BioMed Research International: Biotechnology The latest articles from Hindawi Publishing Corporation © 2015 , Hindawi Publishing Corporation . All rights reserved. Heavy Metal Adsorption onto Kappaphycus sp. from Aqueous Solutions: The Use of Error Functions for Validation of Isotherm and Kinetics Models Thu, 30 Jul 2015 09:44:35 +0000 Biosorption process is a promising technology for the removal of heavy metals from industrial wastes and effluents using low-cost and effective biosorbents. In the present study, adsorption of Pb2+, Cu2+, Fe2+, and Zn2+ onto dried biomass of red seaweed Kappaphycus sp. was investigated as a function of pH, contact time, initial metal ion concentration, and temperature. The experimental data were evaluated by four isotherm models (Langmuir, Freundlich, Temkin, and Dubinin-Radushkevich) and four kinetic models (pseudo-first-order, pseudo-second-order, Elovich, and intraparticle diffusion models). The adsorption process was feasible, spontaneous, and endothermic in nature. Functional groups in the biomass involved in metal adsorption process were revealed as carboxylic and sulfonic acids and sulfonate by Fourier transform infrared analysis. A total of nine error functions were applied to validate the models. We strongly suggest the analysis of error functions for validating adsorption isotherm and kinetic models using linear methods. The present work shows that the red seaweed Kappaphycus sp. can be used as a potentially low-cost biosorbent for the removal of heavy metal ions from aqueous solutions. Further study is warranted to evaluate its feasibility for the removal of heavy metals from the real environment. Md. Sayedur Rahman and Kathiresan V. Sathasivam Copyright © 2015 Md. Sayedur Rahman and Kathiresan V. Sathasivam. All rights reserved. Microbial Diversity for Biotechnology 2014 Tue, 28 Jul 2015 07:50:37 +0000 George Tsiamis, Ameur Cherif, Dimitrios Karpouzas, and Spyridon Ntougias Copyright © 2015 George Tsiamis et al. All rights reserved. Development of Electroactive and Anaerobic Ammonium-Oxidizing (Anammox) Biofilms from Digestate in Microbial Fuel Cells Mon, 27 Jul 2015 08:33:54 +0000 Microbial Fuel cells (MFCs) have been proposed for nutrient removal and energy recovery from different wastes. In this study the anaerobic digestate was used to feed H-type MFC reactors, one with a graphite anode preconditioned with Geobacter sulfurreducens and the other with an unconditioned graphite anode. The data demonstrate that the digestate acts as a carbon source, and even in the absence of anode preconditioning, electroactive bacteria colonise the anodic chamber, producing a maximum power density of 172.2 mW/m2. The carbon content was also reduced by up to 60%, while anaerobic ammonium oxidation (anammox) bacteria, which were found in the anodic compartment of the reactors, contributed to nitrogen removal from the digestate. Overall, these results demonstrate that MFCs can be used to recover anammox bacteria from natural sources, and it may represent a promising bioremediation unit in anaerobic digestor plants for the simultaneous nitrogen removal and electricity generation using digestate as substrate. Enea Gino Di Domenico, Gianluca Petroni, Daniele Mancini, Alberto Geri, Luca Di Palma, and Fiorentina Ascenzioni Copyright © 2015 Enea Gino Di Domenico et al. All rights reserved. Bacterial Diversity and Bioremediation Potential of the Highly Contaminated Marine Sediments at El-Max District (Egypt, Mediterranean Sea) Mon, 27 Jul 2015 08:17:07 +0000 Coastal environments worldwide are threatened by the effects of pollution, a risk particularly high in semienclosed basins like the Mediterranean Sea that is poorly studied from bioremediation potential perspective especially in the Southern coast. Here, we investigated the physical, chemical, and microbiological features of hydrocarbon and heavy metals contaminated sediments collected at El-Max bay (Egypt). Molecular and statistical approaches assessing the structure of the sediment-dwelling bacterial communities showed correlations between the composition of bacterial assemblages and the associated environmental parameters. Fifty strains were isolated on mineral media supplemented by 1% crude oil and identified as a diverse range of hydrocarbon-degrading bacteria involved in different successional stages of biodegradation. We screened the collection for biotechnological potential studying biosurfactant production, biofilm formation, and the capability to utilize different hydrocarbons. Some strains were able to grow on multiple hydrocarbons as unique carbon source and presented biosurfactant-like activities and/or capacity to form biofilm and owned genes involved in different detoxification/degradation processes. El-Max sediments represent a promising reservoir of novel bacterial strains adapted to high hydrocarbon contamination loads. The potential of the strains for exploitation for in situ intervention to combat pollution in coastal areas is discussed. Ranya A. Amer, Francesca Mapelli, Hamada M. El Gendi, Marta Barbato, Doaa A. Goda, Anna Corsini, Lucia Cavalca, Marco Fusi, Sara Borin, Daniele Daffonchio, and Yasser R. Abdel-Fattah Copyright © 2015 Ranya A. Amer et al. All rights reserved. A High Diversity in Chitinolytic and Chitosanolytic Species and Enzymes and Their Oligomeric Products Exist in Soil with a History of Chitin and Chitosan Exposure Sun, 26 Jul 2015 12:45:43 +0000 Chitin is one of the most abundant biomolecules on earth, and its partially de-N-acetylated counterpart, chitosan, is one of the most promising biotechnological resources due to its diversity in structure and function. Recently, chitin and chitosan modifying enzymes (CCMEs) have gained increasing interest as tools to engineer chitosans with specific functions and reliable performance in biotechnological and biomedical applications. In a search for novel CCME, we isolated chitinolytic and chitosanolytic microorganisms from soils with more than ten-years history of chitin and chitosan exposure and screened them for chitinase and chitosanase isoenzymes as well as for their patterns of oligomeric products by incubating their secretomes with chitosan polymers. Of the 60 bacterial strains isolated, only eight were chitinolytic and/or chitosanolytic, while 20 out of 25 fungal isolates were chitinolytic and/or chitosanolytic. The bacterial isolates produced rather similar patterns of chitinolytic and chitosanolytic enzymes, while the fungal isolates produced a much broader range of different isoenzymes. Furthermore, diverse mixtures of oligosaccharides were formed when chitosan polymers were incubated with the secretomes of select fungal species. Our study indicates that soils with a history of chitin and chitosan exposure are a good source of novel CCME for chitosan bioengineering. Malathi Nampally, M. B. Govinda Rajulu, Dominique Gillet, T. S. Suryanarayanan, and Bruno B. Moerschbacher Copyright © 2015 Malathi Nampally et al. All rights reserved. Bacterial Diversity Associated with the Coccolithophorid Algae Emiliania huxleyi and Coccolithus pelagicus f. braarudii Sun, 26 Jul 2015 11:46:22 +0000 Coccolithophores are unicellular calcifying marine phytoplankton that can form large and conspicuous blooms in the oceans and make significant contributions to oceanic carbon cycling and atmospheric CO2 regulation. Despite their importance, the bacterial diversity associated with these algae has not been explored for ecological or biotechnological reasons. Bacterial membership of Emiliania huxleyi and Coccolithus pelagicus f. braarudii cultures was assessed using cultivation and cultivation-independent methods. The communities were species rich compared to other phytoplankton cultures. Community analysis identified specific taxa which cooccur in all cultures (Marinobacter and Marivita). Hydrocarbon-degrading bacteria were found in all cultures. The presence of Acidobacteria, Acidimicrobidae, Schlegelella, and Thermomonas was unprecedented but were potentially explained by calcification associated with coccolith production. One strain of Acidobacteria was cultivated and is closely related to a marine Acidobacteria isolated from a sponge. From this assessment of the bacterial diversity of coccolithophores, a number of biotechnological opportunities are evident, from bioprospecting for novel taxa such as Acidobacteria to helping understand the relationship between obligate hydrocarbonoclastic bacteria occurrence with phytoplankton and to revealing bacterial taxa that have a specific association with algae and may be suitable candidates as a means to improve the efficiency of mass algal cultivation. David H. Green, Virginia Echavarri-Bravo, Debra Brennan, and Mark C. Hart Copyright © 2015 David H. Green et al. All rights reserved. Isolation and Characterization of Phages Infecting Bacillus subtilis Sun, 26 Jul 2015 11:10:47 +0000 Bacteriophages have been suggested as an alternative approach to reduce the amount of pathogens in various applications. Bacteriophages of various specificity and virulence were isolated as a means of controlling food-borne pathogens. We studied the interaction of bacteriophages with Bacillus species, which are very often persistent in industrial applications such as food production due to their antibiotic resistance and spore formation. A comparative study using electron microscopy, PFGE, and SDS-PAGE as well as determination of host range, pH and temperature resistance, adsorption rate, latent time, and phage burst size was performed on three phages of the Myoviridae family and one phage of the Siphoviridae family which infected Bacillus subtilis strains. The phages are morphologically different and characterized by icosahedral heads and contractile (SIOΦ, SUBω, and SPOσ phages) or noncontractile (ARπ phage) tails. The genomes of SIOΦ and SUBω are composed of 154 kb. The capsid of SIOΦ is composed of four proteins. Bacteriophages SPOσ and ARπ have genome sizes of 25 kbp and 40 kbp, respectively. Both phages as well as SUBω phage have 14 proteins in their capsids. Phages SIOΦ and SPOσ are resistant to high temperatures and to the acid (4.0) and alkaline (9.0 and 10.0) pH. Anna Krasowska, Anna Biegalska, Daria Augustyniak, Marcin Łoś, Malwina Richert, and Marcin Łukaszewicz Copyright © 2015 Anna Krasowska et al. All rights reserved. Bioprecipitation of Calcium Carbonate Crystals by Bacteria Isolated from Saline Environments Grown in Culture Media Amended with Seawater and Real Brine Sun, 26 Jul 2015 09:38:01 +0000 The precipitation of calcium carbonate and calcium sulphate by isolated bacteria from seawater and real brine obtained in a desalination plant growth in culture media containing seawater and brine as mineral