoverpa armigera (Hubner). To identify genetic variation of North Western Himalayan population’s, partial (Mid to terminal region) cytochrome c oxidase subunit I (COX-1) gene was amplified and sequenced for three populations collected from Pantnagar, Almora, and Chinyalisaur. The alignment of sequences with other two populations, Nagpur representing central India population and Anhui, China representing complete COX-1 sequence revealed unanimity in middle region with eleven single nucleotide polymorphisms (SNPs) in Nagpur populations. However, the consensus is missing when approaching towards terminal region, which is associated with 15 each SNPs and pair base substitutions in Chinyalisaur populations. In minimum evolution tree, all the five populations were majorly separated into two clades, one comprising of only Nagpur population and the other with rest. Amongst, North Western populations, Chinyalisaur one is promising by farming a separate clade. The pairwise genetic distance ranges from 0.025 to 0.192 with the maximum between H. armigera populations of Nagpur and Chinyalisaur. This genetic isolation of populations can be attributed to a key role of topological barriers of weather and mountain ranges and temporal barriers due to cropping patterns.

Keywords: cytochrome c oxidase subunit I, northwestern Himalayan population, Helicoverpa armigera (Noctuidae: Lepidoptera), phylogenetic relationship, genetic variation

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Authors: Zain Ul Abdin, Mirza Aizaz Asim, Rao Sohail Ahmad Khan, Luqman Amrao, Fiaz Hussain, Hasooba Hira, Saqi Kosar Abbas

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The success of Integrated vector management programmes mainly depends on the correct identification of insect vector species involved in vector borne diseases. Based on molecular data the most important insect species involved as vectors for Zoonotic diseases in Pakistan were identified. The precise and accurate identification of such type of organism is only possible through molecular based techniques like “DNA barcoding”. Morphological species identification in insects at any life stage, is very challenging, therefore, DNA barcoding was used as a tool for rapid and accurate species identification in a wide variety of taxa across the globe and parallel studies revealed that DNA barcoding data can be effectively used in resolving taxonomic ambiguities, detection of cryptic diversity, invasion biology, description of new species etc. A comprehensive survey was carried out for the collection of insects (both adult and immature stages) in district Faisalabad, Pakistan and their DNA was extracted and mitochondrial cytochrome oxidase subunit I (COI-59) barcode sequences was used for molecular identification of immature and adult life stage.This preliminary research work opens new frontiers for developing sustainable insect vectors management programmes for saving lives of mankind from fatal diseases.

Keywords: zoonotic diseases, cytochrome oxidase, and insect vectors, CO1

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Authors: Anguo Chen, Huajie Chen, Caimei Yang, Qihua Hong, Jun Feng

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The experiment was conducted with 360 one-day-old Avian commercial broilers to study the effects of dietary cinnamon extract (CE), garlic extract (GE) and yucca extract (YE) on growth performance and serum biochemical parameters in broilers. The chickens were randomly divided equally into 4 treatment groups, each group with 3 replications, and received the same basal corn-bean diets included a starter from 1 d to 21 d and then a grower until 42 d, added with recommended dose 250 mg/kg CE, 25 mg/kg GE and 10 mg/kg YE to relevant group, respectively. The birds were kept in a stainless steel net coop each replication with 24 h light and were fed and drunk ad libitum. At 21 d and 42 d of age, 6 chicks were respectively picked out from every group and were bled to collect serum samples and intestinal samples for laboratory analysis. The results showed that the average daily gain (ADG) of CE, GE and YE group were increased by 7.20% (P<0.05), 3.43% (P>0.05) and 4.89% (P>0.05), feed gain ratio (F/G) was improved by 9.71% (P<0.05), 3.40% (P>0.05) and 3.40% (P>0.05) compared with the control, respectively. At 21 d of age, the content of serum urea nitrogen (SUN) and serum uric acid (SUA) and the activity of serum xanthine oxidase (SXO) in CE group were reduced by 35.17% (P<0.01), 13.73% (P<0.01) and 16.33% (P<0.05) compared with the control, respectively. At 42 d of age, SUN and SUA level and SXO activity were lowered by 24.35% (P<0.01), 15.49% (P<0.05) and 23.09% (P<0.01), respectively. The SXO activity in CE group was decreased by 14.86% (P<0.01) and 15.34%(P<0.01) compare with GE and YE group, respectively. Also, adding CE, GE and YE into broiler diets resulted in lower UN and UA level of intestinal contents. It is clear that CE was more significantly decreased the SXO activity and SUA levels than GE and YE, especially at the latter period, thereby it may play a more important role in improving the growth performance of broilers.

Keywords: cinnamon extract, broiler, growth performance, serum uric acid, serum xanthine oxidase

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Authors: Anahita Izadyar, My Ni Van, Kayleigh Amber Rodriguez, Ilwoo Seok, Elizabeth E. Hood

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Using a recombinant enzyme derived from corn and a simple modification, we fabricated a facile, fast, and cost-beneficial biosensor to measure glucose. The Nafion/ Plant Produced Mn Peroxidase (PPMP)– glucose oxidase (GOx)- Bovine serum albumin (BSA) /Au electrode showed an excellent amperometric response to detect glucose. This biosensor is capable of responding to a wide range of glucose—20.0 µM−15.0 mM and has a lower detection limit (LOD) of 2.90µM. The reproducibility response using six electrodes is also very substantial and indicates the high capability of this biosensor to detect a wide range of 3.10±0.19µM to 13.2±1.8 mM glucose concentration. Selectivity of this electrode was investigated in an optimized experimental solution contains 10% diet green tea with citrus containing ascorbic acid (AA), and citric acid (CA) in a wide concentration of glucose at 0.02 to 14.0mM with an LOD of 3.10µM. Reproducibility was also investigated using 4 electrodes in this sample and shows notable results in the wide concentration range of 3.35±0.45µM to of 13.0 ± 0.81 mM. We also used other voltammetry methods to evaluate this biosensor. We applied linear sweep voltammetry (LSV) and this technique shows a wide range of 0.10−15.0 mM to detect glucose with a lower detection limit of 19.5µM. The performance and strength of this enzyme biosensor were the simplicity, wide linear ranges, sensitivities, selectivity, and low limits of detection. We expect that the modified biosensor has the potential for monitoring various biofluids.

