World Academy of Science, Engineering and Technology

Authors: Su Mon Saw, Joe Rothnagel

Abstract:

Short open reading frames (sORFs) located in 5’UTR of mRNAs are known as uORFs. Characterization of uORF-encoded peptides (uPEPs) i.e., a subset of short open reading frame encoded peptides (sPEPs) and their translation regulation lead to understanding of causes of genetic disease, proteome complexity and development of treatments. Existence of uORFs within cellular proteome could be detected by LC-MS/MS. The ability of uORF to be translated into uPEP and achievement of uPEP identification will allow uPEP’s characterization, structures, functions, subcellular localization, evolutionary maintenance (conservation in human and other species) and abundance in cells. It is hypothesized that a subset of sORFs are translatable and that their encoded sPEPs are functional and are endogenously expressed contributing to the eukaryotic cellular proteome complexity. This project aimed to investigate whether sORFs encode functional peptides. Liquid chromatography-mass spectrometry (LC-MS) and bioinformatics were thus employed. Due to probable low abundance of sPEPs and small in sizes, the need for efficient peptide enrichment strategies for enriching small proteins and depleting the sub-proteome of large and abundant proteins is crucial for identifying sPEPs. Low molecular weight proteins were extracted using SDS-PAGE from Human Embryonic Kidney (HEK293) cells and Strong Cation Exchange Chromatography (SCX) from secreted HEK293 cells. Extracted proteins were digested by trypsin to peptides, which were detected by LC-MS/MS. The MS/MS data obtained was searched against Swiss-Prot using MASCOT version 2.4 to filter out known proteins, and all unmatched spectra were re-searched against human RefSeq database. ProteinPilot v5.0.1 was used to identify sPEPs by searching against human RefSeq, Vanderperre and Human Alternative Open Reading Frame (HaltORF) databases. Potential sPEPs were analyzed by bioinformatics. Since SDS PAGE electrophoresis could not separate proteins <20kDa, this could not identify sPEPs. All MASCOT-identified peptide fragments were parts of main open reading frame (mORF) by ORF Finder search and blastp search. No sPEP was detected and existence of sPEPs could not be identified in this study. 13 translated sORFs in HEK293 cells by mass spectrometry in previous studies were characterized by bioinformatics. Identified sPEPs from previous studies were <100 amino acids and <15 kDa. Bioinformatics results showed that sORFs are translated to sPEPs and contribute to proteome complexity. uPEP translated from uORF of SLC35A4 was strongly conserved in human and mouse while uPEP translated from uORF of MKKS was strongly conserved in human and Rhesus monkey. Cross-species conserved uORFs in association with protein translation strongly suggest evolutionary maintenance of coding sequence and indicate probable functional expression of peptides encoded within these uORFs. Translation of sORFs was confirmed by mass spectrometry and sPEPs were characterized with bioinformatics.

Keywords: bioinformatics, HEK293 cells, liquid chromatography-mass spectrometry, ProteinPilot, Strong Cation Exchange Chromatography, SDS-PAGE, sPEPs

Procedia PDF Downloads 173

Authors: Saravanan Govindaraju, Kyusik Yun

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The purpose of this study is to develop the chitosan doped curcumin gold cluster nanofiber for wound healing and skin cancer drug delivery applications. Chitosan is a typical marine polysaccharide composed of glucosamine and n-acetyl glucosamine biodegradable and biocompatible polymer. Curcumin is a natural bioactive molecule obtained from Curcuma longo, it mostly occurs in some Asian countries like India and China. It has naturally antioxidant, antimicrobial, wound healing and anticancer property. Due to this advantage, we prepared a combination of natural polymer chitosan with Curcumin and gold nanocluster nanofiber (CH-CUR-AuNCs nanofibers). The prepared nanofiber was characterized by using Fourier transform infrared spectroscopy (FT-IR), and scanning electron microscopy (SEM). Antibacterial studies were performed with E.coli and S.aureus. Antioxidant assay, drug release test, and cytotoxicity will be evaluated. Prepared nanofiber emits low intensity of red fluorescent. The FTIR confirm the presence of chitosan and Curcumin in the nanofiber. In vitro study clearly shows the antibacterial activity against the gram negative and gram positive bacteria. Particularly, synthesised nanofibers provide better antibacterial activity against gram negative than gram positive. Cytotoxicity study also provides better killing rate in cancer cell, biocompatible with normal cell. Prepared CH-CUR-AuNCs nanofibers provide the better killing rate to bacterial strains and cancer cells. Finally, prepared nanofiber can be possible to use for wound healing dressing, patch for skin cancer and other biomedical applications.

Keywords: curcumin, chitosan, gold clusters, nanofibers

Procedia PDF Downloads 251

Authors: A. K. R. Gobinath, He Jianzhong, Kun-Lin Yang

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Fruit waste was used as a feedstock to produce biohydrogen in this study. Fruit waste used in this study was collected from several fruit juice stalls in Singapore. Based on our observation, the fruit waste contained 35-40% orange, 10-20% watermelon, 10-15% apple, 10-15% pineapple, 1-5% mango. They were mixed with water (1:1 ratio based on wet biomass) and blended to attain homogenous mixtures. Later, fruit waste was subjected to one of the following pretreatments: autoclave (121 °C for 20min), microwave (20min) or both. After pretreatment, the total sugar concentration in the hydrolysate was high (>12g/l) when both autoclave and microwave were applied. In contrast, samples without pretreatment measured only less than 2g/l of sugar. While using these hydrolysates as carbon sources, Clostridium strain BOH3 produces 2526-3126 ml/l of hydrogen after 72h of anaerobic fermentation. The hydrogen yield was 295-300 ml/g of sugar which is close to the hydrogen yields from glucose (338 ml/gm) and xylose (330 ml/gm). Our HPLC analysis showed that fruit waste hydrolysate contained oligosugars (25-27%), sucrose (18-23%), fructose (25-30%), glucose (10-15%) and mannose (2-5%). Additionally, pretreatment led to the release of free amino acids (160-512 mg/l), calcium (7.8-12.9 ppm), magnesium (4.32-6.55 ppm), potassium (5.4-65.1 ppm) and sodium (0.4-0.5 ppm) into the hydrolysate. These nutrients were able to support strain-BOH3 to grow and produce high level of hydrogen. Notably, unlike other pretreatment methods (with strong acids and bases), these pretreatment techniques did not generate any inhibitors (e.g. furfural and phenolic acids) to suppress the hydrogen production. Interestingly, strain BOH3 can also ferment pretreated fruit waste slurry and produce hydrogen with a high yield (156-343 ml/gm fruit waste). While fermenting pretreated fruit waste slurry, strain-BOH3 excreted several saccharolytic enzymes majorly xylanase (1.84U/ml), amylase (1.10U/ml), pectinase (0.36U/ml) and cellulase (0.43U/ml). Due to expressions of these enzymes, strain BOH3 was able to directly utilize pretreated fruit waste hydrolysate and produces high-level of hydrogen.

