BioMed Research International: Biotechnology The latest articles from Hindawi Publishing Corporation © 2015 , Hindawi Publishing Corporation . All rights reserved. Renewable Energy and Alternative Fuel Technologies Wed, 25 Mar 2015 07:23:57 +0000 Meisam Tabatabaei, Keikhosro Karimi, Rajeev Kumar, and Ilona Sárvári Horváth Copyright © 2015 Meisam Tabatabaei et al. All rights reserved. A Comparative Study of Almond Biodiesel-Diesel Blends for Diesel Engine in Terms of Performance and Emissions Sun, 22 Mar 2015 13:51:28 +0000 This paper investigates the opportunity of using almond oil as a renewable and alternative fuel source. Different fuel blends containing 10, 30, and 50% almond biodiesel (B10, B30, and B50) with diesel fuel (B0) were prepared and the influence of these blends on emissions and some performance parameters under various load conditions were inspected using a diesel engine. Measured engine performance parameters have generally shown a slight increase in exhaust gas temperature and in brake specific fuel consumption and a slight decrease in brake thermal efficiency. Gases investigated were carbon monoxide (CO) and oxides of nitrogen (). Furthermore, the concentration of the total particulate and the unburned fuel emissions in the exhaust gas were tested. A blend of almond biodiesel with diesel fuel gradually reduced the engine CO and total particulate emissions compared to diesel fuel alone. This reduction increased with more almond biodiesel blended into the fuel. Finally, a slight increase in engine using blends of almond biodiesel was measured. Nidal H. Abu-Hamdeh and Khaled A. Alnefaie Copyright © 2015 Nidal H. Abu-Hamdeh and Khaled A. Alnefaie. All rights reserved. Current Status and Future Potential of Energy Derived from Chinese Agricultural Land: A Review Sun, 22 Mar 2015 11:26:41 +0000 Energy crisis is receiving attention with regard to the global economy and environmental sustainable development. Developing new energy resources to optimize the energy supply structure has become an important measure to prevent energy shortage as well as achieving energy conservation and emission reduction in China. This study proposed the concept of energy agriculture and constructed an energy agricultural technical support system based on the analysis of energy supply and demand and China’s foreign dependence on energy resources, combined with the function of agriculture in the energy field. Manufacturing technology equipment and agricultural and forestry energy, including crop or forestry plants and animal feces, were used in the system. The current status and future potential of China’s marginal land resources, energy crop germplasm resources, and agricultural and forestry waste energy-oriented resources were analyzed. Developing the function of traditional agriculture in food production may promote China’s social, economic, and environmental sustainable development and achieve energy saving and emission reduction. Ningning Zhai, Chunlan Mao, Yongzhong Feng, Tong Zhang, Zhenjie Xing, Yanhong Wang, Shuzhen Zou, Dongxue Yin, Xinhui Han, Guangxin Ren, and Gaihe Yang Copyright © 2015 Ningning Zhai et al. All rights reserved. Catalysis of Rice Straw Hydrolysis by the Combination of Immobilized Cellulase from Aspergillus niger on β-Cyclodextrin-Fe3O4 Nanoparticles and Ionic Liquid Sun, 22 Mar 2015 09:50:48 +0000 Cellulase from Aspergillus niger was immobilized onto β-cyclodextrin-conjugated magnetic particles by silanization and reductive amidation. The immobilized cellulase gained supermagnetism due to the magnetic nanoparticles. Ninety percent of cellulase was immobilized, but the activity of immobilized cellulase decreased by 10%. In this study, ionic liquid (1-butyl-3-methylimidazolium chloride) was introduced into the hydrolytic process because the original reaction was a solid-solid reaction. The activity of immobilized cellulase was improved from 54.87 to 59.11 U g immobilized cellulase−1 at an ionic liquid concentration of 200 mM. Using immobilized cellulase and ionic liquid in the hydrolysis of rice straw, the initial reaction rate was increased from 1.629 to 2.739 g h−1 L−1. One of the advantages of immobilized cellulase is high reusability—it was usable for a total of 16 times in this study. Compared with free cellulase, magnetized cellulase can be recycled by magnetic field and the activity of immobilized cellulase was shown to remain at 85% of free cellulase without denaturation under a high concentration of glucose (15 g L−1). Therefore, immobilized cellulase can hydrolyze rice straw continuously compared with free cellulase. The amount of harvested glucose can be up to twentyfold higher than that from the hydrolysis by free cellulase. Po-Jung Huang, Ken-Lin Chang, Jung-Feng Hsieh, and Shui-Tein Chen Copyright © 2015 Po-Jung Huang et al. All rights reserved. Microalgae as Sustainable Renewable Energy Feedstock for Biofuel Production Sun, 22 Mar 2015 08:58:16 +0000 The world energy crisis and increased greenhouse gas emissions have driven the search for alternative and environmentally friendly renewable energy sources. According to life cycle analysis, microalgae biofuel is identified as one of the major renewable energy sources for sustainable development, with potential to replace the fossil-based fuels. Microalgae biofuel was devoid of the major drawbacks associated with oil crops and lignocelluloses-based biofuels. Algae-based biofuels are technically and economically viable and cost competitive, require no additional lands, require minimal water use, and mitigate atmospheric CO2. However, commercial production of microalgae biodiesel is still not feasible due to the low biomass concentration and costly downstream processes. The viability of microalgae biodiesel production can be achieved by designing advanced photobioreactors, developing low cost technologies for biomass harvesting, drying, and oil extraction. Commercial production can also be accomplished by improving the genetic engineering strategies to control environmental stress conditions and by engineering metabolic pathways for high lipid production. In addition, new emerging technologies such as algal-bacterial interactions for enhancement of microalgae growth and lipid production are also explored. This review focuses mainly on the problems encountered in the commercial production of microalgae biofuels and the possible techniques to overcome these difficulties. Srikanth Reddy Medipally, Fatimah Md. Yusoff, Sanjoy Banerjee, and M. Shariff Copyright © 2015 Srikanth Reddy Medipally et al. All rights reserved. Production and Characterization of Biodiesel Using Nonedible Castor Oil by Immobilized Lipase from Bacillus aerius Sun, 22 Mar 2015 08:47:00 +0000 A novel thermotolerant lipase from Bacillus aerius was immobilized on inexpensive silica gel matrix. The immobilized lipase was used for the synthesis of biodiesel using castor oil as a substrate in a solvent free system at 55°C under shaking in a chemical reactor. Several crucial parameters affecting biodiesel yield such as incubation time, temperature, substrate molar ratio, and amount of lipase were optimized. Under the optimized conditions, the highest biodiesel yield was up to 78.13%. The characterization of synthesized biodiesel was done through FTIR spectroscopy, 1H NMR spectra, and gas chromatography. Sunil Kumar Narwal, Nitin Kumar Saun, Priyanka Dogra, Ghanshyam Chauhan, and Reena Gupta Copyright © 2015 Sunil Kumar Narwal et al. All rights reserved. Improvement of Biogas Production from Orange Peel Waste by Leaching of Limonene Thu, 19 Mar 2015 14:17:43 +0000 Limonene is present in orange peel wastes and is known as an antimicrobial agent, which impedes biogas production when digesting the peels. In this work, pretreatment of the peels to remove limonene under mild condition was proposed by leaching of limonene using hexane as solvent. The pretreatments were carried out with homogenized or chopped orange peel at 20–40°C with orange peel waste and hexane ratio (w/v) ranging from 1 : 2 to 1 : 12 for 10 to 300 min. The pretreated peels were then digested in batch reactors for 33 days. The highest biogas production was achieved by treating chopped orange peel waste and hexane ratio of 12 : 1 at 20°C for 10 min corresponding to more than threefold increase of biogas production from 0.061 to 0.217 m3 methane/kg VS. The solvent recovery was 90% using vacuum filtration and needs further separation using evaporation. The hexane residue in the peel had a negative impact on biogas production as shown by 28.6% reduction of methane and lower methane production of pretreated orange peel waste in semicontinuous digestion system compared to that of untreated peel. Rachma Wikandari, Huong Nguyen, Ria Millati, Claes Niklasson, and Mohammad J. Taherzadeh Copyright © 2015 Rachma Wikandari et al. All rights reserved. Effects of Extrusion Pretreatment Parameters on Sweet Sorghum Bagasse Enzymatic Hydrolysis and Its Subsequent Conversion into Bioethanol Thu, 19 Mar 2015 13:40:51 +0000 Second-generation bioethanol production from sweet sorghum bagasse first extruded at different conditions and then treated with cell wall degrading enzymes and fermented with I. orientalis was determined. The twin extruder parameters tested were barrel temperature, screws speed, and feedstock moisture content using surface response methodology. The best extrusion conditions were 100°C, 200 rpm, and 30% conditioning moisture content. This nonchemical and continuous pretreatment did not generate inhibitory compounds. The extruded feedstocks were saccharified varying the biocatalysis time and solids loading. The best conditions were 20% solids loading and 72 h of enzymatic treatment. These particular conditions converted 70% of the total fibrous carbohydrates into total fermentable C5 and C6 sugars. The extruded enzymatically hydrolyzed sweet sorghum bagasse was fermented with the strain I. orientalis at 12% solids obtaining a yield of 198.1 mL of ethanol per kilogram of bagasse (dw). Erick Heredia-Olea, Esther Pérez-Carrillo, Manuel Montoya-Chiw, and Sergio O. Serna-Saldívar Copyright © 2015 Erick Heredia-Olea et al. All rights reserved. Improving the Thermostability and Optimal Temperature of a Lipase from the Hyperthermophilic Archaeon Pyrococcus furiosus by Covalent Immobilization Sun, 08 Mar 2015 10:05:38 +0000 A recombinant thermostable lipase (Pf2001Δ60) from the hyperthermophilic Archaeon Pyrococcus furiosus (PFUL) was immobilized by hydrophobic interaction on octyl-agarose (octyl PFUL) and by covalent bond on aldehyde activated-agarose in the presence of DTT at pH = 7.0 (one-point covalent attachment) (glyoxyl-DTT PFUL) and on glyoxyl-agarose at pH 10.2 (multipoint covalent attachment) (glyoxyl PFUL). The enzyme’s properties, such as optimal temperature and pH, thermostability, and selectivity, were improved by covalent immobilization. The highest enzyme stability at 70°C for 48 h incubation was achieved for glyoxyl PFUL (around 82% of residual activity), whereas glyoxyl-DTT PFUL maintained around 69% activity, followed by octyl PFUL (27% remaining activity). Immobilization on glyoxyl-agarose improved the optimal temperature to 90°C, while the optimal temperature of octyl PFUL was 70°C. Also, very significant changes in activity with different substrates were found. In general, the covalent bond derivatives were more active than octyl PFUL. The E value also depended substantially on the derivative and the conditions used. It was observed that the reaction of glyoxyl-DTT PFUL using methyl mandelate as a substrate at pH 7 presented the best results for enantioselectivity and enantiomeric excess (ee (%) = 91). Roberta V. Branco, Melissa L. E. Gutarra, Jose M. Guisan, Denise M. G. Freire, Rodrigo V. Almeida, and Jose M. Palomo Copyright © 2015 Roberta V. Branco et al. All rights reserved. Recent Development in Production and Biotechnological Application of Microbial Enzymes Mon, 02 Mar 2015 08:30:26 +0000 Noomen Hmidet, Neelu Nawani, and Sofiane Ghorbel Copyright © 2015 Noomen Hmidet et al. All rights reserved. Genome-Wide Identification, Evolutionary, and Expression Analyses of Histone H3 Variants in Plants Thu, 26 Feb 2015 12:48:15 +0000 Histone variants alter the nucleosome structure and play important roles in chromosome segregation, transcription, DNA repair, and sperm compaction. Histone H3 is encoded by many genes in most eukaryotic species and is the histone that contains the largest variety of posttranslational modifications. Compared with the metazoan H3 variants, little is known about the complex evolutionary history of H3 variants proteins in plants. Here, we study the identification, evolutionary, and expression analyses of histone H3 variants from genomes in major branches in the plant tree of life. Firstly we identified all the histone three related (HTR) genes from the examined genomes, then we classified the four groups variants: centromeric H3, H3.1, H3.3 and H3-like, by phylogenetic analysis, intron information, and alignment. We further demonstrated that the H3 variants have evolved under strong purifying selection, indicating the conservation of HTR proteins. Expression analysis revealed that the HTR has a wide expression profile in maize and rice development and plays important roles in development. Jinteng Cui, Zhanlu Zhang, Yang Shao, Kezhong Zhang, Pingsheng Leng, and Zhe Liang Copyright © 2015 Jinteng Cui et al. All rights reserved. Establishment of Hairy Root Cultures of Rhaponticum carthamoides (Willd.) Iljin for the Production of Biomass and Caffeic Acid Derivatives Tue, 24 Feb 2015 09:51:05 +0000 The aim of the study was to obtain transformed roots of Rhaponticum carthamoides and evaluate their phytochemical profile. Hairy roots were induced from leaf explants by the transformation of Agrobacterium rhizogenes strains A4 and ATCC 15834. The best response (43%) was achieved by infection with A4 strain. The effects of different liquid media (WPM, B5, SH) with full and half-strength concentrations of macro- and micronutrients on biomass accumulation of the best grown hairy root line (RC3) at two different lighting conditions (light or dark) were investigated. The highest biomass (93 g L−1 of the fresh weight after 35 days) was obtained in WPM medium under periodic light. UPLC-PDA-ESI-MS3 and HPLC-PDA analyses of 80% aqueous methanol extracts from the obtained hairy roots revealed the presence of eleven caffeoylquinic acids and their derivatives and five flavonoid glycosides. The production of caffeoylquinic acids and their derivatives was elevated in hairy roots grown in the light. Only light-grown hairy roots demonstrated the capability for the biosynthesis of such flavonoid glycosides as quercetagetin, quercetin, luteolin, and patuletin hexosides. Chlorogenic acid, 3,5-di-O-caffeoylquinic acid and a tentatively identified tricaffeoylquinic acid derivative were detected as the major compounds present in the transformed roots. Ewa Skała, Agnieszka Kicel, Monika A. Olszewska, Anna K. Kiss, and Halina Wysokińska Copyright © 2015 Ewa Skała et al. All rights reserved. MALDI-TOF MS and CD Spectral Analysis for Identification and Structure Prediction of a Purified, Novel, Organic Solvent Stable, Fibrinolytic Metalloprotease from Bacillus cereus B80 Mon, 23 Feb 2015 10:55:41 +0000 The ability to predict protein function from structure is becoming increasingly important; hence, elucidation and determination of protein structure become the major steps in proteomics. The present study was undertaken for identification of metalloprotease produced by Bacillus cereus B80 and recognition of characteristics that can be industrially exploited. The enzyme was purified in three steps combining precipitation and chromatographic methods resulting in 33.5% recovery with 13.1-fold purification of enzyme which was detected as a single band with a molecular mass of 26 kDa approximately in SDS-PAGE and zymogram. The MALDI-TOF MS showed that the enzyme exhibited 70–93% similarity with zinc metalloproteases from various strains Bacillus sp. specifically from Bacillus cereus group. The sequence alignment revealed the presence of zinc-binding region VVVHEMCHMV in the most conserved C terminus region. Secondary structure of the enzyme was obtained by CD spectra and I-TASSER. The enzyme kinetics revealed a Michaelis constant of 0.140 μmol/ml and of 2.11 μmol/min. The application studies showed that the enzyme was able to hydrolyze various proteins with highest affinity towards casein followed by BSA and gelatin. The enzyme exhibited strong fibrinolytic, collagenolytic, and gelatinolytic properties and stability in various organic solvents. Rajshree Saxena and Rajni Singh Copyright © 2015 Rajshree Saxena and Rajni Singh. All rights reserved. Use of Adipose-Derived Mesenchymal Stem Cells in Keratoconjunctivitis Sicca in a Canine Model Mon, 23 Feb 2015 07:14:46 +0000 Keratoconjunctivitis sicca (KCS) or dry eye disease (DED) is an immune-mediated multifactorial disease, with high level of prevalence in humans and dogs. Our aim in this study was to investigate the therapeutic effects of allogeneic adipose-derived mesenchymal stromal cells (Ad-MSCs) implanted around the lacrimal glands in 12 dogs (24 eyes) with KCS, which is refractory to current available treatments. Schirmer tear test (STT) and ocular surface integrity were assessed at 0 (before treatment), 3, 6, and 9 months after treatment. Average STT values and all clinical signs showed a statistically significant change during the follow-up with reduction in all ocular parameters scored: ocular discharge, conjunctival hyperaemia, and corneal changes, and there were no signs of regression or worsening. Implanted cells were well tolerated and were effective reducing clinical signs of KCS with a sustained effect during the study period. None of the animals showed systemic or local complications during the study. To our knowledge, this is the first time in literature that implantation of allogeneic Ad-MSCs around lacrimal glands has been found as an effective therapeutic alternative to treat dogs with KCS. These results could reinforce a good effective solution to be extrapolated to future studies in human. Antonio J. Villatoro, Viviana Fernández, Silvia Claros, Gustavo A. Rico-Llanos, José Becerra, and José A. Andrades Copyright © 2015 Antonio J. Villatoro et al. All rights reserved. Retracted: Microfluidic Method of Pig Oocyte Quality Assessment in Relation to Different Follicular Size Based on Lab-on-Chip Technology Sun, 22 Feb 2015 15:14:24 +0000 BioMed Research International Copyright © 2015 BioMed Research International. All rights reserved. A Novel Aqueous Micellar Two-Phase System Composed of Surfactant and Sorbitol for Purification of Pectinase Enzyme from Psidium guajava and Recycling Phase Components Tue, 10 Feb 2015 13:26:20 +0000 A novel aqueous two-phase system composed of a surfactant and sorbitol was employed for the first time to purify pectinase from Psidium guajava. The influences of different parameters, including the type and concentration of the surfactant and the concentration and composition of the surfactant/sorbitol ratio, on the partitioning behavior and recovery of pectinase were investigated. Moreover, the effects of system pH and the crude load on purification fold and the yield of purified pectinase were studied. The experimental results indicated that the pectinase was partitioned into surfactant-rich top phase, and the impurities were partitioned into the sorbitol-rich bottom phase with the novel method involving an ATPS composed of 26% (w/w) Triton X-100 and 23% (w/w) sorbitol at 54.2% of the TLL crude load of 20% (w/w) at pH 6.0. The enzyme was successfully recovered by this method with a high purification factor of 15.2 and a yield of 98.3%, whereas the phase components were also recovered and recycled at rates above 96%. This study demonstrated that this novel ATPS method can be used as an efficient and economical alternative to the traditional ATPS for the purification and recovery of the valuable enzyme. Mehrnoush Amid, Fara Syazana Murshid, Mohd Yazid Manap, and Muhaini Hussin Copyright © 2015 Mehrnoush Amid et al. All rights reserved. Statistical Optimization of Conditions for Decolorization of Synthetic Dyes by Cordyceps militaris MTCC 3936 Using RSM Wed, 04 Feb 2015 08:31:43 +0000 In the present study, the biobleaching potential of white rot fungus Cordyceps militaris MTCC3936 was investigated. For preliminary screening, decolorization properties of C. militaris were comparatively studied using whole cells in agar-based and liquid culture systems. Preliminary investigation in liquid culture systems revealed 100% decolorization achieved within 3 days of incubation for reactive yellow 18, 6 days for reactive red 31, 7 days for reactive black 8, and 11 days for reactive green 19 and reactive red 74. RSM was further used to study the effect of three independent variables such as pH, incubation time, and concentration of dye on decolorization properties of cell free supernatant of C. militaris. RSM based statistical analysis revealed that dye decolorization by cell free supernatants of C. militaris is more efficient than whole cell based system. The optimized conditions for decolorization of synthetic dyes were identified as dye concentration of 300 ppm, incubation time of 48 h, and optimal pH value as 5.5, except for reactive red 31 (for which the model was nonsignificant). The maximum dye decolorizations achieved under optimized conditions for reactive yellow 18, reactive green 19, reactive red 74, and reactive black 8 were 73.07, 65.36, 55.37, and 68.59%, respectively. Baljinder Kaur, Balvir Kumar, Neena Garg, and Navneet Kaur Copyright © 2015 Baljinder Kaur et al. All rights reserved. Corrigendum to “Molecular Characterization of a Fully Human Chimeric T-Cell Antigen Receptor for Tumor-Associated Antigen EpCAM” Mon, 02 Feb 2015 14:20:45 +0000 Naoto Shirasu, Hiromi Yamada, Hirotomo Shibaguchi, Motomu Kuroki, and Masahide Kuroki Copyright © 2015 Naoto Shirasu et al. All rights reserved. Factors Affecting the Accumulation of Curcumin in Microrhizomes of Curcuma aromatica Salisb Mon, 02 Feb 2015 07:01:38 +0000 Curcuminoids, and mainly curcumin, are potential therapeutic agents for the prevention of various diseases; however, little is known about the factors that influence their accumulation in Curcuma species. In this study, the effects of factors such as sucrose concentration, different ratios of 6-benzylaminopurine (6-BA) and α-naphthalene acetic acid (NAA), and light quality on the accumulation of curcumin and other curcuminoids in Curcuma aromatica were investigated. Microrhizomes grown on media containing 3% sucrose produced more curcumin and other curcuminoids than those grown on higher concentrations. Moreover, when compared to other ratios of 6-BA and NAA, microrhizomes induced on 3% sucrose media supplemented with 3.0 mg/L 6-BA and 0.5 mg/L NAA produced more curcumin and other curcuminoids; however, the amount was less than in microrhizomes grown on 3% sucrose alone. We determined that a 5% sucrose medium supplemented with 3.0 mg/L of 6-BA and 0.5 mg/L of NAA enhanced the levels of curcumin and curcuminoids and that exposure to red light further increased production. Ke Wu, Xiaoxia Zhang, Shulan Sun, and Xiaojing Wang Copyright © 2015 Ke Wu et al. All rights reserved. Engineering Isoprenoid Biosynthesis in Artemisia annua L. for the Production of Taxadiene: A Key Intermediate of Taxol Sun, 01 Feb 2015 13:03:38 +0000 Taxadiene is the first committed precursor to paclitaxel, marketed as Taxol, arguably the most important anticancer agent against ovarian and breast cancer. In Taxus, taxadiene is directly synthesized from geranylgeranyl diphosphate (GGPP) that is the common precursor for diterpenoids and is found in most plants and microbes. In this study, Artemisia annua L., a Chinese medicinal herb that grows fast and is rich in terpenoids, was used as a genetic engineering host to produce taxadiene. The TXS (taxadiene synthase) gene, cloned from Taxus and inserted into pCAMBIA1304, was transformed into Artemisia annua L. using the Agrobacterium tumefaciens-mediated method. Thirty independent transgenic plants were obtained, and GC-MS analysis was used to confirm that taxadiene was produced and accumulated up to 129.7 μg/g dry mass. However, the high expression of TXS did not affect plant growth or photosynthesis in transgenic Artemisia annua L. It is notable that artemisinin is produced and stored in leaves and most taxadiene accumulated in the stem of transgenic Artemisia annua L., suggesting a new way to produce two important compounds in one transgenic plant: leaves for artemisinin and stem for taxadiene. Overall, this study demonstrates that genetic engineering of the taxane biosynthetic pathway in Artemisia annua L. for the production of taxadiene is feasible. Meiya Li, Fusheng Jiang, Xiangli Yu, and Zhiqi Miao Copyright © 2015 Meiya Li et al. All rights reserved. Community Structure of Ammonia-Oxidizing Archaea and Ammonia-Oxidizing Bacteria in Soil Treated with the Insecticide Imidacloprid Sun, 01 Feb 2015 11:30:21 +0000 The purpose of this experiment was to assess the effect of imidacloprid on the community structure of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in soil using the denaturing gradient gel electrophoresis (DGGE) approach. Analysis showed that AOA and AOB community members were affected by the insecticide treatment. However, the calculation of the richness (S) and the Shannon-Wiener index (H) values for soil treated with the field rate (FR) dosage of imidacloprid (1 mg/kg soil) showed no changes in measured indices for the AOA and AOB community members. In turn, the dosage of insecticide (10 mg/kg soil) negatively affected the AOA community, which was confirmed by the decrease of the S and H values in comparison with the values obtained for the control soil. In the case of AOB community, an initial decline followed by the increase of the S and H values was obtained. Imidacloprid decreased the nitrification rate while the ammonification process was stimulated by the addition of imidacloprid. Changes in the community structure of AOA and AOB could be due to an increase in the concentration of N-, known as the most important factor which determines the contribution of these microorganisms to soil nitrification. Mariusz Cycoń and Zofia Piotrowska-Seget Copyright © 2015 Mariusz Cycoń and Zofia Piotrowska-Seget. All rights reserved. Characterization of LpGPAT Gene in Lilium pensylvanicum and Response to Cold Stress Sun, 01 Feb 2015 10:25:36 +0000 LpGPAT was obtained from L. pensylvanicum using RT-PCR and rapid amplification of cDNA ends. The cloned full-length cDNA was 1544 bp; it encoded 410 amino acids and had a molecular size of 46 KDa. The nucleic acid sequence analysis showed that it shared high homology with other known GPATs. SMAT result suggests that there is a PlsC that exists in 176-322 amino acid sequence of LpGAPT; it means LpGPAT protein is a member of the family of acyltransferase and has acyltransferase enzymatic activity. Result of real-time quantitative PCR and semiquantitative PCR support LpGPAT gene is definitely induced by low temperature stress. Shao-kun Sun, Ni-na Yang, Li-jing Chen, Muhammad Irfan, Xing-hua Zhao, and Tian-lai Li Copyright © 2015 Shao-kun Sun et al. All rights reserved. Nitrate Promotes Capsaicin Accumulation in Capsicum chinense Immobilized Placentas Sun, 01 Feb 2015 10:04:55 +0000 In chili pepper’s pods, placental tissue is responsible for the synthesis of capsaicinoids (CAPs), the compounds behind their typical hot flavor or pungency, which are synthesized from phenylalanine and branched amino acids. Placental tissue sections from Habanero peppers (Capsicum chinense Jacq.) were immobilized in a calcium alginate matrix and cultured in vitro, either continuously for 28 days or during two 14-day subculture periods. Immobilized placental tissue remained viable and metabolically active for up to 21 days, indicating its ability to interact with media components. CAPs contents abruptly decreased during the first 7 days in culture, probably due to structural damage to the placenta as revealed by scanning electron microcopy. CAPs levels remained low throughout the entire culture period, even though a slight recovery was noted in subcultured placentas. However, doubling the medium’s nitrate content (from 40 to 80 mM) resulted in an important increment, reaching values similar to those of intact pod’s placentas. These data suggest that isolated pepper placentas cultured in vitro remain metabolically active and are capable of metabolizing inorganic nitrogen sources, first into amino acids and, then, channeling them to CAP synthesis. Jeanny G. Aldana-Iuit, Enrique Sauri-Duch, María de Lourdes Miranda-Ham, Lizbeth A. Castro-Concha, Luis F. Cuevas-Glory, and Felipe A. Vázquez-Flota Copyright © 2015 Jeanny G. Aldana-Iuit et al. All rights reserved. Immunohistological Analysis of ABCD3 Expression in Caucasian and African American Prostate Tumors Sat, 31 Jan 2015 08:57:35 +0000 In a previously published study, we showed that expression of the ABCD3 gene increased with increasing metastatic potential in a panel of prostate cancer cell lines derived from African American and Caucasian American men. Given importance of identifying biomarker(s) that can distinguish indolent versus aggressive prostate tumors, we conducted an immunohistochemical analysis of ABCD3 expression Caucasian and African American prostate tumors. ABCD3 expression in each patient population was compared with clinicopathologic characteristics, Gleason score, and age. ABCD3 expression increased with increasing Gleason score (), age (), and pathology grade () in Caucasian patients. Interestingly, in the AA patients, ABCD3 expression highly increased to the same degree in both low and high Gleason score tumors. Similarly, ABCD3 expression was elevated to the same degree in BPH derived from AA. Our findings demonstrate that increased ABCD3 expression correlates with Gleason Score in CA prostate tumors. However, in AA prostate tumors, ABCD3 expression was higher and was sustained in both low Gleason and high Gleason AA tumors. While the functional role of ABCD3 in prostate cancer is not completely elucidated, this gene warrants further study as a potential biomarker for aggressive prostate. R. Renee Reams, Jacqueline Jones-Triche, Owen T. M. Chan, Brenda Y. Hernandez, Karam F. A. Soliman, and Clayton Yates Copyright © 2015 R. Renee Reams et al. All rights reserved. Biochemical Storage Lesions Occurring in Nonirradiated and Irradiated Red Blood Cells: A Brief Review Thu, 29 Jan 2015 10:23:24 +0000 Red blood cells undergo a series of biochemical fluctuations during 35–42-day storage period at 1°C to 6°C. The sodium/potassium pump is immobilised causing a decrease in intracellular potassium with an increase in cytoplasmic sodium levels, glucose levels decline, and acidosis occurs as a result of low pH levels. The frailty of stored erythrocytes triggers the formation of haemoglobin-containing microparticles and the release of cell-free haemoglobin which may add to transfusion difficulties. Lipid peroxidation, oxidative stress to band 3 structures, and other morphological and structural molecular changes also occur leading to spheroechinocytes and osmotic fragility. These changes that transpire in the red cells during the storage period are referred to as “storage lesions.” It is well documented that gamma irradiation exacerbates storage lesions and the reports of increased potassium levels leading to adverse reactions observed in neonates and infants have been of particular concern. There are, however, remarkably few systematic studies comparing the in vitro storage lesions of irradiated and nonirradiated red cell concentrates and it has been suggested that the impact of storage lesions on leucocyte reduced red blood cell concentrate (RBCC) is incomplete. The review examines storage lesions in red blood cells and their adverse effects in reference to blood transfusion. F. Adams, G. Bellairs, A. R. Bird, and O. O. Oguntibeju Copyright © 2015 F. Adams et al. All rights reserved. Methods for Obtaining and Determination of Squalene from Natural Sources Wed, 28 Jan 2015 08:12:44 +0000 Squalene is a natural dehydrotriterpenic hydrocarbon (C30H50) with six double bonds, known as an intermediate in the biosynthesis of phytosterol or cholesterol in plants or animals. We have briefly reviewed the natural sources for squalene and focused on the main methods and techniques to obtain and to determine it. Some of its applications in different fields of human activity are also mentioned. Ovidiu Popa, Narcisa Elena Băbeanu, Ioana Popa, Sultana Niță, and Cristina Elena Dinu-Pârvu Copyright © 2015 Ovidiu Popa et al. All rights reserved. Lentiviral-Mediated Silencing of Farnesyl Pyrophosphate Synthase through RNA Interference in Mice Thu, 22 Jan 2015 13:47:10 +0000 Farnesyl pyrophosphate synthase (FPPS) plays a vital role in the mevalonate pathway and has been shown to be involved in hypertrophy and cardiovascular diseases. Lentivirus-mediated RNA interference (RNAi) to knock down a gene of interest has become a promising new tool for the establishment of transgenic animals. The interfering fragment, named pLVT202, was chosen from cardiomyocytes tested in vitro and was microinjected into the perivitelline space of zygotes from C57BL/6J mice via a lentivirus vehicle; 20 were identified as carrying copies of the transgene using the polymerase chain reaction (PCR). Real-time PCR and western blotting analysis showed that FPPS was downregulated in multiple tissues in the transgenic mice. The transgenic mouse model provides a novel means of studying the gene function of FPPS. Jian Yang, Chen-Ze Zhao, Bin Chen, Fei Chen, Jie Han, and Shen-Jiang Hu Copyright © 2015 Jian Yang et al. All rights reserved. Zero Discharge Performance of an Industrial Pilot-Scale Plant Treating Palm Oil Mill Effluent Thu, 22 Jan 2015 12:24:03 +0000 Palm oil is one of the most important agroindustries in Malaysia. Huge quantities of palm oil mill effluent (POME) pose a great threat to aqueous environment due to its very high COD. To make full use of discharged wastes, the integrated “zero discharge” pilot-scale industrial plant comprising “pretreatment-anaerobic and aerobic process-membrane separation” was continuously operated for 1 year. After pretreatment in the oil separator tank, 55.6% of waste oil in raw POME could be recovered and sold and anaerobically digested through 2 AnaEG reactors followed by a dissolved air flotation (DAF); average COD reduced to about 3587 mg/L, and biogas production was 27.65 times POME injection which was used to generate electricity. The aerobic effluent was settled for 3 h or/and treated in MBR which could remove BOD3 (30°C) to less than 20 mg/L as required by Department of Environment of Malaysia. After filtration by UF and RO membrane, all organic compounds and most of the salts were removed; RO permeate could be reused as the boiler feed water. RO concentrate combined with anaerobic surplus sludge could be used as biofertilizer. Jin Wang, Qaisar Mahmood, Jiang-Ping Qiu, Yin-Sheng Li, Yoon-Seong Chang, Li-Na Chi, and Xu-Dong Li Copyright © 2015 Jin Wang et al. All rights reserved. Strain Amplification Analysis of an Osteocyte under Static and Cyclic Loading: A Finite Element Study Thu, 15 Jan 