Keywords: plant-produced manganese peroxidase, enzyme-based biosensors, glucose, modified gold electrode, glucose oxidase

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Authors: Farideh K. Khosroushahi, Serkan Erdal, Mucip Geni̇şel

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Soil salinity is one of the major abiotic stress factors that restricts arable land and reduces crop productivity worldwide. High salt concentration adversely affects plant growth and development inducing water deficit, ionic toxicity, nutrient imbalance, and lead to oxidative stress. Although the stimulating role of mammalian sex hormones on various biological and biochemical processes under normal and stress condition have been proven, there is no study regarding with these hormone's effect on modulation of the alternative respiration pathway and AOX gene expression. In this study, changes in alternative respiration pathway in leaves of maize seedlings under salinity and the possible modulating effect of estrogen on these changes were investigated. Maize seedlings were grown in a hydroponic media for 11 days and then were exposed to salt stress for 3 days after being sprayed estrogen. The data obtained from oxygen consumption revealed that salt stress elevated cellular respiration value in the leaves. In addition, a marked increase was observed at alternative respiration level in salt-stressed seedlings. Compared to salt application alone, supplementation with estrogen resulted in a significant rise in alternative oxidase (AOX) activities. Similarly, while salt stress caused to rise in expressions of AOX gene compared to control seedlings, estrogen application resulted in further activation of these genes’ expression compared to stressed-seedlings alone. These data revealed that mitigating role of estrogen against the detrimental effects of salt stress is linked to modulation of alternative respiration pathway.

Keywords: alternative oxidase, estrogen, Ssalt stress, AOX, maize

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Authors: Catherine Joke Adeseko

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Enzymatic browning is an economically important disorder that degrades organoleptic properties and prevent the consumer from purchasing fresh fruit and vegetables. Prevention and control of enzymatic browning in fruit and its product is imperative. Therefore, this study sought to investigate the catalytic effect of polyphenol oxidase (PPO) in the adverse browning of African bush mango (Irvingia gabonensis) fruit peel and pulp. PPO was isolated and purified, and its physicochemical properties, such as the effect of pH with SDS, temperature, and thermodynamic studies, which invariably led to thermal inactivation of purified PPO at 80 °C, were evaluated. The pH and temperature optima of PPO were found at 7.0 and 50, respectively. There was a gradual increase in the activity of PPO as the pH increases. However, the enzyme exhibited a higher activity at neutral pH 7.0, while enzymatic inhibition was observed at acidic region, pH 2.0. The presence of SDS at pH 5.0 downward was found to inhibit the activity of PPO from the peel and pulp of I. gabonensis. The average value of enthalpy (ΔH), entropy (ΔS), and Gibbs free energy (ΔG) obtained at 20 min of incubation and temperature 30 – 80 °C were respectively 39.93 kJ.mol-1, 431.57 J.mol-1 .K-1 and -107.99 kJ.mol-1 for peel PPO, and 37.92 kJ.mol-1, -442.51J.mol-1.K-1, and -107.22 kJ.mol-1 for pulp PPO. Thermal inactivation of PPO from I. gabonensis exhibited a reduction in catalytic activity as the temperature and duration of heat inactivation increases using catechol, reflected by an increment in k value. The half-life of PPO (t1/2) decreases as the incubation temperature increases due to the instability of the enzyme at high temperatures and was higher in pulp than peel. Both D and Z values decrease with increase in temperature. The information from this study suggests processing parameters for controlling PPO in the potential industrial application of I. gabonensis fruit in order to prolong the shelf-life of this fruit for maximum utilization.

Keywords: enzymatic, browning, characterization, activity

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Authors: Maria Ostroukhova, Zhanneta Zalutskaya, Elena Ermilova

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The alternative oxidase (AOX) mediates cyanide-resistant respiration, which bypasses proton-pumping complexes III and IV of the cytochrome pathway to directly transfer electrons from reduced ubiquinone to molecular oxygen. In Chlamydomonas reinhardtii, AOX is a monomeric protein that is encoded by two genes of discrete subfamilies, AOX1 and AOX2. Although AOX has been proposed to play essential roles in stress tolerance of organisms, the role of subfamily AOX2 is largely unknown. In C. reinhardtii, AOX2 was initially identified as one of constitutively low expressed genes. Like other photosynthetic organisms C. reinhardtii cells frequently experience periods of hypoxia. To examine AOX2 transcriptional regulation and role of AOX2 in hypoxia adaptation, real-time PCR analysis and artificial microRNA method were employed. Two experimental approaches have been used to induce the anoxic conditions: dark-anaerobic and light-anaerobic conditions. C. reinhardtii cells exposed to the oxygen deprivation have shown increased AOX2 mRNA levels. By contrast, AOX1 was not an anoxia-responsive gene. In C. reinhardtii, a subset of genes is regulated by transcription factor CRR1 in anaerobic conditions. Notable, the AOX2 promoter region contains the potential motif for CRR1 binding. Therefore, the role of CRR1 in the control of AOX2 transcription was tested. The CRR1-underexpressing strains, that were generated and characterized in this work, exhibited low levels of AOX2 transcripts under anoxic conditions. However, the transformants still slightly induced AOX2 gene expression in the darkness. These confirmed our suggestions that darkness is a regulatory stimulus for AOX genes in C. reinhardtii. Thus, other factors must contribute to AOX2 promoter activity under dark-anoxic conditions. Moreover, knock-down of CRR1 caused a complete reduction of AOX2 expression under light-anoxic conditions. These results indicate that (1) CRR1 is required for AOX2 expression during hypoxia, and (2) AOX2 gene is regulated by CRR1 together with yet-unknown regulatory factor(s). In addition, the AOX2-underexpressing strains were generated. The analysis of amiRNA-AOX2 strains suggested a role of this alternative oxidase in hypoxia adaptation of the alga. In conclusion, the results reported here show that C. reinhardtii AOX2 gene is stress inducible. CRR1 transcriptional factor is involved in the regulation of the AOX2 gene expression in the absence of oxygen. Moreover, AOX2 but not AOX1 functions under oxygen deprivation. This work was supported by Russian Science Foundation (research grant № 16-14-10004).