sources has been studied. However, only bioprecipitation was detected when the bacteria were grown in media with added organic matter. Biomineralization process started rapidly, crystal formation taking place in the beginning a few days after inoculation of media; roughly 90% of total cultivated bacteria showed. Six major colonies with carbonate precipitation capacity dominated bacterial community structure cultivated in heterotrophic platable bacteria medium. Taxonomic identification of these six strains through partial 16S rRNA gene sequences showed their affiliation with Gram-positive Bacillus and Virgibacillus genera. These strains were able to form calcium carbonate minerals, which precipitated as calcite and aragonite crystals and showed bacterial fingerprints or bacteria calcification. Also, carbonic anhydrase activity was observed in three of these isolated bacteria. The results of this research suggest that microbiota isolated from sea water and brine is capable of precipitation of carbonate biominerals, which can occur in situ with mediation of organic matter concentrations. Moreover, calcium carbonate precipitation ability of this microbiota could be of importance in bioremediation of CO2 and calcium in certain environments. G. A. Silva-Castro, I. Uad, A. Gonzalez-Martinez, A. Rivadeneyra, J. Gonzalez-Lopez, and M. A. Rivadeneyra Copyright © 2015 G. A. Silva-Castro et al. All rights reserved. Characterization, Microbial Community Structure, and Pathogen Occurrence in Urban Faucet Biofilms in South China Sun, 26 Jul 2015 08:18:53 +0000 The composition and microbial community structure of the drinking water system biofilms were investigated using microstructure analysis and 454 pyrosequencing technique in Xiamen city, southeast of China. SEM (scanning electron microscope) results showed different features of biofilm morphology in different fields of PVC pipe. Extracellular matrix material and sparse populations of bacteria (mainly rod-shaped and coccoid) were observed. CLSM (confocal laser scanning microscope) revealed different distributions of attached cells, extracellular proteins, α-polysaccharides, and β-polysaccharides. The biofilms had complex bacterial compositions. Differences in bacteria diversity and composition from different tap materials and ages were observed. Proteobacteria was the common and predominant group in all biofilms samples. Some potential pathogens (Legionellales, Enterobacteriales, Chromatiales, and Pseudomonadales) and corrosive microorganisms were also found in the biofilms. This study provides the information of characterization and visualization of the drinking water biofilms matrix, as well as the microbial community structure and opportunistic pathogens occurrence. Huirong Lin, Shuting Zhang, Song Gong, Shenghua Zhang, and Xin Yu Copyright © 2015 Huirong Lin et al. All rights reserved. Diverse Reductive Dehalogenases Are Associated with Clostridiales-Enriched Microcosms Dechlorinating 1,2-Dichloroethane Sun, 26 Jul 2015 07:49:34 +0000 The achievement of successful biostimulation of active microbiomes for the cleanup of a polluted site is strictly dependent on the knowledge of the key microorganisms equipped with the relevant catabolic genes responsible for the degradation process. In this work, we present the characterization of the bacterial community developed in anaerobic microcosms after biostimulation with the electron donor lactate of groundwater polluted with 1,2-dichloroethane (1,2-DCA). Through a multilevel analysis, we have assessed (i) the structural analysis of the bacterial community; (ii) the identification of putative dehalorespiring bacteria; (iii) the characterization of functional genes encoding for putative 1,2-DCA reductive dehalogenases (RDs). Following the biostimulation treatment, the structure of the bacterial community underwent a notable change of the main phylotypes, with the enrichment of representatives of the order Clostridiales. Through PCR targeting conserved regions within known RD genes, four novel variants of RDs previously associated with the reductive dechlorination of 1,2-DCA were identified in the metagenome of the Clostridiales-dominated bacterial community. Giuseppe Merlino, Annalisa Balloi, Massimo Marzorati, Francesca Mapelli, Aurora Rizzi, Davide Lavazza, Francesca de Ferra, Giovanna Carpani, and Daniele Daffonchio Copyright © 2015 Giuseppe Merlino et al. All rights reserved. Expression of Heterologous Cellulases in Thermotoga sp. Strain RQ2 Sun, 26 Jul 2015 07:32:41 +0000 The ability of Thermotoga spp. to degrade cellulose is limited due to a lack of exoglucanases. To address this deficiency, cellulase genes Csac_1076 (celA) and Csac_1078 (celB) from Caldicellulosiruptor saccharolyticus were cloned into T. sp. strain RQ2 for heterologous overexpression. Coding regions of Csac_1076 and Csac_1078 were fused to the signal peptide of TM1840 (amyA) and TM0070 (xynB), resulting in three chimeric enzymes, namely, TM1840-Csac_1078, TM0070-Csac_1078, and TM0070-Csac_1076, which were carried by Thermotoga-E. coli shuttle vectors pHX02, pHX04, and pHX07, respectively. All three recombinant enzymes were successfully expressed in E. coli DH5α and T. sp. strain RQ2, rendering the hosts with increased endo- and/or exoglucanase activities. In E. coli, the recombinant enzymes were mainly bound to the bacterial cells, whereas in T. sp. strain RQ2, about half of the enzyme activities were observed in the culture supernatants. However, the cellulase activities were lost in T. sp. strain RQ2 after three consecutive transfers. Nevertheless, this is the first time heterologous genes bigger than 1 kb (up to 5.3 kb in this study) have ever been expressed in Thermotoga, demonstrating the feasibility of using engineered Thermotoga spp. for efficient cellulose utilization. Hui Xu, Dongmei Han, and Zhaohui Xu Copyright © 2015 Hui Xu et al. All rights reserved. Immobilization of Yarrowia lipolytica Lipase on Macroporous Resin Using Different Methods: Characterization of the Biocatalysts in Hydrolysis Reaction Thu, 09 Jul 2015 09:38:14 +0000 To improve the reusability and organic solvent tolerance of microbial lipase and expand the application of lipase (hydrolysis, esterification, and transesterification), we immobilized marine microbial lipase using different methods and determined the properties of immobilized lipases. Considering the activity and cost of immobilized lipase, the concentration of lipase was fixed at 2 mg/mL. The optimal temperature of immobilized lipases was 40°C and 5°C higher than free lipase. The activities of immobilized lipases were much higher than free lipase at alkaline pH (more than 50% at pH 12). The free lipase lost most activity (35.3%) and immobilized lipases retained more than 46.4% of their initial activity after 3 h heat treatment at 70°C. At alkaline pH, immobilized lipases were more stable than free lipase (more than 60% residue activity at pH 11 for 3 h). Immobilized lipases retained 80% of their activity after 5 cycles and increased enzyme activity (more than 108.7%) after 3 h treatment in tert-butanol. Immobilization of lipase which improved reusability of lipase and provided a chance to expand the application of marine microbial lipase in organic system expanded the application range of lipase to catalyze hydrolysis and esterification in harsh condition. Jingjing Sun, Yiling Chen, Jun Sheng, and Mi Sun Copyright © 2015 Jingjing Sun et al. All rights reserved. Improved Salinity Tolerance in Carrizo Citrange Rootstock through Overexpression of Glyoxalase System Genes Wed, 08 Jul 2015 06:54:54 +0000 Citrus plants are widely cultivated around the world and, however, are one of the most salt stress sensitive crops. To improve salinity tolerance, transgenic Carrizo citrange rootstocks that overexpress glyoxalase I and glyoxalase II genes were obtained and their salt stress tolerance was evaluated. Molecular analysis showed high expression for both glyoxalase genes (BjGlyI and PgGlyII) in 5H03 and 5H04 lines. Under control conditions, transgenic and wild type plants presented normal morphology. In salinity treatments, the transgenic plants showed less yellowing, marginal burn in lower leaves and showed less than 40% of leaf damage compared with wild type plants. The transgenic plants showed a significant increase in the dry weight of shoot but there are no differences in the root and complete plant dry weight. In addition, a higher accumulation of chlorine is observed in the roots in transgenic line 5H03 but in shoot it was lower. Also, the wild type plant accumulated around 20% more chlorine in the shoot compared to roots. These results suggest that heterologous expression of glyoxalase system genes could enhance salt stress tolerance in Carrizo citrange rootstock and could be a good biotechnological approach to improve the abiotic stress tolerance in woody plant species. Ximena Alvarez-Gerding, Rowena Cortés-Bullemore, Consuelo Medina, Jesús L. Romero-Romero, Claudio Inostroza-Blancheteau, Felipe Aquea, and Patricio Arce-Johnson Copyright © 2015 Ximena Alvarez-Gerding et al. All rights reserved. Cyanobacteria: Photoautotrophic Microbial Factories for the Sustainable Synthesis of Industrial Products Thu, 25 Jun 2015 12:16:51 +0000 Cyanobacteria are widely distributed Gram-negative bacteria with a long evolutionary history and the only prokaryotes that perform plant-like oxygenic photosynthesis. Cyanobacteria possess several advantages as hosts for biotechnological applications, including simple growth requirements, ease of genetic manipulation, and attractive platforms for carbon neutral production process. The use of photosynthetic cyanobacteria to directly convert carbon dioxide to biofuels is an emerging area of interest. Equipped with the ability to degrade environmental pollutants and remove heavy metals, cyanobacteria are promising tools for bioremediation and wastewater treatment. Cyanobacteria are characterized by the ability to produce a spectrum of bioactive compounds with antibacterial, antifungal, antiviral, and antialgal properties that are of pharmaceutical and agricultural significance. Several strains of cyanobacteria are also sources of high-value chemicals, for example, pigments, vitamins, and enzymes. Recent advances in biotechnological approaches have facilitated researches directed towards maximizing the production of desired products in cyanobacteria and realizing the potential of these bacteria for various industrial applications. In this review, the potential of cyanobacteria as sources of energy, bioactive compounds, high-value chemicals, and tools for aquatic bioremediation and recent progress in engineering cyanobacteria for these bioindustrial applications are