2015 14:03:13 +0000 Osteocytes, the major type of bone cells which reside in their lacunar and canalicular system within the bone matrix, function as biomechanosensors and biomechanotransducers of the bone. Although biomechanical behaviour of the osteocyte-lacunar-canalicular system has been investigated in previous studies mostly using computational 2-dimensional (2D) geometric models, only a few studies have used the 3-dimensional (3D) finite element (FE) model. In the current study, a 3D FE model was used to predict the responses of strain distributions of osteocyte-lacunar-canalicular system analyzed under static and cyclic loads. The strain amplification factor was calculated for all simulations. Effects on the strain of the osteocyte system were investigated under 500, 1500, 2000, and 3000 microstrain loading magnitudes and 1, 5, 10, 40, and 100 Hz loading frequencies. The maximum strain was found to change with loading magnitude and frequency. It was observed that maximum strain under 3000-microstrain loading was higher than those under 500, 1500, and 2000 microstrains. When the loading strain reached the maximum magnitude, the strain amplification factor of 100 Hz was higher than those of the other frequencies. Data from this 3D FE model study suggests that the strain amplification factor of the osteocyte-lacunar-canalicular system increases with loading frequency and loading strain increasing. Liping Wang, Jianghui Dong, and Cory J. Xian Copyright © 2015 Liping Wang et al. All rights reserved. A Photoperiod-Regulating Gene CONSTANS Is Correlated to Lipid Biosynthesis in Chlamydomonas reinhardtii Thu, 15 Jan 2015 07:44:20 +0000 Background. The regulation of lipid biosynthesis is essential in photosynthetic eukaryotic cells. Thus far, no regulatory genes have been reported in the lipid metabolism pathway. Plant CONSTANS (CO) gene regulates blooming by participating in photoperiod and biological clock. Apart from regulating photoperiod, the Chlamydomonas CO gene also regulates starch content. Results. In this study, the results showed that, under HSM-S condition, cells accumulated more lipids at short-day conditions than at long-day conditions. The silencing of the CrCO gene via RNA interference resulted in an increase in lipid content and an increase in triacylglyceride (TAG) level by 24.5%. CrCO RNAi strains accumulated more lipids at short-day conditions than at long-day conditions. The decrease in CrCO expression resulted in the increased expression of TAG biosynthesis-related genes, such as DGAT2, PAP2, and PDAT3, whereas CIS and FBP1 genes showed a decrease in their mRNA when the CrCO expression was suppressed. On the other hand, the overexpression of CrCO resulted in the decrease in lipid content and TAG level. Conclusions. The results of this study revealed a relationship between CrCO gene and lipid metabolism in Chlamydomonas, suggesting that increasing oil by suppressing CrCO expression in microalgae is feasible. Xiaodong Deng, Xinzhao Fan, Ping Li, and Xiaowen Fei Copyright © 2015 Xiaodong Deng et al. All rights reserved. Phylogenetic Relationship of Phosphate Solubilizing Bacteria according to 16S rRNA Genes Tue, 06 Jan 2015 13:33:02 +0000 Phosphate solubilizing bacteria (PSB) can convert insoluble form of phosphorous to an available form. Applications of PSB as inoculants increase the phosphorus uptake by plant in the field. In this study, isolation and precise identification of PSB were carried out in Malaysian (Serdang) oil palm field (University Putra Malaysia). Identification and phylogenetic analysis of 8 better isolates were carried out by 16S rRNA gene sequencing in which as a result five isolates belong to the Beta subdivision of Proteobacteria, one isolate was related to the Gama subdivision of Proteobacteria, and two isolates were related to the Firmicutes. Bacterial isolates of 6upmr, 2upmr, 19upmnr, 10upmr, and 24upmr were identified as Alcaligenes faecalis. Also, bacterial isolates of 20upmnr and 17upmnr were identified as Bacillus cereus and Vagococcus carniphilus, respectively, and bacterial isolates of 31upmr were identified as Serratia plymuthica. Molecular identification and characterization of oil palm strains as the specific phosphate solubilizer can reduce the time and cost of producing effective inoculate (biofertilizer) in an oil palm field. Mohammad Bagher Javadi Nobandegani, Halimi Mohd Saud, and Wong Mui Yun Copyright © 2015 Mohammad Bagher Javadi Nobandegani et al. All rights reserved. Lipopeptides as the Antifungal and Antibacterial Agents: Applications in Food Safety and Therapeutics Tue, 06 Jan 2015 12:21:06 +0000 A lot of crops are destroyed by the phytopathogens such as fungi, bacteria, and yeast leading to economic losses to the farmers. Members of the Bacillus genus are considered as the factories for the production of biologically active molecules that are potential inhibitors of growth of phytopathogens. Plant diseases constitute an emerging threat to global food security. Many of the currently available antimicrobial agents for agriculture are highly toxic and nonbiodegradable and thus cause extended environmental pollution. Moreover, an increasing number of phytopathogens have developed resistance to antimicrobial agents. The lipopeptides have been tried as potent versatile weapons to deal with a variety of phytopathogens. All the three families of Bacillus lipopeptides, namely, Surfactins, Iturins and Fengycins, have been explored for their antagonistic activities towards a wide range of phytopathogens including bacteria, fungi, and oomycetes. Iturin and Fengycin have antifungal activities, while Surfactin has broad range of potent antibacterial activities and this has also been used as larvicidal agent. Interestingly, lipopeptides being the molecules of biological origin are environmentally acceptable. Khem Raj Meena and Shamsher S. Kanwar Copyright © 2015 Khem Raj Meena and Shamsher S. Kanwar. All rights reserved. Specific Genomic Fingerprints of Phosphate Solubilizing Pseudomonas Strains Generated by Box Elements Mon, 15 Dec 2014 12:08:11 +0000 Primers corresponding to conserved bacterial repetitive of BOX elements were used to show that BOX-DNA sequences are widely distributed in phosphate solubilizing Pseudomonas strains. Phosphate solubilizing Pseudomonas was isolated from oil palm fields (tropical soil) in Malaysia. BOX elements were used to generate genomic fingerprints of a variety of Pseudomonas isolates to identify strains that were not distinguishable by other classification methods. BOX-PCR, that derived genomic fingerprints, was generated from whole purified genomic DNA by liquid culture of phosphate solubilizing Pseudomonas. BOX-PCR generated the phosphate solubilizing Pseudomonas specific fingerprints to identify the relationship between these strains. This suggests that distribution of BOX elements’ sequences in phosphate solubilizing Pseudomonas strains is the mirror image of their genomic structure. Therefore, this method appears to be a rapid, simple, and reproducible method to identify and classify phosphate solubilizing Pseudomonas strains and it may be useful tool for fast identification of potential biofertilizer strains. Mohammad Bagher Javadi Nobandegani, Halimi Mohd Saud, and Wong Mui Yun Copyright © 2014 Mohammad Bagher Javadi Nobandegani et al. All rights reserved. Identification of a Marine Bacillus Strain C5 and Parathion-Methyl Degradation Characteristics of the Extracellular Esterase B1 Thu, 11 Dec 2014 07:01:29 +0000 A bacterial strain C5 that can produce new type of marine esterase was isolated and screened from marine sludge. According to 16S rRNA sequence analysis and physiological and biochemical experiments, the strain was identified as Bacillus subtilis. A single isozyme with a molecular weight of 86 kDa was observed by SDS-PAGE and native-PAGE. On this basis, the mechanism of esterase B1 secreted by strain C5 degrading parathion-methyl was explored, and the effects of temperature and pH on the degradation rate were investigated. From the results, p-nitrophenol was one of the degradation products of B1 degrading parathion-methyl, and the best degradation effect could be achieved at the temperature of 40°C and the neutral pH value. Jianhua Hao, Junzhong Liu, and Mi Sun Copyright © 2014 Jianhua Hao et al. All rights reserved. Barcoding Melting Curve Analysis for Rapid, Sensitive, and Discriminating Authentication of Saffron (Crocus sativus L.) from Its Adulterants Sun, 07 Dec 2014 14:22:31 +0000 Saffron (Crocus sativus L.) is one of the most important and expensive medicinal spice products in the world. Because of its high market value and premium price, saffron is often adulterated through the incorporation of other materials, such as Carthamus tinctorius L. and Calendula officinalis L. flowers, Hemerocallis L. petals, Daucus carota L. fleshy root, Curcuma longa L. rhizomes, Zea may L., and Nelumbo nucifera Gaertn. stigmas. To develop a straightforward, nonsequencing method for rapid, sensitive, and discriminating detection of these adulterants in traded saffron, we report here the application of a barcoding melting curve analysis method (Bar-MCA) that uses the universal chloroplast plant DNA barcoding region trnH-psbA to identify adulterants. When amplified at DNA concentrations and annealing temperatures optimized for the curve analysis, peaks were formed at specific locations for saffron (81.92°C) and the adulterants: D. carota (81.60°C), C. tinctorius (80.10°C), C. officinalis (79.92°C), Dendranthema morifolium (Ramat.) Tzvel. (79.62°C), N. nucifera (80.58°C), Hemerocallis fulva (L.) L. (84.78°C), and Z. mays (84.33°C). The constructed melting curves for saffron and its adulterants have significantly different peak locations or shapes. In conclusion, Bar-MCA could be a faster and more cost-effective method to authenticate saffron and detect its adulterants. Chao Jiang, Liang Cao, Yuan Yuan, Min Chen, Yan Jin, and Luqi Huang Copyright © 2014 Chao Jiang et al. All rights reserved. Cross-Comparison of Leaching Strains Isolated from Two Different Regions: Chambishi and Dexing Copper Mines Sun, 16 Nov 2014 09:41:30 +0000 A cross-comparison of six strains isolated from two different regions, Chambishi copper mine (Zambia, Africa) and Dexing copper mine (China, Asia), was conducted to study the leaching efficiency of low grade copper ores. The strains belong to the three major species often encountered in bioleaching of copper sulfide ores under mesophilic conditions: Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans, and Leptospirillum ferriphilum. Prior to their study in bioleaching, the different strains were characterized and compared at physiological level. The results revealed that, except for copper tolerance, strains within species presented almost similar physiological traits with slight advantages of Chambishi strains. However, in terms of leaching efficiency, native strains always achieved higher cell density and greater iron and copper extraction rates than the foreign microorganisms. In addition, microbial community analysis revealed that the different mixed cultures shared almost the same profile, and At. ferrooxidans strains always outcompeted the other strains. Baba Ngom, Yili Liang, and Xueduan Liu Copyright © 2014 Baba Ngom et al. All rights reserved. Lipolytic Potential of Aspergillus japonicus LAB01: Production, Partial Purification, and Characterisation of an Extracellular Lipase Wed, 29 Oct 2014 12:45:20 +0000 Lipolytic potential of Aspergillus japonicus LAB01 was investigated by describing the catalytic properties and stability of a secreted extracellular lipase. Enzyme production was considered high under room temperature after 4 days using sunflower oil and a combination of casein with sodium nitrate. Lipase was partially purified by 3.9-fold, resulting in a 44.2% yield using ammonium sulphate precipitation (60%) quantified with Superose 12 HR gel filtration chromatography. The activity of the enzyme was maximised at pH 8.5, and the enzyme demonstrated stability under alkaline conditions. The optimum temperature was found to be 45°C, and the enzyme was stable for up to 100 minutes, with more than 80% of initial activity remaining after incubation at this temperature. Partially purified enzyme showed reasonable stability with triton X-100 and was activated in the presence of organic solvents (toluene, hexane, and methanol). Among the tested ions, only Cu2+, Ni2+, and Al3+ showed inhibitory effects. Substrate specificity of the lipase was higher for C14 among various p-nitrophenyl esters assayed. The and values of the purified enzyme for p-nitrophenyl palmitate were 0.13 mM and 12.58 umol/(L·min), respectively. These features render a novel biocatalyst for industrial applications. Lívia Tereza Andrade Souza, Jamil S. Oliveira, Vera L. dos Santos, Wiliam C. B. Regis, Marcelo M. Santoro, and Rodrigo R. Resende Copyright © 2014 Lívia Tereza Andrade Souza et al. All rights reserved. Type 2C Phosphatase 1 of Artemisia annua L. Is a Negative Regulator of ABA Signaling Tue, 28 Oct 2014 09:02:12 +0000 The phytohormone abscisic acid (ABA) plays an important role in plant development and environmental stress response. Additionally, ABA also regulates secondary metabolism such as artemisinin in the medicinal plant Artemisia annua L. Although an earlier study showed that ABA receptor, AaPYL9, plays a positive role in ABA-induced artemisinin content improvement, many components in the ABA signaling pathway remain to be elucidated in Artemisia annua L. To get insight of the function of AaPYL9, we isolated and characterized an AaPYL9-interacting partner, AaPP2C1. The coding sequence of AaPP2C1 encodes a deduced protein of 464 amino acids, with all the features of plant type clade A PP2C. Transcriptional analysis showed that the expression level of AaPP2C1 is increased after ABA, salt, and drought treatments. Yeast two-hybrid and bimolecular fluorescence complementation assays (BiFC) showed that AaPYL9 interacted with AaPP2C1. The P89S, H116A substitution in AaPYL9 as well as G199D substitution or deletion of the third phosphorylation site-like motif in AaPP2C1 abolished this interaction. Furthermore, constitutive expression of AaPP2C1 conferred ABA insensitivity compared with the wild type. In summary, our data reveals that AaPP2C1 is an AaPYL9-interacting partner and involved in the negative modulation of the ABA signaling pathway in A. annua L. Fangyuan Zhang, Xueqing Fu, Zongyou Lv, Qian Shen, Tingxian Yan, Weiming Jiang, Guofeng Wang, Xiaofen Sun, and Kexuan Tang Copyright © 2014 Fangyuan Zhang et al. All rights reserved. Activation of the AT1R/HIF-1α/ACE Axis Mediates Angiotensin II-Induced VEGF Synthesis in Mesenchymal Stem Cells Mon, 20 Oct 2014 11:47:30 +0000 A local renin-angiotensin system (RAS) is expressed in mesenchymal stem cells (MSCs) and regulates stem cell function. The local RAS influences the survival and tissue repairing ability of transplanted stem cells. We have previously reported that angiotensin II (Ang II) pretreatment can significantly increase vascular endothelial growth factor (VEGF) synthesis in MSCs through the ERK1/2 and Akt pathways via the Ang II receptor type 1 (AT1R). However, the role of angiotensin-converting enzyme (ACE) has not been clarified. Furthermore, whether Ang II pretreatment activates hypoxia-inducible factor-1α (HIF-1α) in MSCs has not been elucidated. Our data show that both ACE and HIF-1α are involved in promoting VEGF expression in MSCs, and that both are upregulated by Ang II stimulation. The upregulation of ACE appeared after the rapid degradation of exogenous Ang II, and led to the formation of endogenous Ang II. On the other hand, the ACE inhibitor, captopril, attenuated Ang II-enhanced HIF-1α upregulation, while HIF-1α suppression markedly attenuated ACE expression. This interesting finding suggests an interaction between ACE and HIF-1α. We conclude that Ang II pretreatment, as a trigger, activated the AT1R/HIF-1α/ACE axis that then mediated Ang II-induced VEGF synthesis in MSCs. Chao Liu, Jing-Wen Zhang, Liang Hu, Yi-Chen Song, Lu Zhou, Yue Fan, Hong-Yi Zhu, Yu Wang, and Qing-Ping Li Copyright © 2014 Chao Liu et al. All rights reserved. Serum-Free Medium Optimization Based on Trial Design and Support Vector Regression Tue, 14 Oct 2014 07:59:16 +0000 The Plackett-Burman design and support vector machine (SVM) were reported to be used on many fields such as some feature selections, protein structure prediction, or forecasting of other situations. Here, with suspension adapted Chinese hamster ovary (CHO) cells as the object of study, a serum-free medium for the culture of CHO cells in suspension was optimized by this method. Support vector machine based on genetic algorithm was used to predict the growth rate of CHO and prove the results from the trial designs. Experimental results indicated that ZnSO4, transferrin, and bovine serum albumin (BSA) were important ones. The same conclusion was arrived at when the support vector regression model analyzed the experimental results. With the methods mentioned, the influence of 7 medium supplements on the growth of CHO cells in suspension was evaluated efficiently. Jian Xu, Fang-rong Yan, Zhi-hui Li, Deng Wang, Hai-lin Sheng, and Yu Liu Copyright © 2014 Jian Xu et al. All rights reserved. Freeze-Drying of Plant Tissue Containing HBV Surface Antigen for the Oral Vaccine against Hepatitis B Sun, 12 Oct 2014 07:20:35 +0000 The aim of this study was to develop a freeze-drying protocol facilitating successful processing of plant material containing the small surface antigen of hepatitis B virus (S-HBsAg) while preserving its VLP structure and immunogenicity. Freeze-drying of the antigen in lettuce leaf tissue, without any isolation or purification step, was investigated. Each process step was consecutively evaluated and the best parameters were applied. Several drying profiles and excipients were tested. The profile of 20°C for 20 h for primary and 22°C for 2 h for secondary drying as well as sucrose expressed efficient stabilisation of S-HBsAg during freeze-drying. Freezing rate and postprocess residual moisture were also analysed as important factors affecting S-HBsAg preservation. The process was reproducible and provided a product with VLP content up to 200 µg/g DW. Assays for VLPs and total antigen together with animal immunisation trials confirmed preservation of antigenicity and immunogenicity of S-HBsAg in freeze-dried powder. Long-term stability tests revealed that the stored freeze-dried product was stable at 4°C for one year, but degraded at elevated temperatures. As a result, a basis for an efficient freeze-drying process has been established and a suitable semiproduct for oral plant-derived vaccine against HBV was obtained. Marcin Czyż, Radosław Dembczyński, Roman Marecik, Justyna Wojas-Turek, Magdalena Milczarek, Elżbieta Pajtasz-Piasecka, Joanna Wietrzyk, and Tomasz Pniewski Copyright © 2014 Marcin Czyż et al. All rights reserved. Computational Approaches for Microalgal Biofuel Optimization: A Review Sun, 21 Sep 2014 00:00:00 +0000 The increased demand and consumption of fossil fuels have raised interest in finding renewable energy sources throughout the globe. Much focus has been placed on optimizing microorganisms and primarily microalgae, to efficiently produce compounds that can substitute for fossil fuels. However, the path to achieving economic feasibility is likely to require strain optimization through using available tools and technologies in the fields of systems and synthetic biology. Such approaches invoke a deep understanding of the metabolic networks of the organisms and their genomic and proteomic profiles. The advent of next generation sequencing and other high throughput methods has led to a major increase in availability of biological data. Integration of such disparate data can help define the emergent metabolic system properties, which is of crucial importance in addressing biofuel production optimization. Herein, we review major computational tools and approaches developed and used in order to potentially identify target genes, pathways, and reactions of particular interest to biofuel production in algae. As the use of these tools and approaches has not been fully implemented in algal biofuel research, the aim of this review is to highlight the potential utility of these resources toward their future implementation in algal research. Joseph Koussa, Amphun Chaiboonchoe, and Kourosh Salehi-Ashtiani Copyright © 2014 Joseph Koussa et al. All rights reserved. Purification and Characterization of Alkaline-Thermostable Protease Enzyme from Pitaya (Hylocereus polyrhizus) Waste: A Potential Low Cost of the Enzyme Thu, 18 Sep 2014 07:41:10 +0000 The thermoalkaline protease enzyme from pitaya (Hylocereus polyrhizus) waste was purified by a factor of 221.2 with 71.3% recovery using ammonium sulphate precipitation, gel filtration, and cation exchange chromatography. Gel filtration chromatography together with sodium dodecyl sulphate gel electrophoresis (SDS-PAGE) revealed that the enzyme is monomeric with a molecular weight of 26.7 kDa. The apparent and of the protease were 2.8 mg/mL and 31.20 u/min, respectively. The optimum pH and temperature were 8.0 and 70°C. The enzyme was highly active and stable over a wide pH range (from pH 3.0 to pH 11.0 with the optimum activity at pH 8.0). The protease has broad specificity toward azocasein, casein, hemoglobin, and gelatine. Activity of the enzyme was inhibited by Fe2+ and Zn2+, while protease activity was increased in the presence of Ca2+ and Mg2+ and Cu2+ by factors of 125%, 110%, and 105%, respectively. The alkaline protease showed extreme stability toward surfactants and oxidizing agent. The purified protease exhibited extreme stability in the presence of organic solvents and inhibitors. In addition, the enzyme was relativity stable toward organic solvents and chelating agents, such as ethylenediaminetetraacetic acid (EDTA). The enzyme, derived from pitaya peel, possesses unique characteristics and could be used in various industrial and biotechnological applications. Mehrnoush Amid, Mohd Yazid ABD Manap, and Nor Khanani Zohdi Copyright © 2014 Mehrnoush Amid et al. All rights reserved. Negative Effects of a Nonhost Proteinase Inhibitor of ~19.8 kDa from Madhuca indica Seeds on Developmental Physiology of Helicoverpa armigera (Hübner) Sun, 14 Sep 2014 07:54:05 +0000 An affinity purified trypsin inhibitor from the seed flour extracts of Madhuca indica (MiTI) on denaturing polyacrylamide gel electrophoresis showed that MiTI consisted of a single polypeptide chain with molecular mass of ~19.8 kDa. MiTI inhibited the total proteolytic and trypsin-like activities of the midgut proteinases of Helicoverpa armigera larvae by 87.51% and 76.12%, respectively, at concentration of 5 µg/mL with an IC50 of 1.75 µg/mL against trypsin like midgut proteinases. The enzyme kinetic studies demonstrated that MiTI is a competitive inhibitor with a value of −10 M for Helicoverpa trypsin like midgut proteinases. In vivo experiments with different concentrations of MiTI in artificial diet (0.5, 1.0, and 1.5% w/w) showed an effective downfall in the larval body weight and an increase in larval mortality. The concentration of MiTI in the artificial diet to cause 50% mortality (LD50) of larvae was 1.5% w/w and that to cause reduction in mass of larvae by 50% (ED50) was 1.0% w/w. Nutritional indices observations suggest the toxic and adverse effects of MiTI on the growth and development of H. armigera larvae. The results suggest a strong bioinsecticidal potential of affinity purified MiTI which can be exploited in insect pest management of crop plants. Farrukh Jamal, Dushyant Singh, and Prabhash K. Pandey Copyright © 2014 Farrukh Jamal et al. All rights reserved. The Applicability of Oxidative Stress Biomarkers in Assessing Chromium Induced Toxicity in the Fish Labeo rohita Sun, 14 Sep 2014 07:53:33 +0000 The evaluation of metal’s toxicity in freshwater is one of the imperative areas of research and there is an emergent concern on the development of techniques for detecting toxic effects in aquatic animals. Oxidative stress biomarkers are very useful in assessing the health of aquatic life and more in depth studies are necessary to establish an exact cause effect relationship. Therefore, to study the effectiveness of this approach, a laboratory study was conducted in the fish Labeo rohita as a function of hexavalent chromium and the toxicity indices using a battery of oxidative stress biomarkers such as catalase (CAT), superoxide dismutase (SOD), and glutathione reductase (GR) in the liver, muscle, gills, and brain have been studied along with biometric parameters, behavioral changes, and Cr bioaccumulation. A significant increased HSI was observed in contrast to CF which reduced significantly. SOD, CAT, and GR activity increased significantly in all the tissues of treated fishes. The bioaccumulation of Cr was highest in liver followed by gills, muscle, and brain. This study highlights the significance of using a set of integrated biomarker and advocate to include these parameters in National Water Quality Monitoring Program in areas potentially polluted with metals to assess the health of the ecosystem. Kanchan Kumari, Ankur Khare, and Swati Dange Copyright © 2014 Kanchan Kumari et al. All rights reserved. Simultaneous Coproduction of Hydrogen and Ethanol in Anaerobic Packed-Bed Reactors Thu, 11 Sep 2014 07:31:31 +0000 This study evaluated the use of an anaerobic packed-bed reactor for hydrogen production at different hydraulic retention times (HRT) (1–8 h). Two reactors filled with expanded clay and fed with glucose (3136–3875 mg L−1) were operated at different total upflow velocities: 0.30 cm s−1 (R030) and 0.60 cm s−1 (R060). The effluent pH of the reactors was maintained between 4 and 5 by adding NaHCO3 and HCl solutions. It was observed a maximum hydrogen production rate of 0.92 L H2 h−1 L−1 in R030 at HRT of 1 h. Furthermore, the highest hydrogen yield of 2.39 mol H2 mol−1 glucose was obtained in R060. No clear trend was observed by doubling the upflow velocities at this experiment. High ethanol production was also observed, indicating that the ethanol-pathway prevailed throughout the experiment. Cristiane Marques dos Reis and Edson Luiz Silva Copyright © 2014 Cristiane Marques dos Reis and Edson Luiz Silva. All rights reserved. Cholesterol Assimilation by Lactobacillus Probiotic Bacteria: An In Vitro Investigation Thu, 11 Sep 2014 00:00:00 +0000 Excess cholesterol is associated with cardiovascular diseases (CVD), an important cause of mortality worldwide. Current CVD therapeutic measures, lifestyle and dietary interventions, and pharmaceutical agents for regulating cholesterol levels are inadequate. Probiotic bacteria have demonstrated potential to lower cholesterol levels by different mechanisms, including bile salt hydrolase activity, production of compounds that inhibit enzymes such as 3-hydroxy-3-methylglutaryl coenzyme A, and cholesterol assimilation. This work investigates 11 Lactobacillus strains for cholesterol assimilation. Probiotic strains for investigation were selected from the literature: Lactobacillus reuteri NCIMB 11951, L. reuteri NCIMB 701359, L. reuteri NCIMB 702655, L. reuteri NCIMB 701089, L. reuteri NCIMB 702656, Lactobacillus fermentum NCIMB 5221, L. fermentum NCIMB 8829, L. fermentum NCIMB 2797, Lactobacillus rhamnosus ATCC 53103 GG, Lactobacillus acidophilus ATCC 314, and Lactobacillus plantarum ATCC 14917. Cholesterol assimilation was investigated in culture media and under simulated intestinal conditions. The best cholesterol assimilator was L. plantarum ATCC 14917 (15.18 ± 0.55 mg/1010 cfu) in MRS broth. L. reuteri NCIMB 701089 assimilated over 67% (2254.70 ± 63.33 mg/1010 cfu) of cholesterol, the most of all the strains, under intestinal conditions. This work demonstrates that probiotic bacteria can assimilate cholesterol under intestinal conditions, with L. reuteri NCIMB 701089 showing great potential as a CVD therapeutic. Catherine Tomaro-Duchesneau, Mitchell L. Jones, Divya Shah, Poonam Jain, Shyamali Saha, and Satya Prakash Copyright © 2014 Catherine Tomaro-Duchesneau et al. All rights reserved. Genetic Transformation of Metroxylon sagu (Rottb.) Cultures via Agrobacterium-Mediated and Particle Bombardment Thu, 11 Sep 2014 00:00:00 +0000 Sago palm (Metroxylon sagu) is a perennial plant native to Southeast Asia and exploited mainly for the starch content in its trunk. Genetic improvement of sago palm is extremely slow when compared to other annual starch crops. Urgent attention is needed to improve the sago palm planting material and can be achieved through nonconventional methods. We have previously developed a tissue culture method for sago palm, which is used to provide the planting materials and to develop a genetic transformation procedure. Here, we report the genetic transformation of sago embryonic callus derived from suspension culture using Agrobacterium tumefaciens and gene gun systems. The transformed embryoids cells were selected against Basta (concentration 10 to 30 mg/L). Evidence of foreign genes integration and function of the bar and gus genes were verified via gene specific PCR amplification, gus staining, and dot blot analysis. This study showed that the embryogenic callus was the most suitable material for transformation as compared to the fine callus, embryoid stage, and initiated shoots. The gene gun transformation showed higher transformation efficiency than the ones transformed using Agrobacterium when targets were bombarded once or twice using 280 psi of helium pressure at 6 to 8 cm distance. Evra Raunie Ibrahim, Md. Anowar Hossain, and Hairul Azman Roslan Copyright © 2014 Evra Raunie Ibrahim et al. All rights reserved. Economic Impact of NMMO Pretreatment on Ethanol and Biogas Production from Pinewood Sun, 07 Sep 2014 07:09:13 +0000 Processes for ethanol and biogas (scenario 1) and biomethane (scenario 2) production from pinewood improved by N-methylmorpholine-N-oxide (NMMO) pretreatment were developed and simulated by Aspen plus. These processes were compared with two processes using steam explosion instead of NMMO pretreatment ethanol (scenario 3) and biomethane (scenario 4) production, and the economies of all processes were evaluated by Aspen Process Economic Analyzer. Gasoline equivalent prices of the products including 25% value added tax (VAT) and selling and distribution expenses for scenarios 1 to 4 were, respectively, 1.40, 1.20, 1.24, and 1.04 /l, which are lower than gasoline price. The profitability indexes for scenarios 1 to 4 were 1.14, 0.93, 1.16, and 0.96, respectively. Despite the lower manufacturing costs of biomethane, the profitability indexes of these processes were lower than those of the bioethanol processes, because of higher capital requirements. The results showed that taxing rule is an effective parameter on the economy of the biofuels. The gasoline equivalent prices of the biofuels were 15–37% lower than gasoline; however, 37% of the gasoline price contributes to energy and carbon dioxide tax which are not included in the prices of biofuels based on the Swedish taxation rules. Marzieh Shafiei, Keikhosro Karimi, Hamid Zilouei, and Mohammad J. Taherzadeh Copyright © 2014 Marzieh Shafiei et al. All rights reserved. Fast Synthesis of Multilayer Carbon Nanotubes from Camphor Oil as an Energy Storage Material Tue, 02 Sep 2014 13:03:13 +0000 Among the wide range of renewable energy sources, the ever-increasing demand for electricity storage represents an emerging challenge. Utilizing carbon nanotubes (CNTs) for energy storage is closely being scrutinized due to the promising performance on top of their extraordinary features. In this work, well-aligned multilayer carbon nanotubes were successfully synthesized on a porous silicon (PSi) substrate in a fast process using renewable natural essential oil via chemical vapor deposition (CVD). Considering the influx of vaporized multilayer vertical carbon nanotubes (MVCNTs) to the PSi, the diameter distribution increased as the flow rate decreased in the reactor. Raman spectroscopy results indicated that the crystalline quality of the carbon nanotubes structure exhibits no major variation despite changes in the flow rate. Fourier transform infrared (FT-IR) spectra confirmed the hexagonal structure of the carbon nanotubes because of the presence of a peak corresponding to the carbon double bond. Field emission scanning electron microscopy (FESEM) images showed multilayer nanotubes, each with different diameters with long and straight multiwall tubes. Moreover, the temperature programmed desorption (TPD) method has been used to analyze the hydrogen storage properties of MVCNTs, which indicates that hydrogen adsorption sites exist on the synthesized multilayer CNTs. Amin TermehYousefi, Samira Bagheri, Kawasaki Shinji, Jalal Rouhi, Mohamad Rusop Mahmood, and Shoichiro Ikeda Copyright © 2014 Amin TermehYousefi et al. All rights reserved. L-Methionase: A Therapeutic Enzyme to Treat Malignancies Sun, 31 Aug 2014 10:04:30 +0000 Cancer is an increasing cause of mortality and morbidity throughout the world. L-methionase has potential application against many types of cancers. L-Methionase is an intracellular enzyme in bacterial species, an extracellular enzyme in fungi, and absent in mammals. L-Methionase producing bacterial strain(s) can be isolated by 5,5′-dithio-bis-(2-nitrobenzoic acid) as a screening dye. L-Methionine plays an important role in tumour cells. These cells become methionine dependent and eventually follow apoptosis due to methionine limitation in cancer cells. L-Methionine also plays an indispensable role in gene activation and inactivation due to hypermethylation and/or hypomethylation. Membrane transporters such as GLUT1 and ion channels like Na2+, Ca2+, K+, and Cl− become overexpressed. Further, the α-subunit of ATP synthase plays a role in cancer cells growth and development by providing them enhanced nutritional requirements. Currently, selenomethionine is also used as a prodrug in cancer therapy along with enzyme methionase that converts prodrug into active toxic chemical(s) that causes death of cancerous cells/tissue. More recently, fusion protein (FP) consisting of L-methionase linked to annexin-V has been used in cancer therapy. The fusion proteins have advantage that they have specificity only for cancer cells and do not harm the normal cells. Bhupender Sharma, Sukhdev Singh, and Shamsher S. Kanwar Copyright © 2014 Bhupender Sharma et al. All rights reserved. Erratum to “Quantification of Human and Animal Viruses to Differentiate the Origin of the Fecal Contamination Present in Environmental Samples” Thu, 28 Aug 2014 00:00:00 +0000 Sílvia Bofill-Mas, Marta Rusiñol, Xavier Fernandez-Cassi, Anna Carratalà, Ayalkibet Hundesa, and Rosina Girones Copyright © 2014 Sílvia Bofill-Mas et al. All rights reserved. Characterisation of Potential Antidiabetic-Related Proteins from Pleurotus pulmonarius (Fr.) Quél. (Grey Oyster Mushroom) by MALDI-TOF/TOF Mass Spectrometry Thu, 28 Aug 2014 00:00:00 +0000 Pleurotus pulmonarius has been reported to have a potent remedial effect on diabetic property and considered to be an alternative for type 2 diabetes mellitus treatment. This study aimed to investigate the antidiabetic properties of ammonium sulphate precipitated protein fractions from P. pulmonarius