Keywords: alternative oxidase 2, artificial microRNA approach, chlamydomonas reinhardtii, hypoxia

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Authors: Chetna Tyagi. G. B. V. S. Lakshmi, Ambuj Tripathi, D. K. Avasthi

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Graphene oxide provides a good host matrix for preparing nanocomposites due to the different functional groups attached to its edges and planes. Being biocompatible, it is used in therapeutic applications. As enzyme-based biosensor requires complicated enzyme purification procedure, high fabrication cost and special storage conditions, we need enzyme-less biosensors for use even in a harsh environment like high temperature, varying pH, etc. In this work, we have prepared both enzyme-based and enzyme-less graphene oxide-based biosensors for glucose detection using glucose-oxidase as enzyme and gold nanoparticles, respectively. These samples were characterized using X-ray diffraction, UV-visible spectroscopy, scanning electron microscopy, and transmission electron microscopy to confirm the successful synthesis of the working electrodes. Electrochemical measurements were performed for both the working electrodes using a 3-electrode electrochemical cell. Cyclic voltammetry curves showed the homogeneous transfer of electron on the electrodes in the scan range between -0.2V to 0.6V. The sensing measurements were performed using differential pulse voltammetry for the glucose concentration varying from 0.01 mM to 20 mM, and sensing was improved towards glucose in the presence of gold nanoparticles. Gold nanoparticles in graphene oxide nanocomposite played an important role in sensing glucose in the absence of enzyme, glucose oxidase, as evident from these measurements. The selectivity was tested by measuring the current response of the working electrode towards glucose in the presence of the other common interfering agents like cholesterol, ascorbic acid, citric acid, and urea. The enzyme-less working electrode also showed storage stability for up to 15 weeks, making it a suitable glucose biosensor.

Keywords: electrochemical, enzyme-less, glucose, gold nanoparticles, graphene oxide, nanocomposite

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Authors: Michelle Belinda S. Weerawardana, Gobika Thiripuranathar, Priyani A. Paranagama

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Most of fresh vegetables and fruits available in the retail markets undergo a physiological disorder in its appearance and coloration, which indeed discourages consumer purchase. A loss of millions of dollars yearly to the food industry had been due to this pronounced color reaction called Enzymatic Browning which is driven due to the catalytic activity by an oxidoreductase enzyme, polyphenol oxidase (PPO). The enzyme oxidizes the phenolic compounds which are abundantly available in fruits and vegetables as substrates into quinones, which could react with proteins in its surrounding to generate black pigments, called melanins, which are highly UV-active compounds. Annona muricata (Katu anoda) and Musa acuminata (Ash plantains) is a fruit and a vegetable consumed by Sri Lankans widely due to their high nutritional values, medicinal properties and economical importance. The objective of the present study was to evaluate and determine the effective natural anti-browning inhibitors that could prevent PPO activity in the selected fruit and vegetable. Enzyme extracts from Annona muricata (Katu anoda) and Musa acuminata (Ash plantains), were prepared by homogenizing with analytical grade acetone, and pH of each enzyme extract was maintained at 7.0 using a phosphate buffer. The extracts of inhibitors were prepared using powdered ginger rhizomes and essential oil from the bark of Cinnamomum zeylanicum. Water extracts of ginger were prepared and the essential oil from Ceylon cinnamon bark was extracted using steam distillation method. Since the essential oil is not soluble in water, 0.1µl of cinnamon bark oil was mixed with 0.1µl of Triton X-100 emulsifier and 5.00 ml of water. The effect of each inhibitor on the PPO activity was investigated using catechol (0.1 mol dm-3) as the substrate and two samples of enzyme extracts prepared. The dosages of the prepared Cinnamon bark oil, and ginger (2 samples) which were used to measure the activity were 0.0035 g/ml, 0.091 g/ml and 0.087 g/ml respectively. The measurements of the inhibitory activity were obtained at a wavelength of 525 nm using the UV-visible spectrophotometer. The results evaluated thus revealed that % inhibition observed with cinnamon bark oil, and ginger for Annona muricata was 51.97%, and 60.90% respectively. The effects of cinnamon bark oil, and ginger extract on PPO activity of Musa acuminata were 49.51%, and 48.10%. The experimental findings thus revealed that Cinnamomum zeylanicum bark oil was a more effective inhibitor for PPO enzyme present in Musa acuminata and ginger was effective for PPO enzyme present in Annona muricata. Overall both the inhibitors were proven to be more effective towards the activities of PPO enzyme present in both samples. These inhibitors can thus be corroborated as effective, natural, non-toxic, anti-browning extracts, which when added to the above fruit and vegetable will increase the shelf life and also the acceptance of the product by the consumers.

Keywords: anti-browning agent, enzymatic browning, inhibitory activity, polyphenol oxidase

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Authors: Mannar R. Maurya, Bhawna Uprety, Fernando Avecilla, Pedro Adão, J. Costa Pessoa