discussed. Nyok-Sean Lau, Minami Matsui, and Amirul Al-Ashraf Abdullah Copyright © 2015 Nyok-Sean Lau et al. All rights reserved. Single-Stranded DNA Aptamers against Pathogens and Toxins: Identification and Biosensing Applications Tue, 23 Jun 2015 11:58:35 +0000 Molecular recognition elements (MREs) can be short sequences of single-stranded DNA, RNA, small peptides, or antibody fragments. They can bind to user-defined targets with high affinity and specificity. There has been an increasing interest in the identification and application of nucleic acid molecular recognition elements, commonly known as aptamers, since they were first described in 1990 by the Gold and Szostak laboratories. A large number of target specific nucleic acids MREs and their applications are currently in the literature. This review first describes the general methodologies used in identifying single-stranded DNA (ssDNA) aptamers. It then summarizes advancements in the identification and biosensing application of ssDNA aptamers specific for bacteria, viruses, their associated molecules, and selected chemical toxins. Lastly, an overview of the basic principles of ssDNA aptamer-based biosensors is discussed. Ka Lok Hong and Letha J. Sooter Copyright © 2015 Ka Lok Hong and Letha J. Sooter. All rights reserved. The 482Ser of PPARGC1A and 12Pro of PPARG2 Alleles Are Associated with Reduction of Metabolic Risk Factors Even Obesity in a Mexican-Mestizo Population Mon, 22 Jun 2015 08:51:13 +0000 The aim of this study was to investigate the relationship between functional polymorphisms Gly482Ser in PPARGC1A and Pro12Ala in PPARG2 with the presence of obesity and metabolic risk factors. We included 375 individuals characterized as Mexican-Mestizos and classified by the body mass index (BMI). Body dimensions and distribution of body fat were measured. The HOMA-IR and adiposity indexes were calculated. Adipokines and metabolic profile quantification were performed by ELISA and routine methods. Genetic polymorphisms were determined by polymerase chain reaction restriction fragment length polymorphism analysis. A difference between obese and nonobese subjects in polymorphism PPARGC1A distribution was observed. Among obese individuals, carriers of genotype 482Gly/Gly were observed to have decreased body fat, BMI, and body fat ratio versus 482Ser/Ser carriers and increased resistin and leptin levels in carriers Gly+ phenotype versus Gly− phenotype. Subjects with PPARG2 Ala− phenotype (genotype 12Pro/Pro) showed a decreased HOMA-IR index versus individuals with Ala+ phenotype (genotypes 12Pro/Ala plus 12Ala/Ala). We propose that, in obese Mexican-Mestizos, the combination of alleles 482Ser in PPARGC1A and 12Pro in PPARG2 represents a reduced metabolic risk profile, even when the adiposity indexes are increased. Mónica Vázquez-Del Mercado, Milton-Omar Guzmán-Ornelas, Fernanda-Isadora Corona Meraz, Clara-Patricia Ríos-Ibarra, Eduardo-Alejandro Reyes-Serratos, Jorge Castro-Albarran, Sandra-Luz Ruíz-Quezada, and Rosa-Elena Navarro-Hernández Copyright © 2015 Mónica Vázquez-Del Mercado et al. All rights reserved. Evaluating Insects as Bioindicators of Heavy Metal Contamination and Accumulation near Industrial Area of Gujrat, Pakistan Thu, 18 Jun 2015 12:19:14 +0000 To study the accumulation and contamination of heavy metals (i.e., Cd, Cr, Cu, Ni, and Zn) in soil, air, and water, few insect species were assayed as ecological indicators. Study area comes under industrial zone of district Gujrat of Punjab, Pakistan. Insects used as bioindicators included a libellulid dragonfly (Crocothemis servilia), an acridid grasshopper (Oxya hyla hyla), and a nymphalid butterfly (Danaus chrysippus) near industrial zone of Gujrat. Accumulation of Cd was highest in insect species followed by Cu, Cr, Zn, and Ni at . Hierarchical cluster analysis (HACA) was carried out to study metal accumulation level in all insects. Correlation and regression analysis confirmed HACA observations and declared concentration of heavy metals above permissible limits. Metal concentrations in insects were significantly higher near industries and nallahs in Gujrat and relatively higher concentrations of metals were found in Orthoptera than Odonata and Lepidoptera. The total metal concentrations in insects were pointed significantly higher at sites S3 (Mid of HalsiNala), S9 (End of HalsiNala), and S1 (Start of HalsiNala), whereas lowest value was detected at site S6 (Kalra Khasa) located far from industrial area. HACA indicates that these insect groups are potential indicators of metal contamination and can be used in biomonitoring. Iqra Azam, Sumera Afsheen, Ahmed Zia, Muqaddas Javed, Rashid Saeed, Muhammad Kaleem Sarwar, and Bushra Munir Copyright © 2015 Iqra Azam et al. All rights reserved. A Mutagenesis Assay for Reporter Gene Screening Using Partially Degenerate Oligonucleotides of the Tandems NNT and NNC Thu, 18 Jun 2015 11:30:26 +0000 Not all proteins are tolerable to mutations. Whether a specific protein can be a mutable target is of importance in the biotechnology and pharmaceutical industry. This study reported a novel mutagenesis assay using tandem NNT and NNC oligonucleotides to test the mutability of a candidate gene. These two tandem oligonucleotides avoid the risk of forming nonsense mutations and render flexibility of truncating or expanding the insertion size. As a reporter gene, ZeoR (zeocin resistance gene) was confirmed to have a high tolerance for mutagenesis by this new assay. Huifen Xu, Cuilan Zhou, Andy K. Zhang, Wen Li, Jia Zhang, and Kai Li Copyright © 2015 Huifen Xu et al. All rights reserved. Anaerobic Treatment of Palm Oil Mill Effluent in Pilot-Scale Anaerobic EGSB Reactor Thu, 18 Jun 2015 08:38:30 +0000 Large volumes of untreated palm oil mill effluent (POME) pose threat to aquatic environment due to the presence of very high organic content. The present investigation involved two pilot-scale anaerobic expanded granular sludge bed (EGSB) reactors, continuously operated for 1 year to treat POME. Setting HRT at 9.8 d, the anaerobic EGSB reactors reduced COD from 71179 mg/L to 12341 mg/L and recycled half of sludge by a dissolved air flotation (DAF). The average effluent COD was 3587 mg/L with the consistent COD removal efficiency of 94.89%. Adding cationic polymer (PAM) dose of 30 mg/L to DAF unit and recycling its half of sludge caused granulation of anaerobic sludge. Bacilli and small coccid bacteria were the dominant microbial species of the reactor. The reactor produced 27.65 m3 of biogas per m3 of POME which was utilized for electricity generation. Jin Wang, Qaisar Mahmood, Jiang-Ping Qiu, Yin-Sheng Li, Yoon-Seong Chang, and Xu-Dong Li Copyright © 2015 Jin Wang et al. All rights reserved. In Vitro Assessment of Cadmium Bioavailability in Chinese Cabbage Grown on Different Soils and Its Toxic Effects on Human Health Thu, 18 Jun 2015 08:31:59 +0000 The minimum concentration of cadmium (Cd), by Chinese cabbage grown on Cd contaminated soils that can initiate toxicity in human liver cells using in vitro digestion coupled with Caco-2/HL-7702 cell models was studied. Cadmium bioaccessibility in the gastric phase for yellow soil (YS) cabbage (40.84%) and calcareous soil (CS) cabbage (21.54%) was significantly higher than small intestinal phase with the corresponding values of 21.2% and 11.11%, respectively. Cadmium bioavailability was higher in YS cabbage (5.27%–14.66%) than in CS cabbage (1.12%–9.64%). Cadmium concentrations (>0.74 μg) transported from YS and CS cabbage were able to induce oxidative (MDA, H2O2) stress by inhibiting antioxidant (SOD, GPx) enzyme activities in human liver cells (HL-7702). Additionally the study revealed that the ingestion of Cd contaminated Chinese cabbage grown in acidic soil (yellow soil) weakened the antioxidant defense system under all levels of contamination (2, 6, and 9 mg·kg−1) which ultimately escalated the oxidative stress in liver cells; however, in case of CS cabbage, a marked oxidative stress was observed only at 9 mg kg−1 Cd level of soil. Therefore, it is necessary to monitor Cd concentrations in leafy vegetables grown on acidic soils to minimize human health risk. Rukhsanda Aziz, Muhammad Tariq Rafiq, Zhenli He, Di Liu, Kewang Sun, and Yang Xiaoe Copyright © 2015 Rukhsanda Aziz et al. All rights reserved. Role of Geitlerinema sp. DE2011 and Scenedesmus sp. DE2009 as Bioindicators and Immobilizers of Chromium in a Contaminated Natural Environment Wed, 17 Jun 2015 08:14:20 +0000 The aim of this work was to study the potential of the two phototrophic microorganisms, both isolated from Ebro Delta microbial mats, to be used as bioindicators and immobilizers of chromium. The results obtained indicated that (i) the Minimum Metal Concentration (MMC) significantly affecting Chlorophyll a intensity in Geitlerinema sp. DE2011 and Scenedesmus sp. DE2009 was 0.25 µM and 0.75 µM, respectively, these values being lower than those established by current legislation, and (ii) Scenedesmus sp. DE2009 was able to immobilize chromium externally in extracellular polymeric substances (EPS) and intracellularly in polyphosphate (PP) inclusions. Additionally, this microorganism maintained high viability, including at 500 µM. Based on these results, we postulate that Geitlerinema sp. DE2011 and Scenedesmus sp. DE2009 are good chromium-indicators of cytotoxicity and, further, that Scenedesmus sp. DE2009 plays an important role in immobilizing this metal in a contaminated natural environment. Laia Millach, Antoni Solé, and Isabel Esteve Copyright © 2015 Laia Millach et al. All rights reserved. Microbial Enzymes and Their Applications in Industries and Medicine 2014 Tue, 16 Jun 2015 07:31:50 +0000 Periasamy Anbu, Subash C. B. Gopinath, Bidur Prasad Chaulagain, Thean-Hock Tang, and Marimuthu Citartan Copyright © 2015 Periasamy Anbu et al. All rights reserved. Bioenergy and Biomass Utilization Thu, 11 Jun 2015 12:02:44 +0000 Guangli Cao, Dexun Zou, Ximing Zhang, Lei Zhao, and Guojun Xie Copyright © 2015 Guangli Cao et al. All rights reserved. Codon Optimization Significantly Improves the Expression Level of α-Amylase Gene from Bacillus licheniformis in Pichia pastoris Wed, 10 Jun 2015 07:07:29 +0000 α-Amylase as an important industrial enzyme has been widely used in starch processing, detergent, and paper industries. To improve expression efficiency of recombinant α-amylase from Bacillus licheniformis (B. licheniformis), the α-amylase gene from B. licheniformis was optimized according to the codon usage of Pichia pastoris (P. pastoris) and expressed in P. pastoris. Totally, the codons encoding 305 amino acids were optimized in which a total of 328 nucleotides were changed and the G+C content was increased from 47.6 to 49.2%. The