basidiocarps. Preliminary results demonstrated that 30% (NH4)2SO4 precipitated fraction (F30) inhibited Saccharomyces cerevisiae α-glucosidase activity (24.18%), and 100% (NH4)2SO4 precipitated fraction (F100) inhibited porcine pancreatic α-amylase activity (41.80%). Following RP-HPLC purification, peak 3 from F30 fraction demonstrated inhibition towards α-glucosidase at the same time with meagre inhibition towards α-amylase activity. Characterisation of proteins using MALDI-TOF/TOF MS demonstrated the presence of four different proteins, which could be implicated in the regulation of blood glucose level via various mechanisms. Therefore, this study revealed the presence of four antidiabetic-related proteins which are profilin-like protein, glyceraldehyde-3-phosphate dehydrogenase-like protein, trehalose phosphorylase-like (TP-like) protein, and catalase-like protein. Hence, P. pulmonarius basidiocarps have high potential in lowering blood glucose level, reducing insulin resistance and vascular complications. Nurul Azwa Abd. Wahab, Noorlidah Abdullah, and Norhaniza Aminudin Copyright © 2014 Nurul Azwa Abd. Wahab et al. All rights reserved. Efficient Micropropagation of Highly Economic, Medicinal and Ornamental Plant Lallemantia iberica (Bieb.) Fisch. and C. A. Mey Wed, 27 Aug 2014 06:37:30 +0000 Lallemantia iberica (Bieb.) Fisch. and C. A. Mey is high valued annual ornamental and medicinal plant from Lamiaceae family that prefers dry sunny hillsides, roadsides, slopes, and fallow fields over an altitude of 500–2150 m. It bears beautiful white flowers and bloom from April to June each year. This study reports L. iberica micropropagation using cotyledon node explants isolated from 15-day-old in vitro regenerated plantlets. The cotyledon node explants were cultured on MS medium containing 0.50, 1.00 plus 2.00 mg/L BAP, 0.00, 0.01, and 0.02 mg/L NAA. Maximum shoot regeneration was noted on MS medium containing 0.50 mg/L BAP. Well-developed micropropagated shoots were rooted on MS medium containing 1.00 mg/L IBA. The rooted plants were easily hardened in the growth chamber and acclimatised in greenhouse. Fethi Ahmet Ozdemir, Mehmet Ugur Yildirim, and Mahsa Pourali Kahriz Copyright © 2014 Fethi Ahmet Ozdemir et al. All rights reserved. Negative Regulation of GADD34 on Myofibroblasts during Cutaneous Wound Healing Tue, 19 Aug 2014 08:21:28 +0000 The growth arrest and DNA damage-inducible protein, GADD34, has been proved to be involved in TGF-β signaling pathway and correlates with cell death, which are two important mechanisms in regulating myofibroblast differentiation and apoptosis during tissue repair. But roles of GADD34 in myofibroblasts differentiation and apoptosis remain unknown. To investigate the function of GADD34 in these processes, we subjected WT and GADD34−/− mice to dermal wound healing. Here we show that GADD34−/− mice exhibited accelerated wound closure compared with WT mice. In addition, GADD34−/− mice showed increased number of myofibroblasts, elevated collagen production, and decreased cell apoptosis during wound healing. Moreover, we found that GADD34−/− mice showed increased phosphorylation of Smad3 and lower level of cleaved caspase-3. Thus these results indicate that GADD34 appears to suppress myofibroblast differentiation through inhibiting Smad3-dependent TGFβ signal pathway and promote its apoptosis by activating caspase-3 pathway. Lintao Liu, Naomi Nishio, Sachiko Ito, Yuriko Tanaka, and Ken-ichi Isobe Copyright © 2014 Lintao Liu et al. All rights reserved. Evaluation of Dried Sweet Sorghum Stalks as Raw Material for Methane Production Tue, 19 Aug 2014 06:07:02 +0000 The potential of utilizing dried sweet sorghum stalks as raw material for anaerobic digestion has been evaluated. Two different treatments were tested, a mild thermal and an enzymatic, alone or in combination. Thermal pretreatment was found to decrease the methane yields, whereas one-step enzymatic treatment resulted in a significant increase of 15.1% comparing to the untreated sweet sorghum. Subsequently, in order to increase the total methane production, the combined effect of enzyme load and I/S on methane yields from sweet sorghum was evaluated by employing response surface methodology. The obtained model showed that the maximum methane yield that could be achieved is 296 mL CH4/g VS at I/S ratio of 0.35 with the addition of 11.12 FPU/g sweet sorghum. Leonidas Matsakas, Ulrika Rova, and Paul Christakopoulos Copyright © 2014 Leonidas Matsakas et al. All rights reserved. The Treatment of PPCP-Containing Sewage in an Anoxic/Aerobic Reactor Coupled with a Novel Design of Solid Plain Graphite-Plates Microbial Fuel Cell Thu, 14 Aug 2014 09:03:47 +0000 Synthetic sewage containing high concentrations of pharmaceuticals and personal care products (PPCPs, mg/L level) was treated using an anoxic/aerobic (A/O) reactor coupled with a microbial fuel cell (MFC) at hydraulic retention time (HRT) of 8 h. A novel design of solid plain graphite plates (SPGRPs) was used for the high surface area biodegradation of the PPCP-containing sewage and for the generation of electricity. The average and total nitrogen removal efficiencies achieved were 97.20% and 83.75%, respectively. High removal efficiencies of pharmaceuticals, including acetaminophen, ibuprofen, and sulfamethoxazole, were also obtained and ranged from 98.21% to 99.89%. A maximum power density of 532.61 mW/cm2 and a maximum coulombic efficiency of 25.20% were measured for the SPGRP MFC at the anode. Distinct differences in the bacterial community were presented at various locations including the mixed liquor suspended solids and biofilms. The bacterial groups involved in PPCP biodegradation were identified as Dechloromonas spp., Sphingomonas sp., and Pseudomonas aeruginosa. This design, which couples an A/O reactor with a novel design of SPGRP MFC, allows the simultaneous removal of PPCPs and successful electricity production. Yi-Tang Chang, Chu-Wen Yang, Yu-Jie Chang, Ting-Chieh Chang, and Da-Jiun Wei Copyright © 2014 Yi-Tang Chang et al. All rights reserved. Phytomediated Biostimulation of the Autochthonous Bacterial Community for the Acceleration of the Depletion of Polycyclic Aromatic Hydrocarbons in Contaminated Sediments Thu, 07 Aug 2014 07:16:01 +0000 Polycyclic aromatic hydrocarbons (PAHs) are a large group of organic contaminants causing hazards to organisms including humans. The objective of the study was to validate the vegetation of dredged sediments with Phragmites australis as an exploitable biostimulation approach to accelerate the depletion of PAHs in nitrogen spiked sediments. Vegetation with Phragmites australis resulted in being an efficient biostimulation approach for the depletion of an aged PAHs contamination ( μg PAHs/g dry weight of sediment) in dredged sediments. Phragmites australis accelerated the oxidation of the PAHs by rhizodegradation. The phytobased approach resulted in 58.47% of PAHs depletion. The effects of the treatment have been analyzed in terms of both contaminant depletion and changes in relative abundance of the metabolically active Gram positive and Gram negative PAHs degraders. The metabolically active degraders were quantified both in the sediments and in the root endospheric microbial community. Quantitative real-time PCR reactions have been performed on the retrotranscribed transcripts encoding the Gram positive and Gram negative large subunit (RHD) of the aromatic ring hydroxylating dioxygenases. The Gram positive degraders resulted in being selectively favored by vegetation with Phragmites australis and mandatory for the depletion of the six ring condensed indeno[1,2,3-cd]pyrene and benzo[g,h,i]perylene. Simona Di Gregorio, Alessandro Gentini, Giovanna Siracusa, Simone Becarelli, Hassan Azaizeh, and Roberto Lorenzi Copyright © 2014 Simona Di Gregorio et al. All rights reserved. Enhanced Solid-State Biogas Production from Lignocellulosic Biomass by Organosolv Pretreatment Tue, 05 Aug 2014 09:37:10 +0000 Organosolv pretreatment was used to improve solid-state anaerobic digestion (SSAD) for methane production from three different lignocellulosic substrates (hardwood elm, softwood pine, and agricultural waste rice straw). Pretreatments were conducted at 150 and 180°C for 30 and 60 min using 75% ethanol solution as an organic solvent with addition of sulfuric acid as a catalyst. The statistical analyses showed that pretreatment temperature was the significant factor affecting methane production. Optimum temperature was 180°C for elmwood while it was 150°C for both pinewood and rice straw. Maximum methane production was 152.7, 93.7, and 71.4 liter per kg carbohydrates (CH), which showed up to 32, 73, and 84% enhancement for rice straw, elmwood, and pinewood, respectively, compared to those from the untreated substrates. An inverse relationship between the total methane yield and the lignin content of the substrates was observed. Kinetic analysis of the methane production showed that the process followed a first-order model for all untreated and pretreated lignocelluloses. Safoora Mirmohamadsadeghi, Keikhosro Karimi, Akram Zamani, Hamid Amiri, and Ilona Sárvári Horváth Copyright © 2014 Safoora Mirmohamadsadeghi et al. All rights reserved. Effects of Psychrophilic Storage on Manures as Substrate for Anaerobic Digestion Tue, 05 Aug 2014 07:53:28 +0000 The idea that storage can enhance manure quality as substrate for anaerobic digestion (AD) to recover more methane is evaluated by studying storage time and temperature effects on manure composition. Volatile fatty acids (VFA) and total dissolved organics (CODs) were measured in full scale pig manure storage for a year and in multiple flasks at fixed temperatures, mainly relevant for colder climates. The CODs generation, influenced by the source of the pig manure, was highest initially (0.3 g COD L−1d−1) gradually dropping for 3 months towards a level of COD loss by methane production at 15°C. Methane emission was low (<0.01 g COD L−1d−1) after a brief initial peak. Significant CODs generation was obtained during the warmer season (T > 10°C) in the full scale storage and almost no generation at lower temperatures (4–6°C). CODs consisted mainly of VFA, especially acetate. All VFAs were present at almost constant ratios. The naturally separated manure middle layer without sediment and coarser particles is suitable for sludge bed AD and improved further during an optimal storage time of 1–3 month(s). This implies that high rate AD can be integrated with regular manure slurry handling systems to obtain efficient biogas generation. Wenche Bergland, Carlos Dinamarca, and Rune Bakke Copyright © 2014 Wenche Bergland et al. All rights reserved. Enhanced Ethanol and Biogas Production from Pinewood by NMMO Pretreatment and Detailed Biomass Analysis Mon, 04 Aug 2014 08:30:36 +0000 N-Methyl morpholine-N-oxide (NMMO) is an environmentally friendly and commercially applied cellulose solvent that is suggested for pretreatment of lignocelluloses to improve biofuel productions. However, the underlying mechanisms of the improvements have been poorly understood yet. In an attempt to investigate the mechanisms, pinewood powder and chips were pretreated with 85% (w/w) NMMO at 120°C for 1–15 h. The pretreatment improved ethanol production yield from 7.2% (g/g) for the untreated wood powder to 68.1–86.1% (g/g) and from 1.7% (g/g) for the untreated wood chips to 12.6–51.2% (g/g) of theoretical yield. Similarly, the biogas yields of untreated wood chips and powder were improved from 21 and 66 (mL/g volatile solids) by 3.5–6.8- and 2.6–3.4-folds, respectively. SEM micrographs indicated major increase in the wood porosity by the pretreatment, which would confirm increase in the water swelling capacity as well as enzyme adsorption. The analysis of X-ray diffraction showed considerable reduction in the cellulose crystallinity by the pretreatment, while FTIR spectroscopy results indicated reduction of lignin on the wood surface by the pretreatment. Marzieh Shafiei, Keikhosro Karimi, Hamid Zilouei, and Mohammad J. Taherzadeh Copyright © 2014 Marzieh Shafiei et al. All rights reserved. Repeated Batch Fermentation Biotechnology for the Biosynthesis of Lipid and Gamma-Linolenic Acid by Cunninghamella bainieri 2A1 Thu, 24 Jul 2014 07:09:16 +0000 The biosynthesis of biomedical products including lipid and gamma-linolenic acid (GLA) by Cunninghamella bainieri 2A1 was studied in repeated batch fermentation. Three key process variables, namely, glucose concentration, ammonium tartrate concentration, and harvesting time, were optimized using response surface methodology. Repeated batch fermentation was carried out by the cultivation of Cunninghamella bainieri 2A1 in nitrogen-limited medium with various nitrogen concentration (1–4 g/L) and glucose concentration (20–40 g/L) at three time intervals (12 h, 24 h, and 48 h). Experimental results showed that the highest lipid concentration of 6.2 g/L and the highest GLA concentration of 0.4 g/L were obtained in optimum conditions, where 20.2 g/L glucose, 2.12 g/L ammonium tartrate, and 48 h harvesting time were utilized. Statistical results showed that the interaction between glucose and ammonium tartrate concentration had highly significant effects on lipid and GLA biosynthesis (). Moreover, harvesting time had a significant interaction effect with glucose and ammonium tartrate concentration on lipid production (). Marjan Ganjali Dashti, Peyman Abdeshahian, Wan Mohtar Wan Yusoff, Mohd Sahaid Kalil, and Aidil Abdul Hamid Copyright © 2014 Marjan Ganjali Dashti et al. All rights reserved. Evaluation of Cytotoxic and Antimicrobial Effects of Two Bt Cry Proteins on a GMO Safety Perspective Wed, 23 Jul 2014 00:00:00 +0000 Studies have contested the innocuousness of Bacillus thuringiensis (Bt) Cry proteins to mammalian cells as well as to mammals microbiota. Thus, this study aimed to evaluate the cytotoxic and antimicrobial effects of two Cry proteins, Cry8Ka5 (a novel mutant protein) and Cry1Ac (a widely distributed protein in GM crops). Evaluation of cyto- and genotoxicity in human lymphocytes was performed as well as hemolytic activity coupled with cellular membrane topography analysis in mammal erythrocytes. Effects of Cry8Ka5 and Cry1Ac upon Artemia sp. nauplii and upon bacteria and yeast growth were assessed. The toxins caused no significant effects on the viability ( µg/mL) or to the cellular DNA integrity of lymphocytes (no effects at 1,000 µg/mL). The Cry8Ka5 and Cry1Ac proteins did not cause severe damage to erythrocytes, neither with hemolysis ( µg/mL) nor with alterations in the membrane. Likewise, the Cry8Ka5 and Cry1Ac proteins presented high LC50 (755.11 and >1,000 µg/mL, resp.) on the brine shrimp lethality assay and showed no growth inhibition of the microorganisms tested ( µg/mL). This study contributed with valuable information on the effects of Cry8Ka5 and Cry1Ac proteins on nontarget organisms, which reinforce their potential for safe biotechnological applications. Davi Felipe Farias, Martônio Ponte Viana, Gustavo Ramos de Oliveira, Magda Aparecida Beneventi, Bruno Marques Soares, Claudia Pessoa, Igor Parra Pessoa, Luciano Paulino Silva, Ilka Maria Vasconcelos, Maria Fátima Grossi de Sá, and Ana Fontenele Urano Carvalho Copyright © 2014 Davi Felipe Farias et al. All rights reserved. Efficient Expression of Acetylcholine-Binding Protein from Aplysia californica in Bac-to-Bac System Sun, 20 Jul 2014 11:55:34 +0000 The Bac-to-Bac baculovirus expression system can efficiently produce recombinant proteins, but the system may have to be optimized to achieve high-level expression for different candidate proteins. We reported here the efficient expression of acetylcholine-binding proteins from sea hares Aplysia californica (Ac-AChBP) and a convenient method to monitor protein expression level in this expression system. Three key factors affecting expression of Ac-AChBP were optimized for maximizing the yield, which included the cell density, volume of the infecting baculovirus inoculums, and the culturing time of postinfection. We have found it to reach a high yield of ∼5 mg/L, which needs 55 h incubation after infection at the cell density of 2 × 106 cells/mL with an inoculum volume ratio of 1 : 100. The optimized expression system in this study was also applied for expressing another protein Ls-AChBP from Lymnaea stagnalis successfully. Therefore, this established method is helpful to produce high yields of AChBP proteins for X-ray crystallographic structural and functional studies. Bo Lin, Hailing Meng, Hui Bing, Dongting Zhangsun, and Sulan Luo Copyright © 2014 Bo Lin et al. All rights reserved. Fermentative Polyhydroxybutyrate Production from a Novel Feedstock Derived from Bakery Waste Sun, 20 Jul 2014 08:55:23 +0000 In this study, Halomonas boliviensis was cultivated on bakery waste hydrolysate and seawater in batch and fed-batch cultures for polyhydroxybutyrate (PHB) production. Results demonstrated that bakery waste hydrolysate and seawater could be efficiently utilized by Halomonas boliviensis while PHB contents between 10 and 30% (w/w) were obtained. Furthermore, three methods for bakery waste hydrolysis were investigated for feedstock preparation. These include: (1) use of crude enzyme extracts from Aspergillus awamori, (2) Aspergillus awamori solid mashes, and (3) commercial glucoamylase. In the first method, the resultant free amino nitrogen (FAN) concentration in hydrolysates was 150 and 250 mg L−1 after 20 hours at enzyme-to-solid ratios of 6.9 and 13.1 U g−1, respectively. In both cases, the final glucose concentration was around 130–150 g L−1. In the second method, the resultant FAN and glucose concentrations were 250 mg L−1 and 150 g L−1, respectively. In the third method, highest glucose and lowest FAN concentrations of 170–200 g L−1 and 100 mg L−1, respectively, were obtained in hydrolysates after only 5 hours. The present work has generated promising information contributing to the sustainable production of bioplastic using bakery waste hydrolysate. Daniel Pleissner, Wan Chi Lam, Wei Han, Kin Yan Lau, Lai Chun Cheung, Ming Wui Lee, Ho Man Lei, Kin Yu Lo, Wai Yee Ng, Zheng Sun, Mehmet Melikoglu, and Carol Sze Ki Lin Copyright © 2014 Daniel Pleissner et al. All rights reserved. High Potential Source for Biomass Degradation Enzyme Discovery and Environmental Aspects Revealed through Metagenomics of Indian Buffalo Rumen Thu, 17 Jul 2014 09:40:57 +0000 The complex microbiomes of the rumen functions as an effective system for plant cell wall degradation, and biomass utilization provide genetic resource for degrading microbial enzymes that could be used in the production of biofuel. Therefore the buffalo rumen microbiota was surveyed using shot gun sequencing. This metagenomic sequencing generated 3.9 GB of sequences and data were assembled into 137270 contiguous sequences (contigs). We identified potential 2614 contigs encoding biomass degrading enzymes including glycoside hydrolases (GH: 1943 contigs), carbohydrate binding module (CBM: 23 contigs), glycosyl transferase (GT: 373 contigs), carbohydrate esterases (CE: 259 contigs), and polysaccharide lyases (PE: 16 contigs). The hierarchical clustering of buffalo metagenomes demonstrated the similarities and dissimilarity in microbial community structures and functional capacity. This demonstrates that buffalo rumen microbiome was considerably enriched in functional genes involved in polysaccharide degradation with great prospects to obtain new molecules that may be applied in the biofuel industry. K. M. Singh, Bhaskar Reddy, Dishita Patel, A. K. Patel, Nidhi Parmar, Anand Patel, J. B. Patel, and C. G. Joshi Copyright © 2014 K. M. Singh et al. All rights reserved. Methylamine-Sensitive Amperometric Biosensor Based on (His)6-Tagged Hansenula polymorpha Methylamine Oxidase Immobilized on the Gold Nanoparticles Wed, 16 Jul 2014 11:49:00 +0000 A novel methylamine-selective amperometric bienzyme biosensor based on recombinant primary amine oxidase isolated from the recombinant yeast strain Saccharomyces cerevisiae and commercial horseradish peroxidase is described. Two amine oxidase preparations were used: free enzyme (AMO) and covalently immobilized on the surface of gold nanoparticles (AMO-nAu). Some bioanalytical parameters (sensitivity, selectivity, and storage stability) of the developed biosensors were investigated. The sensitivity for both sensors is high: and  A−1·M−1·m−2 for AMO-nAu biosensor, respectively. The biosensors exhibit the linear range from 15 μM to 150 μM (AMO-nAu) and from 15 μM to 60 μM (AMO). The developed biosensor demonstrated a good selectivity toward methylamine (MA) (signal for dimethylamine and trimethylamine is less than 5% and for ethylamine 15% compared to MA output) and reveals a satisfactory storage stability. The constructed amperometric biosensor was used for MA assay in real samples of fish products in comparison with chemical method. The values obtained with both approaches different methods demonstrated a high correlation. Nataliya Ye. Stasyuk, Oleh V. Smutok, Andriy E. Zakalskiy, Oksana M. Zakalska, and Mykhailo V. Gonchar Copyright © 2014 Nataliya Ye. Stasyuk et al. All rights reserved. Biotemplated Synthesis of Anatase Titanium Dioxide Nanoparticles via Lignocellulosic Waste Material Tue, 15 Jul 2014 00:00:00 +0000 Anatase titanium dioxide nanoparticles (TiO2-NPs) were synthesized by sol-gel method using rice straw as a soft biotemplate. Rice straw, as a lignocellulosic waste material, is a biomass feedstock which is globally produced in high rate and could be utilized in an innovative approach to manufacture a value-added product. Rice straw as a reliable biotemplate has been used in the sol-gel method to synthesize ultrasmall sizes of TiO2-NPs with high potential application in photocatalysis. The physicochemical properties of titanium dioxide nanoparticles were investigated by a number of techniques such as X-ray diffraction analysis (XRD), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), Raman spectroscopy, thermogravimetric analysis (TGA), ultraviolet visible spectra (UV-Vis), and surface area and pore size analysis. All results consensually confirmed that particle sizes of synthesized titanium dioxide were template-dependent, representing decrease in the nanoparticles sizes with increase of biotemplate concentration. Titanium dioxide nanoparticles as small as 13.0 ± 3.3 nm were obtained under our experimental conditions. Additionally, surface area and porosity of synthesized TiO2-NPs have been enhanced by increasing rice straw amount which results in surface modification of nanoparticles and potential application in photocatalysis. Donya Ramimoghadam, Samira Bagheri, and Sharifah Bee Abd Hamid Copyright © 2014 Donya Ramimoghadam et al. All rights reserved. Amino-Functionalization of Carbon Nanotubes by Using a Factorial Design: Human Cardiac Troponin T Immunosensing Application Sun, 13 Jul 2014 08:01:04 +0000 A simple amino-functionalization method for carbon nanotubes and its application in an electrochemical immunosensor for detection of the human cardiac troponin T are described. Amino-functionalized carbon nanotubes allow oriented antibodies immobilization via their Fc regions, improving the performance of an immunosensor. Herein multiwalled carbon nanotubes were amino-functionalized by using the ethylenediamine reagent and assays were designed by fractional factorial study associated with Doehlert matrix. Structural modifications in the carbon nanotubes were confirmed by Fourier transform infrared spectroscopy. After amino-functionalization the carbon nanotubes were attached to screen-printed carbon electrode and a sandwich-type immunoassay was performed for measuring the cardiac troponin T. The electrochemical measurements were obtained through hydrogen peroxide reaction with peroxidase conjugated to the secondary antibody. Under optimal conditions, troponin T immunosensor was evaluated in serum samples, which showed a broad linear range (0.02 to 0.32 ng mL−1) and a low limit of detection, 0.016 ng mL−1. This amino platform can be properly used as clinical tool for cardiac troponin T detection in the acute myocardial infarction diagnosis. Tatianny A. Freitas, Alessandra B. Mattos, Bárbara V. M. Silva, and Rosa F. Dutra Copyright © 2014 Tatianny A. Freitas et al. All rights reserved. Antioxidant Properties of Mushroom Mycelia Obtained by Batch Cultivation and Tocopherol Content Affected by Extraction Procedures Thu, 10 Jul 2014 08:42:36 +0000 The determination of the antioxidant potential of lyophilized mushroom mycelia from 5 strains of the species Pleurotus ostreatus and Coprinus comatus (obtained by submerged cultivation in batch system) was analyzed as ethanolic extracts by evaluating ABTS and the hydroxyl scavenging activity, FRAP method, the chelating capacity, the inhibition of human erythrocyte hemolysis, and the inhibition of xanthine oxidase activity. The main compounds present in all extracts were determined by HPLC chromatography. Overall, results demonstrated that the biologically active substances content is modulated by the extraction method used. The most beneficial extract, characterized by determining the EC50 value, was that of C. comatus M8102, followed by P. ostreatus PQMZ91109. Significant amount of α-tocopherol (179.51 ± 1.51 mg/100 g extract) was determined as well as flavones such as rutin and apigenin. In the P. ostreatus PQMZ91109 extract, 4.8 ± 0.05 mg/100 g extract of tocopherol acetate known to play a significant role as an antioxidant in skin protection against oxidative stress generated by UV rays was determined. The various correlations (–0.9426 for tocopherol content) assessed and the composition of extracts in fluidized bed from the mycelia of the tested species depicted a significant pharmacological potential as well as the possibility of usage in the development of new functional products. Emanuel Vamanu Copyright © 2014 Emanuel Vamanu. All rights reserved. Expression Profiling of Abiotic Stress-Inducible Genes in response to Multiple Stresses in Rice (Oryza sativa L.) Varieties with Contrasting Level of Stress Tolerance Mon, 07 Jul 2014 07:44:14 +0000 The present study considered transcriptional profiles and protein expression analyses from shoot and/or root tissues under three abiotic stress conditions, namely, salinity, dehydration, and cold, as well as following exogenous abscisic acid treatment, at different time points of stress exposure in three indica rice varieties, IR-29 (salt sensitive), Pokkali, and Nonabokra (both salt tolerant). The candidate genes chosen for expression studies were HKT-1, SOS-3, NHX-1, SAPK5, SAPK7, NAC-1, Rab16A, OSBZ8, DREBP2, CRT/DREBP, WRKY24, and WRKY71, along with the candidate proteins OSBZ8, SAMDC, and GST. Gene expression profile revealed considerable differences between the salt-sensitive and salt-tolerant rice varieties, as the expression in the latter was higher even at the constitutive level, whereas it was inducible only by corresponding stress signals in IR-29. Whether in roots or shoots, the transcriptional responses to different stressors peaked following 24 h of stress/ABA exposure, and the transcript levels enhanced gradually with the period of exposure. The generality of stress responses at the transcriptional level was therefore time dependent. Heat map data also showed differential transcript abundance in the three varieties, correlating the observation with transcript profiling. In silico analysis of the upstream regions of all the genes represented the existence of conserved sequence motifs in single or multiple copies that are indispensable to abiotic stress response. Overall, the transcriptome and proteome analysis undertaken in the present study indicated that genes/proteins conferring tolerance, belonging to different functional classes, were overrepresented, thus providing novel insight into the functional basis of multiple stress tolerance in indica rice varieties. The present work will pave the way in future to select gene(s) for overexpression, so as to generate broad spectrum resistance to multiple stresses simultaneously. Supratim Basu and Aryadeep Roychoudhury Copyright © 2014 Supratim Basu and Aryadeep Roychoudhury. All rights reserved. Assessment of Food Processing and Pharmaceutical Industrial Wastes as Potential Biosorbents: A Review Mon, 07 Jul 2014 00:00:00 +0000 There is a growing need for the use of low-cost and ecofriendly adsorbents in water/wastewater treatment applications. Conventional adsorbents as well as biosorbents from different natural and agricultural sources have been extensively studied and reviewed. However, there is a lack of reviews on biosorption utilizing industrial wastes, particularly those of food processing and pharmaceuticals. The current review evaluates the potential of these wastes as biosorbents for the removal of some hazardous contaminants. Sources and applications of these biosorbents are presented, while factors affecting biosorption are discussed. Equilibrium, kinetics, and mechanisms of biosorption are also reviewed. In spite of the wide spread application of these biosorbents in the treatment of heavy metals and dyes, more research is required on other classes of pollutants. In addition, further work should be dedicated to studying scaling up of the process and its economic feasibility. More attention should also be given to enhancing mechanical strength, stability, life time, and reproducibility of the biosorbent. Environmental concerns regarding disposal of consumed biosorbents should be addressed by offering feasible biosorbent regeneration or pollutant immobilization options. Hanan E. M. El-Sayed and Mayyada M. H. El-Sayed Copyright © 2014 Hanan E. M. El-Sayed and Mayyada M. H. El-Sayed. All rights reserved. Potential Use of Halophytes to Remediate Saline Soils Sun, 06 Jul 2014 08:14:27 +0000 Salinity is one of the rising problems causing tremendous yield losses in many regions of the world especially in arid and semiarid regions. To maximize crop productivity, these areas should be brought under utilization where there are options for removing salinity or using the salt-tolerant crops. Use of salt-tolerant crops does not remove the salt and hence halophytes that have capacity to accumulate and exclude the salt can be an effective way. Methods for salt removal include agronomic practices or phytoremediation. The first is cost- and labor-intensive and needs some developmental strategies for implication; on the contrary, the phytoremediation by halophyte is more suitable as it can be executed very easily without those problems. Several halophyte species including grasses, shrubs, and trees can remove the salt from different kinds of salt-affected problematic soils through salt excluding, excreting, or accumulating by their morphological, anatomical, physiological adaptation in their organelle level and cellular level. Exploiting halophytes for reducing salinity can be good sources for meeting the basic needs of people in salt-affected areas as well. This review focuses on the special adaptive features of halophytic plants under saline condition and the possible ways to utilize these plants to remediate salinity. Mirza Hasanuzzaman, Kamrun Nahar, Md. Mahabub Alam, Prasanta C. Bhowmik, Md. Amzad Hossain, Motior M. Rahman, Majeti Narasimha Vara Prasad, Munir Ozturk, and Masayuki Fujita Copyright © 2014 Mirza Hasanuzzaman et al. All rights reserved. Comparison of Behaviour in Different Liquids and in Cells of Gold Nanorods and Spherical Nanoparticles Modified by Linear Polyethyleneimine and Bovine Serum Albumin Tue, 01 Jul 2014 11:36:49 +0000 Gold nanorods (GNRs) are considered one of the most promising forms of nanoparticles for nanobiotechnology; however, the problem of their toxicity is currently not resolved. We synthesised GNRs, modified with linear polyethyleneimine (PEI-GNRs), and examined their physicochemical and some biological properties in comparison with GNRs modified with BSA and spherical gold nanoparticles (sGNPs) modified with the same agents. The influence of the buffer, cell culture media, and serum on hydrodynamic diameter and zeta potential of all GNPs was studied. Simultaneously, the size, shape, and formation of a corona were examined by transmission electron microscopy (TEM). PEI-GNRs and GNPs were nontoxic for BHK-21 and HeLa cells (MTT test). Penetration of all GNPs into BHK-21, melanoma B16, and HeLa cells was examined after 30 min, 3 h, and 24 h of incubation using TEM ultrathin sections. PEI-GNRs and PEI-sGNPs demonstrated fast and active penetration into cells by caveolin-dependent and lipid raft-mediated endocytosis and accumulated in endosomes and lysosomes. BSA-modified GNPs showed prolonged flotation and a significant delay in cell penetration. The results show that the charge of initial NPs determines penetration into cells. Thus, the designed PEI-GNRs were nontoxic and stable in cell culture media and could efficiently penetrate cells. Inna A. Pyshnaya, Kristina V. Razum, Julia E. Poletaeva, Dmitrii V. Pyshnyi, Marina A. Zenkova, and Elena I. Ryabchikova Copyright © 2014 Inna A. Pyshnaya et al. All rights reserved. Antimicrobial Effect of the Triterpene 3β,6β,16β-Trihydroxylup-20(29)-ene on Planktonic Cells and Biofilms from Gram Positive and Gram Negative Bacteria Sun, 29 Jun 2014 10:00:36 +0000 This study evaluated the antimicrobial effect of 3β,6β,16β-trihydroxylup-20(29)-ene (CLF1), a triterpene isolated from Combretum leprosum Mart., in inhibiting the planktonic growth and biofilms of Gram positive bacteria Streptococcus mutans and S. mitis. The antimicrobial activity was assessed by determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The antibiofilm potential was determined by quantifying total biomass and enumerating biofilm-entrapped viable bacteria. In addition, the acute toxicity of CLF1 on Artemia sp. nauplii was also determined. The results showed that CLF1 was able in inhibiting the growth of S. mutans and S. mitis with MIC and MBC of 7.8 μg/mL and 15.6 μg/mL, respectively. CLF1 was highly effective on biofilms of both bacteria. Only 7.8 μg/mL CLF1 was enough to inhibit by 97% and 90% biomass production of S. mutans and S. mitis, respectively. On the other hand, such effects were not evident on Gram negative Pseudomonas aeruginosa and Klebsiella oxytoca. The toxicity tests showed that the LC50 of CLF1 was 98.19 μg/mL. Therefore, CLF1 isolated from C. leprosum may constitute an important natural agent for the development of new therapies for caries and other infectious diseases caused by S. mutans and S. mitis. Francisco Flávio Vasconcelos Evaristo, Maria Rose Jane R. Albuquerque, Hélcio Silva dos Santos, Paulo Nogueira Bandeira, Fábio do Nascimento Ávila, Bruno Rocha da Silva, Ariana Azevedo Vasconcelos, Érica de Menezes Rabelo, Luiz Gonzaga Nascimento-Neto, Francisco Vassiliepe Sousa Arruda, Mayron Alves Vasconcelos, Victor Alves Carneiro, Benildo Sousa Cavada, and Edson Holanda Teixeira Copyright © 2014 Francisco Flávio Vasconcelos Evaristo et al. All rights reserved. Mannosylated Chitosan Nanoparticles for Delivery of Antisense Oligonucleotides for Macrophage Targeting Thu, 26 Jun 2014 13:19:15 +0000 The therapeutic potential of antisense oligonucleotides (ASODN) is primarily dependent upon its safe and efficient delivery to specific cells overcoming degradation and maximizing cellular uptake in vivo. The present study focuses on