Keywords: autoclave pretreatment, biohydrogen production, clostridial fermentation, fruit waste, and microwave pretreatment

Procedia PDF Downloads 520

Authors: Pawinee Innark, Hudsaya Punyanitikul, Chanuluk Khanobdee, Chatchawan Jantasuriyarat, Sompid Samipak

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One of the most devastating diseases in cucumber is downy mildew caused by the fungus Pseudoperonospora cubensis. To enable the use of marker-assisted breeding for resistance cultivars, sixty six microsatellite markers were used to map (quantitative trait loci) QTLs for DM resistance. Total of 315 F2 population from the cross between DM-resistant inbred line CSL0067 and susceptible CSL0139 were evaluated for downy mildew resistance in cotyledon, first and second true leaf at 7, 10, and 14 day after inoculation. The QTL analysis revealed that the downy mildew resistant genes were controlled by multiple recessive genes. From eight linkage groups (LG 1.1, 1.2, 2, 3, 4, 5.1, 5.2 and 6), fourteen QTL positions were detected on 4 linkage groups (LG 1.1, 2, 5.1 and 6) with the log of odd scores ranged from 3.538 to 9.165. Among them, Cot7_5.1_2 and Cot10_5.1 had major-effect QTL with the R2 values of 10.9 and 12.5%, respectively. The flanking markers for Cot7_5.1_2 were SSR19172 - SSR07531 markers and for Cot10_5.1 were SSR03943 - SSR00772. Besides QTLs on chromosome 1, 5 and 6 that were previously reported, this study also revealed a QTL for DM resistance on chromosome 2 that can be used as a new source in cucumber breeding program.

Keywords: cucumber, DNA marker, downy mildew, QTL

Procedia PDF Downloads 234

Authors: Achmad Buhori, Reza Imam Pratama, Tissa Wiraatmaja, Wanti Megawati

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Data from many countries indicates that there is a marked increase of dental caries. The increases in caries appear to occur in lower socioeconomic groups. It is possible that the benefits of prevention of dental caries are not reaching these groups. However, there is a way to decrease dental caries by adding 5% of bromelain and calcium as an active agent in toothpaste. Bromelain can break glutamine-alanine bond and arginine-alanine bond which is a constituent of amino acid that causes dental plague which is one of the factors of dental caries. Calcium help rebuilds the teeth by strengthening and repairing enamel. Bromelain can be found from the extraction of pineapple (Ananas comosus) cobs (88.86-94.22 % of bromelain recovery during extraction based on the enzyme unit) and calcium can be taken from eggshell (95% of dry eggshell consist of calcium). The aim of this experiment is to make a toothpaste which contains bromelain and calcium as an effective, cheap, and healthy way to decrease dental caries around the world.

Keywords: bromelain, calcium, dental caries, dental plague, toothpaste

Procedia PDF Downloads 247

Authors: Yifang Gong

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Pulse diagnosis is one of the most important diagnosis methods in traditional Chinese medicine. It is also the trickiest method to learn. It is known as that it can only to be sensed not explained. This becomes a serious threat to the survival of this diagnostic method. However, there are a large amount of experiences accumulated during the several thousand years of practice of Chinese doctors. A pulse pattern called 'Slippery pulse' is one of the indications of pregnancy. A simple fluid dynamic model is proposed to simulate the effects of the existence of a placenta. The placenta is modeled as an extra plenum in an extremely simplified fluid network model. It is found that because of the existence of the extra plenum, indeed the pulse pattern shows a secondary peak in one pulse period. As for the author’s knowledge, this work is the first time to show the link between Pulse diagnoses and basic physical principle. Key parameters which might affect the pattern are also investigated.

Keywords: Chinese medicine, flow network, pregnancy, pulse

Procedia PDF Downloads 364

Authors: Mona Alharbi

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Melioidosis has emerged as a lethal disease. Unfortunately, the molecular mechanisms of virulence and pathogenicity of Burkholderia pseudomallei remain unknown. However, proteomics research has selected putative targets in B. pseudomallei that might play roles in the B. pseudomallei virulence. BPSL 2418 putative protein has been predicted as a free methionine sulfoxide reductase and interestingly there is a link between the level of the methionine sulfoxide in pathogen tissues and its virulence. Therefore in this work, we describe the cloning expression, purification, and crystallization of BPSL 2418 and the solution of its 3D structure using X-ray crystallography. Also, we aimed to identify the substrate binding and reduced forms of the enzyme to understand the role of BPSL 2418. The gene encoding BPSL2418 from B. pseudomallei was amplified by PCR and reclone in pETBlue-1 vector and transformed into E. coli Tuner DE3 pLacI. BPSL2418 was overexpressed using E. coli Tuner DE3 pLacI and induced by 300μM IPTG for 4h at 37°C. Then BPS2418 purified to better than 95% purity. The pure BPSL2418 was crystallized with PEG 4000 and PEG 6000 as precipitants in several conditions. Diffraction data were collected to 1.2Å resolution. The crystals belonged to space group P2 21 21 with unit-cell parameters a = 42.24Å, b = 53.48Å, c = 60.54Å, α=γ=β= 90Å. The BPSL2418 binding MES was solved by molecular replacement with the known structure 3ksf using PHASER program. The structure is composed of six antiparallel β-strands and four α-helices and two loops. BPSL2418 shows high homology with the GAF domain fRMsrs enzymes which suggest that BPSL2418 might act as methionine sulfoxide reductase. The amino acids alignment between the fRmsrs including BPSL 2418 shows that the three cysteines that thought to catalyze the reduction are fully conserved. BPSL 2418 contains the three conserved cysteines (Cys⁷⁵, Cys⁸⁵ and Cys¹⁰⁹). The active site contains the six antiparallel β-strands and two loops where the disulfide bond formed between Cys⁷⁵ and Cys¹⁰⁹. X-ray structure of free methionine sulfoxide binding and native forms of BPSL2418 were solved to increase the understanding of the BPSL2418 catalytic mechanism.