recombinants were cultured in 96-deep-well microplates and screened by a new plate assay method. Compared with the wild-type gene, the optimized gene is expressed at a significantly higher level in P. pastoris after methanol induction for 168 h in 5- and 50-L bioreactor with the maximum activity of 8100 and 11000 U/mL, which was 2.31- and 2.62-fold higher than that by wild-type gene. The improved expression level makes the enzyme a good candidate for α-amylase production in industrial use. Jian-Rong Wang, Yang-Yuan Li, Dan-Ni Liu, Jing-Shan Liu, Peng Li, Li-Zhi Chen, and Shu-De Xu Copyright © 2015 Jian-Rong Wang et al. All rights reserved. Improvement in Saccharification Yield of Mixed Rumen Enzymes by Identification of Recalcitrant Cell Wall Constituents Using Enzyme Fingerprinting Tue, 09 Jun 2015 08:19:41 +0000 Identification of recalcitrant factors that limit digestion of forages and the development of enzymatic approaches that improve hydrolysis could play a key role in improving the efficiency of meat and milk production in ruminants. Enzyme fingerprinting of barley silage fed to heifers and total tract indigestible fibre residue (TIFR) collected from feces was used to identify cell wall components resistant to total tract digestion. Enzyme fingerprinting results identified acetyl xylan esterases as key to the enhanced ruminal digestion. FTIR analysis also suggested cross-link cell wall polymers as principal components of indigested fiber residues in feces. Based on structural information from enzymatic fingerprinting and FTIR, enzyme pretreatment to enhance glucose yield from barley straw and alfalfa hay upon exposure to mixed rumen-enzymes was developed. Prehydrolysis effects of recombinant fungal fibrolytic hydrolases were analyzed using microassay in combination with statistical experimental design. Recombinant hemicellulases and auxiliary enzymes initiated degradation of plant structural polysaccharides upon application and improved the in vitro saccharification of alfalfa and barley straw by mixed rumen enzymes. The validation results showed that microassay in combination with statistical experimental design can be successfully used to predict effective enzyme pretreatments that can enhance plant cell wall digestion by mixed rumen enzymes. Ajay Badhan, Yu-Xi Wang, Robert Gruninger, Donald Patton, Justin Powlowski, Adrian Tsang, and Tim A. McAllister Copyright © 2015 Ajay Badhan et al. All rights reserved. Molecular Identification of a Newly Isolated Bacillus subtilis BI19 and Optimization of Production Conditions for Enhanced Production of Extracellular Amylase Tue, 09 Jun 2015 08:08:51 +0000 A study was carried out with a newly isolated bacterial strain yielding extracellular amylase. The phylogenetic tree constructed on the basis of 16S rDNA gene sequences revealed this strain as clustered with the closest members of Bacillus sp. and identified as Bacillus subtilis BI19. The effect of various fermentation conditions on amylase production through shake-flask culture was investigated. Rice flour (1.25%) as a cheap natural carbon source was found to induce amylase production mostly. A combination of peptone and tryptone as organic and ammonium sulfate as inorganic nitrogen sources gave highest yield. Maximum production was obtained after 24 h of incubation at 37°C with an initial medium pH 8.0. Addition of surfactants like Tween 80 (0.25 g/L) and sodium lauryl sulfate (0.2 g/L) resulted in 28% and 15% increase in enzyme production, respectively. Amylase production was 3.06 times higher when optimized production conditions were used. Optimum reaction temperature and pH for crude amylase activity were 50°C and 6.0, respectively. The crude enzyme showed activity and stability over a fair range of temperature and pH. These results suggest that B. subtilis BI19 could be exploited for production of amylase at relatively low cost and time. Biplab Kumar Dash, M. Mizanur Rahman, and Palash Kumar Sarker Copyright © 2015 Biplab Kumar Dash et al. All rights reserved. Production and Characterization of Lipases by Two New Isolates of Aspergillus through Solid-State and Submerged Fermentation Tue, 09 Jun 2015 07:43:00 +0000 Due to the numerous applications of lipases in industry, there is a need to study their characteristics, because lipases obtained from different sources may present different properties. The aim of this work was to accomplish the partial characterization of lipases obtained through submerged fermentation and solid-state fermentation by two species of Aspergillus. Fungal strains were isolated from a diesel-contaminated soil and selected as good lipases producers. Lipases obtained through submerged fermentation presented optimal activities at 37°C and pH 7.2 and those obtained through solid-state fermentation at 35°C and pH 6.0. The enzymes produced by submerged fermentation were more temperature-stable than those obtained by solid-state fermentation, presenting 72% of residual activity after one hour of exposition at 90°C. Lipases obtained through submerged fermentation had 80% of stability in acidic pH and those obtained through solid-state fermentation had stability greater than 60% in alkaline pH. Luciane Maria Colla, Aline M. M. Ficanha, Juliana Rizzardi, Telma Elita Bertolin, Christian Oliveira Reinehr, and Jorge Alberto Vieira Costa Copyright © 2015 Luciane Maria Colla et al. All rights reserved. Statistical Optimization of Laccase Production and Delignification of Sugarcane Bagasse by Pleurotus ostreatus in Solid-State Fermentation Tue, 09 Jun 2015 06:56:06 +0000 Laccases are oxidative enzymes related to the degradation of phenolic compounds, including lignin units, with concomitant reduction of oxygen to water. Delignification is a necessary pretreatment step in the process of converting plant biomass into fermentable sugars. The objective of this work was to optimize the production of laccases and to evaluate the delignification of sugarcane bagasse by Pleurotus ostreatus in solid-state fermentation. Among eight variables (pH, water activity, temperature, and concentrations of CuSO4, (NH4)2SO4, KH2PO4, asparagine, and yeast extract), copper sulfate and ammonium sulfate concentrations were demonstrated to significantly influence laccase production. The replacement of ammonium sulfate by yeast extract and the addition of ferulic acid as inducer provided increases of 5.7- and 2.0-fold, respectively, in laccase activity. Optimization of laccase production as a function of yeast extract, copper sulfate, and ferulic acid concentrations was performed by response surface methodology and optimal concentrations were 6.4 g/L, 172.6 μM, and 1.86 mM, respectively. Experimentally, the maximum laccase activity of 151.6 U/g was produced at the 5th day of solid-state fermentation. Lignin content in sugarcane bagasse was reduced from 31.89% to 26.36% after 5 days and to 20.79% after 15 days by the biological treatment of solid-state fermentation. Susan Grace Karp, Vincenza Faraco, Antonella Amore, Luiz Alberto Junior Letti, Vanete Thomaz Soccol, and Carlos Ricardo Soccol Copyright © 2015 Susan Grace Karp et al. All rights reserved. Biotechnological Aspects and Perspective of Microbial Keratinase Production Tue, 09 Jun 2015 06:52:14 +0000 Keratinases are proteolytic enzymes predominantly active when keratin substrates are available that attack disulfide bridges in the keratin to convert them from complex to simplified forms. Keratinases are essential in preparation of animal nutrients, protein supplements, leather manufacture, textile processing, detergent formulation, feather meal processing for feed and fertilizer, the pharmaceutical and biomedical industries, and waste management. Accordingly, it is necessary to develop a method for continuous production of keratinase from reliable sources that can be easily managed. Microbial keratinase is less expensive than conventionally produced keratinase and can be obtained from fungi, bacteria, and actinomycetes. In this overview, the expansion of information about microbial keratinases and important considerations in keratinase production are discussed. Subash C. B. Gopinath, Periasamy Anbu, Thangavel Lakshmipriya, Thean-Hock Tang, Yeng Chen, Uda Hashim, A. Rahim Ruslinda, and M. K. Md. Arshad Copyright © 2015 Subash C. B. Gopinath et al. All rights reserved. Purification and Characterization of a Polyextremophilic α-Amylase from an Obligate Halophilic Aspergillus penicillioides Isolate and Its Potential for Souse with Detergents Tue, 09 Jun 2015 06:42:45 +0000 An extracellular α-amylase from the obligate halophilic Aspergillus penicillioides TISTR3639 strain was produced and enriched to apparent homogeneity by ammonium sulfate precipitation and Sephadex G100 gel filtration column chromatography. The mass of the purified amylase was estimated to be 42 kDa by SDS-PAGE. With soluble starch as the substrate it had a specific activity of 118.42 Umg−1 and and values of 1.05 µmolmin−1mg−1 and 5.41 mgmL−1, respectively. The enzyme was found to have certain polyextremophilic characteristics, with an optimum activity at pH 9, 80°C, and 300 gL−1 NaCl. The addition of CaCl2 at 2 mM was found to slightly enhance the amylase activity, while ZnCl2, FeCl2, or EDTA at 2 mM was strongly or moderately inhibitory, respectively, suggesting the requirement for a (non-Fe2+ or Zn2+) divalent cation. The enzyme retained more than 80% of its activity when incubated with three different laundry detergents and had a better performance compared to a commercial amylase and three detergents in the presence of increasing NaCl concentrations up to 300 gL−1. Accordingly, it has a good potential for use as an α-amylase in a low water activity (high salt concentration) and at high pH and temperatures. Imran Ali, Ali Akbar, Mohammad Anwar, Sehanat Prasongsuk, Pongtharin Lotrakul, and Hunsa Punnapayak Copyright © 2015 Imran Ali et al. All rights reserved. Proteases of Wood Rot Fungi with Emphasis on the Genus Pleurotus Tue, 09 Jun 2015 06:41:17 +0000 Proteases are present in all living organisms and they play an important role in physiological conditions. Cell growth and death, blood clotting, and immune defense are all examples of the importance of proteases in maintaining homeostasis. There is growing interest in proteases due to their use for industrial purposes. The search for proteases with specific characteristics is designed to reduce production costs and to find suitable properties for certain industrial sectors, as well as good producing organisms. Ninety percent of commercialized proteases are obtained from microbial sources and proteases from macromycetes have recently gained prominence in the search for new enzymes with specific characteristics. The production