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The reaction of the tripodal tetradentate dibasic ligand 6,6'–(2–(pyridin–2–yl)ethylazanediyl)bis(methylene)bis(2,4–di–tert–butylphenol), H2L1 I, with [VIVO(acac)2] in CH3CN gives the VVO–complex, [VVO(acac)(L1)] 1. Crystallization of 1 in CH3CN at ~0 ºC, gives dark blue crystals of 1, while at room temperature it affords dark green crystals of [{VVO(L1)}2µ–O] 3. Upon prolonged treatment of 1 in MeOH [VVO(OMe)(MeOH)(L1)] 2 is obtained. All three complexes are analyzed by single–crystal X–ray diffraction, depicting distorted octahedral geometry around vanadium. In the reaction of H2L1 with VIVOSO4 partial hydrolysis of the tripodal ligand results in elimination of the pyridyl fragment of L1 and the formation of H[VVO2(L2)] 4, containing the ONO tridentate ligand 6,6'–azanediylbis(methylene)bis(2,4–di–tert–butylphenol), H2L2 II. Compound 4, which was not fully characterized, undergoes dimerization in acetone yielding the hydroxido–bridged [{VVO(L2)}2µ–(HO)2] 5, having distorted octahedral geometry around each vanadium. In contrast, from a solution of 4 in acetonitrile, the dinuclear compound [{VVO(L2)}2µ–O] 6 is obtained, with trigonal bipyramidal geometry around each vanadium. The methoxido complex 2 is successfully employed as a functional catechol–oxidase mimic in the oxidation of catechol to o–quinone under air. The process is confirmed to follow a Michaelis–Menten type kinetics with respect to catechol, the Vmax and KM values obtained being 7.66×10–6 M min -1 and 0.0557 M, respectively, and the turnover frequency is 0.0541 min–1. Complex 2 is also used as a catalyst precursor for the oxidative bromination of thymol in aqueous medium. The selectivity shows quite interesting trends, namely when not using excess of primary oxidizing agent, H2O2 the para mono–brominated product corresponds to ~93 % of the products and no dibromo derivative is formed.

Keywords: oxidovanadium (V) complexes, tripodal ligand, crystal structure, catechol oxidase mimetic activity

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Authors: Makhosi Buthelezi

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Mitochondrial DNA cytochrome c oxidase I (COI) gene analyses linked the South African groundnut leaf miner (GLM) to the Australian soya bean moth Aproaerema simplexella (Walker) and Indian Aproaerema modicella (Deventer). Thus, the genetic relatedness of GLM, A. simplexela, and A. modicella was examined by performing mitochondrial and nuclear (COI, cytochrome oxidase subunit II (COII), mitochondrial cytochrome b (CYTB), nuclear ribosomal 28S (28S) and intergenic spacer elongation factor-1 alpha ( EF-1 ALPHA) on 44 specimens collected from South Africa, four from Mozambique, and three each from single locations in India and Australia. Phylogenetic analyses were conducted using the Maximum Parsimony (MP) and Neighbour-Joining (NJ) methods. All of the datasets of the five DNA gene regions that were sequenced were also analyzed using the Basic Local Alignment Search Tool (BLAST) to find the closest matches for inclusion in the phylogenetic trees as outgroups and for purposes of information. In the phylogenetic trees for COI, COII, cytb and EF-1 ALPHA, a similar pattern was observed in the way that the sequences assembled into different groups; i.e., some sequences of A. simplexella from Australia were grouped separately from the others, but some Australian sequences grouped with those of the GLM from South Africa, India, and Mozambique. In the phylogenetic tree for 28S, all sequences from South Africa, Australia, India, and Mozambique grouped together and formed one group. For COI, genetic pairwise distance ranged from 0.97 to 3.60 %, for COII it ranged from 0.19% to 2.32%, for cytb it ranged from 0.25 to 9.77% and for EF-1 ALPHA it ranged 0.48 to 6.99%. Results of this study indicate that these populations are genetically related and presumably constitute a single species. Thus, further molecular and morphological studies need to be undertaken in order to resolve this apparent conundrum on the taxonomy of these populations.

Keywords: aproaerema modicella, aproaerema simplexella, mitochondrial DNA, nuclear DNA

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Authors: Haroon Khan, Chul Min Kim, Sung Yeol Kim, Sanket Goel, Prabhat K. Dwivedi, Ashutosh Sharma, Gyu Man Kim

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Enzymatic biofuel cells (EBFCs) have drawn the attention of researchers due to its demanding application in medical implants. In EBFCs, electricity is produced with the help of redox enzymes. In this study, we report the fabrication of membraneless EBFC with new design of electrodes to overcome microchannel related limitations. The device consists of double layer of electrodes on both sides of Y-shaped microchannel to reduce the effect of oxygen depletion layer and diffusion of fuel and oxidant at the end of microchannel. Moreover, the length of microchannel was reduced by half keeping the same area of multiwalled carbon nanotubes (MWCNT) electrodes. Polydimethylsiloxane (PDMS) stencils were used to pattern MWCNT electrodes on etched Indium Tin Oxide (ITO) glass. PDMS casting was used to fabricate microchannel of the device. Both anode and cathode were modified with glucose oxidase and laccase. Furthermore, these enzymes were covalently bound to carboxyl MWCNTs with the help of EDC/NHS. Glucose used as fuel was oxidized by glucose oxidase at anode while oxygen was reduced to water at the cathode side. The resulted devices were investigated with the help of polarization curves obtained from Chronopotentiometry technique by using potentiostat. From results, we conclude that the performance of double layer EBFC is improved 15 % as compared to single layer EBFC delivering maximum power density of 71.25 µW cm-2 at a cell potential of 0.3 V and current density of 250 µA cm-2 at micro channel height of 450-µm and flow rate of 25 ml hr-1. However, the new device was stable only for three days after which its power output was rapidly dropped by 75 %. This work demonstrates that the power output of membraneless EBFC is improved comparatively, but still efforts will be needed to make the device stable over long period of time.