Keywords: X-Ray Crystallography, BPSL2418, Burkholderia pseudomallei, Melioidosis

Procedia PDF Downloads 228

Authors: Nurhayati Br Tarigan, Rizkita Rachmi Esyanti

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Stevia (Stevia rebaudiana Bertoni) has a great potential to be used as a natural sweetener because it contains steviol glycoside, which is approximately 100 - 300 times sweeter than sucrose, yet low calories. Vegetative and generative propagation of S. rebaudiana is inefficient to produce stevia biomass and stevioside. One of alternative for stevia propagation is in vitro shoot culture. This research was conducted to optimize the best medium for shoot growth and to compare the bioconversion efficiency and stevioside production of S. rebaudiana shoot culture cultivated in thin layer culture (TLC), recipient for automated temporary immersion system (TIS RITA®) bioreactor, and bubble column bioreactor. The result showed that 1 ppm of Kinetin produced a healthy shoot and the highest number of leaves compared to BAP. Shoots were then cultivated in TLC, TIS RITA® bioreactor, and bubble column bioreactor. Growth medium efficiency was determined by yield and productivity. TLC produced the highest growth medium efficiency of S. rebaudiana, the yield was 0.471 ± 0.117 gbiomass.gsubstrate-1, and the productivity was 0.599 ± 0.122 gbiomass.Lmedium-1.day-1. While TIS RITA® bioreactor produced the lowest yield and productivity, 0.182 ± 0.024 gbiomass.gsubstrate-1 and 0.041 ± 0.0002 gbiomass.Lmedium-1.day-1 respectively. The yield of bubble column bioreactor was 0.354 ± 0.204 gbiomass.gsubstrate-1 and the productivity was 0,099 ± 0,009 gbiomass.Lmedium-1.day-1. The stevioside content from the highest to the lowest was obtained from stevia shoot which was cultivated on TLC, TIS RITA® bioreactor, and bubble column bioreactor; the content was 93,44 μg/g, 42,57 μg/g, and 23,03 μg/g respectively. All three systems could be used to produce stevia shoot biomass, but optimization on the number of nutrition and oxygen intake was required in each system.

Keywords: bubble column, growth medium efficiency, Stevia rebaudiana, stevioside, TIS RITA®, TLC

Procedia PDF Downloads 255

Authors: Abobkar Saad, Qasmia Abdalla, Fatma Emhemed

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Eighty-four of Calligonum comosum were cultured on Murashige and Skoog medium on every combination supplemented with different concentrations of IAA, BA, Zeatin, and GA3. When 84 seeds were inoculated on MS free hormones, different types of cells contain dense cytoplasm were observed ater 23 days and long thick wall cells arranged in layers. In case of using MS +BA(0.5mg/L), different types and shapes of parenchyma cells contain dense cytoplasm were detected after four weeks. In the case of using MS + BA(1mg/L) + GA3 (3mg/L), thick wall parenchyma cells contain dense cytoplasm after 19 days, but many layers of parenchyma cells contain dense cytoplasm after 28 days. When MS +kin(0.5mg/L) a thick cells wall as Sclereids were observed after 29 days. No any response were observed on Zeatin (0.5, 1 mg/L).

Keywords: anatomy, Calligonum comosum, in vitro, aeeds

Procedia PDF Downloads 404

Authors: Amel Hamma, Allesandro Pegoretti

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Kenaf fibres, with two aspect ratios, were melt compounded with two types of biopolymers named starch grafted polypropylene, and then blends compression molded to form plates of 1 mm thick. Results showed that processing induced variation of fibres length which is quantified by optical microscopy observations. Young modulus, stress at break and impact resistance values of starch-grafted-polypropylenes were remarkably improved by kenaf fibres for both matrixes and demonstrated best values when G906PJ were used as matrix. These results attest the good interfacial bonding between the matrix and fibres even in the absence of any interfacial modification. Vicat Softening Point and storage modules were also improved due to the reinforcing effect of fibres. Moreover, short-term tensile creep tests have proven that kenaf fibres remarkably improve the creep stability of composites. The creep behavior of the investigated materials was successfully modeled by the four parameters Burgers model.

Keywords: creep behaviour, kenaf fibres, mechanical properties, starch-grafted-polypropylene

Procedia PDF Downloads 216

Authors: Yuriko Takayama, Norihiro Kato

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Quorum sensing (QS) is a cell-to-cell communication system in bacteria to regulate expression of target genes. In gram-negative bacteria, activation on QS is controlled by a concentration increase of N-acylhomoserine lactone (AHL), which can diffuse in and out of the cell. Effective control of QS is expected to avoid virulence factor production in infectious pathogens, biofilm formation, and antibiotic production because various cell functions in gram-negative bacteria are controlled by AHL-mediated QS. In this research, we applied cyclodextrins (CDs) as artificial hosts for the AHL signal to reduce the AHL concentration in the culture broth below its threshold for QS activation. The AHL-receptor complex induced under the high AHL concentration activates transcription of the QS-target gene. Accordingly, artificial reduction of the AHL concentration is one of the effective strategies to inhibit the QS. A hydrophobic cavity of the CD can interact with the acyl-chain of the AHL due to hydrophobic interaction in aqueous media. We studied N-hexanoylhomoserine lactone (C6HSL)-mediated QS in Serratia marcescens; accumulation of C6HSL is responsible for regulation of the expression of pig cluster. Inhibitory effects of added CDs on QS were demonstrated by determination of prodigiosin amount inside cells after reaching stationary phase, because production of prodigiosin depends on the C6HSL-mediated QS. By adding approximately 6 wt% hydroxypropyl-β-CD (HP-β-CD) in Luria-Bertani (LB) medium prior to inoculation of S. maecescens AS-1, the intracellularly accumulated prodigiosin was drastically reduced to 7-10%, which was determined after the extraction of prodigiosin in acidified ethanol. The AHL retention ability of HP-β-CD was also demonstrated by Chromobacterium violacuem CV026 bioassay. The CV026 strain is an AHL-synthase defective mutant that activates QS solely by adding AHLs from outside of cells. A purple pigment violacein is induced by activation of the AHL-mediated QS. We demonstrated that the violacein production was effectively suppressed when the C6HSL standard solution was spotted on a LB agar plate dispersing CV026 cells and HP-β-CD. Physico-chemical analysis was performed to study the affinity between the immobilized CD and added C6HSL using a quartz crystal microbalance (QCM) sensor. The COOH-terminated self-assembled monolayer was prepared on a gold electrode of 27-MHz AT-cut quartz crystal. Mono(6-deoxy-6-N, N-diethylamino)-β-CD was immobilized on the electrode using water-soluble carbodiimide. The C6HSL interaction with the β-CD cavity was studied by injecting the C6HSL solution to a cup-type sensor cell filled with buffer solution. A decrement of resonant frequency (ΔFs) clearly showed the effective C6HSL complexation with immobilized β-CD and its stability constant for MBP-SpnR-C6HSL complex was on the order of 102 M-1. The CD has high potential for engineered control of QS because it is safe for human use.