of proteases from saprophytic basidiomycetes has led to the identification of various classes of proteases. The genus Pleurotus has been extensively studied because of its ligninolytic enzymes. The characteristics of this genus are easy cultivation techniques, high yield, low nutrient requirements, and excellent adaptation. There are few studies in the literature about proteases of Pleurotus spp. This review gathers together information about proteases, especially those derived from basidiomycetes, and aims at stimulating further research about fungal proteases because of their physiological importance and their application in various industries such as biotechnology and medicine. Fabíola Dorneles Inácio, Roselene Oliveira Ferreira, Caroline Aparecida Vaz de Araujo, Tatiane Brugnari, Rafael Castoldi, Rosane Marina Peralta, and Cristina Giatti Marques de Souza Copyright © 2015 Fabíola Dorneles Inácio et al. All rights reserved. Optimization of Culture Conditions for Production of the Anti-Leukemic Glutaminase Free L-Asparaginase by Newly Isolated Streptomyces olivaceus NEAE-119 Using Response Surface Methodology Tue, 09 Jun 2015 06:39:51 +0000 Among the antitumor drugs, bacterial enzyme L-asparaginase has been employed as the most effective chemotherapeutic agent in pediatric oncotherapy especially for acute lymphoblastic leukemia. Glutaminase free L-asparaginase producing actinomycetes were isolated from soil samples collected from Egypt. Among them, a potential culture, strain NEAE-119, was selected and identified on the basis of morphological, cultural, physiological, and biochemical properties together with 16S rRNA sequence as Streptomyces olivaceus NEAE-119 and sequencing product (1509 bp) was deposited in the GenBank database under accession number KJ200342. The optimization of different process parameters for L-asparaginase production by Streptomyces olivaceus NEAE-119 using Plackett-Burman experimental design and response surface methodology was carried out. Fifteen variables (temperature, pH, incubation time, inoculum size, inoculum age, agitation speed, dextrose, starch, L-asparagine, KNO3, yeast extract, K2HPO4, MgSO47H2O, NaCl, and FeSO47H2O) were screened using Plackett-Burman experimental design. The most positive significant independent variables affecting enzyme production (temperature, inoculum age, and agitation speed) were further optimized by the face-centered central composite design-response surface methodology. Noura El-Ahmady El-Naggar, Hassan Moawad, Nancy M. El-Shweihy, and Sara M. El-Ewasy Copyright © 2015 Noura El-Ahmady El-Naggar et al. All rights reserved. Lipase-Secreting Bacillus Species in an Oil-Contaminated Habitat: Promising Strains to Alleviate Oil Pollution Tue, 09 Jun 2015 06:32:07 +0000 Lipases are of great interest for different industrial applications due to their diversity and versatility. Among different lipases, microbial lipases are preferable due to their broad substrate specificity, and higher stability with lower production costs compared to the lipases from plants and animals. In the past, a vast number of bacterial species have been reported as potential lipases producers. In this study, the lipases-producing bacterial species were isolated from an oil spillage area in the conventional night market. Isolated species were identified as Bacillus species by biochemical tests which indicate their predominant establishment, and further screened on the agar solid surfaces using lipid and gelatin as the substrates. Out of the ten strains tested, four potential strains were subjected to comparison analysis of the lipolytic versus proteolytic activities. Strain 10 exhibited the highest lipolytic and proteolytic activity. In all the strains, the proteolytic activity is higher than the lipolytic activity except for strain 8, suggesting the possibility for substrate-based extracellular gene induction. The simultaneous secretion of both the lipase and protease is a mean of survival. The isolated bacterial species which harbour both lipase and protease enzymes could render potential industrial-based applications and solve environmental issues. Li Pin Lee, Hudzaifah Mohamed Karbul, Marimuthu Citartan, Subash C. B. Gopinath, Thangavel Lakshmipriya, and Thean-Hock Tang Copyright © 2015 Li Pin Lee et al. All rights reserved. Response of Arbuscular Mycorrhizal Fungi to Hydrologic Gradients in the Rhizosphere of Phragmites australis (Cav.) Trin ex. Steudel Growing in the Sun Island Wetland Wed, 03 Jun 2015 12:43:58 +0000 Within the rhizosphere, AM fungi are a sensitive variable to changes of botanic and environmental conditions, and they may interact with the biomass of plant and other microbes. During the vegetative period of the Phragmites australis growing in the Sun Island Wetland (SIW), the variations of AM fungi colonization were studied. Root samples of three hydrologic gradients generally showed AM fungi colonization, suggesting that AM fungi have the ability for adaptation to flooded habitats. There were direct and indirect hydrological related effects with respect to AM fungi biomass, which interacted simultaneously in the rhizosphere. Though water content in soil and reed growth parameters were both positively associated with AM fungi colonization, only the positive correlations between reed biomass parameters and the colonization could be expected, or both the host plant biomass and the AM fungi could be beneficial. The variations in response of host plant to the edaphic and hydrologic conditions may influence the effectiveness of the plant-mycorrhizal association. This study included a hydrologic component to better assess the role and distribution of AM fungi in wetland ecosystems. And because of that, the range of AM fungi was extended, since they actually showed a notable adaptability to hydrologic gradients. Li Wang, Jieting Wu, Fang Ma, Jixian Yang, Shiyang Li, Zhe Li, and Xue Zhang Copyright © 2015 Li Wang et al. All rights reserved. Biochemical Modulation of Lipid Pathway in Microalgae Dunaliella sp. for Biodiesel Production Wed, 03 Jun 2015 09:52:51 +0000 Exploitation of renewable sources of energy such as algal biodiesel could turn energy supplies problem around. Studies on a locally isolated strain of Dunaliella sp. showed that the mean lipid content in cultures enriched by 200 mg L−1 myoinositol was raised by around 33% (1.5 times higher than the control). Similarly, higher lipid productivity values were achieved in cultures treated by 100 and 200 mg L−1 myoinositol. Fluorometry analyses (microplate fluorescence and flow cytometry) revealed increased oil accumulation in the Nile red-stained algal samples. Moreover, it was predicted that biodiesel produced from myoinositol-treated cells possessed improved oxidative stability, cetane number, and cloud point values. From the genomic point of view, real-time analyses revealed that myoinositol negatively influenced transcript abundance of AccD gene (one of the key genes involved in lipid production pathway) due to feedback inhibition and that its positive effect must have been exerted through other genes. The findings of the current research are not to interprete that myoinositol supplementation could answer all the challenges faced in microalgal biodiesel production but instead to show that “there is a there there” for biochemical modulation strategies, which we achieved, increased algal oil quantity and enhanced resultant biodiesel quality. Ahmad Farhad Talebi, Masoud Tohidfar, Seyedeh Mahsa Mousavi Derazmahalleh, Alawi Sulaiman, Azhari Samsu Baharuddin, and Meisam Tabatabaei Copyright © 2015 Ahmad Farhad Talebi et al. All rights reserved. Development of Microsatellite Markers Derived from Expressed Sequence Tags of Polyporales for Genetic Diversity Analysis of Endangered Polyporus umbellatus Wed, 03 Jun 2015 07:21:06 +0000 A large scale of EST sequences of Polyporales was screened in this investigation in order to identify EST-SSR markers for various applications. The distribution of EST sequences and SSRs in five families of Polyporales was analyzed, respectively. Mononucleotide was the most abundant type, followed by trinucleotide. Among five families, Ganodermataceae occupied the most SSR markers, followed by Coriolaceae. Functional prediction of SSR marker-containing EST sequences in Ganoderma lucidum obtained three main groups, namely, cellular component, biological process, and molecular function. Thirty EST-SSR primers were designed to evaluate the genetic diversity of 13 natural Polyporus umbellatus accessions. Twenty one EST-SSRs were polymorphic with average PIC value of 0.33 and transferability rate of 71%. These 13 P. umbellatus accessions showed relatively high genetic diversity. The expected heterozygosity, Nei’s gene diversity, and Shannon information index were 0.41, 0.39, and 0.57, respectively. Both UPGMA dendrogram and principal coordinate analysis (PCA) showed the same cluster result that divided the 13 accessions into three or four groups. Yuejin Zhang, Yuanyuan Chen, Ruihong Wang, Ailin Zeng, Michael K. Deyholos, Jia Shu, and Hongbo Guo Copyright © 2015 Yuejin Zhang et al. All rights reserved. Production by Tobacco Transplastomic Plants of Recombinant Fungal and Bacterial Cell-Wall Degrading Enzymes to Be Used for Cellulosic Biomass Saccharification Tue, 02 Jun 2015 11:36:50 +0000 Biofuels from renewable plant biomass are gaining momentum due to climate change related to atmospheric CO2 increase. However, the production cost of enzymes required for cellulosic biomass saccharification is a major limiting step in this process. Low-cost production of large amounts of recombinant enzymes by transgenic plants was proposed as an alternative to the conventional microbial based fermentation. A number of studies have shown that chloroplast-based gene expression offers several advantages over nuclear transformation due to efficient transcription and translation systems and high copy number of the transgene. In this study, we expressed in tobacco chloroplasts microbial genes encoding five cellulases and a polygalacturonase. Leaf extracts containing the recombinant enzymes showed the ability to degrade various cell-wall components under different conditions, singly and in combinations. In addition, our group also tested a previously described thermostable xylanase in combination with a cellulase and a polygalacturonase to study the cumulative effect on the depolymerization of a complex plant substrate. Our results demonstrate the feasibility of using transplastomic tobacco leaf extracts to convert cell-wall polysaccharides into reducing sugars, fulfilling a major prerequisite of large scale availability of a variety of cell-wall degrading enzymes for biofuel