designing mannosylated low molecular weight (LMW) chitosan nanoconstructs for safe ODNs delivery by macrophage targeting. Mannose groups were coupled with LMW chitosan and characterized spectroscopically. Mannosylated chitosan ODN nanoparticles (MCHODN NPs) were formulated by self-assembled method using various ratio (moles of amine groups of MCH to phosphate moieties of ODNs) and characterized for gel retardation assay, physicochemical characteristics, cytotoxicity and transfection efficiency, and antisense assay. Complete complexation of MCH/ODN was achieved at charge ratio of 1:1 and above. On increasing the ratio of MCH/ODN, particle size of the NPs decreased whereas zeta potential (ZV) increased. MCHODN NPs displayed much higher transfection efficiency into Raw 264.7 cells (bears mannose receptors) than Hela cells and no significant toxicity was observed at all MCH concentrations. Antisense assay revealed that reduction in lipopolysaccharide (LPS) induced serum TNF- is due to antisense activity of TJU-2755 ODN (sequence complementary to 3′-UTR of TNF-). These results suggest that MCHODN NPs are acceptable choice to improve transfection efficiency in vitro and in vivo. Gyati Shilakari Asthana, Abhay Asthana, Dharm Veer Kohli, and Suresh Prasad Vyas Copyright © 2014 Gyati Shilakari Asthana et al. All rights reserved. Phytochemicals from Kaempferia angustifolia Rosc. and Their Cytotoxic and Antimicrobial Activities Wed, 25 Jun 2014 11:00:34 +0000 Phytochemical investigation on rhizomes of Kaempferia angustifolia has afforded a new abietene diterpene, kaempfolienol (1) along with crotepoxide (2), boesenboxide (3), 2′-hydroxy-4,4′,6′-trimethoxychalcone (4), zeylenol (5), 6-methylzeylenol (6), (24S)-24-methyl-5-lanosta-9(11), 25-dien-3-ol (7), sucrose, -sitosterol, and its glycoside (8). The structures of the compounds were elucidated on the basis of spectroscopic methods (IR, MS, and NMR). Isolation of 6-methylzeylenol (6), (24S)-24-methyl-5-lanosta-9(11), 25-dien-3-ol (7), and -sitosterol-3-O--D-glucopyranoside (8) from this plant species has never been reported previously. The spectroscopic data of (7) is firstly described in this paper. Cytotoxic screening indicated that most of the pure compounds tested showed significant activity with (4) showing the most potent activity against HL-60 (human promyelocytic leukemia) and MCF-7 (human breast cancer) cell lines. However, all extracts and most of the pure compounds tested were found to be inactive against HT-29 (human colon cancer) and HeLa (human cervical cancer) cell lines. Similarly, none of the extracts or compounds showed activity in the antimicrobial testing. Sook Wah Tang, Mohd Aspollah Sukari, Bee Keat Neoh, Yunie Soon Yu Yeap, Ahmad Bustamam Abdul, Nurolaini Kifli, and Gwendoline Cheng Lian Ee Copyright © 2014 Sook Wah Tang et al. All rights reserved. Oil Palm Frond Juice as Future Fermentation Substrate: A Feasibility Study Mon, 23 Jun 2014 11:25:31 +0000 Oil palm frond (OPF) juice is a potential industrial fermentation substrate as it has high sugars content and the OPF are readily available daily. However, maximum sugars yield and storage stability of the OPF juice are yet to be determined. This study was conducted to determine the effect of physical pretreatment and storage duration of OPF petiole on sugars yield. Storage stability of OPF juice at different storing conditions was also investigated. It was found that OPF petiole squeezed by hydraulic pressing machine gave the highest sugars recovery at almost 40 g/kg, accounting for a recovery yield of 88%. Storage of OPF petiole up to 72 hrs prior to squeezing reduced the free sugars by 11 g/kg. Concentrated OPF juice with 95% water removal had the best storage stability at both 4 and , when it was stored for 10 days. Moreover, concentrated OPF syrup prepared by thermal processing did not give any Maillard effect on microbial growth. Based on our results, OPF juice meets all the criteria as a good fermentation substrate as it is renewable, consistently available, and easy to be obtained, it does not inhibit microbial growth and product formation, and it contains no impurities. Che Mohd Hakiman Che Maail, Hidayah Ariffin, Mohd Ali Hassan, Umi Kalsom Md Shah, and Yoshihito Shirai Copyright © 2014 Che Mohd Hakiman Che Maail et al. All rights reserved. Development and Characterization of Polyphenon 60 and Caffeine Microemulsion for Enhanced Antibacterial Activity Sun, 22 Jun 2014 12:21:50 +0000 Green tea catechins and caffeine have exhibited antibacterial activity; however, their use is limited by lack of stability and effective delivery systems. Polyphenon 60 (P60) and caffeine were encapsulated in a single microemulsion (ME) formulation with an objective to lower the minimum inhibitory concentrations (MICs) of the individual agents against selected pathogens (S. epidermidis and E. coli). Combination of two natural compounds would advocate two different mechanisms on the bacterial growth thereby providing for better antibacterial activity. Thermodynamically stable ME was developed and characterized with an average particle size of 17.58 nm, further confirmed by TEM analysis. Antibacterial studies included chequerboard microdilution assay to determine the MIC and fractional inhibitory concentration (FIC) of both the natural compounds individually and in combination. MIC and FIC results indicated that the combination of the above two natural compounds was proficient in lowering the MICs of individual agents. Results of DPPH assay indicated that ME system preserved the long term antioxidative potential of P60 and caffeine. The cytotoxicity of the optimized formulation on Vero cell line by MTT assay was found to be nontoxic to mammalian cells. Sonal Gupta, Rakhi Bansal, Javed Ali, Reema Gabrani, and Shweta Dang Copyright © 2014 Sonal Gupta et al. All rights reserved. Evaluation of Antibacterial, Antifungal, and Antioxidant Activities of Safflower Natural Dyes during Flowering Sun, 22 Jun 2014 06:23:21 +0000 Two Carthamus tinctorius varieties (Jawhara and 104) were studied in order to investigate their natural dyes contents and biological activities. Obtained results showed that quinochalcone contents and antioxidant activities varied considerably as function of flowering stages. So flowers at fructification stage contained the highest carthamin content with the strongest antioxidant capacity with all assays (FRAP, DPPH, and chelating power methods). In parallel, we showed a decrease in the content of precarthamin. The quantitative variation of these molecules could be due to colour change of C. tinctorius flowers. Correlation analysis indicated that the ABTS method showed the highest correlation coefficients with carthamin and precarthamin contents, that is, 0.886 and 0.973, respectively. Concerning the regional effect, the contents of precarthamin and carthamin varied significantly at studied regions with the optimum production given by samples of Beja (902.41 μg/g DW and 42.05 μg/g DW, respectively, at flowering stage). During flowering, the antimicrobial activity of these two natural dyes increased where the maximum inhibitory effect mentioned with carthamin mainly against E. coli (iz = 25.89 mm) at fructification stage. Therefore, the increased frequency of resistance to commonly used antibiotics leads to the search for new effective natural drugs at food and pharmaceutical industries. Nidhal Salem, Kamel Msaada, Salem Elkahoui, Giuseppe Mangano, Sana Azaeiz, Imen Ben Slimen, Sarra Kefi, Giorgio Pintore, Ferid Limam, and Brahim Marzouk Copyright © 2014 Nidhal Salem et al. All rights reserved. A Rat Model of Thrombosis in Common Carotid Artery Induced by Implantable Wireless Light-Emitting Diode Device Thu, 19 Jun 2014 12:50:15 +0000 This work has developed a novel approach to form common carotid artery (CCA) thrombus in rats with a wireless implantable light-emitting diode (LED) device. The device mainly consists of an external controller and an internal LED assembly. The controller was responsible for wirelessly transmitting electrical power. The internal LED assembly served as an implant to receive the power and irradiate light on CCA. The thrombus formation was identified with animal sonography, 7T magnetic resonance imaging, and histopathologic examination. The present study showed that a LED assembly implanted on the outer surface of CCA could induce acute occlusion with single irradiation with 6 mW/cm2 LED for 4 h. If intermittent irradiation with 4.3–4.5 mW/cm2 LED for 2 h was shut off for 30 min, then irradiation for another 2 h was applied; the thrombus was observed to grow gradually and was totally occluded at 7 days. Compared with the contralateral CCA without LED irradiation, the arterial endothelium in the LED-irradiated artery was discontinued. Our study has shown that, by adjusting the duration of irradiation and the power intensity of LED, it is possible to produce acute occlusion and progressive thrombosis, which can be used as an animal model for antithrombotic drug development. Jih-Chao Yeh, Kuo-Lun Huang, Yung-Chin Hsiao, Yu-Han Hsu, Yun-Han Lin, Shyh-Liang Lou, and Tsong-Hai Lee Copyright © 2014 Jih-Chao Yeh et al. All rights reserved. Plant-Derived Antimicrobials Reduce E. coli O157:H7 Virulence Factors Critical for Colonization in Cattle Gastrointestinal Tract In Vitro Thu, 19 Jun 2014 11:39:45 +0000 This study investigated the effect of subinhibitory concentrations (SIC) of five plant-derived antimicrobials (PDAs), namely, trans cinnamaldehyde, eugenol, carvacrol, thymol, and β-resorcylic acid, on E. coli O157:H7 (EHEC) attachment and invasion of cultured bovine colonic (CO) and rectoanal junction (RAJ) epithelial cells. In addition, PDAs’ effect on EHEC genes critical for colonization of cattle gastrointestinal tract (CGIT) was determined in bovine rumen fluid (RF) and intestinal contents (BICs). Primary bovine CO and RAJ epithelial cells were established and were separately inoculated with three EHEC strains with or without (control) SIC of each PDA. Following incubation, EHEC that attached and invaded the cells were determined. Furthermore, the expression of EHEC genes critical for colonization in cattle was investigated using real-time, quantitative polymerase chain reaction in RF and BICs. All the PDAs decreased EHEC invasion of CO and RAJ epithelial cells (). The PDAs also downregulated () the expression of EHEC genes critical for colonization in CGIT. Results suggest that the PDAs could potentially be used to control EHEC colonization in cattle; however follow-up in vivo studies in cattle are warranted. Sangeetha Ananda Baskaran and Kumar Venkitanarayanan Copyright © 2014 Sangeetha Ananda Baskaran and Kumar Venkitanarayanan. All rights reserved. Isolation, Screening, and Identification of Cellulolytic Bacteria from Natural Reserves in the Subtropical Region of China and Optimization of Cellulase Production by Paenibacillus terrae ME27-1 Thu, 19 Jun 2014 07:02:30 +0000 From different natural reserves in the subtropical region of China, a total of 245 aerobic bacterial strains were isolated on agar plates containing sugarcane bagasse pulp as the sole carbon source. Of the 245 strains, 22 showed hydrolyzing zones on agar plates containing carboxymethyl cellulose after Congo-red staining. Molecular identification showed that the 22 strains belonged to 10 different genera, with the Burkholderia genus exhibiting the highest strain diversity and accounting for 36.36% of all the 22 strains. Three isolates among the 22 strains showed higher carboxymethyl cellulase (CMCase) activity, and isolate ME27-1 exhibited the highest CMCase activity in liquid culture. The strain ME27-1 was identified as Paenibacillus terrae on the basis of 16S rRNA gene sequence analysis as well as physiological and biochemical properties. The optimum pH and temperature for CMCase activity produced by the strain ME27-1 were 5.5 and 50°C, respectively, and the enzyme was stable at a wide pH range of 5.0–9.5. A 12-fold improvement in the CMCase activity (2.08 U/mL) of ME27-1 was obtained under optimal conditions for CMCase production. Thus, this study provided further information about the diversity of cellulose-degrading bacteria in the subtropical region of China and found P. terrae ME27-1 to be highly cellulolytic. Yan-Ling Liang, Zheng Zhang, Min Wu, Yuan Wu, and Jia-Xun Feng Copyright © 2014 Yan-Ling Liang et al. All rights reserved. Combination of the Auxins NAA, IBA, and IAA with GA3 Improves the Commercial Seed-Tuber Production of Potato (Solanum tuberosum L.) under In Vitro Conditions Tue, 17 Jun 2014 10:39:00 +0000 The study compared the effects of medium containing various concentrations of α-naphthaleneacetic acid (NAA), indole-3-acetic acid (IAA), and indole-3-butyric acid (IBA), alone or in combination with gibberellic acid (GA3) in micropropagation of three potato (Solanum tuberosum L.) cultivars Pasinler, Granola, and Caspar using binodal stem cuttings. The results testified improved regeneration on medium containing variants of NAA, IAA, and IBA plus GA3 on all cultivars. The minimum days to shoot induction on three cultivars ranged 4.25–5 d on medium containing 0.25 mg L−1   mg L−1 NAA. The longest shoots (11.8 cm), maximum number of nodes (13.50), and maximum number of leaves (11.00) were recorded on cv. Caspar on medium containing 1 mg L−1   mg L−1 GA3. The minimum time to root induction (12.25 d) was noted on cv. Pasinler on the same medium. All of the regenerated shoots could be easily rooted. The results showed that the combined effect of various concentrations of NAA, IAA, and IBA plus GA3 was more pronounced compared to the auxins used alone. The results of this research are of significant importance for potato breeders. Ahmet Metin Kumlay Copyright © 2014 Ahmet Metin Kumlay. All rights reserved. Antibacterial Activity of Leaf Extracts of Baeckea frutescens against Methicillin-Resistant Staphylococcus aureus Mon, 16 Jun 2014 08:32:56 +0000 This study was based on screening antibacterial activity of the ethanol extract of Baeckea frutescens L. against MRSA clinical isolates, analyzes the potential antibacterial compound, and assesses the cytotoxicity effect of the extract in tissue culture. Leaves of Baeckea frutescens L. were shade dried, powdered, and extracted using solvent ethanol. Preliminary phytochemical screening of the crude extracts revealed the presence of alkaloids, flavonoids, steroids, terpenoids, phenols, and carbohydrates. The presence of these bioactive constituents is related to the antibacterial activity of the plant. Disc diffusion method revealed a high degree of activity against microorganisms. The results confirm that Baeckea frutescens L. can be used as a source of drugs to fight infections caused by susceptible bacteria. Somayeh Razmavar, Mahmood Ameen Abdulla, Salmah Binti Ismail, and Pouya Hassandarvish Copyright © 2014 Somayeh Razmavar et al. All rights reserved. Acetylcholinesterase Inhibition by Biofumigant (Coumaran) from Leaves of Lantana camara in Stored Grain and Household Insect Pests Sun, 15 Jun 2014 09:33:25 +0000 Recent studies proved that the biofumigants could be an alternative to chemical fumigants against stored grain insect pests. For this reason, it is necessary to understand the mode of action of biofumigants. In the present study the prospectus of utilising Lantana camara as a potent fumigant insecticide is being discussed. Inhibition of acetylcholinesterase (AChE) by Coumaran, an active ingredient extracted from the plant L. camara, was studied. The biofumigant was used as an enzyme inhibitor and acetylthiocholine iodide as a substrate along with Ellman’s reagent to carry out the reactions. The in vivo inhibition was observed in both dose dependent and time dependent in case of housefly, and the nervous tissue (ganglion) and the whole insect homogenate of stored grain insect exposed to Coumaran. The possible mode of action of Coumaran as an acetylcholinesterase inhibitor is discussed. Yallappa Rajashekar, Anjanappa Raghavendra, and Nandagopal Bakthavatsalam Copyright © 2014 Yallappa Rajashekar et al. All rights reserved. Characterization and Potential Use of Cuttlefish Skin Gelatin Hydrolysates Prepared by Different Microbial Proteases Sun, 15 Jun 2014 07:37:13 +0000 Composition, functional properties, and in vitro antioxidant activities of gelatin hydrolysates prepared from cuttlefish skin were investigated. Cuttlefish skin gelatin hydrolysates (CSGHs) were obtained by treatment with crude enzyme preparations from Bacillus licheniformis NH1, Bacillus mojavensis A21, Bacillus subtilis A26, and commercial alcalase. All CSGHs had high protein contents, 74.3–78.3%, and showed excellent solubility (over 90%). CSGH obtained by alcalase demonstrated high antioxidant activities monitored by β-carotene bleaching, DPPH radical scavenging, lipid peroxidation inhibition, and reducing power activity. Its antioxidant activity remained stable or increased in a wide range of pH (1–9), during heating treatment (100°C for 240 min) and after gastrointestinal digestion simulation. In addition, alcalase-CSGH was incorporated into turkey meat sausage to determine its effect on lipid oxidation during 35 days of storage period. At 0.5 mg/g, alcalase-CSGH delayed lipid oxidation monitored by TBARS and conjugated diene up to 10 days compared to vitamin C. The results reveal that CSGHs could be used as food additives possessing both antioxidant activity and functional properties. Mourad Jridi, Imen Lassoued, Rim Nasri, Mohamed Ali Ayadi, Moncef Nasri, and Nabil Souissi Copyright © 2014 Mourad Jridi et al. All rights reserved. Overexpression of D-Xylose Reductase (xyl1) Gene and Antisense Inhibition of D-Xylulokinase (xyiH) Gene Increase Xylitol Production in Trichoderma reesei Wed, 11 Jun 2014 10:05:26 +0000 T. reesei is an efficient cellulase producer and biomass degrader. To improve xylitol production in Trichoderma reesei strains by genetic engineering, two approaches were used in this study. First, the presumptive D-xylulokinase gene in T. reesei (xyiH), which has high homology to known fungi D-xylulokinase genes, was silenced by transformation of T. reesei QM9414 strain with an antisense construct to create strain S6-2-2. The expression of the xyiH gene in the transformed strain S6-2-2 decreased at the mRNA level, and D-xylulokinase activity decreased after 48 h of incubation. This led to an increase in xylitol production from undetectable levels in wild-type T. reesei QM9414 to 8.6 mM in S6-2-2. The T. reesei Δxdh is a xylose dehydrogenase knockout strain with increased xylitol production compared to the wild-type T. reesei QM9414 (22.8 mM versus undetectable). The copy number of the xylose reductase gene (xyl1) in T. reesei Δxdh strain was increased by genetic engineering to create a new strain Δ9-5-1. The Δ9-5-1 strain showed a higher xyl1 expression and a higher yield of xylose reductase, and xylitol production was increased from 22.8 mM to 24.8 mM. Two novel strains S6-2-2 and Δ9-5-1 are capable of producing higher yields of xylitol. T. reesei has great potential in the industrial production of xylitol. Yuanyuan Hong, Mehdi Dashtban, Greg Kepka, Sanfeng Chen, and Wensheng Qin Copyright © 2014 Yuanyuan Hong et al. All rights reserved. Microfluidic Method of Pig Oocyte Quality Assessment in relation to Different Follicular Size Based on Lab-on-Chip Technology Mon, 09 Jun 2014 12:35:39 +0000 Since microfollicular environment and the size of the follicle are important markers influencing oocyte quality, the aim of this study is to present the spectral characterization of oocytes isolated from follicles of various sizes using lab-on-chip (LOC) technology and to demonstrate how follicle size may affect oocyte quality. Porcine oocytes (each, ) recovered from follicles of different sizes, for example, from large (>5 mm), medium (3–5 mm), and small (<3 mm), were analyzed after preceding in vitro maturation (IVM). The LOC analysis was performed using a silicon-glass sandwich with two glass optical fibers positioned “face-to-face.” Oocytes collected from follicles of different size classes revealed specific and distinguishable spectral characteristics. The absorbance spectra (microspectrometric specificity) for oocytes isolated from large, medium, and small follicles differ significantly () and the absorbance wavelengths were between 626 and 628 nm, between 618 and 620 nm, and less than 618 nm, respectively. The present study offers a parametric and objective method of porcine oocyte assessment. However, up to now this study has been used to evidence spectral markers associated with follicular size in pigs, only. Further investigations with functional-biological assays and comparing LOC analyses with fertilization and pregnancy success and the outcome of healthy offspring must be performed. Bartosz Kempisty, Rafał Walczak, Paweł Antosik, Patrycja Sniadek, Marta Rybska, Hanna Piotrowska, Dorota Bukowska, Jan Dziuban, Michał Nowicki, Jędrzej M. Jaśkowski, Maciej Zabel, and Klaus-Peter Brüssow Copyright © 2014 Bartosz Kempisty et al. All rights reserved. Indole Alkaloids from Marine Sources as Potential Leads against Infectious Diseases Thu, 05 Jun 2014 12:13:29 +0000 Indole alkaloids comprise a large and complex class of natural products found in a variety of marine sources. Infectious diseases remain a major threat to public health, and in the absence of long-term protective vaccines, the control of these infectious diseases is based on a small number of chemotherapeutic agents. Furthermore, the emerging resistance against these drugs makes it urgently necessary to discover and develop new, safe and, effective anti-infective agents. In this regard, the aim of this review is to highlight indole alkaloids from marine sources which have been shown to demonstrate activity against infectious diseases. Paulo H. B. França, Daniel P. Barbosa, Daniel L. da Silva, Êurica A. N. Ribeiro, Antônio E. G. Santana, Bárbara V. O. Santos, José M. Barbosa-Filho, Jullyana S. S. Quintans, Rosana S. S. Barreto, Lucindo J. Quintans-Júnior, and João X. de Araújo-Júnior Copyright © 2014 Paulo H. B. França et al. All rights reserved. Exogenous Proline and Glycine Betaine Mediated Upregulation of Antioxidant Defense and Glyoxalase Systems Provides Better Protection against Salt-Induced Oxidative Stress in Two Rice (Oryza sativa L.) Varieties Tue, 03 Jun 2014 08:21:14 +0000 The present study investigates the roles of exogenous proline (Pro, 5 mM) and glycine betaine (GB, 5 mM) in improving salt stress tolerance in salt sensitive (BRRI dhan49) and salt tolerant (BRRI dhan54) rice (Oryza sativa L.) varieties. Salt stresses (150 and 300 mM NaCl for 48 h) significantly reduced leaf relative water (RWC) and chlorophyll (chl) content and increased endogenous Pro and increased lipid peroxidation and H2O2 levels. Ascorbate (AsA), glutathione (GSH) and GSH/GSSG, ascorbate peroxidae (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), glutathione reductase (GR), glutathione peroxidase (GPX), catalase (CAT), and glyoxalase I (Gly I) activities were reduced in sensitive variety and these were increased in tolerant variety due to salt stress. The glyoxalase II (Gly II), glutathione S-transferase (GST), and superoxide dismutase (SOD) activities were increased in both cultivars by salt stress. Exogenous Pro and GB application with salt stress improved physiological parameters and reduced oxidative damage in both cultivars where BRRI dhan54 showed better tolerance. The result suggests that exogenous application of Pro and GB increased rice seedlings’ tolerance to salt-induced oxidative damage by upregulating their antioxidant defense system where these protectants rendered better performance to BRRI dhan54 and Pro can be considered as better protectant than GB. Mirza Hasanuzzaman, Md. Mahabub Alam, Anisur Rahman, Md. Hasanuzzaman, Kamrun Nahar, and Masayuki Fujita Copyright © 2014 Mirza Hasanuzzaman et al. All rights reserved. Plant-Made Biologics Mon, 02 Jun 2014 12:20:32 +0000 Qiang Chen, Luca Santi, and Chenming Zhang Copyright © 2014 Qiang Chen et al. All rights reserved. Simultaneous Heterotrophic Nitrification and Aerobic Denitrification by Chryseobacterium sp. R31 Isolated from Abattoir Wastewater Mon, 02 Jun 2014 07:36:43 +0000 A heterotrophic carbon utilizing microbe (R31) capable of simultaneous nitrification and denitrification (SND) was isolated from wastewater of an Indian slaughterhouse. From an initial COD value of 583.0 mg/L, 95.54% was removed whilst, from a starting -N concentration of 55.7 mg/L, 95.87% was removed after 48 h contact. The concentrations of the intermediates hydroxylamine, nitrite, and nitrate were low, thus ensuring nitrogen removal. Aerobic denitrification occurring during ammonium removal by R31 was confirmed by utilization of both nitrate and nitrite as nitrogen substrates. Glucose and succinate were superior while acetate and citrate were poor substrates for nitrogen removal. Molecular phylogenetic identification, supported by chemotaxonomic and physiological properties, assigned R31 as a close relative of Chryseobacterium haifense. The -N utilization rate and growth of strain R31 were found to be higher at C/N = 10 in comparison to those achieved with C/N ratios of 5 and 20. Monod kinetic coefficients, half saturation concentration , maximum rate of substrate utilization , yield coefficient, and endogenous decay coefficient indicated potential application of R31 in large-scale SND process. This is the first report on concomitant carbon oxidation, nitrification, and denitrification in the genus Chryseobacterium and the associated kinetic coefficients. Pradyut Kundu, Arnab Pramanik, Arpita Dasgupta, Somnath Mukherjee, and Joydeep Mukherjee Copyright © 2014 Pradyut Kundu et al. All rights reserved. Antibacterial and Antioxidant Activities of Derriobtusone A Isolated from Lonchocarpus obtusus Sun, 01 Jun 2014 06:48:47 +0000 This study evaluated the effect of derriobtusone A, a flavonoid isolated from Lonchocarpus obtusus, on two important pathogenic bacteria, Staphylococcus aureus and Escherichia coli, as well as its antioxidant activity and toxicity. Planktonic growth assays were performed, and the inhibition of biofilm formation was evaluated. In addition, antioxidant activity was assessed by DPPH radical scavenging assay, ferrous ion chelating assay, ferric-reducing antioxidant power assay, and β-carotene bleaching assay. Toxicity was evaluated by the brine shrimp lethality test. Results showed that derriobtusone A completely inhibited the planktonic growth of S. aureus at 250 and 500 μg/mL; however, it did not have the same activity on E. coli. Derriobtusone A reduced the biomass and colony-forming unit (cfu) of S. aureus biofilm at concentrations of 250 and 500 μg/mL. In various concentrations, it reduced the biofilm biomass of E. coli, and, in all concentrations, it weakly reduced the cfu. Derriobtusone A showed highly efficient antioxidant ability in scavenging DPPH radical and inhibiting β-carotene oxidation. The compound showed no lethality to Artemia sp. nauplii. In conclusion, derriobtusone A may be an effective molecule against S. aureus and its biofilm, as well as a potential antioxidant compound with no toxicity. Mayron Alves Vasconcelos, Francisco Vassiliepe Sousa Arruda, Daniel Barroso de Alencar, Silvana Saker-Sampaio, Maria Rose Jane Ribeiro Albuquerque, Hélcio Silva dos Santos, Paulo Nogueira Bandeira, Otília Deusdênia Loiola Pessoa, Benildo Sousa Cavada, Mariana Henriques, Maria Olivia Pereira, and Edson Holanda Teixeira Copyright © 2014 Mayron Alves Vasconcelos et al. All rights reserved. Manufacturing Economics of Plant-Made Biologics: Case Studies in Therapeutic and Industrial Enzymes Thu, 29 May 2014 12:20:07 +0000 Production of recombinant biologics in plants has received considerable attention as an alternative platform to traditional microbial and animal cell culture. Industrially relevant features of plant systems include proper eukaryotic protein processing, inherent safety due to lack of adventitious agents, more facile scalability, faster production (transient systems), and potentially lower costs. Lower manufacturing cost has been widely claimed as an intuitive feature of the platform by the plant-made biologics community, even though cost information resides within a few private companies and studies accurately documenting such an advantage have been lacking. We present two technoeconomic case studies representing plant-made enzymes for diverse applications: human butyrylcholinesterase produced indoors for use as a medical countermeasure and cellulases produced in the field for the conversion of cellulosic biomass into ethanol as a fuel extender. Production economics were modeled based on results reported with the latest-generation expression technologies on Nicotiana host plants. We evaluated process unit operations and calculated bulk active and per-dose or per-unit costs using SuperPro Designer modeling software. Our analyses indicate that substantial cost advantages over alternative platforms can be achieved with plant systems, but these advantages are molecule/product-specific and depend on the relative cost-efficiencies of alternative sources of the same product. Daniel Tusé, Tiffany Tu, and Karen A. McDonald Copyright © 2014 Daniel Tusé et al. All rights reserved. Interaction of Polybrominated Diphenyl Ethers and Aerobic Granular Sludge: Biosorption and Microbial Degradation Thu, 29 May 2014 11:47:41 +0000 As a new category of persistent organic pollutants, polybrominated diphenyl ethers (PBDEs) have become ubiquitous global environmental contaminants. No literature is available on the aerobic biotransformation of decabromodiphenyl ether (BDE-209). Herein, we investigated the interaction of PBDEs with aerobic granular sludge. The results show that the removal of BDE-209 from wastewater is mainly via biosorption onto aerobic granular sludge. The uptake capacity increased when temperature, contact time, and sludge dosage increased or solution pH dropped. Ionic strength had a negative influence on BDE-209 adsorption. The modified pseudo first-order kinetic model was appropriate to describe the adsorption kinetics. Microbial debromination of BDE-209 did not occur during the first 30 days of operation. Further study found that aerobic microbial degradation of 4,4′-dibromodiphenyl ether happened with the production of lower BDE congeners. Shou-Qing Ni, Qingjie Cui, and Zhen Zheng Copyright © 2014 Shou-Qing Ni et al. All rights reserved. Basal Transcription Factor 3 Plays an Important Role in Seed Germination and Seedling Growth of Rice Thu, 29 May 2014 09:31:02 +0000 BTF3 has been recognized to be involved in plant growth and development. But its function remains mostly unknown during seed germination and seedling stage. Here, we have analyzed OsBTF3-related sequences in Oryza sativa L. subspecies, japonica, which resembles with the conserved domain of a nascent polypeptide associated complex (NAC) with different homologs of OsBTF3 and human BTF3. Inhibition of Osj10gBTF3 has led to considerable morphological changes during seed germination and seedling growth. Germination percentage was not influenced by the application of GA3, ABA, and NaCl but all concentrations caused wild-type (WT) seeds to germinate more rapidly than the RNAi (Osj10gBTF3Ri) transgenic lines. Seedling inhibition was more severe in the Osj10gBTF3Ri seedlings compared with their WT especially when treated with 100 or 200 M GA3; 50% reduction in shoots was observed in Osj10gBTF3Ri seedlings. The expression of Osj3g1BTF3, Osj3g2BTF3 and Osj10gBTF3 was primarily constitutive and generally modulated by NaCl, ABA, and GA3 stresses in both Osj10gBTF3Ri lines and WT at the early seedling stage, suggesting that Osj3g1BTF3 and Osj10gBTF3 are much similar but different from Osj3g2BTF3 in biological function. These results show that OsBTF3 plays an important role in seed germination and seedling growth gives a new perception demonstrating that more multifaceted regulatory functions are linked with BTF3 in plants. Wenyi Wang, Mengyun Xu, Ya Wang, and Muhammad Jamil Copyright © 2014 Wenyi Wang et al. All rights reserved. Particulate Size of Microalgal Biomass Affects Hydrolysate Properties and Bioethanol Concentration Thu, 29 May 2014 08:23:52 +0000 Effective optimization of microalgae-to-bioethanol process systems hinges on an in-depth characterization of key process parameters relevant to the overall bioprocess engineering. One of the such important variables is the biomass particle size distribution and the effects on saccharification levels and bioethanol titres. This study examined the effects of three different microalgal biomass particle size ranges, 35 μm ≤ ≤ 90 μm, 125 μm ≤ ≤ 180 μm, and 295 μm ≤ ≤ 425 μm, on the degree of enzymatic hydrolysis and bioethanol production. Two scenarios were investigated: single enzyme hydrolysis (cellulase) and double enzyme hydrolysis (cellulase and cellobiase). The glucose yield from biomass in the smallest particle size range (35 μm ≤ ≤ 90 μm) was the highest, 134.73 mg glucose/g algae, while the yield from biomass in the larger particle size range (295 μm ≤ ≤ 425 μm) was 75.45 mg glucose/g algae. A similar trend was observed for bioethanol yield, with the highest yield of 0.47 g EtOH/g glucose obtained from biomass in the smallest particle size range. The results have shown that the microalgal biomass particle size has a significant effect on enzymatic hydrolysis and bioethanol yield. Razif Harun, Michael K. Danquah, and Selvakumar Thiruvenkadam Copyright © 2014 Razif Harun et al. All rights reserved. Effect of Algae and Plant Lectins on Planktonic Growth and Biofilm Formation in Clinically Relevant Bacteria and Yeasts Wed, 28 May 2014 19:29:20 +0000 This study aimed to evaluate the abilities of plant and algae lectins to inhibit planktonic growth and biofilm formation in bacteria and yeasts. Initially, ten lectins were tested on Staphylococcus epidermidis, Staphylococcus aureus, Klebsiella oxytoca, Pseudomonas aeruginosa, Candida albicans, and C. tropicalis at concentrations of 31.25 to 250 μg/mL. The lectins from Cratylia floribunda (CFL), Vatairea macrocarpa (VML), Bauhinia bauhinioides (BBL), Bryothamnion seaforthii (BSL), and Hypnea musciformis (HML) showed activities against at least one microorganism. Biofilm formation in the presence of the lectins was also evaluated; after 24 h of incubation with the lectins, the biofilms were analyzed by quantifying the biomass (by crystal violet staining) and by enumerating the viable cells (colony-forming units). The lectins reduced the biofilm biomass and/or the number of viable cells to differing degrees depending on the microorganism tested, demonstrating the different characteristics of the lectins. These findings indicate that the lectins tested in this study may be natural alternative antimicrobial agents; however, further studies are required to better elucidate the functional use of these proteins. Mayron Alves Vasconcelos, Francisco Vassiliepe Sousa Arruda, Victor Alves Carneiro, Helton Colares Silva, Kyria Santiago Nascimento, Alexandre Holanda Sampaio, Benildo Cavada, Edson Holanda Teixeira, Mariana Henriques, and Maria Olivia Pereira Copyright © 2014 Mayron Alves Vasconcelos et al. All rights reserved. Jacobsen Catalyst as a Cytochrome P450 Biomimetic Model for the Metabolism of Monensin A Wed, 28 May 2014 13:24:01 +0000 Monensin A is a commercially