Keywords: acylhomoserine lactone, cyclodextrin, intracellular signaling, quorum sensing

Procedia PDF Downloads 221

Authors: Norihiro Kato, Yuriko Takayama

Abstract:

Some gram-negative bacteria enable the simultaneous activation of gene expression involved in N-acylhomoserine lactone (AHL) dependent cell-to-cell communication system. Such regulatory system for the bacterial group behavior is termed as quorum sensing (QS) because a diffusible AHL signal can accumulate around the cell during the increase of the cell density and trigger activation of the sequential QS process. By blocking the QS, the expression of diverse genes related to infection, antibiotic production, and biofilm formation is inhibited. Conditioning of QS by regulation of the DNA-receptor-AHL interaction is a potential target for enhancing host defenses against pathogenicity. We focused on engineered application of transcriptional regulator SpnR produced in opportunistic human pathogen Serratia marcescens. The SpnR can interact with AHL signals at an N-terminal domain and also with a promoter region of a QS target gene at a C-terminal domain. As the initial process of the QS activation, the SpnR forms a complex with the AHL to enhance the expression of pig cluster; the SpnR normally acts as an activator for the expression of the QS-dependent gene. In this research, we attempt to artificially control QS by changing the role of SpnR. The QS-dependent prodigiosin production is expected to inhibit by externally added SpnR in the culture broth of AS-1 strain because the AHL concentration was kept below the threshold by AHL-SpnR complex formation. Maltose-binding protein (MBP)-tagged SpnR (MBP-SpnR) was overexpressed in Escherichia coli and purified using an affinity chromatography equipped with an amylose resin column. The specific interaction between AHL and MBP-SpnR was demonstrated by quartz crystal microbalance (QCM) sensor. AHL with amino end-group was coupled with COOH-terminated self-assembled monolayer prepared on a gold electrode of 27-MHz quartz crystal sensor using water-soluble carbodiimide. After the injection of MBP-SpnR into a cup-type sensor cell filled with the buffer solution, time course of resonant frequency change (ΔFs) was determined. A decrease of ΔFs clearly showed the uptake of MBP-SpnR onto the AHL-immobilized electrode. Furthermore, no binding affinity was observed after the heat-inactivation of MBP-SpnR at 80ºC. These results suggest that MBP-SpnR possesses a specific affinity for AHL. MBP-SpnR was added to the culture medium as an AHL trap to study inhibitory effects on intracellularly accumulated prodigiosin. With approximately 2 µM MBP-SpnR, the amount of prodigiosin induced was half that of the control without any additives. In conclusion, the function of SpnR could be switched by adding it to the cell culture. Exogenously added MBP-SpnR possesses high affinity for AHL derived from cells and acts as an inhibitor of AHL-mediated QS.

Keywords: intracellular signaling, microbial biotechnology, quorum sensing, transcriptional regulator

Procedia PDF Downloads 255

Authors: Keyur Raval, Steffen Krohn, Bruno Moerschbacher

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Chitin, the biopolymer of the N-acetylglucosamine, is the most abundant biopolymer on the planet after cellulose. Industrially, chitin is isolated and purified from the shell residues of shrimps. A deacetylated derivative of chitin i.e. chitosan has more market value and applications owing to it solubility and overall cationic charge compared to the parent polymer. This deacetylation on an industrial scale is performed chemically using alkalis like sodium hydroxide. This reaction not only is hazardous to the environment owing to negative impact on the marine ecosystem. A greener option to this process is the enzymatic process. In nature, the naïve chitin is converted to chitosan by chitin deacetylase (CDA). This enzymatic conversion on the industrial scale is however hampered by the crystallinity of chitin. Thus, this enzymatic action requires the substrate i.e. chitin to be soluble which is technically difficult and an energy consuming process. We in this project wanted to address this shortcoming of CDA. In lieu of this, we have modeled a fusion protein with CDA and an auxiliary protein. The main interest being to increase the accessibility of the enzyme towards crystalline chitin. A similar fusion work with chitinases had improved the catalytic ability towards insoluble chitin. In the first step, suitable partners were searched through the protein data bank (PDB) wherein the domain architecture were sought. The next step was to create the models of the fused product using various in silico techniques. The models were created by MODELLER and evaluated for properties such as the energy or the impairment of the binding sites. A fusion PCR has been designed based on the linker sequences generated by MODELLER and would be tested for its activity towards insoluble chitin.