industry. Paolo Longoni, Sadhu Leelavathi, Enrico Doria, Vanga Siva Reddy, and Rino Cella Copyright © 2015 Paolo Longoni et al. All rights reserved. Nanofibrillated Cellulose and Copper Nanoparticles Embedded in Polyvinyl Alcohol Films for Antimicrobial Applications Tue, 02 Jun 2015 11:26:48 +0000 Our long-term goal is to develop a hybrid cellulose-copper nanoparticle material as a functional nanofiller to be incorporated in thermoplastic resins for efficiently improving their antimicrobial properties. In this study, copper nanoparticles were first synthesized through chemical reduction of cupric ions on TEMPO nanofibrillated cellulose (TNFC) template using borohydride as a copper reducing agent. The resulting hybrid material was embedded into a polyvinyl alcohol (PVA) matrix using a solvent casting method. The morphology of TNFC-copper nanoparticles was analyzed by transmission electron microscopy (TEM); spherical copper nanoparticles with average size of 9.2 ± 2.0 nm were determined. Thermogravimetric analysis and antimicrobial performance of the films were evaluated. Slight variations in thermal properties between the nanocomposite films and PVA resin were observed. Antimicrobial analysis demonstrated that one-week exposure of nonpathogenic Escherichia coli DH5α to the nanocomposite films results in up to 5-log microbial reduction. Tuhua Zhong, Gloria S. Oporto, Jacek Jaczynski, and Changle Jiang Copyright © 2015 Tuhua Zhong et al. All rights reserved. Improving Biomethane Production and Mass Bioconversion of Corn Stover Anaerobic Digestion by Adding NaOH Pretreatment and Trace Elements Tue, 02 Jun 2015 11:09:19 +0000 This research applied sodium hydroxide (NaOH) pretreatment and trace elements to improve biomethane production when using corn stover for anaerobic digestion. Full-factor experimental tests identified the best combination of trace elements with the NaOH pretreatment, indicating that the best combination was with 1.0, 0.4, and 0.4 mg·L−1·d−1 of elements Fe, Co, and Ni, respectively. The cumulative biomethane production adding NaOH pretreatment and trace elements was 11,367 mL; total solid bioconversion rate was 55.7%, which was 41.8%–62.2% higher than with NaOH-pretreatment alone and 22.2%–56.3% higher than with untreated corn stover. The best combination was obtained 5–9 days shorter than T90 and maintained good system operation stability. Only a fraction of the trace elements in the best combination was present in the resulting solution; more than 85% of the total amounts added were transferred into the solid fraction. Adding 0.897 g of Fe, 0.389 g of Co, and 0.349 g of Ni satisfied anaerobic digestion needs and enhanced biological activity at the beginning of the operation. The results showed that NaOH pretreatment and adding trace elements improve corn stover biodegradability and enhance biomethane production. ChunMei Liu, HaiRong Yuan, DeXun Zou, YanPing Liu, BaoNing Zhu, and XiuJin Li Copyright © 2015 ChunMei Liu et al. All rights reserved. Enzymatic Saccharification of Lignocellulosic Residues by Cellulases Obtained from Solid State Fermentation Using Trichoderma viride Tue, 02 Jun 2015 10:23:26 +0000 The aim of this study was to verify the viability of lignocellulosic substrates to obtain renewable energy source, through characterization of the cellulolytic complex, which was obtained by solid state fermentation using Trichoderma viride. Enzymatic activity of the cellulosic complex was measured during saccharification of substrates filter paper, eucalyptus sawdust, and corncob, and compared with the activity of commercial cellulase. The characterization of the enzymes was performed by a 22 Full Factorial Design, where the pH and temperature were the variables of study. Enzymatic saccharification of different substrates appearedviable until 12 to be viable until 12 h; after this period the activity decreased for both enzymatic forms (cellulolytic complex and commercial cellulase). The enzymatic activity of the commercial cellulase was favored with the use of corncob as substrate, while the cellulolytic complex does not show any difference in its specificity by the substrates studied. The largest activities of both enzymes were obtained in the temperature and pH range between 40°C and 50°C and 4.8 and 5.2, respectively. The cellulolytic complex obtained appeared to be viable for the saccharification of lignocellulosic residues compared with the commercial cellulase. Tanara Sartori, Heloisa Tibolla, Elenizi Prigol, Luciane Maria Colla, Jorge Alberto Vieira Costa, and Telma Elita Bertolin Copyright © 2015 Tanara Sartori et al. All rights reserved. Stem Cells for Cutaneous Wound Healing Tue, 02 Jun 2015 06:30:11 +0000 Optimum healing of a cutaneous wound involves a well-orchestrated cascade of biological and molecular processes involving cell migration, proliferation, extracellular matrix deposition, and remodelling. When the normal biological process fails for any reason, this healing process can stall resulting in chronic wounds. Wounds are a growing clinical burden on healthcare systems and with an aging population as well as increasing incidences of obesity and diabetes, this problem is set to increase. Cell therapies may be the solution. A range of cell based approaches have begun to cross the rift from bench to bedside and the supporting data suggests that the appropriate administration of stem cells can accelerate wound healing. This review examines the main cell types explored for cutaneous wound healing with a focus on clinical use. The literature overwhelmingly suggests that cell therapies can help to heal cutaneous wounds when used appropriately but we are at risk of clinical use outpacing the evidence. There is a need, now more than ever, for standardised methods of cell characterisation and delivery, as well as randomised clinical trials. Giles T. S. Kirby, Stuart J. Mills, Allison J. Cowin, and Louise E. Smith Copyright © 2015 Giles T. S. Kirby et al. All rights reserved. Advances in the Development of Biotherapeutics Wed, 20 May 2015 13:35:53 +0000 Pedro H. Oliveira, Juergen Mairhofer, Paula M. Alves, Alvaro R. Lara, and Cleo Kontoravdi Copyright © 2015 Pedro H. Oliveira et al. All rights reserved. Development of an IP-Free Biotechnology Platform for Constitutive Production of HPV16 L1 Capsid Protein Using the Pichia pastoris PGK1 Promoter Mon, 18 May 2015 12:28:31 +0000 The human papillomavirus (HPV) L1 major capsid protein, which forms the basis of the currently available vaccines against cervical cancer, self-assembles into virus-like particles (VLPs) when expressed heterologously. We report the development of a biotechnology platform for HPV16 L1 protein expression based on the constitutive PGK1 promoter () from the methylotrophic yeast Pichia pastoris. The L1 gene was cloned under regulation of into pPGKΔ3 expression vector to achieve intracellular expression. In parallel, secretion of the L1 protein was obtained through the use of an alternative vector called pPGKΔ3α, in which a codon optimized α-factor signal sequence was inserted. We devised a work-flow based on the detection of the L1 protein by dot blot, colony blot, and western blot to classify the positive clones. Finally, intracellular HPV VLPs assembly was demonstrated for the first time in yeast cells. This study opens up perspectives for the establishment of an innovative platform for the production of HPV VLPs or other viral antigens for vaccination purposes, based on constitutive expression in P. pastoris. F. C. Mariz, E. C. Coimbra, A. L. S. Jesus, L. M. Nascimento, F. A. G. Torres, and A. C. Freitas Copyright © 2015 F. C. Mariz et al. All rights reserved. Expression and Characterization of Geobacillus stearothermophilus SR74 Recombinant α-Amylase in Pichia pastoris Mon, 18 May 2015 12:09:17 +0000 Geobacillus stearothermophilus SR74 is a locally isolated thermophilic bacteria producing thermostable and thermoactive α-amylase. Increased production and commercialization of thermostable α-amylase strongly warrant the need of a suitable expression system. In this study, the gene encoding the thermostable α-amylase in G. stearothermophilus SR74 was amplified, sequenced, and subcloned into P. pastoris GS115 strain under the control of a methanol inducible promoter, alcohol oxidase (AOX). Methanol induced recombinant expression and secretion of the protein resulted in high levels of extracellular amylase production. YPTM medium supplemented with methanol (1% v/v) was the best medium and once optimized, the maximum recombinant α-amylase SR74 achieved in shake flask was 28.6 U mL−1 at 120 h after induction. The recombinant 59 kDa α-amylase SR74 was purified 1.9-fold using affinity chromatography with a product yield of 52.6% and a specific activity of 151.8 U mg−1. The optimum pH of α-amylase SR74 was 7.0 and the enzyme was stable between pH 6.0–8.0. The purified enzyme was thermostable and thermoactive, exhibiting maximum activity at 65°C with a half-life (t1/2) of 88 min at 60°C. In conclusion, thermostable α-amylase SR74 from G. stearothermophilus SR74 would be beneficial for industrial applications, especially in liquefying saccrification. Sivasangkary Gandhi, Abu Bakar Salleh, Raja Noor Zaliha Raja Abd Rahman, Thean Chor Leow, and Siti Nurbaya Oslan Copyright © 2015 Sivasangkary Gandhi et al. All rights reserved. The J-Domain of Heat Shock Protein 40 Can Enhance the Transduction Efficiency of Arginine-Rich Cell-Penetrating Peptides Sun, 17 May 2015 13:56:58 +0000 Sense and antisense oligonucleotide pairs encoding cell-penetrating peptides PTD , DPV3A, E162, pVEC, R11, and TP13 were used to construct two sets of pET22b-CPP-DsRed and pET22b-CPP-J-DsRed vectors for CPP-DsRed and CPP-J-DsRed recombinant proteins expression. PTD-DsRed, DPV3A-DsRed, PTD-J-DsRed, and DPV3A-J-DsRed recombinant proteins were expressed in a soluble form. PTD-J-DsRed and DPV3A-J-DsRed recombinant proteins were able to escape from E. coli host cells into the culture medium. The membrane-penetrating activity of PTD-J-DsRed and DPV3A-J-DsRed recombinant proteins to mammalian cells was more effective than that of PTD-DsRed and DPV3A-DsRed. The route of the cellular membrane translocation of these recombinant proteins is suggested via macropinocytosis followed by an endosomal escape pathway. Tzu-Yin Lin, Yu-Hsiu Su, Kun-Hsiung Lee, and Chin-Kai Chuang Copyright © 2015 Tzu-Yin Lin et al. All rights reserved. Gene Delivery into Plant Cells for Recombinant Protein Production Sun, 17 May 2015 13:55:28 +0000 Recombinant proteins are primarily produced from cultures of mammalian, insect, and bacteria cells. In recent years, the development of deconstructed virus-based vectors has allowed plants to become a viable platform for