Keywords: EBFC, glucose, MWCNT, microfluidic

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Authors: Miao Zhang, Limin Liu, Feng Zhi, Panpan Niu, Mengya Yang, Xuemei Zhu, Ying Diao, Jun Wang, Ying Zhao

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Background and Aims: Clinical studies have demonstrated that serum semicarbazide-sensitive amine oxidase (SSAO) activities positively correlate with the progression of atherosclerosis. The aim of the present study is to investigate the effect of SSAO inactivation on the development of atherosclerosis. Methods: Female LDLr knockout (KO) mice were given the Western-type diet for 6 and 9 weeks to induce the formation of early and advanced lesions, and semicarbazide (SCZ, 0.125%) was added into the drinking water to inactivate SSAO in vivo. Results: Despite no impact on plasma total cholesterol levels, abrogation of SSAO by SCZ not only resulted in the enlargement of both early (1.5-fold, p=0.0043) and advanced (1.8-fold, p=0.0013) atherosclerotic lesions, but also led to reduced/increased lesion contents of macrophages/smooth muscle cells (SMCs) (macrophage: ~0.74-fold, p=0.0002(early)/0.0016(advanced); SMC: ~1.55-fold, p=0.0003(early) /0.0001(advanced)), respectively. Moreover, SSAO inactivation inhibited the migration of circulating monocytes into peripheral tissues and reduced the amount of circulating Ly6Chigh monocytes (0.7-fold, p=0.0001), which may account for the reduced macrophage content in lesions. In contrast, the increased number of SMCs in lesions of SCZ-treated mice is attributed to an augmented synthetic vascular SMC phenotype switch as evidenced by the increased proliferation of SMCs and accumulation of collagens in vivo. Conclusion: SSAO inactivation by SCZ promotes the phenotypic switch of SMCs and the development of atherosclerosis. The enzymatic activity of SSAO may thus represent a potential target in the prevention and/or treatment of atherosclerosis.

Keywords: atherosclerosis, phenotype switch of smooth muscle cells, SSAO/VAP-1, semicarbazide

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Authors: M. Y. Abubakar, Ipinjolu J. K., Yuzine B. Esa, Magawata I., Hassan W. A., Turaki A. A.

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Catfish (Heterobranchus spp.) is a major freshwater fish that are widely distributed in Nigeria waters and are gaining rapid aquaculture expansion. However, indiscriminate artificial crossbreeding of the species with others poses a threat to their biodiversity. There is a paucity of information about the genetic variability, hence this insight on the genetic variability is badly needed, not only for the species conservation but for aquaculture expansion. In this study, we tested the level of Genetic diversity, population differentiation and phylogenetic relationship analysis on 35 individuals of two populations of Heterobranchus bidorsalis and 29 individuals of three populations of Heterobranchus longifilis using the mitochondrial cytochrome c oxidase subunit I (mtDNA COI) gene sequence. Nucleotide sequences of 650 bp fragment of the COI gene of the two species were compared. In the whole 4 and 5 haplotypes were distinguished in the populations of H. bidorsalis & H. longifilis with accession numbers (MG334168 - MG334171 & MG334172 to MG334176) respectively. Haplotypes diversity indices revealed a range of 0.59 ± 0.08 to 0.57 ± 0.09 in H. bidorsalis and 0.000 to 0.001051 ± 0.000945 in H. longifilis population, respectively. Analysis of molecular variance (AMOVA) revealed no significant variation among H. bidorsalis population of the Niger & Benue Rivers, detected significant genetic variation was between the Rivers of Niger, Kaduna and Benue population of H. longifilis. Two main clades were recovered, showing a clear separation between H. bidorsalis and H. longifilis in the phylogenetic tree. The mtDNA COI genes studied revealed high gene flow between populations with no distinct genetic differentiation between the populations as measured by the fixation index (FST) statistic. However, a proportion of population-specific haplotypes was observed in the two species studied, suggesting a substantial degree of genetic distinctiveness for each of the population investigated. These findings present the description of the species character and accessions of the fish’s genetic resources, through gene sequence submitted in Genetic database. The data will help to protect their valuable wild resource and contribute to their recovery and selective breeding in Nigeria.

Keywords: AMOVA, genetic diversity, Heterobranchus spp., mtDNA COI, phylogenetic tree

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Authors: Kedar S. Prabhavalkar, Nimmy Baby Poovanpallil

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Lamotrigine is approved for maintenance treatment of bipolar I disorder. However, its role in the treatment of acute bipolar depression is not well clear. Its efficacy in the treatment of major depressive disorders including refractory unipolar depression suggested the use of lamotrigine as an augmentation drug for acute bipolar depression. The present study aims to evaluate and perform a comparative analysis of the therapeutic effects of lamotrigine, an epileptic mood stabilizer, when used alone and in combination with duloxetine in treating acute bipolar depression at different doses of lamotrigine. Male swiss albino mice were used. For evaluation of efficacy of combination, immobility period was analyzed 30 min after the treatment from forced swim and tail suspension tests. Further amount of sucrose consumed in sucrose preference test was estimated. The combination of duloxetine and lamotrigine showed potentiation of antidepressant activity in acute models. Decrease in immobility time and increase in the amount of sucrose consumption in stressed mice were higher in combined group compared to lamotrigine monotherapy group. Brain monoamine levels were also attenuated more with combination compared to monotherapy. Results of the present study suggest potential role of lamotrigine and duloxetine combination in the treatment of acute bipolar depression.

Keywords: lamotrigine, duloxetine, acute bipolar depression, augmentation

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Authors: Susmita Goswami, Joyeeta Mitra, Indu Gaur, Neha Bhadauria, Shilpi Shilpi, Prabir K. Paul

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'Malbhog’, an indigenous banana variety, much prized for its flavour and delicacy suffers production losses due to Fusarium oxysporum f.sp. cubense. The pathogen enters young plants through feeder roots causing wilting of plants ultimately leading to death of plants. The pathogen spreads rapidly to other plants in the field. In eastern part of India, this variety escapes the onslaught of the pathogen when either co-cultivated or rotated with Amorphophallus campanulatus (yam). The present study provides an insight into the physiological aspect of the biocontrol by yam. In vitro application of sterile aqueous extract of yam tuber (100gm/100ml distilled water and its 1:10 and 1:100 dilutions) were mixed with PDA media which was substantially inoculated with spores of Fusarium oxysporum f.sp. cubense. The extract could significantly reduce germination of pathogen spores. Banana variety susceptible to Fusarium sp was raised in soil rite under aseptic conditions. Spores of the pathogen (106 spores/ml) were inoculated into the soil rite. The plants were spread with aqueous extract of yam. The control plants were treated with sterilized distilled water. The activity of phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO) and peroxidase (POX) were estimated in leaves and roots at interval of 24 hours for 5 days after treatment. The incidence of wilt disease was recorded after two weeks. The results demonstrated that yam extract could induce significant activity of PAL, PPO and POX along with accumulation of phenols in both roots and leaves of banana plants. However, significantly high activity of enzymes and phenol accumulation was observed in roots. The disease incidence was significantly low in yam treated plants. The results clearly demonstrated the control of the pathogen due to induction of defense mechanism in the host by the extract. The observed control of the pathogen in the field could possibly be due to induction of such defense responses in host by exudates leached into the soil from yam tubers. Yam extract could be a potential source of environment-friendly biocide against Panama wilt of banana.