Keywords: chitin deacetylase, modeling, chitin binding domain, chitinases

Procedia PDF Downloads 233

Authors: Vijay Kumar, Mack Moyo, Johannes Van Staden

Abstract:

Lachenalia viridiflora is a critically endangered bulbous plant with high potential on the international floriculture market. In the present study, an efficient protocol for in vitro plantlet regeneration through somatic embryogenesis was developed. Embryogenic callus was established on Murashige and Skoog (MS) basal medium supplemented with various concentrations and combinations of picloram and thidiazuron (TDZ). A high number of SEs (28.5 ± 1.49) with at different developmental stages of somatic embryos (SEs: globular embryos, torpedo and cotyledon embryo with bipolar characteristics) was obtained on Murashige and Skoog (MS) (Murashige and Skoog 1962) medium with 2.5 μM picloram, and 1.0 μM TDZ. Histological and scanning electron microscopic (SEM) analysis confirmed the presence of somatic embryos. Mature somatic embryos germinated and developed into plantlets after 6 weeks on half/full strength MS medium. High plant regeneration frequency (91.11 %) was achieved on full-strength MS medium supplemented with 5 μM phloroglucinol (PG). Well-developed healthy plantlets were successfully acclimatized in the greenhouse with a survival rate of 80%. The result of this study is beneficial in the mass propagation of high-quality Lachenalia viridiflora clonal plants for the commercial horticultural market and also provides a platform for future genetic transformation studies on the plant.

Keywords: horticultural plant, Lachenalia viridiflora, phloroglucinol, somatic embryogenesis, thidiazuron

Procedia PDF Downloads 611

Authors: Ambika Selvaraj

Abstract:

Magnetic iron oxide nanoparticles (IO) of < 20nm (superparamagnetic) become promising tool in cancer therapy, and integrated nanodevices for cancer detection and screening. The obstacles include particle heterogeneity and cost. It can be overcome by developing monodispersed nanoparticles in economical approach. We have successfully synthesized < 7 nm IO by low temperature controlled technique, in which Fe0 is sandwiched between stabilizer and Fe2+. Size analysis showed the excellent size control from 31 nm at 33°C to 6.8 nm at 10°C. Resultant monodispersed IO were found to be stable for > 50 reuses, proved its applicability in biomedical applications.

Keywords: low temperature synthesis, hybrid iron nanoparticles, cancer therapy, biomedical applications

Procedia PDF Downloads 322

Authors: Pratibha Srivastava, Ayyamperumal Jeyaprakash, Gary Steck

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Exotic, invasive tephritid fruit flies (Diptera: Tephritidae) are a major concern to fruit and vegetable production in the USA. Timely detection and identification of these agricultural pests facilitate the possibility of eradication from newly invaded areas. They spread primarily as larvae in infested fruits carried in commerce or personal baggage. Identification of larval stages to species level is difficult but necessary to determine pest loads and their pathways into the USA. The main focus of this study is the New World genus, Anastrepha. Many of its constituent taxa are pests of major economic importance. This study is significant for national quarantine use, as morphological diagnostics to separate larvae of the various members remain poorly developed. Elongation factor 1 alpha sequences were amplified from Anastrepha fraterculus specimens collected from South America (Ecuador and Peru). Phylogenetic analysis was performed to characterize the Anastrepha fraterculus complex at a molecular level.

Keywords: anastrepha, diptera, elongation factor, fruit fly

Procedia PDF Downloads 193

Authors: Muna Jalal Ali, Essam A. Makky, Mashitah M. Yusoff

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Objectives: The aim of this study to evaluate the antimicrobial susceptibility of combined toothpaste with medicinal plants and the relations between the commercial toothpaste to its price and the patient age as well. Materials and Methods: Oral isolates of different patients aged 3 to 60 years were obtained, purified, and tested against four different ethnomedicinal plant extracts for antimicrobial activity. A total of 10 different commercial toothpastes (different brands and prices) were collected from the market, and the combined action of the medicinal plants and toothpaste was studied. Results: We found a higher bacterial population in the age group of 3–40 years than the group of 40–60 years, with approximately 44% and 32%, respectively. The combined action of ethanolic extract (alone) against oral isolates showed a synergistic effect, with 32.20, 30.50, and 25.42% for combinations A (Ci/Ca), B (Ci/Ca/P), and C (Ci/Ca/P/N), respectively. By contrast, the combined action of ethnomedicinal plants with 10 different toothpastes improved the antimicrobial sensitivity by 60, 100, and 0% for combinations A, B, and C respectively. Clinical relevance: The ethanolic extract of only combinations A and B with commercial toothpaste showed high antibacterial activity against oral isolates and the effectiveness of toothpaste is not related to the price.

Keywords: microbial evolution, oral isolates, ethnomedicinal plants, antimicrobial activity, toothpaste

Procedia PDF Downloads 292

Authors: Friday G. Okibe, Edit B. Agbaji, Victor O. Ajibola, Christain C. Onoyima

Abstract:

Controlled drug delivery systems reduce dose-dependent toxicity associated with potent drugs, including anticancer drugs. In this research, hydroxyapatite (HA) and hydroxyapatite-sodium alginate nanocomposites (HASA) were successfully prepared and characterized using Fourier Transform Infrared spectroscopy (FTIR) and Scanning Electron Microscopy (SEM). The FTIR result showed absorption peaks characteristics of pure hydroxyapatite (HA), and also confirmed the chemical interaction between hydroxyapatite and sodium alginate in the formation of the composite. Image analysis from SEM revealed nano-sized hydroxyapatite and hydroxyapatite-sodium alginate nanocomposites with irregular morphologies. Particle size increased with the formation of the nanocomposites relative to pure hydroxyapatite, with no significant change in particles morphologies. Drug loading and in-vitro drug release study were carried out using synthetic body fluid as the release medium, at pH 7.4 and 37 °C and under perfect sink conditions. The result shows that drug loading is highest for pure hydroxyapatite and decreased with increasing quantity of sodium alginate. However, the release study revealed that HASA-5%wt and HASA-20%wt presented better release profile than pure hydroxyapatite, while HASA-33%wt and HASA-50%wt have poor release profiles. This shows that Methotrexate-loaded hydroxyapatite-sodium alginate if prepared under optimal conditions is a potential carrier for effective delivery of Methotrexate.