recombinant protein production, with advantages in versatility, speed, cost, scalability, and safety over the current production paradigms. In this paper, we review the recent progress in the methodology of agroinfiltration, a solution to overcome the challenge of transgene delivery into plant cells for large-scale manufacturing of recombinant proteins. General gene delivery methodologies in plants are first summarized, followed by extensive discussion on the application and scalability of each agroinfiltration method. New development of a spray-based agroinfiltration and its application on field-grown plants is highlighted. The discussion of agroinfiltration vectors focuses on their applications for producing complex and heteromultimeric proteins and is updated with the development of bridge vectors. Progress on agroinfiltration in Nicotiana and non-Nicotiana plant hosts is subsequently showcased in context of their applications for producing high-value human biologics and low-cost and high-volume industrial enzymes. These new advancements in agroinfiltration greatly enhance the robustness and scalability of transgene delivery in plants, facilitating the adoption of plant transient expression systems for manufacturing recombinant proteins with a broad range of applications. Qiang Chen and Huafang Lai Copyright © 2015 Qiang Chen and Huafang Lai. All rights reserved. Diabetes and Stem Cell Function Sun, 17 May 2015 13:54:38 +0000 Diabetes mellitus is one of the most common serious metabolic diseases that results in hyperglycemia due to defects of insulin secretion or insulin action or both. The present review focuses on the alterations to the diabetic neuronal tissues and skeletal muscle, including stem cells in both tissues, and the preventive effects of physical activity on diabetes. Diabetes is associated with various nervous disorders, such as cognitive deficits, depression, and Alzheimer’s disease, and that may be caused by neural stem cell dysfunction. Additionally, diabetes induces skeletal muscle atrophy, the impairment of energy metabolism, and muscle weakness. Similar to neural stem cells, the proliferation and differentiation are attenuated in skeletal muscle stem cells, termed satellite cells. However, physical activity is very useful for preventing the diabetic alteration to the neuronal tissues and skeletal muscle. Physical activity improves neurogenic capacity of neural stem cells and the proliferative and differentiative abilities of satellite cells. The present review proposes physical activity as a useful measure for the patients in diabetes to improve the physiological functions and to maintain their quality of life. It further discusses the use of stem cell-based approaches in the context of diabetes treatment. Shin Fujimaki, Tamami Wakabayashi, Tohru Takemasa, Makoto Asashima, and Tomoko Kuwabara Copyright © 2015 Shin Fujimaki et al. All rights reserved. Early Implementation of QbD in Biopharmaceutical Development: A Practical Example Sun, 17 May 2015 13:42:48 +0000 In drug development, the “onus” of the low R&D efficiency has been put traditionally onto the drug discovery process (i.e., finding the right target or “binding” functionality). Here, we show that manufacturing is not only a central component of product success, but also that, by integrating manufacturing and discovery activities in a “holistic” interpretation of QbD methodologies, we could expect to increase the efficiency of the drug discovery process as a whole. In this new context, early risk assessment, using developability methodologies and computational methods in particular, can assist in reducing risks during development in a cost-effective way. We define specific areas of risk and how they can impact product quality in a broad sense, including essential aspects such as product efficacy and patient safety. Emerging industry practices around developability are introduced, including some specific examples of applications to biotherapeutics. Furthermore, we suggest some potential workflows to illustrate how developability strategies can be introduced in practical terms during early drug development in order to mitigate risks, reduce drug attrition and ultimately increase the robustness of the biopharmaceutical supply chain. Finally, we also discuss how the implementation of such methodologies could accelerate the access of new therapeutic treatments to patients in the clinic. Jesús Zurdo, Andreas Arnell, Olga Obrezanova, Noel Smith, Ramón Gómez de la Cuesta, Thomas R. A. Gallagher, Rebecca Michael, Yvette Stallwood, Caroline Ekblad, Lars Abrahmsén, and Ingmarie Höidén-Guthenberg Copyright © 2015 Jesús Zurdo et al. All rights reserved. Physicochemical and Biological Characterization of a Biosimilar Trastuzumab Sun, 17 May 2015 13:03:30 +0000 According to the World Health Organization, the incidence of malignant neoplasms and endocrine, blood, and immune disorders will increase in the upcoming decades along with the demand of affordable treatments. In response to this need, the development of biosimilar drugs is increasing worldwide. The approval of biosimilars relies on the compliance with international guidelines, starting with the demonstration of similarity in their physicochemical and functional properties against the reference product. Subsequent clinical studies are performed to demonstrate similar pharmacological behavior and to diminish the uncertainty related to their safety and efficacy. Herein we present a comparability exercise between a biosimilar trastuzumab and its reference product, by using a hierarchical strategy with an orthogonal approach, to assess the physicochemical and biological attributes with potential impact on its pharmacokinetics, pharmacodynamics, and immunogenicity. Our results showed that the high degree of similarity in the physicochemical attributes of the biosimilar trastuzumab with respect to the reference product resulted in comparable biological activity, demonstrating that a controlled process is able to provide consistently the expected product. These results also constitute the basis for the design of subsequent delimited pharmacological studies, as they diminish the uncertainty of exhibiting different profiles. Carlos A. López-Morales, Mariana P. Miranda-Hernández, L. Carmina Juárez-Bayardo, Nancy D. Ramírez-Ibáñez, Alexis J. Romero-Díaz, Nelly Piña-Lara, Víctor R. Campos-García, Néstor O. Pérez, Luis F. Flores-Ortiz, and Emilio Medina-Rivero Copyright © 2015 Carlos A. López-Morales et al. All rights reserved. Extraction Optimization for Obtaining Artemisia capillaris Extract with High Anti-Inflammatory Activity in RAW 264.7 Macrophage Cells Thu, 14 May 2015 13:34:10 +0000 Plant extracts have been used as herbal medicines to treat a wide variety of human diseases. We used response surface methodology (RSM) to optimize the Artemisia capillaris Thunb. extraction parameters (extraction temperature, extraction time, and ethanol concentration) for obtaining an extract with high anti-inflammatory activity at the cellular level. The optimum ranges for the extraction parameters were predicted by superimposing 4-dimensional response surface plots of the lipopolysaccharide- (LPS-) induced PGE2 and NO production and by cytotoxicity of A. capillaris Thunb. extracts. The ranges of extraction conditions used for determining the optimal conditions were extraction temperatures of 57–65°C, ethanol concentrations of 45–57%, and extraction times of 5.5–6.8 h. On the basis of the results, a model with a central composite design was considered to be accurate and reliable for predicting the anti-inflammation activity of extracts at the cellular level. These approaches can provide a logical starting point for developing novel anti-inflammatory substances from natural products and will be helpful for the full utilization of A. capillaris Thunb. The crude extract obtained can be used in some A. capillaris Thunb.-related health care products. Mi Jang, Seung-Weon Jeong, Bum-Keun Kim, and Jong-Chan Kim Copyright © 2015 Mi Jang et al. All rights reserved. The Impact of LEP G-2548A and LEPR Gln223Arg Polymorphisms on Adiposity, Leptin, and Leptin-Receptor Serum Levels in a Mexican Mestizo Population Sun, 10 May 2015 14:35:33 +0000 The polymorphisms in leptin (LEP G-2548A) and leptin-receptor (LEPR Gln223Arg) seem to influence obesity and lipid metabolism among others. The aim of this study was to investigate the effect of these polymorphisms on adiposity, leptin (sLeptin), and leptin-receptor (sLeptin-receptor) serum concentrations as well as inflammation markers. We included 382 adults originally from Western Mexico. They were genotyped by PCR-RFLP. Obese individuals showed higher sLeptin ( ng/mL) but lower sLeptin-receptor ( ng/mL) levels than normal weight ones ( ng/mL,  ng/mL, resp.), . Obese subjects carriers of Arg/Arg genotype had more () sLeptin-receptor ( ng/mL) and less () sLeptin ( ng/mL) levels than Gln/Gln genotype ( ng/mL,  ng/mL, resp.). Body fat mass was lower (P from 0.003 to 0.045) for A/A () or Arg/Arg () genotypes with respect to G/G () and G/A () or Gln/Gln () and Gln/Arg () genotypes carriers. Our results suggest that LEP -2548A and LEPR 223Arg could be genetic markers of less body fat mass accumulation in obese subjects from Western Mexico. Efraín Chavarria-Avila, Mónica Vázquez-Del Mercado, Eduardo Gomez-Bañuelos, Sandra-Luz Ruiz-Quezada, Jorge Castro-Albarran, Lizeth Sánchez-López, Beatriz Teresita Martín-Marquez, and Rosa-Elena Navarro-Hernández Copyright © 2015 Efraín Chavarria-Avila et al. All rights reserved. Novel 3′-Processing Integrase Activity Assay by Real-Time PCR for Screening and Identification of HIV-1 Integrase Inhibitors Thu, 07 May 2015 12:12:36 +0000 The 3′-end processing (3′P) of each viral long terminal repeat (LTR) during human immunodeficiency virus type-1 (HIV-1) integration is a vital step in the HIV life cycle. Blocking the 3′P using 3′P inhibitor has recently become an attractive strategy for HIV-1 therapeutic intervention. Recently, we have developed a novel real-time PCR based assay for the detection of 3′P activity in vitro. The methodology usually involves biotinylated HIV-1 LTR, HIV-1 integrase (IN), and specific primers and probe. In this novel assay, we designed the HIV-1 LTR substrate based on a sequence with a homology to HIV-1 LTR labeled at its 3′ end with biotin on the sense strand. Two nucleotides at the 3′ end were subsequently removed by IN activity. Only two nucleotides labeled biotin were captured on an avidin-coated tube; therefore, inhibiting the binding of primers and probe results in late signals in the real-time PCR. This novel assay has successfully detected both the 3′P activity of HIV-1 IN and the anti-IN