Keywords: Amorphophallus campanulatus, banana, Fusarium oxysporum f.sp. cubense, phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO), peroxidase (POX)

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Authors: Hanouf A. S. Al Nuwaysir, Nadine Moubayed, Abir Ben Bacha, Islem Abid

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Consumption of waste water continues to cause significant problems for human health in both developed and developing countries. Many regulations have been implied by different world authorities controlling water quality for the presence of coliforms used as standard indicators of water quality deterioration and historically leading health protection concept. In this study, the European directive for the detection of Escherichia coli, ISO 9308-1, was applied to examine and monitor coliforms in water samples collected from Wadi Hanifa and neighboring wells, Riyadh governorate, kingdom of Saudi Arabia, which is used for irrigation and industrial purposes. Samples were taken from different locations for 8 months consecutively, chlorine concentration ranging from 0.1- 0.4 mg/l, was determined using the DPD FREE CHLORINE HACH kit. Water samples were then analyzed following the ISO protocol which relies on the membrane filtration technique (0.45µm, pore size membrane filter) and a chromogenic medium TTC, a lactose based medium used for the detection and enumeration of total coliforms and E.coli. Data showed that the number of bacterial isolates ranged from 60 to 300 colonies/100ml for well and surface water samples respectively; where higher numbers were attributed to the surface samples. Organisms which apparently ferment lactose on TTC agar plates, appearing as orange colonies, were selected and additionally cultured on EMB and MacConkey agar for a further differentiation among E.coli and coliform bacteria. Two additional biochemical tests (Cytochrome oxidase and indole from tryptophan) were also investigated to detect and differentiate the presence of E.coli from other coliforms, E. coli was identified in an average of 5 to 7colonies among 25 selected colonies.On the other hand, a more rapid, specific and sensitive analytical molecular detection namely single colony PCR was also performed targeting hha gene to sensitively detect E.coli, giving more accurate and time consuming identification of colonies considered presumptively as E.coli. Comparative methodologies, such as ultrafiltration and direct DNA extraction from membrane filters (MoBio, Grermany) were also applied; however, results were not as accurate as the membrane filtration, making it a technique of choice for the detection and enumeration of water coliforms, followed by sufficiently specific enzymatic confirmatory stage.

Keywords: coliform, cytochrome oxidase, hha primer, membrane filtration, single colony PCR

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Authors: Iulianna Taritsa, Kuldeep Neote, Eric Fossel

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Lysosome-induced immunogenic cell death (LIICD) is a powerful mechanism of targeting cancer cells that kills circulating malignant cells and primes the host’s immune cells against future remission. Current immunotherapies for cancer are limited in preventing recurrence – a gap that can be bridged by training the immune system to recognize cancer neoantigens. Lysosomal leakage can be induced therapeutically to traffic antigens from dying cells to dendritic cells, which can later present those tumorigenic antigens to T cells. Previous research has shown that oxidative agents administered in the tumor microenvironment can initiate LIICD. We generated a fusion protein between an oxidative agent known as xanthine oxidase (XO) and a mini-antibody specific for EGFR/HER2-sensitive breast tumor cells. The anti-EGFR single domain antibody fragment is uniquely sourced from llama, which is functional without the presence of a light chain. These llama micro-antibodies have been shown to be better able to penetrate tissues and have improved physicochemical stability as compared to traditional monoclonal antibodies. We demonstrate that the fusion protein created is stable and can induce early markers of immunogenic cell death in an in vitro human breast cancer cell line (SkBr3). Specifically, we measured overall cell death, as well as surface-expressed calreticulin, extracellular ATP release, and HMGB1 production. These markers are consensus indicators of ICD. Flow cytometry, luminescence assays, and ELISA were used respectively to quantify biomarker levels between treated versus untreated cells. We also included a positive control group of SkBr3 cells dosed with doxorubicin (a known inducer of LIICD) and a negative control dosed with cisplatin (a known inducer of cell death, but not of the immunogenic variety). We looked at each marker at various time points after cancer cells were treated with the XO/antibody fusion protein, doxorubicin, and cisplatin. Upregulated biomarkers after treatment with the fusion protein indicate an immunogenic response. We thus show the potential for this fusion protein to induce an anticancer effect paired with an adaptive immune response against EGFR/HER2+ cells. Our research in human cell lines here provides evidence for the success of the same therapeutic method for patients and serves as the gateway to developing a new treatment approach against breast cancer.

Keywords: apoptosis, breast cancer, immunogenic cell death, lysosome

Procedia PDF Downloads 179

Authors: R. I. You, C. L. Ho, M. S. Dai, H. M. Hung, S. F. Yen, C. S. Chen, T. Y. Chao

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Neutrophils are the most abundant innate immune cells to against invading microorganisms. Numerous data shown neutrophils have plasticity in response to physiological and pathological conditions. Tumor-associated neutrophils (TAN) exist in distinct types of tumor and play an important role in cancer biology. Different transcriptomic profiles of neutrophils in tumor and non-tumor samples have been identified. Several miRNAs have been recognized as regulators of gene expression in neutrophil, which may have key roles in neutrophil activation. However, the miRNAs expression patterns in TAN are not well known. To address this question, magnetic bead isolated neutrophils from tumor-bearing mice were used in this study. We analyzed production of reactive oxygen species (ROS) by luminol-dependent chemiluminescence assay. The expression of miRNAs targeting NADPH oxidase, ROS generation and autophagy was explored using quantitative real-time polymerase chain reaction. Our data suggest that tumor environment influence neutrophil develop to differential states of activation via miRNAs regulation.