Keywords: drug-delivery, hydroxyapatite, methotrexate, nanocomposites, sodium alginate

Procedia PDF Downloads 259

Authors: M. T. Asadollahzadeh, A. Ghasemian, A. R. Saraeian, H. Resalati, P. R. Lennartsson, M. J. Taherzadeh

Abstract:

Since filamentous fungi are capable of assimilating several types of sugars (hexoses and pentoses), they are potential candidates for bioconversion of spent sulfite liquor (SSL). Three filamentous fungi such as Aspergillus oryzae, Mucor indicus, and Rhizopus oryzae were investigated in this work. The SSL was diluted in order to obtain concentrations of 50, 60, 70, 80, and 90% and supplemented with two types of nutrients. The results from cultivations in shake flask showed that A. oryzae and M. indicus were not able to grow in pure SSL and SSL90% while R. oryzae could grow only in SSL50% and SSL60%. Cultivation with A. oryzae resulted in the highest yield of produced fungal biomass, while R. oryzae cultivation resulted in the lowest fungal biomass yield. Although, the mediums containing yeast extract, (NH₄)₂SO₄, KH₂PO₄, CaCl₂∙2H₂O, and MgSO₄∙7H₂O as nutrients supplementations produced higher fungal biomass compared to the mediums containing NH₄H₂PO₄ and ammonia, but there was no significant difference between two types of nutrients in terms of sugars and acetic acid consumption rate. The sugars consumption in M. indicus cultivation was faster than A. oryzae and R. oryzae cultivation. Acetic acid present in SSL was completely consumed during cultivation of all fungi. M. indicus was the best and fastest ethanol producer from SSL among the fungi examined, when yeast extract and salts were used as nutrients supplementations. Furthermore, no further improvement in ethanol concentration and rate of sugars consumption was obtained in medium supplemented with NH₄H₂PO₄ and ammonia compared to medium containing yeast extract, (NH₄)₂SO₄, KH₂PO₄, CaCl₂∙2H₂O, and MgSO₄∙7H₂O. On the other hand, the higher dilution of SSL resulted in a better fermentability, and better consumption of sugars and acetic acid.

Keywords: ethanol, filamentous fungi, fungal biomass, spent sulfite liquor

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Authors: M. Peña Aguilar Juan, E. Nava Galván Claudia, Pastrana Palma Alberto

Abstract:

In this work was developed and implemented a thermal fogging disinfection system to counteract pathogens from poultry feces in agribusiness farms, to reduce mortality rates and increase biosafety in them. The control system consists of two phases for the conditioning of the farm during the sanitary break. In the first phase, viral and bacterial inactivation was performed by treating the stool dry cleaning, along with the development of a specialized product that foster the generation of temperatures above 55 °C in less than 24 hr, for virus inactivation. In the second phase, a process for disinfection by fogging was implemented, along with the development of a specialized disinfectant that guarantee no risk for the operators’ health or birds. As a result of this process, it was possible to minimize the level of mortality of chickens on farms from 12% to 5.49%, representing a reduction of 6.51% in the death rate, through the formula applied to the treatment of poultry litter based on oxidising agents used as antiseptics, hydrogen peroxide solutions, glacial acetic acid and EDTA in order to act on bacteria, viruses, micro bacteria and spores.

Keywords: innovation, triple helix, poultry farms, biosecurity

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Authors: Sergio S. De Jesus, Aline Santana, Rubens Maciel Filho

Abstract:

The objective of this study was to choose and determine a green solvent system with similar extraction efficiencies as the traditional Bligh and Dyer method. Sunflower seed oil was extracted using Bligh and Dyer method with 2-methyltetrahydrofuran and isoamyl using alcohol ratios of 1:1; 2:1; 3:1; 1:2; 3:1. At the same time comparative experiments was performed with chloroform and methanol ratios of 1:1; 2:1; 3:1; 1:2; 3:1. Comparison study was done using 5 replicates (n=5). Statistical analysis was performed using Microsoft Office Excel (Microsoft, USA) to determine means and Tukey’s Honestly Significant Difference test for comparison between treatments (α = 0.05). The results showed that using classic method with methanol and chloroform presented the extraction oil yield with the values of 31-44% (w/w) and values of 36-45% (w/w) using green solvents for extractions. Among the two extraction methods, 2 methyltetrahydrofuran and isoamyl alcohol ratio 2:1 provided the best results (45% w/w), while the classic method using chloroform and methanol with ratio of 3:1 presented a extraction oil yield of 44% (w/w). It was concluded that the proposed extraction method using 2-methyltetrahydrofuran and isoamyl alcohol in this work allowed the same efficiency level as chloroform and methanol.

Keywords: extraction, green solvent, lipids, sugarcane

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Authors: Yasinta Ratna Esti Wulandari, Vivitri Dewi Prasasty, Adrianus Rio, Cindy Geniola

Abstract:

Mulberry is a plant that widely cultivated around the world, mostly for silk industry. In recent years, the study showed that the mulberry leaves have an anti-diabetic effect which mostly comes from the compound known as 1-deoxynojirimycin (DNJ). DNJ is a very potent α-glucosidase inhibitor. It will decrease the degradation rate of carbohydrates in digestive tract, leading to slower glucose absorption and reducing the post-prandial glucose level significantly. The mulberry leaves also known as the best source of DNJ. Since then, the DNJ in mulberry leaves had received a considerable attention, because of the increased number of diabetic patients and the raise of people awareness to find a more natural cure for diabetic. The DNJ content in mulberry leaves varied depend on the mulberry species, leaf’s age, and the plant’s growth environment. Few of the mulberry varieties that were cultivated in Indonesiaare Morus alba var. kanva-2, M. alba var. multicaulis, M. bombycis var. lembang, and M. cathayana. The lack of data concerning phytochemicals contained in the Indonesian mulberry leaves are restraining their use in the medicinal field. The aim of this study is to fully utilize the use of mulberry leaves cultivated in Indonesia as a medicinal herb in local, national, or global community, by determining the DNJ and other phytochemical contents in them. This study used eight leaf samples which are the young leaves and mature leaves of both Morus alba var. kanva-2, M. alba var. multicaulis, M. bombycis var. lembang, and M. cathayana. The DNJ content was analyzed using reverse phase high performance liquid chromatography (HPLC). The stationary phase was silica C18 column and the mobile phase was acetonitrile:acetic acid 0.1% 1:1 with elution rate 1 mL/min. Prior to HPLC analysis the samples were derivatized with FMOC to ensure the DNJ detectable by VWD detector at 254 nm. Results showed that the DNJ content in samples are ranging from 2.90-0.07 mg DNJ/ g leaves, with the highest content found in M. cathayana mature leaves (2.90 ± 0.57 mg DNJ/g leaves). All of the mature leaf samples also found to contain higher amount of DNJ from their respective young leaf samples. The phytochemicals in leaf samples was tested using qualitative test. Result showed that all of the eight leaf samples contain alkaloids, phenolics, flavonoids, tannins, and terpenes. The presence of this phytochemicals contribute to the therapeutic effect of mulberry leaves. The pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS) analysis was also performed to the eight samples to quantitatively determine their phytochemicals content. The pyrolysis temperature was set at 400 °C, with capillary column Phase Rtx-5MS 60 × 0.25 mm ID stationary phase and helium gas mobile phase. Few of the terpenes found are known to have anticancer and antimicrobial properties. From all the results, all of four samples of mulberry leaves which are cultivated in Indonesia contain DNJ and various phytochemicals like alkaloids, phenolics, flavonoids, tannins, and terpenes which are beneficial to our health.