activity by Raltegravir and sodium azide agent. This real-time PCR assay has been shown to be effective and inexpensive for a high-throughput screening of novel IN inhibitors. Supachai Sakkhachornphop, Weeraya Thongkum, and Chatchai Tayapiwatana Copyright © 2015 Supachai Sakkhachornphop et al. All rights reserved. Antioxidant Activity and Total Phenolic and Flavonoid Content of Various Solvent Extracts from In Vivo and In Vitro Grown Trifolium pratense L. (Red Clover) Wed, 06 May 2015 15:12:22 +0000 In the present study the extracts of in vivo and in vitro grown plants as well as callus tissue of red clover were tested for their antioxidant activities, using different extraction solvent and different antioxidant assays. The total flavonoid and phenolic contents as well as extraction yield of the extracts were also investigated to determine their correlation with the antioxidant activity of the extracts. Among all the tested extracts the highest amounts of total phenolic and total flavonoids content were found in methanol extract of in vivo grown plants. The antioxidant activity of tested samples followed the order in vivo plant extract > callus extract > in vitro extract. The highest reducing power, 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical scavenging, and chelating power were found in methanol extracts of in vivo grown red clover, while the chloroform fraction of in vivo grown plants showed the highest 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, superoxide anion radical scavenging and hydrogen peroxide scavenging compared to the other tested extracts. A significant correlation was found between the antioxidant activity of extracts and their total phenolic and total flavonoid content. According to the findings, the extract of in vitro culture of red clover especially the callus tissue possesses a comparable antioxidant activity to the in vivo cultured plants’ extract. Arash Khorasani Esmaeili, Rosna Mat Taha, Sadegh Mohajer, and Behrooz Banisalam Copyright © 2015 Arash Khorasani Esmaeili et al. All rights reserved. Benchtop Technologies for Circulating Tumor Cells Separation Based on Biophysical Properties Tue, 21 Apr 2015 06:04:13 +0000 Circulating tumor cells (CTCs) are tumor cells that have detached from primary tumor site and are transported via the circulation system. The importance of CTCs as prognostic biomarker is leveraged when multiple studies found that patient with cutoff of 5 CTCs per 7.5 mL blood has poor survival rate. Despite its clinical relevance, the isolation and characterization of CTCs can be quite challenging due to their large morphological variability and the rare presence of CTCs within the blood. Numerous methods have been employed and discussed in the literature for CTCs separation. In this paper, we will focus on label free CTCs isolation methods, in which the biophysical and biomechanical properties of cells (e.g., size, deformability, and electricity) are exploited for CTCs detection. To assess the present state of various isolation methods, key performance metrics such as capture efficiency, cell viability, and throughput will be reported. Finally, we discuss the challenges and future perspectives of CTC isolation technologies. Wan Shi Low and Wan Abu Bakar Wan Abas Copyright © 2015 Wan Shi Low and Wan Abu Bakar Wan Abas. All rights reserved. Peripheral Blood Mononuclear Cell Proteome Changes in Patients with Myelodysplastic Syndrome Thu, 16 Apr 2015 10:31:43 +0000 Our aim was to search for proteome changes in peripheral blood mononuclear cells (PBMCs) of MDS patients with refractory cytopenia with multilineage dysplasia. PBMCs were isolated from a total of 12 blood samples using a Histopaque-1077 solution. The proteins were fractioned, separated by 2D SDS-PAGE (pI 4–7), and double-stained. The proteomes were compared and statistically processed with Progenesis SameSpots; then proteins were identified by nano-LC-MS/MS. Protein functional association and expression profiles were analyzed using the EnrichNet application and Progenesis SameSpots hierarchical clustering software, respectively. By comparing the cytosolic, membrane, and nuclear fractions of the two groups, 178 significantly (, ANOVA) differing spots were found, corresponding to 139 unique proteins. Data mining of the Reactome and KEGG databases using EnrichNet highlighted the possible involvement of the identified protein alterations in apoptosis, proteasome protein degradation, heat shock protein action, and signal transduction. Western blot analysis revealed underexpression of vinculin and advanced fragmentation of fermitin-3 in MDS patients. To the best of our knowledge, this is the first time that proteome changes have been identified in the mononuclear cells of MDS patients. Vinculin and fermitin-3, the proteins involved in cell adhesion and integrin signaling, have been shown to be dysregulated in MDS. Klara Pecankova, Pavel Majek, Jaroslav Cermak, and Jan E. Dyr Copyright © 2015 Klara Pecankova et al. All rights reserved. Transgenic Carrot Expressing Fusion Protein Comprising M. tuberculosis Antigens Induces Immune Response in Mice Thu, 09 Apr 2015 11:48:23 +0000 Tuberculosis remains one of the major infectious diseases, which continues to pose a major global health problem. Transgenic plants may serve as bioreactors to produce heterologous proteins including antibodies, antigens, and hormones. In the present study, a genetic construct has been designed that comprises the Mycobacterium tuberculosis genes cfp10, esat6 and dIFN gene, which encode deltaferon, a recombinant analog of the human γ-interferon designed for expression in plant tissues. This construct was transferred to the carrot (Daucus carota L.) genome by Agrobacterium-mediated transformation. This study demonstrates that the fusion protein CFP10-ESAT6-dIFN is synthesized in the transgenic carrot storage roots. The protein is able to induce both humoral and cell-mediated immune responses in laboratory animals (mice) when administered either orally or by injection. It should be emphasized that M. tuberculosis antigens contained in the fusion protein have no cytotoxic effect on peripheral blood mononuclear cells. Natalia V. Permyakova, Alla A. Zagorskaya, Pavel A. Belavin, Elena A. Uvarova, Olesya V. Nosareva, Andrey E. Nesterov, Anna A. Novikovskaya, Evgeniy L. Zav’yalov, Mikhail P. Moshkin, and Elena V. Deineko Copyright © 2015 Natalia V. Permyakova et al. All rights reserved. Effect of Various Concentrations of Caffeine, Pentoxifylline, and Kallikrein on Hyperactivation of Frozen Bovine Semen Thu, 09 Apr 2015 09:18:13 +0000 Caffeine, pentoxifylline, and kallikrein are substances that affect the efficiency of sperms in the fertilization process; however, they have not been adequately studied. The present study aimed to examine the influence of caffeine, kallikrein, and pentoxifylline on sperm motility in bovine as well as investigate their optimum concentrations for increasing the movement of sperms in bovine. Frozen bovine sperms were thawed in universal IVF medium supplemented with 1, 5, and 10 mM caffeine or pentoxifylline or 1, 4, and 8 U/mL kallikrein and were then incubated for 30 min. Treated semen parameters were analyzed using a computer assisted semen analyzer (CASA). Data analysis showed that the mean values concerning progression and motility of sperm increased in caffeine and pentoxifylline treatments when compared with the kallikrein group. The obtained results revealed that kallikrein is not necessary for the improvement of bovine sperm motility. Additionally, our results revealed that 5 mM from caffeine was the best concentration added to the medium, followed by 1 or 5 mM from pentoxifylline. Therefore, it is concluded from the present study that caffeine has hyperactivation efficacy at 5 mM concentration compared to other treatments. Ibrahim A. H. Barakat, Mohamed A. Danfour, Fatma A. M. Galewan, and Mohamed A. Dkhil Copyright © 2015 Ibrahim A. H. Barakat et al. All rights reserved. Improvement of Starch Digestion Using α-Amylase Entrapped in Pectin-Polyvinyl Alcohol Blend Wed, 08 Apr 2015 13:23:52 +0000 Polyvinyl alcohol (PVA) and pectin blends were used to entrap α-amylase (Termamyl) using glutaraldehyde as a cross-linker. The effect of glutaraldehyde concentration (0.25, 0.5, 0.75, 1.0, and 1.25%) on the activity of the immobilized enzyme and rate of enzyme released was tested during a 24 h period. Characteristics of the material, such as scanning electron microscopy (SEM), tensile strength (TS), elongation, and rate of dissolution in water (pH 5.7), ruminal buffering solution (pH 7.0), and reactor containing 0.1 mol L−1 sodium phosphate buffer (pH 6.5), were also analyzed. SEM results showed that the surfaces of the pectin/PVA/amylase films were highly irregular and rough. TS values increased as a function of glutaraldehyde concentration, whereas percentage of elongation (%E) decreased. Pectin/PVA/amylase films presented similar values of solubility in the tested solvents. The material obtained with 0.25% glutaraldehyde performed best with repeated use (active for 24 h), in a phosphate buffer reactor. By contrast, the material obtained with 1.25% glutaraldehyde presented higher performance during in vitro testing using an artificial rumen. The results suggest that pectin/PVA/amylase is a highly promising material for biotechnological applications. Maurício Cruz, Kátia Fernandes, Cristine Cysneiros, Reginaldo Nassar, and Samantha Caramori Copyright © 2015 Maurício Cruz et al. All rights reserved. Metabolic Engineering of Escherichia coli for Poly(3-hydroxybutyrate) Production under Microaerobic Condition Tue, 07 Apr 2015 16:54:14 +0000 The alcohol dehydrogenase promoter and mixed acid fermentation pathway deficient mutants of Escherichia coli were employed to produce poly(3-hydroxybutyrate) (P3HB) under microaerobic condition. The E. coli mutant with ackA-pta, poxB, ldhA, and adhE deletions accumulated 0.67 g/L P3HB, up to 78.84% of cell dry weight in tube cultivation. The deletion of pyruvate formate-lyase gene pflB drastically decreased P3HB production and P3HB content to 0.09 g/L and 24.44%, respectively. Overexpressing pflB via the plasmid in its knocked out mutant restored cell growth and P3HB accumulation, indicating the importance of the pyruvate formate-lyase in microaerobic carbon metabolism. The engineered E. coli BWapld (pWYC09) produced 5.00 g/L P3HB from 16.50 g/L glucose in 24 h batch fermentation, and P3HB production yield from glucose was 0.30 g/g, which reached up to 63% of maximal theoretical yield. Xiao-Xing Wei, Wei-Tao Zheng, Xue Hou, Jian Liang, and Zheng-Jun Li Copyright © 2015 Xiao-Xing Wei et al. 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.