important natural product isolated from Streptomyces cinnamonensins that is primarily employed to treat coccidiosis. Monensin A selectively complexes and transports sodium cations across lipid membranes and displays a variety of biological properties. In this study, we evaluated the Jacobsen catalyst as a cytochrome P450 biomimetic model to investigate the oxidation of monensin A. Mass spectrometry analysis of the products from these model systems revealed the formation of two products: 3-O-demethyl monensin A and 12-hydroxy monensin A, which are the same ones found in in vivo models. Monensin A and products obtained in biomimetic model were tested in a mitochondrial toxicity model assessment and an antimicrobial bioassay against Staphylococcus aureus, S. aureus methicillin-resistant, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Escherichia coli. Our results demonstrated the toxicological effects of monensin A in isolated rat liver mitochondria but not its products, showing that the metabolism of monensin A is a detoxification metabolism. In addition, the antimicrobial bioassay showed that monensin A and its products possessed activity against Gram-positive microorganisms but not for Gram-negative microorganisms. The results revealed the potential of application of this biomimetic chemical model in the synthesis of drug metabolites, providing metabolites for biological tests and other purposes. Bruno Alves Rocha, Anderson Rodrigo Moraes de Oliveira, Murilo Pazin, Daniel Junqueira Dorta, Andresa Piacezzi Nascimento Rodrigues, Andresa Aparecida Berretta, Ana Paula Ferranti Peti, Luiz Alberto Beraldo de Moraes, Norberto Peporine Lopes, Stanislav Pospíšil, Paul Jonathan Gates, and Marilda das Dores Assis Copyright © 2014 Bruno Alves Rocha et al. All rights reserved. Glioprotective Effects of Ashwagandha Leaf Extract against Lead Induced Toxicity Wed, 28 May 2014 12:12:22 +0000 Withania somnifera (Ashwagandha), also known as Indian Ginseng, is a well-known Indian medicinal plant due to its antioxidative, antistress, antigenotoxic, and immunomodulatory properties. The present study was designed to assess and establish the cytoprotective potential of Ashwagandha leaf aqueous extract against lead induced toxicity. Pretreatment of C6 cells with 0.1% Ashwagandha extract showed cytoprotection against 25 μM to 400 μM concentration of lead nitrate. Further pretreatment with Ashwagandha extract to lead nitrate exposed cells (200 μM) resulted in normalization of glial fibrillary acidic protein (GFAP) expression as well as heat shock protein (HSP70), mortalin, and neural cell adhesion molecule (NCAM) expression. Further, the cytoprotective efficacy of Ashwagandha extract was studied in vivo. Administration of Ashwagandha extract provided significant protection to lead induced altered antioxidant defense that may significantly compromise normal cellular function. Ashwagandha also provided a significant protection to lipid peroxidation (LPx) levels, catalase, and superoxide dismutase (SOD) but not reduced glutathione (GSH) contents in brain tissue as well as peripheral organs, liver and kidney, suggesting its ability to act as a free radical scavenger protecting cells against toxic insult. These results, thus, suggest that Ashwagandha water extract may have the potential therapeutic implication against lead poisoning. Praveen Kumar, Raghavendra Singh, Arshed Nazmi, Dinesh Lakhanpal, Hardeep Kataria, and Gurcharan Kaur Copyright © 2014 Praveen Kumar et al. All rights reserved. Application of PCR-ELISA in Molecular Diagnosis Tue, 27 May 2014 12:29:42 +0000 Polymerase chain reaction-enzyme linked immunosorbent assay (PCR-ELISA) is an immunodetection method that can quantify PCR product directly after immobilization of biotinylated DNA on a microplate. This method, which detects nucleic acid instead of protein, is a much more sensitive method compared to conventional PCR method, with shorter analytical time and lower detection limit. Its high specificity and sensitivity, together with its semiquantitative ability, give it a huge potential to serve as a powerful detection tool in various industries such as medical, veterinary, and agricultural industries. With the recent advances in PCR-ELISA, it is envisaged that the assay is more widely recognized for its fast and sensitive detection limit which could improve overall diagnostic time and quality. Mei Jean Sue, Swee Keong Yeap, Abdul Rahman Omar, and Sheau Wei Tan Copyright © 2014 Mei Jean Sue et al. All rights reserved. Retracted: Preventative and Therapeutic Probiotic Use in Allergic Skin Conditions: Experimental and Clinical Findings Mon, 26 May 2014 12:07:48 +0000 BioMed Research International Copyright © 2014 BioMed Research International. All rights reserved. Quantitative Evaluation of E1 Endoglucanase Recovery from Tobacco Leaves Using the Vacuum Infiltration-Centrifugation Method Mon, 26 May 2014 06:36:14 +0000 As a production platform for recombinant proteins, plant leaf tissue has many advantages, but commercialization of this technology has been hindered by high recovery and purification costs. Vacuum infiltration-centrifugation (VI-C) is a technique to obtain extracellularly-targeted products from the apoplast wash fluid (AWF). Because of its selective recovery of secreted proteins without homogenizing the whole tissue, VI-C can potentially reduce downstream production costs. Lab scale experiments were conducted to quantitatively evaluate the VI-C method and compared to homogenization techniques in terms of product purity, concentration, and other desirable characteristics. From agroinfiltrated Nicotiana benthamiana leaves, up to 81% of a truncated version of E1 endoglucanase from Acidothermus cellulolyticus was recovered with VI-C versus homogenate extraction, and average purity and concentration increases of 4.2-fold and 3.1-fold, respectively, were observed. Formulas were developed to predict recovery yields of secreted protein obtained by performing multiple rounds of VI-C on the same leaf tissue. From this, it was determined that three rounds of VI-C recovered 97% of the total active recombinant protein accessible to the VI-C procedure. The results suggest that AWF recovery is an efficient process that could reduce downstream processing steps and costs for plant-made recombinant proteins. Nathaniel J. Kingsbury and Karen A. McDonald Copyright © 2014 Nathaniel J. Kingsbury and Karen A. McDonald. All rights reserved. Production and Biochemical Characterization of a High Maltotetraose (G4) Producing Amylase from Pseudomonas stutzeri AS22 Mon, 26 May 2014 00:00:00 +0000 Amylase production and biochemical characterization of the crude enzyme preparation from Pseudomonas stutzeri AS22 were evaluated. The highest α-amylase production was achieved after 24 hours of incubation in a culture medium containing 10 g/L potato starch and 5 g/L yeast extract, with initial pH 8.0 at 30°C under continuous agitation at 200 rpm. The optimum temperature and pH for the crude α-amylase activity were 60°C and 8.0, respectively. The effect of different salts was evaluated and it was found that both α-amylase production and activity were Ca2+-dependent. The amylolytic preparation was found to catalyze exceptionally the formation of very high levels of maltotetraose from starch (98%, w/w) in the complete absence of glucose since the initial stages of starch hydrolysis (15 min) and hence would have a potential application in the manufacturing of maltotetraose syrups. Hana Maalej, Hanen Ben Ayed, Olfa Ghorbel-Bellaaj, Moncef Nasri, and Noomen Hmidet Copyright © 2014 Hana Maalej et al. All rights reserved. N-Glycosylation Modification of Plant-Derived Virus-Like Particles: An Application in Vaccines Sun, 25 May 2014 12:46:39 +0000 Plants have been developed as an alternative system to mammalian cells for production of recombinant prophylactic or therapeutic proteins for human and animal use. Effective plant expression systems for recombinant proteins have been established with the optimal combination of gene expression regulatory elements and control of posttranslational processing of recombinant glycoproteins. In plant, virus-like particles (VLPs), viral “empty shells” which maintain the same structural characteristics of virions but are genome-free, are considered extremely promising as vaccine platforms and therapeutic delivery systems. Unlike microbial fermentation, plants are capable of carrying out N-glycosylation as a posttranslational modification of glycoproteins. Recent advances in the glycoengineering in plant allow human-like glycomodification and optimization of desired glycan structures for enhancing safety and functionality of recombinant pharmaceutical glycoproteins. In this review, the current plant-derived VLP approaches are focused, and N-glycosylation and its in planta modifications are discussed. Hyun-Soon Kim, Jae-Heung Jeon, Kyung Jin Lee, and Kisung Ko Copyright © 2014 Hyun-Soon Kim et al. All rights reserved. Production and Enhancement of Omega-3 Fatty Acid from Mortierella alpina CFR-GV15: Its Food and Therapeutic Application Wed, 21 May 2014 11:35:15 +0000 Mortierella sp. has been known to produce polyunsaturated fatty acids (PUFAs) such as GLA and AA under normal growth medium conditions. Similarly, under the stress condition, this fungus produces EPA and DHA in their mycelial biomass. Among the 67 soil samples screened from the Western Ghats of India, 11 Mortierella isolates showed the presence of omega-6 and omega-3 fatty acid, mainly GLA, AA, EPA, and DHA in starch, yeast-extract medium. Nile red and TTC strains were used for screening their qualitative oleaginesity. Among the representative isolates, when Mortierella sp. is grown in a fat-producing basal medium, a maximum lipid content of 42.0 ± 1.32% in its mycelia, 6.72 ± 0.5% EPA, and 4.09 ± 0.1% DHA was obtained. To understand the Mortierella sp. CFR-GV15, to the species level, its morphology was seen under the light microscope and scanning electron microscope, respectively. These microscopic observations showed that isolate Mortierella sp. CFR-GV15 produced coenocytic hyphae. Later on, its 18S rRNA and the internal transcribed spacer (ITS) sequences were cloned, sequenced, and analyzed phylogenetically to 18S rRNA and ITS1 and ITS4 sequences of related fungi. This newly isolated Mortierella alpina CFR-GV15 was found to be promising culture for the development of an economical method for commercial production of omega-3 fatty acid for food and therapeutical application. Ganesan Vadivelan and Govindarajulu Venkateswaran Copyright © 2014 Ganesan Vadivelan and Govindarajulu Venkateswaran. All rights reserved. Effect of Different Pretreatment of Sugar Cane Bagasse on Cellulase and Xylanases Production by the Mutant Penicillium echinulatum 9A02S1 Grown in Submerged Culture Tue, 20 May 2014 09:34:44 +0000 The main limitation to the industrial scale hydrolysis of cellulose is the cost of cellulase production. This study evaluated cellulase and xylanase enzyme production by the cellulolytic mutant Penicillium echinulatum 9A02S1 using pretreated sugar cane bagasse as a carbon source. Most cultures grown with pretreated bagasse showed similar enzymatic activities to or higher enzymatic activities than cultures grown with cellulose or untreated sugar cane bagasse. Higher filter paper activity (1.253 ± 0.147 U·mL−1) was detected in the medium on the sixth day of cultivation when bagasse samples were pretreated with sodium hydroxide, hydrogen peroxide, and anthraquinone. Endoglucanase enzyme production was also enhanced by pretreatment of the bagasse. Nine cultures grown with bagasse possessed higher β-glucosidase activities on the sixth day than the culture grown with cellulose. The highest xylanase activity was observed in cultures with cellulose and with untreated sugar cane bagasse. These results indicate that pretreated sugar cane bagasse may be able to serve as a partial or total replacement for cellulose in submerged fermentation for cellulase production using P. echinulatum, which could potentially reduce future production costs of enzymatic complexes capable of hydrolyzing lignocellulosic residues to form fermented syrups. Marli Camassola and Aldo J. P. Dillon Copyright © 2014 Marli Camassola and Aldo J. P. Dillon. All rights reserved. The Antiacetylcholinesterase and Antileishmanial Activities of Canarium patentinervium Miq. Thu, 15 May 2014 00:00:00 +0000 In continuation of our natural and medicinal research programme on tropical rainforest plants, a bioassay guided fractionation of ethanolic extract of leaves of Canarium patentinervium Miq. (Burseraceae Kunth.) led to the isolation of scopoletin (1), scoparone (2), (+)-catechin (3), vomifoliol (4), lioxin (5), and syringic acid (6). All the compounds exhibited antiacetylcholinesterase activity with syringic acid, a phenolic acid exhibiting good AChE inhibition (IC50 29.53 ± 0.19 μg/mL). All compounds displayed moderate antileishmanial activity with scopoletin having the highest antileishmanial activity (IC50 163.30 ± 0.32 μg/mL). Given the aforementioned evidence, it is tempting to speculate that Canarium patentinervium Miq. represents an exciting scaffold from which to develop leads for treatment of neurodegenerative and parasitic diseases. R. Mogana, A. Adhikari, S. Debnath, S. Hazra, B. Hazra, K. Teng-Jin, and C. Wiart Copyright © 2014 R. Mogana et al. All rights reserved. Purification, Characterization, and Potential of Saline Waste Water Remediation of a Polyextremophilic α-Amylase from an Obligate Halophilic Aspergillus gracilis Wed, 14 May 2014 11:25:30 +0000 An obligate halophilic Aspergillus gracilis which was isolated from a hypersaline man-made saltern from Thailand was screened for its potential of producing extracellular α-amylase in the previous studies. In this study the α-amylase was extracted and purified by the help of column chromatography using Sephadex G-100 column. Presence of amylase was verified by SDS-PAGE analysis, showing a single band of approximately 35 kDa. The specific activity of the enzyme was found to be 131.02 U/mg. The Lineweaver-Burk plot showed the and values of 8.36 U/mg and 6.33 mg/mL, respectively. The enzyme was found to have the best activity at 5 pH, 60°C, and 30% of NaCl concentration, showing its polyextremophilic nature. The use of various additives did not show much variation in the activity of enzyme, showing its resilience against inhibitors. The enzyme, when tested for its use for synthetic waste water remediation by comparing its activity with commercial amylase in different salt concentrations showed that the α-amylase from A. gracilis was having better performance at increasing salt concentrations than the commercial one. This shows its potential to be applied in saline waste water and other low water activity effluents for bioremediation. Imran Ali, Ali Akbar, Benjawan Yanwisetpakdee, Sehanat Prasongsuk, Pongtharin Lotrakul, and Hunsa Punnapayak Copyright © 2014 Imran Ali et al. All rights reserved. Inhibitory Effect on In Vitro LDL Oxidation and HMG Co-A Reductase Activity of the Liquid-Liquid Partitioned Fractions of Hericium erinaceus (Bull.) Persoon (Lion’s Mane Mushroom) Tue, 13 May 2014 09:44:40 +0000 Oxidation of low-density lipoprotein (LDL) has been strongly suggested as the key factor in the pathogenesis of atherosclerosis. Mushrooms have been implicated in having preventive effects against chronic diseases due especially to their antioxidant properties. In this study, in vitro inhibitory effect of Hericium erinaceus on LDL oxidation and the activity of the cholesterol biosynthetic key enzyme, 3-hydroxy-3-methyl glutaryl coenzyme A (HMG Co-A) reductase, was evaluated using five liquid-liquid solvent fractions consisting of methanol : dichloromethane (M : DCM), hexane (HEX), dichloromethane (DCM), ethyl acetate (EA), and aqueous residue (AQ). The hexane fraction showed the highest inhibition of oxidation of human LDL as reflected by the increased lag time (100 mins) for the formation of conjugated diene (CD) at 1 µg/mL and decreased production (68.28%, IC50 0.73 mg/mL) of thiobarbituric acid reactive substances (TBARS) at 1 mg/mL. It also mostly inhibited (59.91%) the activity of the HMG Co-A reductase at 10 mg/mL. The GC-MS profiling of the hexane fraction identified the presence of myconutrients: inter alia, ergosterol and linoleic acid. Thus, hexane fraction of Hericium erinaceus was found to be the most potent in vitro inhibitor of both LDL oxidation and HMG Co-A reductase activity having therapeutic potential for the prevention of oxidative stress-mediated vascular diseases. Mohammad Azizur Rahman, Noorlidah Abdullah, and Norhaniza Aminudin Copyright © 2014 Mohammad Azizur Rahman et al. All rights reserved. Phytochemical Evaluation, Antimicrobial Activity, and Determination of Bioactive Components from Leaves of Aegle marmelos Sun, 11 May 2014 08:59:36 +0000 The therapeutic value of Aegle marmelos Correa (Rutaceae), commonly known as ‘‘Bael,’’ has been recognized as a component of traditional medication for the treatment of various human ailments. The plant, though, being highly explored, still lacks sufficient evidences for the best variety possessing the highest degree of medicinal values. The present study is focused on phytochemical screening of aqueous and methanolic leaf extracts of 18 varieties/accessions of A. marmelos. The crude extracts of A. marmelos revealed the presence of several biologically active phytochemicals with the highest quantity of alkaloids, flavonoids, and phenols in Pant Aparna variety. The antibacterial efficacy was investigated against pathogenic bacterial strains and the highest inhibitory activity of aqueous extract was obtained against S. epidermidis, whereas methanolic extract was found to be most potent against S. aureus at 40 mg/mL concentration. However, in aqueous : ethanol, the best results were observed against E. aerogenes followed by K. pneumonia and S. epidermidis. The MIC of aqueous and methanol extract of Aegle marmelos ranged from 10 mg/mL to 40 mg/mL whereas in aqueous : ethanol it ranged between 40 mg/mL and 160 mg/mL. The GC-MS analysis revealed the presence of many bioactive compounds such as flavonoids, alcohols, aldehydes, aromatic compounds, fatty acid methyl esters, terpenoids, phenolics, and steroids that can be postulated for antibacterial activity. Farina Mujeeb, Preeti Bajpai, and Neelam Pathak Copyright © 2014 Farina Mujeeb et al. All rights reserved. Norovirus Narita 104 Virus-Like Particles Expressed in Nicotiana benthamiana Induce Serum and Mucosal Immune Responses Sun, 11 May 2014 08:37:40 +0000 Narita 104 virus is a human pathogen belonging to the norovirus (family Caliciviridae) genogroup II. Noroviruses cause epidemic gastroenteritis worldwide. To explore the potential of developing a plant-based vaccine, a plant optimized gene encoding Narita 104 virus capsid protein (NaVCP) was expressed transiently in Nicotiana benthamiana using a tobacco mosaic virus expression system. NaVCP accumulated up to approximately 0.3 mg/g fresh weight of leaf at 4 days postinfection. Initiation of hypersensitive response-like symptoms followed by tissue necrosis necessitated a brief infection time and was a significant factor limiting expression. Transmission electron microscopy of plant-derived NaVCP confirmed the presence of fully assembled virus-like particles (VLPs). In this study, an optimized method to express and partially purify NaVCP is described. Further, partially purified NaVCP was used to immunize mice by intranasal delivery and generated significant mucosal and serum antibody responses. Thus, plant-derived Narita 104 VLPs have potential for use as a candidate subunit vaccine or as a component of a multivalent subunit vaccine, along with other genotype-specific plant-derived VLPs. Lolita George Mathew, Melissa M. Herbst-Kralovetz, and Hugh S. Mason Copyright © 2014 Lolita George Mathew et al. All rights reserved. Immobilization of a Pleurotus ostreatus Laccase Mixture on Perlite and Its Application to Dye Decolourisation Thu, 08 May 2014 16:18:58 +0000 In the present study, a crude laccase preparation from Pleurotus ostreatus was successfully immobilized on perlite, a cheap porous silica material, and tested for Remazol Brilliant Blue R (RBBR) decolourisation in a fluidized bed recycle reactor. Results showed that RBBR decolourisation is mainly due to enzyme action despite the occurrence of dye adsorption-related enzyme inhibition. Fine tuning of immobilization conditions allowed balancing the immobilization yield and the resulting rate of decolourisation, with the adsorption capacity of the solid biocatalyst. In the continuous lab scale reactor, a maximum conversion degree of 56.1% was achieved at reactor space-time of 4.2 h. Stability and catalytic parameters of the immobilized laccases were also assessed in comparison with the soluble counterparts, revealing an increase in stability, despite a reduction of the catalytic performances. Both effects are most likely ascribable to the occurrence of multipoint attachment phenomena. Cinzia Pezzella, Maria Elena Russo, Antonio Marzocchella, Piero Salatino, and Giovanni Sannia Copyright © 2014 Cinzia Pezzella et al. All rights reserved. Potent Protein Glycation Inhibition of Plantagoside in Plantago major Seeds Wed, 07 May 2014 12:19:37 +0000 Plantagoside (5,7,4′,5′-tetrahydroxyflavanone-3′-O-glucoside) and its aglycone (5,7,3′,4′,5′-pentahydroxyflavanone), isolated from a 50% ethanol extract of Plantago major seeds (Plantaginaceae), were established to be potent inhibitors of the Maillard reaction. These compounds also inhibited the formation of advanced glycation end products in proteins in physiological conditions and inhibited protein cross-linking glycation. These results indicate that P. major seeds have potential therapeutic applications in the prevention of diabetic complications. Nobuyasu Matsuura, Tadashi Aradate, Chihiro Kurosaka, Makoto Ubukata, Shiho Kittaka, Yuri Nakaminami, Kanae Gamo, Hiroyuki Kojima, and Mitsuharu Ohara Copyright © 2014 Nobuyasu Matsuura et al. All rights reserved. A Simple and Efficient Method to Isolate LTR Sequences of Plant Retrotransposon Tue, 06 May 2014 12:59:22 +0000 Retrotransposons (RTNs) have important roles in the formation of plant genome size, structure, and evolution. Ubiquitous distributions, abundant copy numbers, high heterogeneities, and insertional polymorphisms of RTNs have made them as excellent sources for molecular markers development. However, the wide application of RTNs-based molecular markers is restricted by the scarcity of the LTR (long terminal repeat) sequences information. A new, simple, and efficient method to isolate LTR sequences of RTNs was presented based on the degenerate RNase H nested primers and PPT (polypurine tract) primer of RTNs in tree peony. This method combined the characteristics and advantages of high-efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR), annealing control primer (ACP) system, and suppression PCR method. Nineteen LTR sequences were isolated using this new method in tree peony and the applicability of the LTR sequences based markers was validated by further SSAP analysis. The results showed that the new method is simple, of low-cost, and highly efficient, which is just conducted by three rounds of PCR and does not need any restriction enzymes and adapters, much less the hybridizations. This new method is rapid, economical, and cost- and time-saving, which could be easily used to isolate LTR sequences of RTNs. Da-Long Guo, Xiao-Gai Hou, and Xi Zhang Copyright © 2014 Da-Long Guo et al. All rights reserved. Highly Effective Renaturation of a Streptokinase from Streptococcus pyogenes DT7 as Inclusion Bodies Overexpressed in Escherichia coli Mon, 05 May 2014 10:25:51 +0000 The streptokinase (SK) is emerging as an important thrombolytic therapy agent in the treatment of patients suffering from cardiovascular diseases. We reported highly effective renaturation of a SK from S. pyogeness DT7 overexpressed in E. coli, purification, and biochemical characterization. A gene coding for the SK was cloned from S. pyogeness DT7. Because accumulation of active SK is toxic to the host cells, we have expressed it in the form of inclusion bodies. The mature protein was overexpressed in E. coli BL21 DE3/pESK under the control of the strong promoter tac induced by IPTG with a level of 60% of the total cell proteins. The activity of the rSK, renatured in phosphate buffer supplemented with Triton X-100 and glycerol, was covered with up to 41 folds of its initial activity. The purified of protein was identified with MALDI-TOF mass spectrometry through four peptide fragments, which showed 100% identification to the corresponding peptides of the putative SK from GenBank. Due to overexpression and highly effective renaturation of large amounts of inclusion bodies, the recombinant E. coli BL21 DE3/pESK system could be potentially applied for large-scale production of SK used in the therapy of acute myocardial infarction. Sy Le Thanh Nguyen, Dinh Thi Quyen, and Hong Diep Vu Copyright © 2014 Sy Le Thanh Nguyen et al. All rights reserved. Integrative Effects of Feeding Aspergillus awamori and fructooligosaccharide on Growth Performance and Digestibility in Broilers: Promotion Muscle Protein Metabolism Sun, 04 May 2014 14:04:10 +0000 This study was conducted to show the effect of Aspergillus awamori (AA), fructooligosaccharide (FOS), and combined Aspergillus awamori and fructooligosaccharide (AA + FOS) on growth, digestibility, blood parameters, and expression of some growth-related genes. A total of 60 broiler chicks at the age of 15 d were divided into a control group () and 3 treatment groups. The control group was fed a basal diet, and the treatment groups were fed basal diets supplemented with 0.05% AA, 0.05% FOS, and combined of 0.05% AA and 0.05% FOS. Results from measurement of growth performance and digestibility revealed a significant increase in the body weight gain with improved feed conversion rate in the experimental groups. Interestingly, dry matter digestibility (DMD) and crude protein utilization (CPU) were improved. In addition, plasma total cholesterol and low density lipoprotein-cholesterol (LDL-C) were decreased, while plasma high density lipoprotein-cholesterol (HDL-C) was increased by feeding AA, FOS, and AA + FOS. Expressions of growth hormone secretagogue receptor (GHSR), insulin-like growth factor 1 (IGF-1), and insulin-like growth factor 1 receptor (IGF1R) were increased in experimental groups. In conclusion, the supplementation of either Aspergillus awamori or fructooligosaccharide or both improves digestibility and growth performance probably by promoting skeletal muscle protein metabolism. Ahmed A. Saleh, Khairy Amber, Mohammed A. El-Magd, Mostafa S. Atta, Ahmed A. Mohammed, Mohamed M. Ragab, and Hanaa Abd El-Kader Copyright © 2014 Ahmed A. Saleh et al. All rights reserved. Snapback Primer Mediated Clamping PCR for Detection of EGFR and KRAS Mutations in NSCLC Patients by High Resolution Melting Analysis Sun, 04 May 2014 13:56:45 +0000 Assays for detecting somatic mutations are requested with higher sensitivity and more convenience. Here, we describe snapback primer mediated allele clamping enrichment polymerase chain reaction (SPACE-PCR), a novel form of PCR that amplifies minority alleles selectively from mixtures. We replaced regular PCR with SPACE-PCR before sequencing or genotyping assays to improve mutation detection sensitivity by up to 100-fold in detecting EGFR and KRAS somatic mutations. Combined SPACE-PCR with analysis of snapback primer by high resolution melting (SPACE-HRM), the high sensitive system that enables a closed-tube detection of mutations after isolating mutants has been established, as low as 1/105–1/1000 mutant samples can be diagnosed. And finally, in a double-blind experiment of 150 cases of non-small-cell lung cancer (NSCLC) patients, compared with direct DNA sequencing and ADX-EGFR/KRAS mutation detection kit, up to 25% of the PCR-direct sequencing negative cases turned out to be positive in SPACE-HRM mutation tests; the specificity is 100%. Results demonstrated that the SPACE-HRM system we set up is a high sensitive assay that can be used for EGFR and KRAS allele enrichment and reliable detection. We anticipate that the method will be employed in multiple applications in the clinic, including diagnosis, scanner recurrence monitoring, and treatment management. Haiyan Sun, Yang Yang, Lixin Yang, Bo Su, Gening Jiang, Ke Fei, and Daru Lu Copyright © 2014 Haiyan Sun et al. All rights reserved. Morphogenesis and Production of Enzymes by Penicillium echinulatum in Response to Different Carbon Sources Wed, 30 Apr 2014 09:12:52 +0000 The effect of different carbon sources on morphology and cellulase and xylanase production of Penicillium echinulatum was evaluated in this work. Among the six carbon sources studied, cellulose and sugar cane bagasse were the most suitable for the production of filter paper activity, endoglucanases, xylanases, and β-glucosidases. However, sucrose and glucose showed β-glucosidase activities similar to those obtained with the insoluble sources. The polyacrylamide gels proved the enzymatic activity, since different standards bands were detected in the media mentioned above. Regarding morphology, it was observed that the mycelium in a dispersed form provided the greatest enzymatic activity, possibly due to greater interaction between the substrate and hyphae. These data are important in understanding the physiology of fungi and could contribute to obtaining enzyme with potential application in the technology of second generation ethanol. Willian Daniel Hahn Schneider, Laísa dos Reis, Marli Camassola, and Aldo José Pinheiro Dillon Copyright © 2014 Willian Daniel Hahn Schneider et al. All rights reserved. Watermelon (Citrullus lanatus (Thunb.) Matsum. and Nakai) Juice Modulates Oxidative Damage Induced by Low Dose X-Ray in Mice Tue, 29 Apr 2014 13:20:43 +0000 Watermelon is a natural product that contains high level of antioxidants and may prevent oxidative damage in tissues due to free radical generation following an exposure to ionizing radiation. The present study aimed to investigate the radioprotective effects of watermelon (Citrullus lanatus (Thunb.) Matsum. and Nakai) juice against oxidative damage induced by low dose X-ray exposure in mice. Twelve adult male ICR mice were randomly divided into two groups consisting of radiation (Rx) and supplementation (Tx) groups. Rx received filtered tap water, while Tx was supplemented with 50% (v/v) watermelon juice for 28 days ad libitum prior to total body irradiation by 100 μGy X-ray on day 29. Brain, lung, and liver tissues were assessed for the levels of malondialdehyde (MDA), apurinic/apyrimidinic (AP) sites, glutathione (GSH), and superoxide dismutase (SOD) inhibition activities. Results showed significant reduction of MDA levels and AP sites formation of Tx compared to Rx . Mice supplemented with 50% watermelon juice restore the intracellular antioxidant activities by significantly increased SOD inhibition activities and GSH levels compared to Rx. These findings may postulate that supplementation of 50% watermelon (Citrullus lanatus (Thunb.) Matsum. and Nakai) juice could modulate oxidative damage induced by low dose X-ray exposure. Mohd Khairul Amran Mohammad, Muhamad Idham Mohamed, Ainul Mardhiyah Zakaria, Hairil Rashmizal Abdul Razak, and Wan Mazlina Md. Saad Copyright © 2014 Mohd Khairul Amran Mohammad et al. All rights reserved. In Vivo Antistress and Antioxidant Effects of Fermented and Germinated Mung Bean Tue, 29 Apr 2014 09:40:52 +0000 Mung bean has been traditionally used to alleviate heat stress. This effect may be contributed by the presence of flavonoids and γ-aminobutyric acid (GABA). On the other hand, fermentation and germination have been practised to enhance the nutritional and antioxidant properties of certain food products. The main focus of current study was to compare the antistress effect of none-process, fermented and germinated mung bean extracts. Acute and chronic restraint stresses were observed to promote the elevation of serum biochemical markers including cholesterol, triglyceride, total protein, liver enzymes, and glucose. Chronic cold restraint stress was observed to increase theadrenal gland weight, brain 5-hydroxytryptamine (5-HT), and malondialdehyde (MDA) level while reducing brain antioxidant enzyme level. However, these parameters were found reverted in mice treated with diazepam, high concentration of fermented mung bean and high concentration of germinated mung bean. Moreover, enhanced level of antioxidant on the chronic stress mice was observed in fermented and germinated mung bean treated groups. In comparison between germinated and fermented mung bean, fermented mung bean always showed better antistress and antioxidant effects throughout this study. Swee Keong Yeap, Boon Kee Beh, Norlaily Mohd Ali, Hamidah Mohd Yusof, Wan Yong Ho, Soo Peng Koh, Noorjahan Banu Alitheen, and Kamariah Long Copyright © 2014 Swee Keong Yeap et al. All rights reserved. A Review on Antibacterial, Antiviral, and Antifungal Activity of Curcumin Tue, 29 Apr 2014 07:34:15 +0000 Curcuma longa L. (Zingiberaceae family) and its polyphenolic compound curcumin have been subjected to a variety of antimicrobial investigations due to extensive traditional uses and low side effects. Antimicrobial activities for curcumin and rhizome extract of C. longa against different bacteria, viruses, fungi, and parasites have been reported. The promising results for antimicrobial activity of curcumin made it a good candidate to enhance the inhibitory effect of existing antimicrobial agents through synergism. Indeed, different investigations have been done to increase the antimicrobial activity of curcumin, including synthesis of different chemical derivatives to increase its water solubility as well ass cell up take of curcumin. This review aims to summarize previous antimicrobial studies of curcumin towards its application in the future studies as a natural antimicrobial agent. Soheil Zorofchian Moghadamtousi, Habsah Abdul Kadir, Pouya Hassandarvish, Hassan Tajik, Sazaly Abubakar, and Keivan Zandi Copyright © 2014 Soheil Zorofchian Moghadamtousi et al. All rights reserved. Biotechnology in Environmental Monitoring and Pollution Abatement Wed, 23 Apr 2014 07:13:00 +0000 Kannan Pakshirajan, Eldon R. Rene, and Aiyagari Ramesh Copyright © 2014 Kannan Pakshirajan et al. All rights reserved. The Interaction Pattern between a Homology Model of 40S Ribosomal S9 Protein of Rhizoctonia solani and 1-Hydroxyphenaize by Docking Study Tue, 22 Apr 2014 14:03:16 +0000 1-Hydroxyphenazine (1-OH-PHZ), a natural product from Pseudomonas aeruginosa strain SD12, was earlier reported to have potent antifungal activity against Rhizoctonia solani. In the present work, the antifungal activity of 1-OH-PHZ on 40S ribosomal S9 protein was validated by molecular docking approach. 