Keywords: Morus, 1-deoxynojirimycin, HPLC, Py-GC-MS

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Authors: Maletsema Alina Mofokeng, Hussein Shimelis, Mark Laing, Pangirayi Tongoona

Abstract:

Sorghum is one of the most important cereal crops grown for food, feed and bioenergy. Knowledge of genetic diversity is important for conservation of genetic resources and improvement of crop plants through breeding. The objective of this study was to assess the level of genetic diversity among sorghum genotypes using microsatellite markers. A total of 103 accessions of sorghum genotypes obtained from the Department of Agriculture, Forestry and Fisheries, the African Centre for Crop Improvement and Agricultural Research Council-Grain Crops Institute collections in South Africa were estimated using 30 microsatellite markers. For all the loci analysed, 306 polymorphic alleles were detected with a mean value of 6.4 per locus. The polymorphic information content had an average value of 0.50 with heterozygosity mean value of 0.55 suggesting an important genetic diversity within the sorghum genotypes used. The unweighted pair group method with arithmetic mean clustering based on Euclidian coefficients revealed two major distinct groups without allocating genotypes based on the source of collection or origin. The genotypes 4154.1.1.1, 2055.1.1.1, 4441.1.1.1, 4442.1.1.1, 4722.1.1.1, and 4606.1.1.1 were the most diverse. The sorghum genotypes with high genetic diversity could serve as important sources of novel alleles for breeding and strategic genetic conservation.

Keywords: Genetic Diversity, Genotypes, Microsatellites, Sorghum

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Authors: William Kelly, Sorelle Veigne, Xianhua Li, Zuyi Huang, Shyamsundar Subramanian, Eugene Schaefer

Abstract:

The ambr15™ bioreactor is a single-use microbioreactor for cell line development and process optimization. The ambr system offers fully automatic liquid handling with the possibility of fed-batch operation and automatic control of pH and oxygen delivery. With operating conditions for large scale biopharmaceutical production properly scaled down, micro bioreactors such as the ambr15™ can potentially be used to predict the effect of process changes such as modified media or different cell lines. In this study, gassing rates and dilution rates were varied for a semi-continuous cell culture system in the ambr15™ bioreactor. The corresponding changes to metabolite production and consumption, as well as cell growth rate and therapeutic protein production were measured. Conditions were identified in the ambr15™ bioreactor that produced metabolic shifts and specific metabolic and protein production rates also seen in the corresponding larger (5 liter) scale perfusion process. A Dynamic Flux Balance model was employed to understand and predict the metabolic changes observed. The DFB model-predicted trends observed experimentally, including lower specific glucose consumption when CO₂ was maintained at higher levels (i.e. 100 mm Hg) in the broth. A Computational Fluid Dynamic (CFD) model of the ambr15™ was also developed, to understand transfer of O₂ and CO₂ to the liquid. This CFD model predicted gas-liquid flow in the bioreactor using the ANSYS software. The two-phase flow equations were solved via an Eulerian method, with population balance equations tracking the size of the gas bubbles resulting from breakage and coalescence. Reasonable results were obtained in that the Carbon Dioxide mass transfer coefficient (kLa) and the air hold up increased with higher gas flow rate. Volume-averaged kLa values at 500 RPM increased as the gas flow rate was doubled and matched experimentally determined values. These results form a solid basis for optimizing the ambr15™, using both CFD and FBA modelling approaches together, for use in microscale simulations of larger scale cell culture processes.

Keywords: cell culture, computational fluid dynamics, dynamic flux balance analysis, microbioreactor

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Authors: S. V. Prabhakar Vattikuti, Chan Byon

Abstract:

In this paper, we report a facile synthetic strategy of randomly disturbed Bi₂S₃ nanorods on WS₂ nanosheets, which are synthesized via a controlled hydrothermal method without surfactant under an inert atmosphere. We developed a simple hydrothermal method for the formation of heterostructured of Bi₂S₃/WS₂ with a large scale (>95%). The structural features, composition, and morphology were characterized by XRD, SEM-EDX, TEM, HRTEM, XPS, UV-vis spectroscopy, N₂ adsorption-desorption, and TG-DTA measurements. The heterostructured Bi₂S₃/WS₂ composite has significant photocatalytic efficiency toward the photodegradation of organic dye. The time-dependent UV-vis absorbance spectroscopy measurement was consistent with the enhanced photocatalytic degradation of rhodamine B (RhB) under visible light irradiation with the diminishing carrier recombination for the Bi₂S₃/WS₂ photocatalyst. Due to their marked synergistic effects, the supported Bi₂S₃ nanorods on WS₂ nanosheet heterostructures exhibit significant visible-light photocatalytic activity and stability for the degradation of RhB. A possible reaction mechanism is proposed for the Bi₂S₃/WS₂ composite.

Keywords: photocatalyst, heterostructures, transition metal disulfides, organic dye, nanorods

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Authors: Muhamad Afiq Faisal, Yahaya, Mohd Faizal, Hamzah

Abstract:

Genetic modification technology allows scientists to alter the genetic information of a particular organism. The technology allows the production of genetically modified organism (GMO) that has the enhanced property compared to the unmodified organism. The application of such technology is not only in agriculture industry, it is now has been applied extensively in biopharmaceutical industry such as transgenic vaccines. In Malaysia, Biosafety Act 2007 has been enacted in which all GMO-based products must be labeled with adequate information before being marketed. This paper aims to determine the awareness level amongst Malaysian consumers on the GM products available in the market and the efficiency of information supplied in the GM product labeling. The result of the survey will serve as a guideline for Malaysia government agency bodies to provide comprehensive yet efficient information to consumers for the purpose of GM product labeling in the near future. In conclusion, the efficiency of information delivery plays a vital role in ensuring that the information is being conveyed clearly to Malaysian consumers during the selection process of GM products available in the market.