Keywords: tumor-associated neutrophil, miRNAs, neutrophil, ROS

Procedia PDF Downloads 440

Authors: B. M. Md-Zain, N. A. Rahman, M. A. B. Abdul-Latiff, W. M. R. Idris

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We conducted molecular research to portray phylogenetic relationships of Malaysian primates particularly in the genus of Macaca. We have sequenced cytochrome C oxidase subunit I (COI) of mitochondrial DNA of several individuals from M. fascicularis and M. arctoides. PCR amplifications were performed and COI DNA sequences were aligned using ClustalW. Phylogenetic trees were constructed using distance analyses by employing neighbor-joining algorithm (NJ). We managed to sequence 700 bp of COI DNA sequences. The tree topology showed that M. fascicularis did not clump based on phyleogeography division in Peninsular Malaysia. Individuals from Negeri Sembilan merged together with samples from Perak and Penang into one clade. In addition, phylogenetic analyses indicated that M. arctoides was classified into sinica group instead of fascicularis group supported by genetic distance data. COI gene is an effective locus to clarify phylogenetic position of M. arctoides but not in discriminating M. fascicularis population in Peninsular Malaysia.

Keywords: cercopithecine, long-tailed macaque, Macaca fascicularis, Macaca arctoides

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Authors: C. I. Boshoff, S. Steenkamp-Jonker

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Cystic echinococcosis (CE), caused by Echinococcus granulosus is an important parasitic infection in livestock worldwide, with severe zoonotic potential. It is important to understand the variability of Echinococcus granulosus, as genotype variations may influence lifecycle patterns, development rate, and transmission. Cystic Echinococcus samples were collected from domestic animals in Eastern Cape Province, South Africa. A molecular study was performed on 14 hydatid cysts obtained from caprine, ovine and bovine livers in order to determine the Echinococcus granulosus strain present in these hosts. The sequencing of the mitochondrial cytochrome C oxidase subunit I (coxI) gene of the hydatid cysts produced sequences of 400 bp for each sample analysed. These sequences were aligned with those present in GenBank and a phylogenetic tree was constructed. Based on coxI genotype the isolates could be grouped into E. granulosus sensu stricto. The findings of the study represent a pilot molecular study on Echinococcus from domestic animals undertaken in South Africa.

Keywords: Echinococcus granulosus, genotypes, livestock, South Africa

Procedia PDF Downloads 408

Authors: Rini Jarial, Puranjan Mishra, Lakhveer Singh, Sveta Thakur, A. W. Zularisam, Mimi Sakinah

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The aim of the present study was to assess the biological properties of Camellia sinensis and to identify its functional compounds. The methanolic leaves-extract (MLE) of commercial green tea (Camellia sinensis) was assessed for anti-bacterial activities by measuring inhibition zones against a panel of pathogenic bacterial strains using agar diffusion method. The flavonoid (5.0 to 8.0 mg/ml) and protein content (10 to 15 mg/ml) of the MLE were recorded. MLE at a concentration of 25 μg/ml showed marked anti-bacterial activity against all bacterial strains (11-30 mm zone of inhibition) and was maximum against Staphylococcus aureus (30 mm). The MLE of Camellia sinensis had the best MIC values of 2.25 and 0.56 mg/ml against S. aureus and Enterobacter sp., respectively. The MLE also possessed good anti-lipolytic activity (65%) against a Porcine pancreatic lipase (PPL) and cholesterol oxidase inhibition (79%). The present study provided strong experimental evidences that the MLE of Camellia sinensis is not only a potent source of natural anti-oxidants and anti-bacterial activity but also possesses efficient cholesterol degradation and anti-lipolytic activities that might be beneficial in the body weight management.

Keywords: anti-oxidant, anti-bacterial activity, anti-lipolytic activity, Camellia sinensis, phyto-chemicals

Procedia PDF Downloads 264

Authors: Toshiyuki Sato, Kenta Iitani, Koji Toma, Takahiro Arakawa, Kohji Mitsubayashi

Abstract:

A 2-dimensional imaging system (Sniff-camera) for gaseous ethanol emissions from a human palm skin was constructed and demonstrated. This imaging system measures gaseous ethanol concentrations as intensities of chemiluminescence (CL) by luminol reaction induced by alcohol oxidase and luminol-hydrogen peroxide system. A conversion of ethanol distributions and concentrations to 2-dimensional CL was conducted on an enzyme-immobilized mesh substrate in a dark box, which contained a luminol solution. In order to visualize ethanol emissions from human palm skin, we developed highly sensitive and selective imaging system for transpired gaseous ethanol at sub ppm-levels. High sensitivity imaging allows us to successfully visualize the emissions dynamics of transdermal gaseous ethanol. The intensity of each pixel on the palm shows the reflection of ethanol concentrations distributions based on the metabolism of oral alcohol administration. This imaging system is significant and useful for the assessment of ethanol measurement of the palmar skin.

Keywords: sniff-camera, gas-imaging, ethanol vapor, human body gas

Procedia PDF Downloads 344

Authors: Wilailak Siripornadulsil, Siriyanapat Tasaku, Jutamas Buahorm, Surasak Siripornadulsil

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The objectives of this study were to isolate LAB from various sources, dietary supplement, Thai traditional fermented food, and freshwater fish and to characterize their potential as probiotic cultures. Out of 1,558 isolates, 730 were identified as LAB based on isolation on MRS agar supplemented with a bromocresol purple indicator and CaCO3 and gram-positive, catalase and oxidase negative characteristics. Eight isolates showed the potential probiotic properties including tolerance to acid, bile salt and heat, proteolytic, amylolytic and lipolytic activities and oxalate-degrading capability. They all showed the antimicrobial activity against some Gram-negative and Gram-positive pathogenic bacteria. Based on 16S rDNA sequence analysis, they were identified as Enterococcus faecalis BT2 and MG30, Leconostoc mesenteroides SW64 and Pediococcus pentosaceous BD33, CF32, NP6, PS34 and SW5. The health beneficial effects and food safety will be further investigated and developed as a probiotic or protective culture used in Nile tilapia belly flap meat fermentation.