1-OH-PHZ showed interaction with two polar contacts with residues, Arg69 and Phe19, which inhibits the synthesis of fungal protein. Our study reveals that 1-OH-PHZ can be a potent inhibitor of 40S ribosomal S9 protein of R. solani that may be a promising approach for the management of fungal diseases. Seema Dharni, Sanchita, Abdul Samad, Ashok Sharma, and Dharani Dhar Patra Copyright © 2014 Seema Dharni et al. All rights reserved. Lentiviral Protein Transduction with Genome-Modifying HIV-1 Integrase-I-PpoI Fusion Proteins: Studies on Specificity and Cytotoxicity Tue, 22 Apr 2014 00:00:00 +0000 Rare-cutting endonucleases, such as the I-PpoI, can be used for the induction of double strand breaks (DSBs) in genome editing and targeted integration based on homologous recombination. For therapeutic approaches, the specificity and the pattern of off-target effects are of high importance in these techniques. For its applications, the endonuclease needs to be transported into the target cell nucleus, where the mechanism of transport may affect its function. Here, we have studied the lentiviral protein transduction of the integrase (IN)-PpoI fusion protein using the cis-packaging method. In genome-wide interaction studies, IN-fusion proteins were verified to bind their target sequence containing 28S ribosomal RNA (rRNA) genes with a 100-fold enrichment, despite the well-documented behavior of IN to be tethered into various genomic areas by host-cell factors. In addition, to estimate the applicability of the method, DSB-induced cytotoxic effects with different vector endonuclease configurations were studied in a panel of cells. Varying the amount and activity of endonuclease enabled the adjustment of ratio between the induced DSBs and transported DNA. In cell studies, certain cancerous cell lines were especially prone to DSBs in rRNA genes, which led us to test the protein transduction in a tumour environment in an in vivo study. In summary, the results highlight the potential of lentiviral vectors (LVVs) for the nuclear delivery of endonucleases. Vesa Turkki, Diana Schenkwein, Oskari Timonen, Tiia Husso, Hanna P. Lesch, and Seppo Ylä-Herttuala Copyright © 2014 Vesa Turkki et al. All rights reserved. Combinatorial Control of Transgene Expression by Hypoxia-Responsive Promoter and MicroRNA Regulation for Neural Stem Cell-Based Cancer Therapy Thu, 17 Apr 2014 14:11:28 +0000 Owing to their strong migratory capacity, tumor tropism, and tumor inhibitory effect, neural stem cells (NSCs) have recently emerged as one of the most attractive gene delivery vectors for cancer therapy. However, further animal studies found that proportional NSC vectors were distributed to nontarget organs after intravenous injection and the nonspecific transgene expression led to significant cytotoxic effects in these organs. Hence, an expression cassette that controls the transgene expression within NSC vectors in a tumor site-specific manner is desired. Considering hypoxia as a hallmark of tumor microenvironment, we have developed a novel NSC vector platform coupling transcriptional targeting with microRNA (miRNA) regulation for tumor hypoxia targeting. This combinatorial vector employed a hypoxia-responsive promoter and repeated targeting sequences of an miRNA that is enriched in NSCs but downregulated upon hypoxia induction to control the transgene expression. This resulted in significantly improved hypoxic selectivity over the use of a control vector without miRNA regulation. Thus, incorporating miRNA regulation into a transcriptional targeting vector adds an extra layer of security to prevent off-target transgene expression and should be useful for the development of NSC vectors with high targeting specifcity for cancer therapy. Yumei Luo and Detu Zhu Copyright © 2014 Yumei Luo and Detu Zhu. All rights reserved. Coastal Biotechnology: Facing the Global and the Regional Changes Thu, 17 Apr 2014 10:05:48 +0000 Song Qin, Wei Zhang, and Hanzhi Lin Copyright © 2014 Song Qin et al. All rights reserved. Biodegradation and Utilization of Organophosphorus Pesticide Malathion by Cyanobacteria Thu, 17 Apr 2014 08:48:18 +0000 Three strains of filamentous Cyanobacteria were used to study their growth and utilization of organophosphorus pesticide malathion. A sharp decrease in the growth of the algal strains was observed by increasing the concentration of malathion. Amongst them Nostoc muscorum tolerated different concentrations and was recorded as the highest efficient strain for biodegradation (91%) of this compound. Moreover, carbohydrate and protein content of their cells overtopped the other strains especially at higher concentrations. The algal strains were further subjected to grow under P-limitation in absence and presence of malathion. Although, the algal growth under P-limitation recorded a very poor level, a massive enhanced growth and phosphorous content of cells were obtained when the P-limited medium was amended with malathion. This study clarified that N. muscorum with its capability to utilize malathion as a sole phosphorous source is considered as an inexpensive and efficient biotechnology for remediation of organophosphorus pesticide from contaminated wastewater. Wael M. Ibrahim, Mohamed A. Karam, Reda M. El-Shahat, and Asmaa A. Adway Copyright © 2014 Wael M. Ibrahim et al. All rights reserved. Production and Cytotoxicity of Extracellular Insoluble and Droplets of Soluble Melanin by Streptomyces lusitanus DMZ-3 Wed, 16 Apr 2014 13:49:37 +0000 A Streptomyces lusitanus DMZ-3 strain with potential to synthesize both insoluble and soluble melanins was detected. Melanins are quite distinguished based on their solubility for varied biotechnological applications. The present investigation reveals the enhanced production of insoluble and soluble melanins in tyrosine medium by a single culture. Streptomyces lusitanus DMZ-3 was characterized by 16S rRNA gene analysis. An enhanced production of 5.29 g/L insoluble melanin was achieved in a submerged bioprocess following response surface methodology. Combined interactive effect of temperature (50°C), pH (8.5), tyrosine (2.0 g/L), and beef extract (0.5 g/L) were found to be critical variables for enhanced production in central composite design analysis. An optimized indigenous slant culture system was an innovative approach for the successful production (264 mg/L) of pure soluble melanin from the droplets formed on the surface of the culture. Both insoluble and soluble melanins were confirmed and characterized by Chemical, reactions, UV, FTIR, and TLC analysis. First time, cytotoxic study of melanin using brine shrimps was reported. Maximum cytotoxic activity of soluble melanin was Lc50-0.40 µg/mL and insoluble melanin was Lc50-0.80 µg/mL. D. N. Madhusudhan, Bi Bi Zainab Mazhari, Syed G. Dastager, and Dayanand Agsar Copyright © 2014 D. N. Madhusudhan et al. All rights reserved. In Vitro and In Vivo Leishmanicidal Activity of Astronium fraxinifolium (Schott) and Plectranthus amboinicus (Lour.) Spreng against Leishmania (Viannia) braziliensis Wed, 16 Apr 2014 12:23:24 +0000 The aim of the present work was to evaluate antileishmanial activity of Astronium fraxinifolium and Plectranthus amboinicus. For the in vitro tests, essential oil of P. amboinicus (OEPA) and ethanolic extracts from A. fraxinifolium (EEAF) were incubated with 106  promastigotes of L. (Viannia) braziliensis. The OEPA was able to reduce the parasite growth after 48 h; nonetheless, all the EEAFs could totally abolish the parasite growth. For the in vivo studies, BALB/c mice were infected subcutaneously (s.c.) with 107  L. braziliensis promastigotes. Treatment was done by administering OEPA intralesionally (i.l.) for 14 days. No difference was found in lesion thickness when those animals were compared with the untreated animals. Further, golden hamsters were infected s.c. with 106  L. braziliensis promastigotes. The first protocol of treatment consisted of ethanolic leaf extract from A. fraxinifolium (ELEAF) administered i.l. for 4 days and a booster dose at the 7th day. The animals showed a significant reduction of lesion thickness in the 6th week, but it was not comparable to the animals treated with Glucantime. The second protocol consisted of 15 daily intralesional injections. The profiles of lesion thickness were similar to the standard treatment. In conclusion, in vivo studies showed a high efficacy when the infected animals were intralesionally treated with leaf ethanolic extract from A. fraxinifolium. Silvio César Gomes de Lima, Maria Jania Teixeira, José Evaldo Gonçalves Lopes Júnior, Selene Maia de Morais, Alba Fabiola Torres, Milena Aguiar Braga, Raphael Oliveira Rodrigues, Gilvandete Maria Pinheiro Santiago, Alice Costa Martins, and Aparecida Tiemi Nagao-Dias Copyright © 2014 Silvio César Gomes de Lima et al. All rights reserved. Aerobic Sludge Granulation in a Full-Scale Sequencing Batch Reactor Tue, 15 Apr 2014 14:02:21 +0000 Aerobic granulation of activated sludge was successfully achieved in a full-scale sequencing batch reactor (SBR) with 50,000 m3 d−1 for treating a town’s wastewater. After operation for 337 days, in this full-scale SBR, aerobic granules with an average SVI30 of 47.1 mL g−1, diameter of 0.5 mm, and settling velocity of 42 m h−1 were obtained. Compared to an anaerobic/oxic plug flow (A/O) reactor and an oxidation ditch (OD) being operated in this wastewater treatment plant, the sludge from full-scale SBR has more compact structure and excellent settling ability. Denaturing gradient gel electrophoresis (DGGE) analysis indicated that Flavobacterium sp., uncultured beta proteobacterium, uncultured Aquabacterium sp., and uncultured Leptothrix sp. were just dominant in SBR, whereas uncultured bacteroidetes were only found in A/O and OD. Three kinds of sludge had a high content of protein in extracellular polymeric substances (EPS). X-ray fluorescence (XRF) analysis revealed that metal ions and some inorganics from raw wastewater precipitated in sludge acted as core to enhance granulation. Raw wastewater characteristics had a positive effect on the granule formation, but the SBR mode operating with periodic feast-famine, shorter settling time, and no return sludge pump played a crucial role in aerobic sludge granulation. Jun Li, Li-Bin Ding, Ang Cai, Guo-Xian Huang, and Harald Horn Copyright © 2014 Jun Li et al. All rights reserved. Fungal Laccases Degradation of Endocrine Disrupting Compounds Tue, 15 Apr 2014 00:00:00 +0000 Over the past decades, water pollution by trace organic compounds (ng/L) has become one of the key environmental issues in developed countries. This is the case of the emerging contaminants called endocrine disrupting compounds (EDCs). EDCs are a new class of environmental pollutants able to mimic or antagonize the effects of endogenous hormones, and are recently drawing scientific and public attention. Their widespread presence in the environment solicits the need of their removal from the contaminated sites. One promising approach to face this challenge consists in the use of enzymatic systems able to react with these molecules. Among the possible enzymes, oxidative enzymes are attracting increasing attention because of their versatility, the possibility to produce them on large scale, and to modify their properties. In this study five different EDCs were treated with four different fungal laccases, also in the presence of both synthetic and natural mediators. Mediators significantly increased the efficiency of the enzymatic treatment, promoting the degradation of substrates recalcitrant to laccase oxidation. The laccase showing the best performances was chosen to further investigate its oxidative capabilities against micropollutant mixtures. Improvement of enzyme performances in nonylphenol degradation rate was achieved through immobilization on glass beads. Gemma Macellaro, Cinzia Pezzella, Paola Cicatiello, Giovanni Sannia, and Alessandra Piscitelli Copyright © 2014 Gemma Macellaro et al. All rights reserved. Soybean Seeds: A Practical Host for the Production of Functional Subunit Vaccines Mon, 14 Apr 2014 00:00:00 +0000 Soybean seeds possess several inherent qualities that make them an ideal host for the production of biopharmaceuticals when compared with other plant-based and non-plant-based recombinant expression systems (e.g., low cost of production, high protein to biomass ratio, long-term stability of seed proteins under ambient conditions, etc.). To demonstrate the practicality and feasibility of this platform for the production of subunit vaccines, we chose to express and characterize a nontoxic form of S. aureus enterotoxin B (mSEB) as a model vaccine candidate. We show that soy-mSEB was produced at a high vaccine to biomass ratio and represented ~76 theoretical doses of human vaccine per single soybean seed. We localized the model vaccine candidate both intracellularly and extracellularly and found no difference in mSEB protein stability or accumulation relative to subcellular environment. We also show that the model vaccine was biochemically and immunologically similar to native and recombinant forms of the protein produced in a bacterial expression system. Immunization of mice with seed extracts containing mSEB mounted a significant immune response within 14 days of the first injection. Taken together, our results highlight the practicality of soybean seeds as a potential platform for the production of functional subunit vaccines. Laura C. Hudson, Renu Garg, Kenneth L. Bost, and Kenneth J. Piller Copyright © 2014 Laura C. Hudson et al. All rights reserved. Biologic Propensities and Phytochemical Profile of Vangueria madagascariensis J. F. Gmelin (Rubiaceae): An Underutilized Native Medicinal Food Plant from Africa Thu, 10 Apr 2014 14:05:00 +0000 Vangueria madagascariensis (VM), consumed for its sweet-sour fruits, is used as a biomedicine for the management of diabetes and bacterial infections in Africa. The study aims to assess the potential of VM on α-amylase, α-glucosidase, glucose movement, and antimicrobial activity. The antioxidant properties were determined by measuring the FRAP, iron chelating activity, and abilities to scavenge DPPH, HOCl, ∙OH, and NO radicals. Leaf decoction, leaf methanol, and unripe fruit methanol extracts were observed to significantly inhibit α-amylase. Active extracts against α-glucosidase were unripe fruit methanol, unripe fruit decoction, leaf decoction, and ripe fruit methanol, which were significantly lower than acarbose. Kinetic studies revealed a mixed noncompetitive type of inhibition. Leaf methanolic extract was active against S. aureus and E. coli. Total phenolic content showed a strong significant positive correlation () with FRAP. Methanolic leaf extract showed a more efficient NO scavenging potential and was significantly lower than ascorbic acid. Concerning ∙OH-mediated DNA degradation, only the methanol extracts of leaf, unripe fruit, and ripe fruit had IC50 values which were significantly lower than α-tocopherol. Given the dearth of information on the biologic propensities of VM, this study has established valuable primary information which has opened new perspectives for further pharmacological research. Nelvana Ramalingum and M. Fawzi Mahomoodally Copyright © 2014 Nelvana Ramalingum and M. Fawzi Mahomoodally. All rights reserved. Arrabidaea chica Hexanic Extract Induces Mitochondrion Damage and Peptidase Inhibition on Leishmania spp. Wed, 09 Apr 2014 08:54:22 +0000 Currently available leishmaniasis treatments are limited due to severe side effects. Arrabidaea chica is a medicinal plant used in Brazil against several diseases. In this study, we investigated the effects of 5 fractions obtained from the crude hexanic extract of A. chica against Leishmania amazonensis and L. infantum, as well as on the interaction of these parasites with host cells. Promastigotes were treated with several concentrations of the fractions obtained from A. chica for determination of their minimum inhibitory concentration (MIC). In addition, the effect of the most active fraction (B2) on parasite’s ultrastructure was analyzed by transmission electron microscopy. To evaluate the inhibitory activity of B2 fraction on Leishmania peptidases, parasites lysates were treated with the inhibitory and subinhibitory concentrations of the B2 fraction. The minimum inhibitory concentration of B2 fraction was 37.2 and 18.6 μg/mL for L. amazonensis and L. infantum, respectively. Important ultrastructural alterations as mitochondrial swelling with loss of matrix content and the presence of vesicles inside this organelle were observed in treated parasites. Moreover, B2 fraction was able to completely inhibit the peptidase activity of promastigotes at pH 5.5. The results presented here further support the use of A. chica as an interesting source of antileishmanial agents. Igor A. Rodrigues, Mariana M. B. Azevedo, Francisco C. M. Chaves, Celuta S. Alviano, Daniela S. Alviano, and Alane B. Vermelho Copyright © 2014 Igor A. Rodrigues et al. All rights reserved. Chitinase from a Novel Strain of Serratia marcescens JPP1 for Biocontrol of Aflatoxin: Molecular Characterization and Production Optimization Using Response Surface Methodology Wed, 09 Apr 2014 07:50:43 +0000 Chitinase is one of the most important mycolytic enzymes with industrial significance, and produced by a number of organisms. A chitinase producing isolate Serratia marcescens JPP1 was obtained from peanut hulls in Jiangsu Province, China, and exhibited antagonistic activity against aflatoxins. In this study, we describe the optimization of medium composition with increased production of chitinase for the selected bacteria using statistical methods: Plackett-Burman design was applied to find the key ingredients, and central composite design of response surface methodology was used to optimize the levels of key ingredients for the best yield of chitinase. Maximum chitinase production was predicted to be 23.09 U/mL for a 2.1-fold increase in medium containing 12.70 g/L colloidal chitin, 7.34 g/L glucose, 5.00 g/L peptone, 1.32 g/L (NH4)2SO4, 0.7 g/L K2HPO4, and 0.5 g/L MgSO4·7H2O. Polymerase chain reaction (PCR) amplification of the JPP1 chitinase gene was performed and obtained a 1,789 bp nucleotide sequence; its open reading frame encoded a protein of 499 amino acids named as ChiBjp. Kai Wang, Pei-sheng Yan, and Li-xin Cao Copyright © 2014 Kai Wang et al. All rights reserved. Evaluation of Drought Tolerance of the Vietnamese Soybean Cultivars Provides Potential Resources for Soybean Production and Genetic Engineering Mon, 07 Apr 2014 14:02:02 +0000 Drought is one of the greatest constraints to soybean production in many countries, including Vietnam. Although a wide variety of the newly produced cultivars have been produced recently in Vietnam through classical breeding to cope with water shortage, little knowledge of their molecular and physiological responses to drought has been discovered. This study was conducted to quickly evaluate drought tolerance of thirteen local soybean cultivars for selection of the best drought-tolerant cultivars for further field test. Differences in drought tolerance of cultivars were assessed by root and shoot lengths, relative water content, and drought-tolerant index under both normal and drought conditions. Our data demonstrated that DT51 is the strongest drought-tolerant genotype among all the tested cultivars, while the highest drought-sensitive phenotype was observed with MTD720. Thus, DT51 could be subjected to further yield tests in the field prior to suggesting it for use in production. Due to their contrasting drought-tolerant phenotypes, DT51 and MTD720 provide excellent genetic resources for further studies underlying mechanisms regulating drought responses and gene discovery. Our results provide vital information to support the effort of molecular breeding and genetic engineering to improve drought tolerance of soybean. Nguyen Binh Anh Thu, Quang Thien Nguyen, Xuan Lan Thi Hoang, Nguyen Phuong Thao, and Lam-Son Phan Tran Copyright © 2014 Nguyen Binh Anh Thu et al. All rights reserved. Streptomyces flavogriseus HS1: Isolation and Characterization of Extracellular Proteases and Their Compatibility with Laundry Detergents Sun, 06 Apr 2014 14:13:56 +0000 The present study describes the isolation of a new protease producing Streptomyces strain HS1 and the biochemical characterization of the secreted proteases. By sequencing of its noted 16S rDNA, HS1 strain was found to have a 100% identity with Streptomyces flavogriseus. The highest protease production was found using FermII media. In these conditions maximum protease production (99 U/mL) was obtained after 96 h incubation at 30°C and 150 rpm. HS1 strain produced at least five proteases as revealed by zymogram technique. The enzyme preparation exhibited activity over a broad range of pH (5–11) and temperature (25–70°C). Optimum activity was observed at a pH of 7.0 and a temperature of 50°C. Proteolytic activity was significantly unaffected by Ca2+ and Mg2+. EDTA and PMSF highly decreased the original activity. The crude extracellular proteases showed high stability when used as a detergent additive. These properties offer an interesting potential for enzymatic hydrolysis at the industrial level. Sofiane Ghorbel, Maher Kammoun, Hala Soltana, Moncef Nasri, and Noomen Hmidet Copyright © 2014 Sofiane Ghorbel et al. All rights reserved. Antioxidant Activity of Essential Oil and Extracts of Valeriana jatamansi Roots Sun, 06 Apr 2014 11:47:37 +0000 Valeriana jatamansi is an indigenous medicinal plant used in the treatment of a number of diseases. In the present study, chemical composition of the essential oil was determined by GC-MS. Seven major components were identified in Valeriana jatamansi essential oil, namely, β-vatirenene, β-patchoulene, dehydroaromadendrene, β-gurjunene, patchoulic alcohol, β-guaiene, and α-muurolene. Methanolic, aqueous, and chloroform extracts of Valeriana jatamansi roots were also prepared and analyzed for their polyphenols and flavonoid content. Antioxidant activity of essential oil and different extracts of Valeriana jatamansi roots was determined by DPPH radical scavenging and chelation power assay. A linear correlation has been obtained by comparing the antioxidant activity and polyphenols and flavonoid content of the extracts. Results indicated that antioxidant activity of methanolic extract could be attributed to the presence of rich amount of polyphenols and flavonoid. Essential oil of Valeriana jatamansi roots showed moderate antioxidant activity. Sakshima Thusoo, Sahil Gupta, Rasleen Sudan, Jaspreet Kour, Sahil Bhagat, Rashid Hussain, and Madhulika Bhagat Copyright © 2014 Sakshima Thusoo et al. All rights reserved. A Plant-Produced Antigen Elicits Potent Immune Responses against West Nile Virus in Mice Thu, 03 Apr 2014 11:49:36 +0000 We described the rapid production of the domain III (DIII) of the envelope (E) protein in plants as a vaccine candidate for West Nile Virus (WNV). Using various combinations of vector modules of a deconstructed viral vector expression system, DIII was produced in three subcellular compartments in leaves of Nicotiana benthamiana by transient expression. DIII expressed at much higher levels when targeted to the endoplasmic reticulum (ER) than that targeted to the chloroplast or the cytosol, with accumulation level up to 73 μg DIII per gram of leaf fresh weight within 4 days after infiltration. Plant ER-derived DIII was soluble and readily purified to > 95% homogeneity without the time-consuming process of denaturing and refolding. Further analysis revealed that plant-produced DIII was processed properly and demonstrated specific binding to an anti-DIII monoclonal antibody that recognizes a conformational epitope. Furthermore, subcutaneous immunization of mice with 5 and 25 μg of purified DIII elicited a potent systemic response. This study provided the proof of principle for rapidly producing immunogenic vaccine candidates against WNV in plants with low cost and scalability. Junyun He, Li Peng, Huafang Lai, Jonathan Hurtado, Jake Stahnke, and Qiang Chen Copyright © 2014 Junyun He et al. All rights reserved. Optimization of Extraction and Enrichment of Steroidal Alkaloids from Bulbs of Cultivated Fritillaria cirrhosa Tue, 01 Apr 2014 09:03:49 +0000 The bulbs of cultivated Fritillaria cirrhosa (BCFC) are used in China both for food and folk medicine due to its powerful biological activities. The aim of this study is to optimize the extraction and enrichment conditions of alkaloids from BCFC. Firstly, the orthogonal experimental design was used to optimize and evaluate four variables (ethanol concentration, solid-liquid ratio, extraction time, and temperature). Thereafter, resin adsorption was as a means to enrich alkaloids. Among 16 tested resins, H-103 resin presented higher adsorption capacity and desorption ratio. The equilibrium experimental data of the adsorption of total alkaloids, imperialine, and peimisine were well-fitted to the pseudo-first-order kinetics model, Langmuir and Freundlich isotherms models. Finally, in order to optimize the parameters for purifying alkaloids, dynamic adsorption and desorption tests were carried out. After one run treatment with H-103 resin, the contents of total alkaloids, imperialine, and peimisine in the product were 21.40-, 18.31-, and 22.88-fold increased with recovery yields of 94.43%, 90.57%, and 96.16%, respectively. Dongdong Wang, Shu Wang, Qingdan Du, Nanyi Wang, Simei Liu, Xiaoxia Wang, and Jinghui Jiang Copyright © 2014 Dongdong Wang et al. All rights reserved. A Noncellulosomal Mannanase26E Contains a CBM59 in Clostridium cellulovorans Thu, 27 Mar 2014 14:12:20 +0000 A multicomponent enzyme-complex prevents efficient degradation of the plant cell wall for biorefinery. In this study, the method of identifying glycoside hydrolases (GHs) to degrade hemicelluloses was demonstrated. The competence of C. cellulovorans, which changes to be suitable for degradation of each carbon source, was used for the method. C. cellulovorans was cultivated into locust bean gum (LBG) that is composed of galactomannan. The proteins produced by C. cellulovorans were separated into either fractions binding to crystalline cellulose or not. Proteins obtained from each fraction were further separated by SDS-PAGE and were stained with Coomassie Brilliant Blue and were detected for mannanase activity. The proteins having the enzymatic activity for LBG were cut out and were identified by mass spectrometry. As a result, four protein bands were classified into glycosyl hydrolase family 26 (GH26) mannanases. One of the identified mannanases, Man26E, contains a carbohydrate-binding module (CBM) family 59, which binds to xylan, mannan, and Avicel. Although mannose and galactose are the same as a hexose, the expression patterns of the proteins from C. cellulovorans were quite different. More interestingly, zymogram for mannanase activity showed that Man26E was detected in only LBG medium. Kosuke Yamamoto and Yutaka Tamaru Copyright © 2014 Kosuke Yamamoto and Yutaka Tamaru. All rights reserved. Antioxidants, Phytochemicals, and Cytotoxicity Studies on Phaleria macrocarpa (Scheff.) Boerl Seeds Thu, 27 Mar 2014 12:42:01 +0000 In recent years, the utilization of certain medicinal plants as therapeutic agents has drastically increased. Phaleria macrocarpa (Scheff.) Boerl is frequently used in traditional medicine. The present investigation was undertaken with the purpose of developing pharmacopoeial standards for this species. Nutritional values such as ash, fiber, protein, fat, and carbohydrate contents were investigated, and phytochemical screenings with different reagents showed the presence of flavonoids, glycosides, saponin glycosides, phenolic compounds, steroids, tannins, and terpenoids. Our results also revealed that the water fraction had the highest antioxidant activity compared to the methanol extract and other fractions. The methanol and the fractionated extracts (hexane, chloroform, ethyl acetate, and water) of P. macrocarpa seeds were also investigated for their cytotoxic effects on selected human cancer cells lines (MCF-7, HT-29, MDA-MB231, Ca Ski, and SKOV-3) and a normal human fibroblast lung cell line (MRC-5). Information from this study can be applied for future pharmacological and therapeutic evaluations of the species, and may assist in the standardization for quality, purity, and sample identification. To the best of our knowledge, this is the first report on the phytochemical screening and cytotoxic effect of the crude and fractionated extracts of P. macrocarpa seeds on selected cells lines. Ma Ma Lay, Saiful Anuar Karsani, Behrooz Banisalam, Sadegh Mohajer, and Sri Nurestri Abd Malek Copyright © 2014 Ma Ma Lay et al. All rights reserved. Therapeutic Effects of PADRE-BAFF Autovaccine on Rat Adjuvant Arthritis Tue, 25 Mar 2014 07:26:49 +0000 B cell activating factor (BAFF) is a cytokine of tumor necrosis factor family mainly produced by monocytes and dendritic cells. BAFF can regulate the proliferation, differentiation, and survival of B lymphocytes by binding with BAFF-R on B cell membrane. Accumulating evidences showed that BAFF played crucial roles and was overexpressed in various autoimmune diseases such as systemic lupus erythematous (SLE) and rheumatoid arthritis (RA). This suggests that BAFF may be a therapeutic target for these diseases. In the present study, we developed a BAFF therapeutic vaccine by coupling a T helper cell epitope AKFVAAWTLKAA (PADRE) to the N terminus of BAFF extracellular domains (PADRE-BAFF) and expressed this fusion protein in Escherichia coli. The purified vaccine can induce high titer of neutralizing BAFF antibodies and ameliorate the syndrome of complete Freund’s adjuvant (CFA) induced rheumatoid arthritis in rats. Our data indicated that the BAFF autovaccine may be a useful candidate for the treatment of some autoimmune diseases associated with high level of BAFF. Guo-dong Feng, Xiao-chang Xue, Mei-li Gao, Xian-feng Wang, Zhen Shu, Nan Mu, Yuan Gao, Zeng-lu Wang, Qiang Hao, Wei-na Li, Meng Li, Cun Zhang, Wei Zhang, and Ying-qi Zhang Copyright © 2014 Guo-dong Feng et al. All rights reserved. Bacteriostatic Antimicrobial Combination: Antagonistic Interaction between Epsilon-Viniferin and Vancomycin against Methicillin-Resistant Staphylococcus aureus Mon, 24 Mar 2014 16:30:35 +0000 Stilbenoids have been considered as an alternative phytotherapeutic treatment against methicillin-resistant Staphylococcus aureus (MRSA) infection. The combined effect of ε-viniferin and johorenol A with the standard antibiotics, vancomycin and linezolid, was assessed against MRSA ATCC 33591 and HUKM clinical isolate. The minimum inhibitory concentration (MIC) value of the individual tested compounds and the fractional inhibitory concentration index (FICI) value of the combined agents were, respectively, determined using microbroth dilution test and microdilution checkerboard (MDC) method. Only synergistic outcome from checkerboard test will be substantiated for its rate of bacterial killing using time-kill assay. The MIC value of ε-viniferin against ATCC 33591 and johorenol A against both strains was 0.05 mg/mL whereas HUKM strain was susceptible to 0.1 mg/mL of ε-viniferin. MDC study showed that only combination between ε-viniferin and vancomycin was synergistic against ATCC 33591 (FICI 0.25) and HUKM (FICI 0.19). All the other combinations (ε-viniferin-linezolid, johorenol A-vancomycin, and johorenol A-linezolid) were either indifferent or additive against both strains. However, despite the FICI value showing synergistic effect for ε-viniferin-vancomycin, TKA analysis displayed antagonistic interaction with bacteriostatic action against both strains. As conclusion, ε-viniferin can be considered as a bacteriostatic stilbenoid as it antagonized the bactericidal activity of vancomycin. These findings therefore disputed previous report that ε-viniferin acted in synergism with vancomycin but revealed that it targets similar site in close proximity to vancomycin’s action, possibly at the bacterial membrane protein. Hence, this combination has a huge potential to be further studied and developed as an alternative treatment in combating MRSA in future. Dayang Fredalina Basri, Lee Wee Xian, Nur Indah Abdul Shukor, and Jalifah Latip Copyright © 2014 Dayang Fredalina Basri et al. All rights reserved. From Structure to Catalysis: Recent Developments in the Biotechnological Applications of Lipases Mon, 24 Mar 2014 06:51:48 +0000 Microbial lipases are highly appreciated as biocatalysts due to their peculiar characteristics such as the ability to utilize a wide range of substrates, high activity and stability in organic solvents, and regio- and/or enantioselectivity. These enzymes are currently being applied in a variety of biotechnological processes, including detergent preparation, cosmetics and paper production, food processing, biodiesel and biopolymer synthesis, and the biocatalytic resolution of pharmaceutical derivatives, esters, and amino acids. However, in certain segments of industry, the use of lipases is still limited by their high cost. Thus, there is a great interest in obtaining low-cost, highly active, and stable lipases that can be applied in several different industrial branches. Currently, the design of specific enzymes for each type of process has been used as an important tool to address the limitations of natural enzymes. Nowadays, it is possible to “order” a “customized” enzyme that has ideal properties for the development of the desired bioprocess. This review aims to compile recent advances in the biotechnological application of lipases focusing on various methods of enzyme improvement, such as protein engineering (directed evolution and rational design), as well as the use of structural data for rational modification of lipases in order to create higher active and selective biocatalysts. Cristiane D. Anobom, Anderson S. Pinheiro, Rafael A. De-Andrade, Erika C. G. Aguieiras, Guilherme C. Andrade, Marcelo V. Moura, Rodrigo V. Almeida, and Denise M. Freire Copyright © 2014 Cristiane D. Anobom et al. All rights reserved. Genetic Analysis of Diversity within a Chinese Local Sugarcane Germplasm Based on Start Codon Targeted Polymorphism Mon, 24 Mar 2014 00:00:00 +0000 In-depth information on sugarcane germplasm is the basis for its conservation and utilization. Data on sugarcane molecular markers are limited for the Chinese sugarcane germplasm collections. In the present study, 20 start codon targeted (SCoT) marker primers were designed to assess the genetic diversity among 107 sugarcane accessions within a local sugarcane germplasm collection. These primers amplified 176 DNA fragments, of which 163 were polymorphic (92.85%). Polymorphic information content (PIC) values ranged from 0.783 to 0.907 with a mean of 0.861. Unweighted pair group method of arithmetic averages (UPGMA) cluster analysis of the SCoT marker data divided the 107 sugarcane accessions into six clusters at 0.674 genetic similarity coefficient level. Relatively abundant genetic diversity was observed among ROC22, ROC16, and ROC10, which occupied about 80% of the total sugarcane acreage in China, indicating their potential breeding value on Mainland China. Principal component analysis (PCA) partitioned the 107 sugarcane accessions into two major groups, the Domestic Group and the Foreign Introduction Group. Each group was further divided based on institutions, where the sugarcane accessions were originally developed. The knowledge of genetic diversity among the local sugarcane germplasm provided foundation data for managing sugarcane germplasm, including construction of a core collection and regional variety distribution and subrogation. Youxiong Que, Yongbao Pan, Yunhai Lu, Cui Yang, Yuting Yang, Ning Huang, and