Keywords: genetic modification technology, genetically modified organisms, genetically modified organism products labeling, Biosafety Act 2007

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Authors: Titin Handayani, Endang Yuniastuti

Abstract:

Kepuh (Sterculia foetida L.) is a potential plant contain mainly oil seeds that can be used as a source of alternative bioenergy and medicine. The main problem of kepuh cultivation is the limited supply of seed plants. Seeds development were very easy, but to produce fruit have to wait for approximately 5 years. The objective of this research was to obtain kepuh plants through direct in vitro regeneration. Hypocotyls and shoot tips explants were excised from sterile germinated seedlings and placed on shoot induction medium containing basal salts of Murashige and Skoog (MS) and various concentrations of plant growth regulators. The results showed that shoots induction from the apical and axillary buds on MS medium + 1.5 and 2 mg/L BAP and 0.5 and 1 mg/L IAA was growth very slowly. Increasing of BAP concentrations was increased shoot formation. The first subcultures were increased the rate of shoots growth on MS medium supplemented with 2 mg/L BAP and 0.5 mg/L IAA. The second of shoots subculture on MS medium + 1.5 to 2 mg/L BAP + 0.5 mg/L IAA was increased the number of shoots up to 4.8 in average. The best medium of shoots elongation were MS + 1 mgL-1 kinetin + 5 mg/L GA3. The highest percentage of roots (65%) occurred on MS medium with 5 mg/L IBA which average number of roots was 3.1. High percentages of survival and plants of normal appearance were obtained after five weeks of acclimatization.

Keywords: Kepuh, Sterculia foetida L, shoot multiplication, rooting, acclimatization, bioenergy, medicine

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Authors: Mona Hejazi, Ali Motie Nasrabadi

Abstract:

Epilepsy is a persistent neurological disorder that affects more than 50 million people worldwide. Hence, there is a necessity to introduce an efficient prediction model for making a correct diagnosis of the epileptic seizure and accurate prediction of its type. In this study we consider how the Effective Connectivity (EC) patterns obtained from intracranial Electroencephalographic (EEG) recordings reveal information about the dynamics of the epileptic brain and can be used to predict imminent seizures, as this will enable the patients (and caregivers) to take appropriate precautions. We use this definition because we believe that effective connectivity near seizures begin to change, so we can predict seizures according to this feature. Results are reported on the standard Freiburg EEG dataset which contains data from 21 patients suffering from medically intractable focal epilepsy. Six channels of EEG from each patients are considered and effective connectivity using Directed Transfer Function (DTF) and Granger Causality (GC) methods is estimated. We concentrate on effective connectivity standard deviation over time and feature changes in five brain frequency sub-bands (Alpha, Beta, Theta, Delta, and Gamma) are compared. The performance obtained for the proposed scheme in predicting seizures is: average prediction time is 50 minutes before seizure onset, the maximum sensitivity is approximate ~80% and the false positive rate is 0.33 FP/h. DTF method is more acceptable to predict epileptic seizures and generally we can observe that the greater results are in gamma and beta sub-bands. The research of this paper is significantly helpful for clinical applications, especially for the exploitation of online portable devices.

Keywords: effective connectivity, Granger causality, directed transfer function, epilepsy seizure prediction, EEG

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Authors: Sungging Pintowantoro, Yuli Setiyorini, Rochman Rochim, Agung Purniawan

Abstract:

The bacterial infections are frequent and undesired occurrences after bone fracture treatment. One approach to reduce the incidence of bone fracture infection is the additional of microbial agents into bone cement. In this study, the synthesis of bone cement from re-cycles biowaste was successfully conducted completed with anti-bacterial function. The re-cycle of biowaste using microwave assisted was done in our previous studies in order to produce some of powder (calcium carbonate, carbonated-hydroxyapatite and chitosan). The ratio of these powder combined with methylmethacrylate (MMA) as the matrix in bone cement were investigated using XRD, FTIR, SEM-EDX, hardness test and anti-bacterial test, respectively. From the XRD, FTIR and EDX were resulted the formation of carbonated-hydroxyapatite, calcium carbonate and chitosan. The morphology was revealed porous structure both C2H3K1L and C2H1K3L, respectively. The antibacterial activity was tested against Staphylococcus aureus (S. aureus) for 24 hours. The inhibition of S. aureus was clearly shown, the hollow zone was resulted in various distance 14.2mm, 7.5mm, and 7.7mm, respectively. The hardness test was depicted in various results, however, C2H1K3L can be achived 36.84HV which is closed to dry cancelous bone 35HV. In general, this study results was promising materials to use as bone cement materials.

Keywords: biomaterials, biowaste recycling, materials processing, microwave processing

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Authors: Hiroshi Nakayama, Yuji Ito

Abstract:

Synthetic cannabinoids have attracted much public attention recently in Japan. 1-pentyl-3-(1-naphthoyl)-indole (JWH-018), 1-pentyl-2-methyl-3-(1-naphthoyl) indole (JWH-015), 1-(5-fluoropentyl)-3- (1-(2,2,3,3- tetramethylcyclopropyl)) indole (XLR-11) and 1-methyl-3- (1-admantyl) indole (JWH-018 adamantyl analog) are known as synthetic cannabinoids and are also considered dangerous illegal drugs in Japan. It has become necessary to develop sensitive and useful methods for detection of synthetic cannabinoids. We produced two monoclonal antibodies (MAb) against synthetic cannabinoids, named NT1 (IgG1) and NT2 (IgG1), using Hybridoma technology. The cross-reactivity of these produced MAbs was evaluated using a competitive enzyme-linked immunosorbent assay (ELISA). In the results, we found both of these antibodies recognize many kinds of synthetic cannabinoids analog. However, neither of these antibodies recognizes naphtoic acid, 1-methyl-indole and indole known as a raw material of synthetic cannabinoid. Thus, the MAbs produced in this study could be a useful tool for the detection of synthetic cannabinoids.

Keywords: ELISA, monoclonal antibody, sensor, synthetic cannabinoid

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