Keywords: probiotic, lactic acid bacteria, pathogen, protective culture

Procedia PDF Downloads 362

Authors: M. J. Norashirene, Y. Zakiah, S. Nurdiana, I. Nur Hilwani, M. H. Siti Khairiyah, M. J. Muhamad Arif

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In this study, six bacterial isolates of a slightly thermophilic organism from the Sg. Klah hot spring, Malaysia were successfully isolated and designated as M7T55D1, M7T55D2, M7T55D3, M7T53D1, M7T53D2 and M7T53D3 respectively. The bacterial isolates were screened for their cellulose hydrolytic ability on Carboxymethlycellulose agar medium. The isolated bacterial strains were identified morphologically, biochemically and molecularly with the aid of 16S rDNA sequencing. All of the bacteria showed their optimum growth at a slightly alkaline pH of 7.5 with a temperature of 55°C. All strains were Gram-negative, non-spore forming type, strictly aerobic, catalase-positive and oxidase-positive with the ability to produce thermostable cellulase. Based on BLASTn results, bacterial isolates of M7T55D2 and M7T53D1 gave the highest homology (97%) with similarity to Tepidimonas ignava while isolates M7T55D1, M7T55D3, M7T53D2 and M7T53D3 showed their closest homology (97%-98%) with Tepidimonas thermarum. These cellulolytic thermophiles might have a commercial potential to produce valuable thermostable cellulase.

Keywords: cellulase, cellulolytic, thermophiles, 16S rDNA gene

Procedia PDF Downloads 320

Authors: D. Sengani, N. Potgieter, P. E. L. Mojapelo

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Three bacteria species which comprise of Gram negative and Gram positive microorganisms were isolated and identified on the basis of morpho-cultural study, catalase tests, oxidase tests and biochemical characteristics were found belonging to different genera including Burkholderia cepacia, Pasteurella pneumotropica and Enterococcus faecalis. The main objective of this study was to isolate and identify PCB degrading bacteria from PCB contaminated soils and test them for their degradation ability of PCBs in natural habitat conditions. The results indicated an overall decrease of PCB concentration level with the gradient average ranging from 1.5 to 1.8 respectively. Enterococcus faecalis removed as much as 32% of PCBs in the contaminated soil samples. Whereas Pasteurella pneumotropica could remove 24% of PCBs, Burkholderia cepacia 21% of PCBs and the mixed culture removed 23%. Data showed that the three bacterial strains could tolerate high concentration of PCBs. The results provided the evidence that naturally occurring bacteria in soil contaminated with PCBs have the potential to degrade PCBs. Statistical analysis showed that there was a significant positive correlation between bacteria growth and treatment with a coefficient of (r) =0.1459 and p value < 0.001.

Keywords: bacteria, bioaccumulation, biodegradation, bioremediation, polychlorinated biphenyls

Procedia PDF Downloads 222

Authors: Habu Musa, Nusaibah Binti Syd Ali

Abstract:

Oil palm industries particularly in Malaysia and Indonesia are being devastated by Ganoderma disease caused by Ganoderma spp. To date, this disease has been causing serious oil palm yield losses and collapse of oil palm trees, thus affecting its contribution to the producer’s economy. Research on sustainable and eco-friendly remedy to counter Ganoderma disease is on the upsurge to avoid the current control measures via synthetic fungicides. Trichoderma species have been the most studied and valued microbes as biological control agents in an effort to combat a wide range of plant diseases sustainably. Therefore, in this current study, the potential of Trichoderma spp. (Trichoderma asperellum, Trichoderma harzianum, and Trichoderma virens) as a consortium approach was evaluated as biological control agents against Ganoderma disease on oil palm. The consortium of Trichoderma spp. applied found to be the most effective treatment in suppressing Ganoderma disease with 83.03% and 89.16% from the foliar and bole symptoms respectively. Besides, it exhibited tremendous enhancement in the oil palm seedling vegetative growth parameters. Also, it had highly induced significant activity of peroxidase, polyphenol oxidase and total phenolic content was recorded in the consortium treatment compared to the control treatment. Disease development was slower in the seedlings treated with consortium of Trichoderma spp. compared to the positive control, which exhibited with the highest percentage of disease severity.

Keywords: biological control, ganoderma disease, trichoderma, disease severity

Procedia PDF Downloads 254

Authors: Dalia. G. Aseel, E. E. Hafez, S. M. Hammad

Abstract:

Viral RNAs of both potato leaf roll virus (PLRV) and potato virus Y (PVY) were extracted from infected potato leaves collected from different Egyptian regions. Differential Display Polymerase Chain Reaction (DD-PCR) using (Endogluconase, β-1,3-glucanases, Chitinase, Peroxidase and Polyphenol oxidase) primers (forward strand) for was performed. The obtained data revealed different banding patterns depending on the viral type and the region of infection. Regarding PLRV, a 58 up regulated and 19 down regulated genes were detected, while, 31 up regulated and 14 down regulated genes were observed in case of PVY. Based on the nucleotide sequencing, variable phylogenetic relationships were reported for the three sequenced genes coding for: Induced stolen tip protein, Disease resistance RPP-like protein and non-specific lipid-transfer protein. In a complementary approach, using the quantitative Real-time PCR, the expressions of PRs genes understudy were estimated in the infected leaves by PLRV and PVY of three potato cultivars (Spunta, Diamont and Cara). The infection with both viruses inhibited the expressions of the five PRs genes. On the contrary, infected leaves by PLRV or PVY elevated the expression of some defense genes. This interaction also may be enhanced and/or inhibited the expression of some genes responsible for the plant defense mechanisms.

Keywords: PLRV, PVY, PR genes, DD-PCR, qRT-PCR, sequencing

Procedia PDF Downloads 314