Liping Xu Copyright © 2014 Youxiong Que et al. All rights reserved. Mitogenic Effects of Phosphatidylcholine Nanoparticles on MCF-7 Breast Cancer Cells Thu, 20 Mar 2014 17:38:47 +0000 Lecithins, mainly composed of the phospholipids phosphatidylcholines (PC), have many different uses in the pharmaceutical and clinical field. PC are involved in structural and biological functions as membrane trafficking processes and cellular signaling. Considering the increasing applications of lecithin-based nanosystems for the delivery of therapeutic agents, the aim of the present work was to determine the effects of phosphatidylcholine nanoparticles over breast cancer cellular proliferation and signaling. PC dispersions at 0.01 and 0.1% (w/v) prepared in buffer pH 7.0 and 5.0 were studied in the MCF-7 breast cancer cell line. Neutral 0.1% PC-derived nanoparticles induced the activation of the MEK-ERK1/2 pathway, increased cell viability and induced a 1.2 fold raise in proliferation. These biological effects correlated with the increase of epidermal growth factor receptor (EGFR) content and its altered cellular localization. Results suggest that nanoparticles derived from PC dispersion prepared in buffer pH 7.0 may induce physicochemical changes in the plasma membrane of cancer cells which may affect EGFR cellular localization and/or activity, increasing activation of the MEK-ERK1/2 pathway and inducing proliferation. Results from the present study suggest that possible biological effects of delivery systems based on lecithin nanoparticles should be taken into account in pharmaceutical formulation design. Yamila B. Gándola, Sebastián E. Pérez, Pablo E. Irene, Ana I. Sotelo, Johanna G. Miquet, Gerardo R. Corradi, Adriana M. Carlucci, and Lorena Gonzalez Copyright © 2014 Yamila B. Gándola et al. All rights reserved. The Design of a Quantitative Western Blot Experiment Sun, 16 Mar 2014 12:19:16 +0000 Western blotting is a technique that has been in practice for more than three decades that began as a means of detecting a protein target in a complex sample. Although there have been significant advances in both the imaging and reagent technologies to improve sensitivity, dynamic range of detection, and the applicability of multiplexed target detection, the basic technique has remained essentially unchanged. In the past, western blotting was used simply to detect a specific target protein in a complex mixture, but now journal editors and reviewers are requesting the quantitative interpretation of western blot data in terms of fold changes in protein expression between samples. The calculations are based on the differential densitometry of the associated chemiluminescent and/or fluorescent signals from the blots and this now requires a fundamental shift in the experimental methodology, acquisition, and interpretation of the data. We have recently published an updated approach to produce quantitative densitometric data from western blots (Taylor et al., 2013) and here we summarize the complete western blot workflow with a focus on sample preparation and data analysis for quantitative western blotting. Sean C. Taylor and Anton Posch Copyright © 2014 Sean C. Taylor and Anton Posch. All rights reserved. Cloning and Expression of a Cytosolic HSP90 Gene in Chlorella vulgaris Thu, 13 Mar 2014 08:28:28 +0000 Heat shock protein 90 (HSP90), a highly conserved molecular chaperone, plays essential roles in folding, keeping structural integrity, and regulating the subset of cytosolic proteins. We cloned the cDNA of Chlorella vulgaris HSP90 (named CvHSP90) by combining homology cloning with rapid amplification of cDNA ends (RACE). Sequence analysis indicated that CvHSP90 is a cytosolic member of the HSP90 family. Quantitative RT-PCR was applied to determine the expression level of messenger RNA (mRNA) in CvHSP90 under different stress conditions. C. vulgaris was kept in different temperatures (5–45°C) for 1 h. The mRNA expression level of CvHSP90 increased with temperature from 5 to 10°C, went further from 35 to 40°C, and reached the maximum at 40°C. On the other hand, for C. vulgaris kept at 35°C for different durations, the mRNA expression level of CvHSP90 increased gradually and reached the peak at 7 h and then declined progressively. In addition, the expression level of CvHSP90 at 40 or 45 in salinity () was almost fourfold of that at 25 in salinity () for 2 h. Therefore, CvHSP90 may be a potential biomarker to monitor environment changes. Zhengyi Liu, Lei Zhang, Yang Pu, Zhaopu Liu, Zhiling Li, Yushan Zhao, and Song Qin Copyright © 2014 Zhengyi Liu et al. All rights reserved. Plant Virus Expression Vector Development: New Perspectives Thu, 13 Mar 2014 00:00:00 +0000 Plant made biologics have elicited much attention over recent years for their potential in assisting those in developing countries who have poor access to modern medicine. Additional applications such as the stockpiling of vaccines against pandemic infectious diseases or potential biological warfare agents are also under investigation. Plant virus expression vectors represent a technology that enables high levels of pharmaceutical proteins to be produced in a very short period of time. Recent advances in research and development have brought about the generation of superior virus expression systems which can be readily delivered to the host plant in a manner that is both efficient and cost effective. This review presents recent innovations in plant virus expression systems and their uses for producing biologics from plants. Kathleen Hefferon Copyright © 2014 Kathleen Hefferon. All rights reserved. Biotechnology and Green Chemistry Wed, 12 Mar 2014 16:40:57 +0000 Bernardo Dias Ribeiro, Isabel Marrucho, Luciana Gonçalves, and Maria Alice Z. Coelho Copyright © 2014 Bernardo Dias Ribeiro et al. All rights reserved. Chemical Constituents from the Fruits of Forsythia suspensa and Their Antimicrobial Activity Wed, 12 Mar 2014 09:36:36 +0000 Lignans and phenylethanoid glycosides purified from Forsythia suspensa were reported to display various bioactivities in the previous literature, including the antimicrobial activity. Therefore, the present research is aimed to purify and identify the chemical constituents of the methanol extracts of fruits of F. suspensa. The methanol extracts of fruits of F. suspensa were fractionated and further purified with the assistance of column chromatography to afford totally thirty-four compounds. Among these isolates, 3β-acetoxy-20α-hydroxyursan-28-oic acid (1) was reported from the natural sources for the first time. Some of the purified principles were subjected to the antimicrobial activity examinations against Escherichia coli to explore new natural lead compounds. Ping-Chung Kuo, Guo-Feng Chen, Mei-Lin Yang, Ya-Hua Lin, and Chi-Chung Peng Copyright © 2014 Ping-Chung Kuo et al. All rights reserved. Comparative Evaluation of Recombinant Protein Production in Different Biofactories: The Green Perspective Wed, 12 Mar 2014 09:01:16 +0000 In recent years, the production of recombinant pharmaceutical proteins in heterologous systems has increased significantly. Most applications involve complex proteins and glycoproteins that are difficult to produce, thus promoting the development and improvement of a wide range of production platforms. No individual system is optimal for the production of all recombinant proteins, so the diversity of platforms based on plants offers a significant advantage. Here, we discuss the production of four recombinant pharmaceutical proteins using different platforms, highlighting from these examples the unique advantages of plant-based systems over traditional fermenter-based expression platforms. Matilde Merlin, Elisa Gecchele, Stefano Capaldi, Mario Pezzotti, and Linda Avesani Copyright © 2014 Matilde Merlin et al. All rights reserved. A Purified Recombinant Lipopeptide as Adjuvant for Cancer Immunotherapy Tue, 11 Mar 2014 13:33:37 +0000 Synthetic lipopeptides have been widely used as vaccine adjuvants to enhance immune responses. The present study demonstrated that the tryptic N-terminal fragment of the lipoprotein rlipo-D1E3 (lipo-Nter) induces superior antitumor effects compared to a synthetic lipopeptide. The lipo-Nter was purified and formulated with protein or peptide vaccines to determine if lipo-Nter could be used as a novel adjuvant and could induce antitumor immunity in a cervical cancer model. Purified lipo-Nter activated the maturation of bone marrow-derived dendritic cells (BM-DCs), leading to the secretion of TNF-α through TLR2/6 but not TLR1/2. A recombinant mutant HPV16 E7 (rE7m) protein was mixed with lipo-Nter to immunize the mice; the anti-E7 antibody titers were increased, and the T helper cells were skewed toward the Th1 fate (increased IL-2 and decreased IL-5 secretion). Single-dose injection of rE7m and lipo-Nter inhibited tumor growth, but the injection of rE7m alone did not. Accordingly, lipo-Nter also enhanced the antitumor immunity of the E7-derived peptide but not the synthetic lipopeptide (Pam3CSK4). We demonstrated that the lipo-Nter of a bacterial-derived recombinant lipoprotein is a novel adjuvant that could be used for the development of a new generation of vaccines. Ying-Chyi Song, Hsueh-Hung Liu, I-Hua Chen, Hsin-Wei Chen, Pele Chong, Chih-Hsiang Leng, and Shih-Jen Liu Copyright © 2014 Ying-Chyi Song et al. All rights reserved. Effect of Age-Related Cartilage Turnover on Serum C-Telopeptide of Collagen Type II and Osteocalcin Levels in Growing Rabbits with and without Surgically Induced Osteoarthritis Wed, 05 Mar 2014 13:23:09 +0000 This study aims to determine the effect of age-related cartilage turnover on the serum C-telopeptide of type II collagen (CTX-II) and osteocalcin (OC) levels in growing rabbits with and without surgically induced osteoarthritis. Twenty-four New Zealand male 3-month-old rabbits were randomized into three operated groups (n = 6 per group, with surgically induced osteroarthritis in the right knee; after blood sampling, the knees were harvested following euthanization at 2, 3, and 6 months after surgery) and a control group (n = 6, blood samples were obtained monthly between 3 and 15 months). Histomorphologically, the medial femoral condyles, particularly the central parts, harbored the most severe osteoarthritic changes among the operated rabbits. The serum levels of CTX-II and OC decreased in the controls from 3 to 11 months and then remained stable. No significant differences in the serum CTX-II and OC levels between the osteoarthritic rabbits and controls were observed. The osteoarthritic-to-normal ratios (ONRs, the ratios of serum CTX-II or OC levels in osteoarthritic rabbits to those of the controls at same ages) enabled an overall assessment of osteoarthritis and age-related cartilage turnover. Elevated CTX-II ONRs were observed in rabbits with mild to advanced osteoarthritis. However, the OC ONRs were unhelpful in assessing osteoarthritic growing rabbits. Chung-Cheng Huang, Chen-Chang Lee, Ching-Jen Wang, Feng-Sheng Wang, Hsuan-Ying Huang, Shu-Hang Ng, Chia-Yi Tseng, and Sheung-Fat Ko Copyright © 2014 Chung-Cheng Huang et al. All rights reserved. Dissolution of Arsenic Minerals Mediated by Dissimilatory Arsenate Reducing Bacteria: Estimation of the Physiological Potential for Arsenic Mobilization Sun, 02 Mar 2014 11:22:27 +0000 The aim of this study was characterization of the isolated dissimilatory arsenate reducing bacteria in the context of their potential for arsenic removal from primary arsenic minerals through reductive dissolution. Four strains, Shewanella sp. OM1, Pseudomonas sp. OM2, Aeromonas sp. OM4, and Serratia sp. OM17, capable of anaerobic growth with As (V) reduction, were isolated from microbial mats from an ancient gold mine. All of the isolated strains: (i) produced siderophores that promote dissolution of minerals, (ii) were resistant to dissolved arsenic compounds, (iii) were able to use the dissolved arsenates as the terminal electron acceptor, and (iii) were able to use copper minerals containing arsenic minerals (e.g., enargite) as a respiratory substrate. Based on the results obtained in this study, we postulate that arsenic can be released from some As-bearing polymetallic minerals (such as copper ore concentrates or middlings) under reductive conditions by dissimilatory arsenate reducers in indirect processes. Drewniak Lukasz, Rajpert Liwia, Mantur Aleksandra, and Sklodowska Aleksandra Copyright © 2014 Drewniak Lukasz et al. All rights reserved. Current Biotechnological Advancements on Sustainable Metal and Nutrient Removal Thu, 27 Feb 2014 13:52:25 +0000 Li-Yuan Chai, Chong-Jian Tang, Qaisar Mahmood, and Xian-Wei Liu Copyright © 2014 Li-Yuan Chai et al. All rights reserved. Antioxidant Activity of Extract and Its Major Constituents from Okra Seed on Rat Hepatocytes Injured by Carbon Tetrachloride Thu, 27 Feb 2014 12:07:59 +0000 The antioxidant activities and protective effects of total phenolic extracts (TPE) and their major components from okra seeds on oxidative stress induced by carbon tetrachloride (CCl4) in rat hepatocyte cell line were investigated. The major phenolic compounds were identified as quercetin 3-O-glucosyl () glucoside (QDG) and quercetin 3-O-glucoside (QG). TPE, QG, and QDG from okra seeds exhibited excellent reducing power and free radical scavenging capabilities including α, α-diphenyl-β-picrylhydrazyl (DPPH), superoxide anions, and hydroxyl radical. Overall, DPPH radical scavenging activity and reducing power of QG and QDG were higher than those of TPE while superoxide and hydroxyl radical scavenging activities of QG and TPE were higher than those of QDG. Furthermore, TPE, QG, and QDG pretreatments significantly alleviated the cytotoxicity of CCl4 on rat hepatocytes, with attenuated lipid peroxidation, increased SOD and CAT activities, and decreased GPT and GOT activities. The protective effects of TPE and QG on rat hepatocytes were stronger than those of QDG. However, the cytotoxicity of CCl4 on rat hepatocytes was not affected by TPE, QG, and QDG posttreatments. It was suggested that the protective effects of TPE, QG, and QDG on rat hepatocyte against oxidative stress were related to the direct antioxidant capabilities and the induced antioxidant enzymes activities. Lianmei Hu, Wenlan Yu, Ying Li, Nagendra Prasad, and Zhaoxin Tang Copyright © 2014 Lianmei Hu et al. All rights reserved. Effect of Acute Gamma Irradiation on Curcuma alismatifolia Varieties and Detection of DNA Polymorphism through SSR Marker Tue, 25 Feb 2014 05:53:55 +0000 The effects of eight different doses (0, 10, 20, 25, 35, 40, 60, and 100 Gy) of acute gamma irradiation on 44 (three varieties of Curcuma alismatifolia: Chiang Mai Red, Sweet Pink, Kimono Pink, and one Curcuma hybrid (Doi Tung 554) individual plants were investigated. Radiation sensitivity tests revealed that the LD50 values of the varieties were achieved at 21 Gy for Chiang Mai Red, 23 Gy for Sweet Pink, 25 Gy for Kimono Pink, and 28 Gy for Doi Tung 554. From the analysis of variance (ANOVA), significant variations were observed for vegetative traits, flowering development, and rhizome characteristics among the four varieties of Curcuma alismatifolia and dose levels as well as the dose × variety interaction. In irradiated plants, the leaf length, leaf width, inflorescence length, the number of true flowers, the number of pink bracts, number of shoots, plant height, rhizome size, number of storage roots, and number of new rhizomes decreased significantly () as the radiation dose increased. The cophenetic correlation coefficient (CCC) between genetic dissimilarity matrix estimated from the morphological characters and the UPGMA clustering method was , showing a proof fit. In terms of genetic variation among the acutely irradiated samples, the number of presumed alleles revealed by simple sequence repeats ranged from two to seven alleles with a mean value of 3.1, 4.5, and 5.3 alleles per locus for radiation doses of 0, 10, and 20 Gy, respectively. The average values of the effective number of alleles, Nei’s gene diversity, and Shannon’s information index were 2.5–3.2, 0.51–0.66, and 0.9–1.3, respectively. The constructed dendrogram grouped the entities into seven clusters. Principal component analysis (PCA) supported the clustering results. Consequently, it was concluded that irradiation with optimum doses of gamma rays efficiently induces mutations in Curcuma alismatifolia varieties. Sima Taheri, Thohirah Lee Abdullah, Zaiton Ahmad, and Nur Ashikin Psyquay Abdullah Copyright © 2014 Sima Taheri et al. All rights reserved. Microwave Assisted Enzymatic Kinetic Resolution of (±)-1-Phenyl-2-propyn-1-ol in Nonaqueous Media Sun, 23 Feb 2014 07:10:45 +0000 Kinetic resolution of 1-phenyl-2-propyn-1-ol, an important chiral synthon, was studied through trans-esterification with acyl acetate to investigate synergism between microwave irradiation and enzyme catalysis. Lipases from different microbial origins were employed for the kinetic resolution of (R/S)-1-phenyl-2-propyn-1-ol, among which Candida antarctica lipase B, immobilized on acrylic resin (Novozym 435), was found to be the best catalyst in n-hexane as solvent. Vinyl acetate was the most effective among different acyl esters studied. The effect of various parameters was studied in a systematic manner. Definite synergism between microwave and enzyme was observed. The initial rate was improved around 1.28 times under microwave irradiation than conventional heating. Under optimum conditions, maximum conversion (48.78%) and high enantiomeric excess (93.25%) were obtained in 2 h. From modeling studies, it is concluded that the reaction follows the Ping-Pong bi-bi mechanism with dead end alcohol inhibition. Kinetic parameters were obtained by using nonlinear regression. This process is green, clean, and easily scalable as compared to the chemical process. Saravanan Devendran and Ganapati D. Yadav Copyright © 2014 Saravanan Devendran and Ganapati D. Yadav. All rights reserved. In Vitro Antiviral Activity of Circular Triple Helix Forming Oligonucleotide RNA towards Feline Infectious Peritonitis Virus Replication Thu, 20 Feb 2014 16:38:20 +0000 Feline Infectious Peritonitis (FIP) is a severe fatal immune-augmented disease in cat population. It is caused by FIP virus (FIPV), a virulent mutant strain of Feline Enteric Coronavirus (FECV). Current treatments and prophylactics are not effective. The in vitro antiviral properties of five circular Triple-Helix Forming Oligonucleotide (TFO) RNAs (TFO1 to TFO5), which target the different regions of virulent feline coronavirus (FCoV) strain FIPV WSU 79-1146 genome, were tested in FIPV-infected Crandell-Rees Feline Kidney (CRFK) cells. RT-qPCR results showed that the circular TFO RNAs, except TFO2, inhibit FIPV replication, where the viral genome copy numbers decreased significantly by 5-fold log10 from 1014 in the virus-inoculated cells to 109 in the circular TFO RNAs-transfected cells. Furthermore, the binding of the circular TFO RNA with the targeted viral genome segment was also confirmed using electrophoretic mobility shift assay. The strength of binding kinetics between the TFO RNAs and their target regions was demonstrated by NanoITC assay. In conclusion, the circular TFOs have the potential to be further developed as antiviral agents against FIPV infection. Oi Kuan Choong, Parvaneh Mehrbod, Bimo Ario Tejo, and Abdul Rahman Omar Copyright © 2014 Oi Kuan Choong et al. All rights reserved. Production of Biodiesel from Chlorella sp. Enriched with Oyster Shell Extracts Mon, 17 Feb 2014 08:35:56 +0000 This study investigated the cultivation of the marine microalga Chlorella sp. without supplying an inorganic carbon source, but instead with enriching the media with extracts of oyster shells pretreated by a high-pressure homogenization process. The pretreated oyster shells were extracted by a weak acid, acetic acid, that typically has harmful effects on cell growth and also poses environmental issues. The concentration of the residual dissolved carbon dioxide in the medium was sufficient to maintain cell growth at 32 ppm and pH 6.5 by only adding 5% (v/v) of oyster shell extracts. Under this condition, the maximum cell density observed was 2.74 g dry wt./L after 27 days of cultivation. The total lipid content was also measured as 18.1 (%, w/w), and this value was lower than the 23.6 (%, w/w) observed under nitrogen deficient conditions or autotrophic conditions. The fatty acid compositions of the lipids were also measured as 10.9% of C16:1 and 16.4% of C18:1 for the major fatty acids, which indicates that the biodiesel from this culture process should be a suitable biofuel. These results suggest that oyster shells, environmental waste from the food industry, can be used as a nutrient and carbon source with seawater, and this reused material should be important for easily scaling up the process for an outdoor culture system. Cheol Soon Choi, Woon Yong Choi, Do Hyung Kang, and Hyeon Yong Lee Copyright © 2014 Cheol Soon Choi et al. All rights reserved. Impact Assessment of Cadmium Toxicity and Its Bioavailability in Human Cell Lines (Caco-2 and HL-7702) Sun, 16 Feb 2014 13:58:20 +0000 Cadmium (Cd) is a widespread environmental toxic contaminant, which causes serious health-related problems. In this study, human intestinal cell line (Caco-2 cells) and normal human liver cell line (HL-7702 cells) were used to investigate the toxicity and bioavailability of Cd to both cell lines and to validate these cell lines as in vitro models for studying Cd accumulation and toxicity in human intestine and liver. Results showed that Cd uptake by both cell lines increased in a dose-dependent manner and its uptake by Caco-2 cells (720.15 µg mg−1 cell protein) was significantly higher than HL-7702 cells (229.01 µg mg−1 cell protein) at 10 mg L−1. A time- and dose-dependent effect of Cd on cytotoxicity assays (LDH release, MTT assay) was observed in both Cd-treated cell lines. The activities of antioxidant enzymes and differentiation markers (SOD, GPX, and AKP) of the HL-7702 cells were higher than those of Caco-2 cells, although both of them decreased significantly with raising Cd levels. The results from the present study indicate that Cd above a certain level inhibits cellular antioxidant activities and HL-7702 cells are more sensitive to Cd exposure than Caco-2 cells. However, Cd concentrations <0.5 mg L−1 pose no toxic effects on both cell lines. Rukhsanda Aziz, M. T. Rafiq, Jie Yang, Di Liu, Lingli Lu, Zhenli He, M. K. Daud, Tingqiang Li, and Xiaoe Yang Copyright © 2014 Rukhsanda Aziz et al. All rights reserved. Enhancement of Biodiesel Production from Marine Alga, Scenedesmus sp. through In Situ Transesterification Process Associated with Acidic Catalyst Thu, 13 Feb 2014 12:04:43 +0000 The aim of this study was to increase the yield of biodiesel produced by Scenedesmus sp. through in situ transesterification by optimizing various process parameters. Based on the orthogonal matrix analysis for the acidic catalyst, the effects of the factors decreased in the order of reaction temperature (47.5%) > solvent quantity (26.7%) > reaction time (17.5%) > catalyst amount (8.3%). Based on a Taguchi analysis, the effects of the factors decreased in the order of solvent ratio (34.36%) > catalyst (28.62%) > time (19.72%) > temperature (17.32%). The overall biodiesel production appeared to be better using NaOH as an alkaline catalyst rather than using H2SO4 in an acidic process, at 55.07 ± 2.18% (based on lipid weight) versus 48.41 ± 0.21%. However, in considering the purified biodiesel, it was found that the acidic catalyst was approximately 2.5 times more efficient than the alkaline catalyst under the following optimal conditions: temperature of 70°C (level 2), reaction time of 10 hrs (level 2), catalyst amount of 5% (level 3), and biomass to solvent ratio of 1 : 15 (level 2), respectively. These results clearly demonstrated that the acidic solvent, which combined oil extraction with in situ transesterification, was an effective catalyst for the production of high-quantity, high-quality biodiesel from a Scenedesmus sp. Ga Vin Kim, WoonYong Choi, DoHyung Kang, ShinYoung Lee, and HyeonYong Lee Copyright © 2014 Ga Vin Kim et al. All rights reserved. Degradation Potential of Protocatechuate 3,4-Dioxygenase from Crude Extract of Stenotrophomonas maltophilia Strain KB2 Immobilized in Calcium Alginate Hydrogels and on Glyoxyl Agarose Wed, 12 Feb 2014 12:14:44 +0000 Microbial intradiol dioxygenases have been shown to have a great potential for bioremediation; however, their structure is sensitive to various environmental and chemical agents. Immobilization techniques allow for the improvement of enzyme properties. This is the first report on use of glyoxyl agarose and calcium alginate as matrixes for the immobilization of protocatechuate 3,4-dioxygenase. Multipoint attachment of the enzyme to the carrier caused maintenance of its initial activity during the 21 days. Immobilization of dioxygenase in calcium alginate or on glyoxyl agarose resulted in decrease in the optimum temperature by 5°C and 10°C, respectively. Entrapment of the enzyme in alginate gel shifted its optimum pH towards high-alkaline pH while immobilization of the enzyme on glyoxyl agarose did not influence pH profile of the enzyme. Protocatechuate 3,4-dioygenase immobilized in calcium alginate showed increased activity towards 2,5-dihydroxybenzoate, caffeic acid, 2,3-dihydroxybenzoate, and 3,5-dihydroxybenzoate. Slightly lower activity of the enzyme was observed after its immobilization on glyoxyl agarose. Entrapment of the enzyme in alginate gel protected it against chelators and aliphatic alcohols while its immobilization on glyoxyl agarose enhanced enzyme resistance to inactivation by metal ions. Urszula Guzik, Katarzyna Hupert-Kocurek, Marta Krysiak, and Danuta Wojcieszyńska Copyright © 2014 Urszula Guzik et al. All rights reserved. Chemodynamics of Methyl Parathion and Ethyl Parathion: Adsorption Models for Sustainable Agriculture Thu, 06 Feb 2014 07:27:14 +0000 The toxicity of organophosphate insecticides for nontarget organism has been the subject of extensive research for sustainable agriculture. Pakistan has banned the use of methyl/ethyl parathions, but they are still illegally used. The present study is an attempt to estimate the residual concentration and to suggest remedial solution of adsorption by different types of soils collected and characterized for physicochemical parameters. Sorption of pesticides in soil or other porous media is an important process regulating pesticide transport and degradation. The percentage removal of methyl parathion and ethyl parathion was determined through UV-Visible spectrophotometer at 276 nm and 277 nm, respectively. The results indicate that agricultural soil as compared to barren soil is more efficient adsorbent for both insecticides, at optimum batch condition of pH 7. The equilibrium between adsorbate and adsorbent was attained in 12 hours. Methyl parathion is removed more efficiently (by seven orders of magnitude) than ethyl parathion. It may be attributed to more available binding sites and less steric hindrance of methyl parathion. Adsorption kinetics indicates that a good correlation exists between distribution coefficient (Kd) and soil organic carbon. A general increase in Kd is noted with increase in induced concentration due to the formation of bound or aged residue. Noshabah Tabassum, Uzaira Rafique, Khaled S. Balkhair, and Muhammad Aqeel Ashraf Copyright © 2014 Noshabah Tabassum et al. All rights reserved. Use of Synthetic Single-Stranded Oligonucleotides as Artificial Test Soiling for Validation of Surgical Instrument Cleaning Processes Mon, 03 Feb 2014 06:58:16 +0000 Surgical instruments are often strongly contaminated with patients’ blood and tissues, possibly containing pathogens. The reuse of contaminated instruments without adequate cleaning and sterilization can cause postoperative inflammation and the transmission of infectious diseases from one patient to another. Thus, based on the stringent sterility requirements, the development of highly efficient, validated cleaning processes is necessary. Here, we use for the first time synthetic single-stranded DNA (ssDNA_ODN), which does not appear in nature, as a test soiling to evaluate the cleaning efficiency of routine washing processes. Stainless steel test objects were coated with a certain amount of ssDNA_ODN. After cleaning, the amount of residual ssDNA_ODN on the test objects was determined using quantitative real-time PCR. The established method is highly specific and sensitive, with a detection limit of 20 fg, and enables the determination of the cleaning efficiency of medical cleaning processes under different conditions to obtain optimal settings for the effective cleaning and sterilization of instruments. The use of this highly sensitive method for the validation of cleaning processes can prevent, to a significant extent, the insufficient cleaning of surgical instruments and thus the transmission of pathogens to patients. Nadja Wilhelm, Nadja Perle, Robert Simmoteit, Christian Schlensak, Hans P. Wendel, and Meltem Avci-Adali Copyright © 2014 Nadja Wilhelm et al. All rights reserved. Induction of Apoptotic Effects of Antiproliferative Protein from the Seeds of Borreria hispida on Lung Cancer (A549) and Cervical Cancer (HeLa) Cell Lines Wed, 29 Jan 2014 13:33:18 +0000 A 35 KDa protein referred to as F3 was purified from the seeds of Borreria hispida by precipitation with 80% ammonium sulphate and gel filtration on Sephadex G-100 column. RP-HPLC analysis of protein fraction (F3) on an analytical C-18 column produced a single peak, detected at 220 nm. F3 showed an apparent molecular weight of 35 KDa by SDS PAGE and MALDI-TOF-MS analyses. Peptide mass fingerprinting analysis of F3 showed the closest homology with the sequence of 1-aminocyclopropane-1-carboxylate deaminase of Pyrococcus horikoshii. The protein (F3) exhibited significant cytotoxic activity against lung (A549) and cervical (HeLa) cancer cells in a dose-dependent manner at concentrations ranging from 10 µg to 1000 µg/mL, as revealed by the MTT assay. Cell cycle analysis revealed the increased growth of sub-G0 population in both cell lines exposed to a concentration of 1000 µg/mL of protein fraction F3 as examined from flow cytometry. This is the first report of a protein from the seeds of Borreria hispida with antiproliferative and apoptotic activity in lung (A549) and cervical (HeLa) cancer cells. S. Rupachandra and D. V. L. Sarada Copyright © 2014 S. Rupachandra and D. V. L. Sarada. All rights reserved. Demonstration of Redox Potential of Metschnikowia koreensis for Stereoinversion of Secondary Alcohols/1,2-Diols Mon, 27 Jan 2014 14:07:47 +0000 The present work reports the Metschnikowia koreensis-catalyzed one-pot deracemization of secondary alcohols/1,2-diols and their derivatives with in vivo cofactor regeneration. Reaction is stereoselective and proceeds with sequential oxidation of (R)-secondary alcohols to the corresponding ketones and the reduction of the ketones to (S)-secondary alcohols. Method is applicable to a repertoire of racemic aryl secondary alcohols and 1,2-diols establishing a wide range of substrate specificity of M. koreensis. This ecofriendly method afforded the product in high yield (88%) and excellent optical purity (>98% ee), minimizing the requirement of multistep reaction and expensive cofactor. Vachan Singh Meena, Linga Banoth, and U. C. Banerjee Copyright © 2014 Vachan Singh Meena et al. All rights reserved. Characteristics, Process Parameters, and Inner Components of Anaerobic Bioreactors Thu, 23 Jan 2014 14:02:01 +0000 The anaerobic bioreactor applies the principles of biotechnology and microbiology, and nowadays it has been used widely in the wastewater treatment plants due to their high efficiency, low energy use, and green energy generation. Advantages and disadvantages of anaerobic process were shown, and three main characteristics of anaerobic bioreactor (AB), namely, inhomogeneous system, time instability, and space instability were also discussed in this work. For high efficiency of wastewater treatment, the process parameters of anaerobic digestion, such as temperature, pH, Hydraulic retention time (HRT), Organic Loading Rate (OLR), and sludge retention time (SRT) were introduced to take into account the optimum conditions for living, growth, and multiplication of bacteria. The inner components, which can improve SRT, and even enhance mass transfer, were also explained and have been divided into transverse inner components, longitudinal inner components, and biofilm-packing material. At last, the newly developed special inner components were discussed and found more efficient and productive. Awad Abdelgadir, Xiaoguang Chen, Jianshe Liu, Xuehui Xie, Jian Zhang, Kai Zhang, Heng Wang, and Na Liu Copyright © 2014 Awad Abdelgadir et al. All rights reserved. Microbial Diversity for Biotechnology Thu, 23 Jan 2014 09:12:23 +0000 George Tsiamis, Dimitrios Karpouzas, Ameur Cherif, and Konstantinos Mavrommatis Copyright © 2014 George Tsiamis et al. All rights reserved. Microtexturing of the Conductive PEDOT:PSS Polymer for Superhydrophobic Organic Electrochemical Transistors Wed, 22 Jan 2014 14:01:47 +0000 Superhydrophobic surfaces are bioinspired, nanotechnology artifacts, which feature a reduced friction coefficient, whereby they can be used for a number of very practical applications including, on the medical side, the manipulation of biological solutions. In this work, we integrated superhydrophobic patterns with the conducting polymer PEDOT:PSS, one of the most used polymers in organic electronics because highly sensitive to ionized species in solution. In doing so, we combined geometry and materials science to obtain an advanced device where, on account of the superhydrophobicity of the system, the solutions of interest can be manipulated and, on account of the conductive PEDOT:PSS polymer, the charged molecules dispersed inside can be quantitatively measured. This original substrate preparation allowed to perform electrochemical measurements on ionized species in solution with decreasing concentration down to 10−7 molar. Moreover, it was demonstrated the ability of the device of realizing specific, combined time and space resolved analysis of the sample. Collectively, these results demonstrate how a tight, interweaving integration of different disciplines can provide realistic tools for the detection of pathologies. The scheme here introduced offers breakthrough capabilities that are expected to radically improve both the pace and the productivity of biomedical research, creating an access revolution. Francesco Gentile, Nicola Coppedè, Giuseppe Tarabella, Marco Villani, Davide Calestani, Patrizio Candeloro, Salvatore Iannotta, and Enzo Di Fabrizio Copyright © 2014 Francesco Gentile et al. All rights reserved. Biochemical and Anatomical Changes and Yield Reduction in Rice (Oryza sativa L.) under Varied Salinity Regimes Wed, 22 Jan 2014 08:21:05 +0000 Five Malaysian rice (Oryza sativa L.) varieties, MR33, MR52, MR211, MR219, and MR232, were tested in pot culture under different salinity regimes for biochemical response, physiological activity, and grain yield. Three different levels of salt stresses, namely, 4, 8, and 12 dS m−1, were used in a randomized complete block design with four replications under glass house conditions. The results revealed that the chlorophyll content, proline, sugar content, soluble protein, free amino acid, and yield per plant of all the genotypes were influenced by different salinity levels. The chlorophyll content was observed to decrease with salinity level but the proline increased with salinity levels in all varieties. Reducing sugar and total sugar increased up to 8 dS m−1 and decreased up to 12 dS m−1. Nonreducing sugar decreased with increasing the salinity levels in all varieties. Soluble protein and free amino acid also decreased with increasing salinity levels. Cortical cells of MR211 and MR232 did not show cell collapse up to 8 dS m−1 salinity levels compared to susceptible checks (IR20 and BRRI dhan29). Therefore, considering all parameters, MR211 and MR232 showed better salinity tolerance among the tested varieties. Both cluster and principal component analyses depict the similar results. M. A. Hakim, Abdul Shukor Juraimi, M. M. Hanafi, Mohd Razi Ismail, Ahmad Selamat, M. Y. Rafii, and M. A. Latif Copyright © 2014 M. A. Hakim et al. All rights reserved. Evaluation of Antioxidant Compounds, Antioxidant Activities, and Mineral Composition of 13 Collected Purslane (Portulaca oleracea L.) Accessions Tue, 21 Jan 2014 09:15:19 +0000 The methanolic extracts of 13 accessions of purslane were analyzed for their total phenol content (TPC), total flavonoid contents (TFC), and total carotenoid contents (TCC) and antioxidant activity of extracts was screened using FRAP assay and DPPH radical scavenging methods. The TPC, TFC, and TCC ranged from to  mg GAE/g DW, to  mg RE/g DW, and to  mg (β-carotene equivalent) BCE/g DW, respectively. The DPPH scavenging (IC50) activity varied between  mg/mL and  mg/mL and FRAP ranged from to  μmol TE/g DW. Among all the measured micro- and macrominerals K content was the highest followed by N, Na, Ca, Mg, P, Fe, Zn, and Mn. The overall findings proved that ornamental purslane was richer in antioxidant properties, whereas common purslane possesses more mineral contents than ornamental ones. Md. Amirul Alam, Abdul Shukor Juraimi, M. Y. Rafii, Azizah Abdul Hamid, Farzad Aslani, M. M. Hasan, Mohd Asraf Mohd Zainudin, and Md. Kamal Uddin Copyright © 2014 Md. Amirul Alam et al. All rights reserved. Construction of a Novel Liver-Targeting Fusion Interferon by Incorporation of a Plasmodium Region I-Plus Peptide Sun, 19 Jan 2014 13:48:46 +0000 Interferon alpha (IFN α) exerts a multiplicity of biological actions including antiviral, immunomodulatory, and antiproliferative effects. Administration of IFN α is the current treatment for chronic hepatitis B; however, therapy outcome has not been completely satisfactory. The systemic effects of IFN α may account for its low in vivo biological activity and multiple adverse events. The purpose of this study was to design a novel liver-targeting fusion interferon (IFN-CSP) by fusing IFN α2b with a Plasmodium region I-plus peptide, thus targeting the drug specifically to the liver. The DNA sequence encoding IFN-CSP was constructed using improved splicing by overlapping extension-PCR method, and then cloned into the pET-21b vector for protein expression in E. coli BL21 (DE3). The recombinant protein was expressed as a His-tagged protein and purified using a combination of Ni affinity and HiTrap affinity chromatography at a purity of over 95%. The final yield of biologically active IFN-CSP was up to 270 mg/L culture. The purified recombinant protein showed anti-HBV activity and liver-targeting potentiality in vitro. These data suggests that the novel fusion interferon IFN-CSP may be an excellent candidate as a liver-targeting anti-HBV agent. Xuemei Lu, Xiaobao Jin, Yanting Huang, Jie Wang, Juan Shen, Fujiang Chu, Hanfang Mei, Yan Ma, and Jiayong Zhu Copyright © 2014 Xuemei Lu et al. All rights reserved. Synergistic Exposure of Rice Seeds to Different Doses of -Ray and Salinity Stress Resulted in Increased Antioxidant Enzyme Activities and Gene-Specific Modulation of TC-NER Pathway Thu, 16 Jan 2014 10:24:21 +0000 Recent reports have underlined the potential of gamma (γ)-rays as tools for seed priming, a process used in seed industry to increase seed vigor and to enhance plant tolerance to biotic/abiotic stresses. However, the impact of γ-rays on key aspects of plant metabolism still needs to be carefully evaluated. In the present study, rice seeds were challenged with different doses of γ-rays and grown in absence/presence of NaCl to assess the impact of these treatments on the early stages of plant life. Enhanced germination efficiency associated with increase in radicle and hypocotyl length was observed, while at later stages no increase in plant tolerance to salinity stress was evident. APX, CAT, and GR were enhanced at transcriptional level and in terms of enzyme activity, indicating the activation of antioxidant defence. The profiles of DNA damage accumulation were obtained using SCGE and the implication of TC-NER pathway in DNA damage sensing and repair mechanisms is discussed. OsXPB2, OsXPD, OsTFIIS, and OsTFIIS-like genes showed differential modulation in seedlings and plantlets in response to γ-irradiation and salinity stress. Altogether, the synergistic exposure to γ-rays and NaCl resulted in enhanced oxidative stress and proper activation of antioxidant mechanisms, thus being compatible with plant survival. Anca Macovei, Bharti Garg, Shailendra Raikwar, Alma Balestrazzi, Daniela Carbonera, Armando Buttafava, Juan Francisco Jiménez Bremont, Sarvajeet Singh Gill, and Narendra Tuteja Copyright © 2014 Anca Macovei et al. All rights reserved. Comparison of Extraction Methods of Chitin from Ganoderma lucidum Mushroom Obtained in Submerged Culture Wed, 15 Jan 2014 16:01:08 +0000 The chitin was isolated from the Ganoderma lucidum submerged cultures mycelium as potential source of chitin under biotechnological processes. The extraction of chitin was carried out through 5 different assays which involved mainly three phases: pulverization of the mushroom, deproteinization of the mycelia with NaOH solution, and a process of decolorization with potassium permanganate and oxalic acid. The chitin contents extracted from 9-day mycelia were 413, 339, 87, 78, and 144 mg/g−1 (milligrams of chitin/grams of dry biomass) for A1, A2, A3, A4, and A5, respectively. Obtained chitin was characterized by X-Ray Diffraction (XRD), by Fourier transform infrared spectroscopy (FTIR), and by thermal analysis (TGA). The results showed that Ganoderma lucidum chitin has similar characteristic of chitin from different fonts. The advantage of the biotechnological processes and the fact that Ganoderma lucidum fungus may be used as a potential raw material for chitin production were demonstrated. Sandra Patricia Ospina Álvarez, David Alexander Ramírez Cadavid, Diana Marcela Escobar Sierra, Claudia Patricia Ossa Orozco, Diego Fernando Rojas Vahos, Paola Zapata Ocampo, and Lucía Atehortúa Copyright © 2014 Sandra Patricia Ospina Álvarez et al. All rights reserved. Production of Fungal Amylases Using Cheap, Readily Available Agriresidues, for Potential Application in Textile Industry Thu, 09 Jan 2014 10:53:52 +0000 The study aimed at isolation and screening of fungal amylase producer, optimization of solid state fermentation conditions for maximum amylase production by the best amylase producer, and characterization of the crude amylases, so produced. Aspergillus fumigatus NTCC1222 showed the highest amylase activity (164.1 U/mL) in secondary screening under SSF conditions and was selected for further studies. The test strain showed maximum amylase production (341.7 U/mL) and supernatant protein concentration (9.7 mg/mL) for incubation period (6 days), temperature (35°C), initial pH (6.0), nutrient salt solution as moistening agent, and beef extract as nitrogen source. Pomegranate peel produced maximum amylase activity, but wheat bran (only slightly lesser amylase activity as compared to that of pomegranate peel) was chosen for further studies, keeping in mind the seasonal availability of pomegranate peel. TLC confirmed the amylase produced to be α-type and 60 kDa was the molecular weight of the partially purified amylase. The enzyme showed maximum enzyme activity at pH 6.0, temperature of 55°C, and incubation time of 60 minutes. UV (616.0 U/mL) and chemical (814.2 U/mL) mutation enhanced amylase activity as compared to wild test strain. The study indicates that Aspergillus fumigatus NTCC1222 can be an important source of amylase and the crude enzyme, hence obtained, can be cost effectively applied in multiple sections of textile wet processing. Shalini Singh, Sanamdeep Singh, Vrinda Bali, Lovleen Sharma, and Jyoti Mangla Copyright © 2014 Shalini Singh et al. All rights reserved. Effect of Temperature on Chinese Rice Wine Brewing with High Concentration Presteamed Whole Sticky Rice Mon, 06 Jan 2014 08:04:27 +0000 Production of high quality Chinese rice wine largely depends on fermentation temperature. However, there is no report on the ethanol, sugars, and acids kinetics in the fermentation mash of Chinese rice wine treated at various temperatures. The effects of fermentation temperatures on Chinese rice wine quality were investigated. The compositions and concentrations of ethanol, sugars, glycerol, and organic acids in the mash of Chinese rice wine samples were determined by HPLC method. The highest ethanol concentration and the highest glycerol concentration both were attained at the fermentation mash treated at 23°C. The highest peak value of maltose (90 g/L) was obtained at 18°C. Lactic acid and acetic acid both achieved maximum values at 33°C. The experimental results indicated that temperature contributed significantly to the ethanol production, acid flavor contents, and sugar contents in the fermentation broth of the Chinese rice wines. Dengfeng Liu, Hong-Tao Zhang, Weili Xiong, Jianhua Hu, Baoguo Xu, Chi-Chung Lin, Ling Xu, and Lihua Jiang Copyright © 2014 Dengfeng Liu et al. All rights reserved. Degradation of Diuron by Phanerochaete chrysosporium: Role of Ligninolytic Enzymes and Cytochrome P450 Tue, 31 Dec 2013 13:31:04 +0000 The white-rot fungus Phanerochaete chrysosporium was investigated for its capacity to degrade the herbicide diuron in liquid stationary cultures. The presence of diuron increased the production of lignin peroxidase in relation to control cultures but only barely affected the production of manganese peroxidase. The herbicide at the concentration of 7 μg/mL did not cause any reduction in the biomass production and it was almost completely removed after 10 days. Concomitantly with the removal of diuron, two metabolites, DCPMU [1-(3,4-dichlorophenyl)-3-methylurea] and DCPU [(3,4-dichlorophenyl)urea], were detected in the culture medium at the concentrations of 0.74 μg/mL and 0.06 μg/mL, respectively. Crude extracellular ligninolytic enzymes were not efficient in the in vitro degradation of diuron. In addition, 1-aminobenzotriazole (ABT), a cytochrome P450 inhibitor, significantly inhibited both diuron degradation and metabolites production. Significant reduction in the toxicity evaluated by the Lactuca sativa L. bioassay was observed in the cultures after 10 days of cultivation. In conclusion, P. chrysosporium can efficiently metabolize diuron without the accumulation of toxic products. Jaqueline da Silva Coelho-Moreira, Adelar Bracht, Aline Cristine da Silva de Souza, Roselene Ferreira Oliveira, Anacharis Babeto de Sá-Nakanishi, Cristina Giatti Marques de Souza, and Rosane Marina Peralta Copyright © 2013 Jaqueline da Silva Coelho-Moreira et al. All rights reserved. Toward Personalized Cell Therapies by Using Stem Cells 2013 Sun, 29 Dec 2013 15:00:07 +0000 Ken-ichi Isobe, Herman S. Cheung, and Ji Wu Copyright © 2013 Ken-ichi Isobe et al. All rights reserved. Coronary CT Angiography in Coronary Artery Disease: Correlation between Virtual Intravascular Endoscopic Appearances and Left Bifurcation Angulation and Coronary Plaques Sat, 28 Dec 2013 08:08:00 +0000 The aim of this study is to investigate the relationship between intraluminal appearances of coronary plaques and left coronary bifurcation angle and plaque components using coronary CT virtual intravascular endoscopy (VIE). Fifty patients suspected of coronary artery disease undergoing coronary CT angiography were included in the study. The left bifurcation angle in patients with diseased left coronary artery which was measured as 94.3° ± 16.5 is significantly larger than that in patients with normal left coronary artery, which was measured as 76.5° ± 15.9 (). Irregular VIE appearances were found in 10 out of 11 patients with mixed plaques in the left anterior descending (LAD) and left circumflex (LCx), while, in 29 patients with calcified plaques in the LAD and LCx, irregular VIE appearances were only noticed in 5 patients. Using 80° as a cut-off value to determine coronary artery disease, smooth VIE appearances were found in 95% of patients (18/19) with left bifurcation angle of less than 80°, while irregular VIE appearances were observed in nearly 50% of patients (15/31) with left bifurcation angle of more than 80°. This preliminary study shows that VIE appearances of the coronary lumen are directly related to the types of plaques. Zhonghua Sun Copyright © 2013 Zhonghua Sun. All rights reserved. Intravenous Transplantation of Mesenchymal Stromal Cells to Enhance Peripheral Nerve Regeneration Thu, 26 Dec 2013 18:13:05 +0000 Peripheral nerve injury is a common and devastating complication after trauma and can cause irreversible impairment or even complete functional loss of the affected limb. While peripheral nerve repair results in some axonal regeneration and functional recovery, the clinical outcome is not optimal and research continues to optimize functional recovery after nerve repair. Cell transplantation approaches are being used experimentally to enhance regeneration. Intravenous infusion of mesenchymal stromal cells (MSCs) into spinal cord injury and stroke was shown to improve functional outcome. However, the repair potential of intravenously transplanted MSCs in peripheral nerve injury has not been addressed yet. Here we describe the impact of intravenously infused MSCs on functional outcome in a peripheral nerve injury model. Rat sciatic nerves were transected followed, by intravenous MSCs transplantation. Footprint analysis was carried out and 21 days after transplantation, the nerves were removed for histology. Labelled MSCs were found in the sciatic nerve lesion site after intravenous injection and regeneration was improved. Intravenously infused MSCs after acute peripheral nerve target the lesion site and survive within the nerve and the MSC treated group showed greater functional improvement. The results of study suggest that nerve repair with cell transplantation could lead to greater functional outcome. Stella M. Matthes, Kerstin Reimers, Insa Janssen, Christina Liebsch, Jeffery D. Kocsis, Peter M. Vogt, and Christine Radtke Copyright © 2013 Stella M. Matthes et al. All rights reserved. Cadmium-Induced Upregulation of Lipid Peroxidation and Reactive Oxygen Species Caused Physiological, Biochemical, and Ultrastructural Changes in Upland Cotton Seedlings Thu, 26 Dec 2013 09:34:31 +0000 Cadmium (Cd) toxicity was investigated in cotton cultivar (ZMS-49) using physiological, ultrastructural, and biochemical parameters. Biomass-based tolerance index decreased, and water contents increased at 500 M Cd. Photosynthetic efficiency determined by chlorophyll fluorescence and photosynthetic pigments declined under Cd stress. Cd contents were more in roots than shoots. A significant decrease in nutrient levels was found in roots and stem. A significant decrease in nutrient levels was found in roots and stems. In response to Cd stress, more MDA and ROS contents were produced in leaves than in other parts of the seedlings. Total soluble proteins were reduced in all parts except in roots at 500 M Cd. Oxidative metabolism was higher in leaves than aerial parts of the plant. There were insignificant alterations in roots and leaves ultrastructures such as a little increase in nucleoli, vacuoles, starch granules, and plastoglobuli in Cd-imposed stressful conditions. Scanning micrographs at 500 M Cd showed a reduced number of stomata as well as near absence of closed stomata. Cd depositions were located in cell wall, vacuoles, and intracellular spaces using TEM-EDX technology. Upregulation of oxidative metabolism, less ultrastructural modification, and Cd deposition in dead parts of cells show that ZMS-49 has genetic potential to resist Cd stress, which need to be explored. Muhammad Daud Khan, Lei Mei, Basharat Ali, Yue Chen, Xin Cheng, and S. J. Zhu Copyright © 2013 Muhammad Daud Khan et al. All rights reserved. Biologic Activity and Biotechnological Development of Natural Products Tue, 24 Dec 2013 08:53:39 +0000 José Carlos Tavares Carvalho, Fabio Ferreira Perazzo, Leandro Machado, and Didier Bereau Copyright © 2013 José Carlos Tavares Carvalho et al. All rights reserved. An Uncommon Plant Growth Regulator, Diethyl Aminoethyl Hexanoate, Is Highly Effective in Tissue Cultures of the Important Medicinal Plant Purple Coneflower (Echinacea purpurea L.) Mon, 23 Dec 2013 18:15:13 +0000 We investigated the effects of various concentrations of diethyl aminoethyl hexanoate (DA-6) on the regeneration and growth of adventitious buds in in vitro purple coneflower cultures. Among the 3 types of explants tested, leaf explants required higher concentrations of DA-6 than petiole and root explants in order to obtain high regeneration rates, while root explants required the lowest concentration of DA-6. Additionally, explants with higher ploidy levels were more sensitive to the addition of DA-6, while explants with lower ploidy levels required higher concentrations of DA-6 to achieve its maximal regeneration rate. Interestingly, the application of a concentration that was conducive to the regeneration of explants with lower ploidy levels was inhibitory to the regeneration of explants with higher ploidy levels. Moreover, during the growth of regenerated buds, DA-6 application significantly improved plant height and weight, root weight, root thickness, root number, primary root length, total root length, and root/top ratio. Differences in the responses of explants to supplementation with DA-6 were also observed among explants with different ploidy levels, with buds having lower ploidy levels responding to lower concentrations of DA-6. Taken together, the results of the present experiments showed that proper application of DA-6 could increase in vitro culture efficiency in purple coneflower. Xiao-Lu Chen, Jun-Jie Zhang, Rong Chen, Qing-Ling Li, Yue-Sheng Yang, and Hong Wu Copyright © 2013 Xiao-Lu Chen et al. All rights reserved. Start-Up Characteristics of a Granule-Based Anammox UASB Reactor Seeded with Anaerobic Granular Sludge Mon, 23 Dec 2013 10:22:50 +0000 The granulation of anammox sludge plays an important role in the high nitrogen removal performance of the anammox reactor. In this study, anaerobic granular sludge was selected as the seeding sludge to start up anammox reactor in order to directly obtain anammox granules. Results showed that the anammox UASB reactor was successfully started up by inoculating anaerobic granular sludge, with substrate capacity of 4435.2 mg/(L·d) and average ammonium and nitrite removal efficiency of 90.36% and 93.29%, respectively. During the start-up course, the granular sludge initially disintegrated and then reaggregated and turned red, suggesting the high anammox performance. Zn-Fe precipitation was observed on the surface of granules during the operation by SEM-EDS, which would impose inhibition to the anammox activity of the granules. Accordingly, it is suggested to relatively reduce the trace metals concentrations, of Fe and Zn in the conventional medium. The findings of this study are expected to be used for a shorter start-up and more stable operation of anammox system. Lei Xiong, Yun-Yan Wang, Chong-Jian Tang, Li-Yuan Chai, Kang-Que Xu, Yu-Xia Song, Mohammad Ali, and Ping Zheng Copyright © 2013 Lei Xiong et al. All rights reserved. Engineered Hypopharynx from Coculture of Epithelial Cells and Fibroblasts Using Poly(ester urethane) as Substratum Sun, 22 Dec 2013 18:11:32 +0000 Porous polymeric scaffolds have been much investigated and applied in the field of tissue engineering research. Poly(ester urethane) (PEU) scaffolds, comprising pores of 1–20 μm in diameter on one surface and ≥200 μm on the opposite surface and in bulk, were fabricated using phase separation method for hypopharyngeal tissue engineering. The scaffolds were grafted with silk fibroin (SF) generated from natural silkworm cocoon to enhance the scaffold’s hydrophilicity and further improve cytocompatibility to both primary epithelial cells (ECs) and fibroblasts of human hypopharynx tissue. Coculture of ECs and fibroblasts was conducted on the SF-grafted PEU scaffold (PEU-SF) to evaluate its in vitro cytocompatibility. After co-culture for 14 days, ECs were lined on the scaffold surface while fibroblasts were distributed in scaffold bulk. The results of in vivo investigation showed that PEU porous scaffold possessed good biocompatibility after it was grafted by silk fibroin. SF grafting improved the cell/tissue infiltration into scaffold bulk. Thus, PEU-SF porous scaffold is expected to be a good candidate to support the hypopharynx regeneration. Zhisen Shen, Jingjing Chen, Cheng Kang, Changfeng Gong, and Yabin Zhu Copyright © 2013 Zhisen Shen et al. All rights reserved. The Uptake Mechanism of Cd(II), Cr(VI), Cu(II), Pb(II), and Zn(II) by Mycelia and Fruiting Bodies of Galerina vittiformis Sun, 22 Dec 2013 18:05:22 +0000 Optimum concentrations of heavy metals like copper, cadmium, lead, chromium, and zinc in soil are essential in carrying out various cellular activities in minimum concentrations and hence help in sustaining all life forms, although higher concentration of these metals is lethal to most of the life forms. Galerina vittiformis, a macrofungus, was found to accumulate these heavy metals into its fleshy fruiting body in the order Pb(II) > Cd(II) > Cu(II) > Zn(II) > Cr(VI) from 50 mg/kg soil. It possesses various ranges of potential cellular mechanisms that may be involved in detoxification of heavy metals and thus increases its tolerance to heavy metal stress, mainly by producing organic acids and phytochelatins (PCs). These components help in repairing stress damaged proteins and compartmentalisation of metals to vacuoles. The stress tolerance mechanism can be deduced by various analytical tools like SEM-EDX, FTIR, and LC-MS. Production of two kinds of phytochelatins was observed in the organism in response to metal stress. Dilna Damodaran, Raj Mohan Balakrishnan, and Vidya K. Shetty Copyright © 2013 Dilna Damodaran et al. All rights reserved. Optimization of Polygalacturonase Production from a Newly Isolated Thalassospira frigidphilosprofundus to Use in Pectin Hydrolysis: Statistical Approach Thu, 19 Dec 2013 11:57:14 +0000 The present study deals with the production of cold active polygalacturonase (PGase) by submerged fermentation using Thalassospira frigidphilosprofundus, a novel species isolated from deep waters of Bay of Bengal. Nonlinear models were applied to optimize the medium components for enhanced production of PGase. Taguchi orthogonal array design was adopted to evaluate the factors influencing the yield of PGase, followed by the central composite design (CCD) of response surface methodology (RSM) to identify the optimum concentrations of the key factors responsible for PGase production. Data obtained from the above mentioned statistical experimental design was used for final optimization study by linking the artificial neural network and genetic algorithm (ANN-GA). Using ANN-GA hybrid model, the maximum PGase activity (32.54 U/mL) was achieved at the optimized concentrations of medium components. In a comparison between the optimal output of RSM and ANN-GA hybrid, the latter favored the production of PGase. In addition, the study also focused on the determination of factors responsible for pectin hydrolysis by crude pectinase extracted from T. frigidphilosprofundus through the central composite design. Results indicated 80% degradation of pectin in banana fiber at 20°C in 120 min, suggesting the scope of cold active PGase usage in the treatment of raw banana fibers. V. P. B. Rekha, Mrinmoy Ghosh, Vijayanand Adapa, Sung-Jong Oh, K. K. Pulicherla, and K. R. S. Sambasiva Rao Copyright © 2013 V. P. B. Rekha et al. All rights reserved. Wound-Healing Potential of Cultured Epidermal Sheets Is Unaltered after Lyophilization: A Preclinical Study in Comparison to Cryopreserved CES Wed, 18 Dec 2013 12:44:44 +0000 Lyophilized Cultured Epidermal Sheets (L-CES) have been reported to be as effective as the cryopreserved CES (F-CES) in treating skin ulcers. However, unlike F-CES, no preclinical study assessing wound-healing effects has been conducted for L-CES. The present study was set out to investigate the microstructure, cytokine profile, and wound-healing effects of L-CES in comparison to those of F-CES. Keratinocytes were cultured to prepare CES, followed by cryopreservation at −70°C and lyophilization. Under microscopic observation, intact cells with apparent intracellular junctions were observed in L-CES. The L-CES, like fresh CES, consisted of three to four well-maintained epidermal layers, as shown by the expression of keratins, involucrin, and p63. There were no differences in the epidermal layer or protein expression between L-CES and F-CES, and both CES were comparable to fresh CES. TGF-, EGF, VEGF, IL-1, and MMPs were detected in L-CES at levels similar to those in F-CES. In a mouse study, wounds treated with L-CES or F-CES completely healed at least 4 days faster than untreated wounds. CES-treated wounds completely healed by day 10, while the untreated wounds did not heal by day 14. Masson’s trichrome staining showed that collagen deposition in the CES-treated wounds was highly increased in the dermis of the wound center compared to that in the control wounds. Thus, this study demonstrates that L-CES is as clinically effective as F-CES for wound treatment. H. Jang, Y. H. Kim, M. K. Kim, K. H. Lee, and S. Jeon Copyright © 2013 H. Jang et al. All rights reserved. Preparative Purification of Recombinant Proteins: Current Status and Future Trends Tue, 17 Dec 2013 13:53:24 +0000 Advances in fermentation technologies have resulted in the production of increased yields of proteins of economic, biopharmaceutical, and medicinal importance. Consequently, there is an absolute requirement for the development of rapid, cost-effective methodologies which facilitate the purification of such products in the absence of contaminants, such as superfluous proteins and endotoxins. Here, we provide a comprehensive overview of a selection of key purification methodologies currently being applied in both academic and industrial settings and discuss how innovative and effective protocols such as aqueous two-phase partitioning, membrane chromatography, and high-performance tangential flow filtration may be applied independently of or in conjunction with more traditional protocols for downstream processing applications. Mayank Saraswat, Luca Musante, Alessandra Ravidá, Brian Shortt, Barry Byrne, and Harry Holthofer Copyright © 2013 Mayank Saraswat et al. All rights reserved. Combined Industrial Wastewater Treatment in Anaerobic Bioreactor Posttreated in Constructed Wetland Mon, 16 Dec 2013 08:42:27 +0000 Constructed wetland (CW) with monoculture of Arundo donax L. was investigated for the posttreatment of anaerobic bioreactor (ABR) treating combined industrial wastewater. Different dilutions of combined industrial wastewater (20, 40, 60, and 80) and original wastewater were fed into the ABR and then posttreated by the laboratory scale CW. The respective removal efficiencies of COD, BOD, TSS, nitrates, and ammonia were 80%, 78–82%, 91.7%, 88–92%, and 100% for original industrial wastewater treated in ABR. ABR was efficient in the removal of Ni, Pb, and Cd with removal efficiencies in the order of Cd (2.7%) > Ni (79%) > Pb (85%). Posttreatment of the ABR treated effluent was carried out in lab scale CW containing A. donax L. CW was effective in the removal of COD and various heavy metals present in ABR effluents. The posttreatment in CW resulted in reducing the metal concentrations to 1.95 mg/L, 0 mg/L, and 0.004 mg/L for Ni, Pb, and Cd which were within the permissible water quality standards for industrial effluents. The treatment strategy was effective and sustainable for the treatment of combined industrial wastewater. Bibi Saima Zeb, Qaisar Mahmood, Saima Jadoon, Arshid Pervez, Muhammad Irshad, Muhammad Bilal, and Zulfiqar Ahmad Bhatti Copyright © 2013 Bibi Saima Zeb et al. All rights reserved. Cyclin B1 Destruction Box-Mediated Protein Instability: The Enhanced Sensitivity of Fluorescent-Protein-Based Reporter Gene System Sun, 15 Dec 2013 16:36:21 +0000 The periodic expression and destruction of several cyclins are the most important steps for the exact regulation of cell cycle. Cyclins are degraded by the ubiquitin-proteasome system during cell cycle. Besides, a short sequence near the N-terminal of cyclin B called the destruction box (D-box; CDB) is also required. Fluorescent-protein-based reporter gene system is insensitive to analysis because of the overly stable fluorescent proteins. Therefore, in this study, we use human CDB fused with both enhanced green fluorescent protein (EGFP) at C-terminus and red fluorescent protein (RFP, DsRed) at N-terminus in the transfected human melanoma cells to examine the effects of CDB on different fluorescent proteins. Our results indicated that CDB-fused fluorescent protein can be used to examine the slight gene regulations in the reporter gene system and have the potential to be the system for screening of functional compounds in the future. Chao-Hsun Yang, Wan-Ting Kuo, Yun-Ting Chuang, Cheng-Yu Chen, and Chih-Chien Lin Copyright © 2013 Chao-Hsun Yang et al. All rights reserved. Fermentative Succinate Production: An Emerging Technology to Replace the Traditional Petrochemical Processes Thu, 12 Dec 2013 18:13:45 +0000 Succinate is a valuable platform chemical for multiple applications. Confronted with the exhaustion of fossil energy resources, fermentative succinate production from renewable biomass to replace the traditional petrochemical process is receiving an increasing amount of attention. During the past few years, the succinate-producing process using microbial fermentation has been made commercially available by the joint efforts of researchers in different fields. In this review, recent attempts and experiences devoted to reduce the production cost of biobased succinate are summarized, including strain improvement, fermentation engineering, and downstream processing. The key limitations and challenges faced in current microbial production systems are also proposed. Yujin Cao, Rubing Zhang, Chao Sun, Tao Cheng, Yuhua Liu, and Mo Xian Copyright © 2013 Yujin Cao et al. All rights reserved. Technological Aptitude and Applications of Leuconostoc mesenteroides Bioactive Strains Isolated from Algerian Raw Camel Milk Wed, 11 Dec 2013 13:45:21 +0000 Two strains (B7 and Z8) of the Leuconostoc mesenteroides subspecies mesenteroides that were isolated from Algerian camel milk from an initial pool of 13 strains and demonstrated a high ability to inhibit the growth of Listeria spp. were selected and characterised at the phenotypic and genotypic levels. Probiotic profiling and inhibition spectra against food borne pathogens in mixed cultures were also investigated. The bacteriocin produced by L. mesenteroides strain B7 was identified as leucocin B by specific PCR. In vitro studies demonstrated that both Leuconostoc mesenteroides strains exhibited a marked probiotic profile, showing high survival at low pH (2-3 and 4) in the presence of 0.5%, 1%, and 2% of bile salts and at pH 3 in the presence of 3 mg/mL pepsin. Susceptibility testing against antimicrobial agents was also performed for both strains. When tested in a mixed culture with Listeria innocua, Listeria ivanovii, or Staphylococcus aureus, strain B7 reduced the numbers of these species by 1.87, 1.78, and 1.38 log units, respectively. Consequently, these two strains were found to possess good probiotic properties in vitro and a high capacity for Listeria spp. inhibition in mixed cultures. Therefore, these strains have a favourable technological aptitude and a potential application as novel probiotic starters. Zineb Benmechernene, Hanane Fatma Chentouf, Bellil Yahia, Ghazi Fatima, Marcos Quintela-Baluja, Pilar Calo-Mata, and Jorge Barros-Velázquez Copyright © 2013 Zineb Benmechernene et al. All rights reserved. Cultivation of Isochrysis galbana in Phototrophic, Heterotrophic, and Mixotrophic Conditions Tue, 10 Dec 2013 14:08:47 +0000 This study compared the growth and biomass production of Isochrysis galbana under hetero-, mixo-, and phototrophic conditions using different organic carbon sources. The growth of I. galbana was inhibited in heterotrophy but was enhanced in mixotrophy compared to that in phototrophy. Subsequently, the influences of organic carbon and environmental factors (light and salinity) on the growth of I. galbana were further investigated. Algal dry weight increased as glycerol concentrations increased from 0 to 200 mmol and the highest algal production occurred at 50 mmol glycerol. At a range of light intensities of 25–200 μmol photons m−2 s−2, the highest algal growth rate occurred at 100 photons μmol m−2 s−2. The growth of I. galbana was significantly affected by photoperiod, and the maximal dry weight was obtained at 12 h light and 12 h dark. In the salinity test, I. galbana could grow in a wide range of salinities from 10 to 65‰, but the 35‰  salinity was optimal. This study suggests that the growth and production of I. galbana can be improved using mixotrophic culture at 50 mmol glycerol in 35‰  salinity. Yousef Alkhamis and Jian G. Qin Copyright © 2013 Yousef Alkhamis and Jian G. Qin. All rights reserved. Genotoxin Induced Mutagenesis in the Model Plant Physcomitrella patens Mon, 09 Dec 2013 17:52:41 +0000 The moss Physcomitrella patens is unique for the high frequency of homologous recombination, haploid state, and filamentous growth during early stages of the vegetative growth, which makes it an excellent model plant to study DNA damage responses. We used single cell gel electrophoresis (comet) assay to determine kinetics of response to Bleomycin induced DNA oxidative damage and single and double strand breaks in wild type and mutant lig4 Physcomitrella lines. Moreover, APT gene when inactivated by induced mutations was used as selectable marker to ascertain mutational background at nucleotide level by sequencing of the APT locus. We show that extensive repair of DSBs occurs also in the absence of the functional LIG4, whereas repair of SSBs is seriously compromised. From analysis of induced mutations we conclude that their accumulation rather than remaining lesions in DNA and blocking progression through cell cycle is incompatible with normal plant growth and development and leads to sensitive phenotype. Marcela Holá, Jaroslav Kozák, Radka Vágnerová, and Karel J. Angelis Copyright © 2013 Marcela Holá et al. All rights reserved.